AActa agriculturae Slovenica • eISSN 1854-1941 • 117 – 4 • Ljubljana, december 2021 117•4 2021ActA AgrIcuLturAe SLoveNIcA Acta agriculturae Slovenica Letnik / Volume 117 · Številka / Number 3 · 2021 eISSN 1854-1941 Glavna in odgovorna urednika / Editors-in-Chief Franc BATIČ, rastlinska pridelava / plant production Jernej OGOREVC, živalska prireja / animal production Področni uredniki / Section Editors Franc BATIČ (botanika in ekologija rastlin / botany and plant ecology), Majda ČERNIČ-ISTENIČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural development), Jure ČOP (pridelovanje krme / fodder production), Zalika ČREPINŠEK (agrometeorolologija / agrometeorology), Marko FLAJŠMAN (poljedelstvo / field crops), Matjaž GLAVAN (urejanje kmetijskih zemljišč / agricultural land management), Helena GRČMAN (pedologija / soil science), Andrej GREGORI (gojenje gob / mushrooms growing), Metka HUDINA (hortikultura / horticulture), Anton IVANČIČ (genetika in biotehnologija / genetics and biotechnology), Jernej JAKŠE (genetika in biotehnologija / genetics and biotechnology), Damjana KASTELEC (statistika / statistics), Aleš KOLMANIČ (poljedelstvo / field crops), Zlata LUTHAR (genetika in biotehnologija / genetics and biotechnology), Andrej LAVRENČIČ (pridelovanje krme / fodder production), Marina PINTAR (urejanje kmeti- jskih zemljišč / agricultural land management), Andrej SIMONČIČ (varstvo rastlin / plant protection), Stanislav TRDAN (var- stvo rastlin / plant protection), Andrej UDOVČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural de- velopment), Andreja URBANEK-KRANJC (fiziologija rastlin / plant physiology), Rajko VIDRIH (živilstvo / food technology), Dominik VODNIK (fiziologija rastlin / plant physiology), Filip VUČANJK (kmetijsko strojništvo / agricultural machinery) Peter DOVČ (živalska biotehnologija / animal biotechnology, populacijske študije / population studies, genomika / genomics), Milena KOVAČ (selekcija in biometrija / selection and biometry), Janez SALOBIR (prehrana / nutrition) Mednarodni uredniški odbor / International Editorial Board Dunja BANDELJ (Koper, Slovenia), Michael BLANKE (Bonn, Germany), Ivan N. FESENKO (Orel, Russia), Marko FLAJŠMAN (Ljubljana, Slovenia), Jürg FUHRER (Liebefeld-Bern, Switzerland), Helena GRČMAN (Ljubljana, Slovenia), Metka HUDINA (Ljubljana, Slovenia), Anton IVANČIČ (Maribor, Slovenia), Lučka KAJFEŽ BOGATAJ (Ljubljana, Slovenia), Damijana KASTELEC (Ljubljana, Slovenia), Iztok Košir (Žalec, Slovenija), Chetan KESWANI (Varanasi, India), Ivan KREFT (Ljubljana, Slovenia), Jaromír LACHMAN (Prague, Czech Republic), Mario LEŠNIK (Maribor, Slovenia), Zlata LUTHAR (Ljubljana, Slovenia), Senad MURTIĆ (Sarajevo, Bosnia and Herzegovina), Alessandro PERESSOTTI (Udine, Italy), Hardy PFANZ (Essen, Germany), Slaven PRODANOVIĆ (Belgrade, Serbia), Naser SABAGHNIA (Maragheg, Iran), Olalekan Sulei- man SAKARIYAWO (Abeokuta, Nigeria), Andrej SIMONČIČ (Ljubljana, Slovenia), Giuseppe SORTINO (Palermo, Italy), Bojan STIPEŠEVIĆ (Osijek, Croatia), Massimo TAGLIAVINI (Bolzano, Italy), Željko TOMANOVIĆ (Beograd, Serbia), Stanislav TRDAN (Ljubljana, Slovenia), Andrej UDOVČ (Ljubljana, Slovenia), Rajko VIDRIH (Ljubljana, Slovenia), Dominik VODNIK (Ljubljana, Slovenia), Alena VOLLMANNOVA (Nitra, Slovak Republic) Drago BABNIK (Ljubljana, Slovenia), Tomaž BARTOL (Ljubljana, Slovenia), Michel BONNEAU (Saint Gilles, Belgium), Milena KOVAČ (Ljubljana, Slovenia), Amarendra Narayan MISRA (Balasore, Orissa, India), Zdenko PUHAN (Zürich, Switzerland), Dejan ŠKORJANC (Maribor, Slovenia), Jernej TURK (Maribor, Slovenia) Tehnični uredniki / Technical Editors Karmen STOPAR, Jure FERLIN, Jože STOPAR Oblikovanje / Graphic art and design Milojka ŽALIK HUZJAN Jezikovni pregled / Proofreading Avtorji v celoti odgovarjajo za vsebino in jezik prispevkov / The authors are responsible for the content and for the language of their contributions. 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Acta agriculturae Slovenica izhaja s finančno pomočjo / is published with the financial support: Javne agencije za raziskovalno dejavnost Republike Slovenije / Slovenian Research Agency. Acta agriculturae Slovenica je vključena v / is included into: Scopus (SJR, SNIP), DOAJ, WOS Zoological Records, CrossRef, CAB Abstracts, FSTA, Google Scholar, dLib, COBISS. Ovitek: Oblika plodov preučevanih sort pistacije (črke označujejo sorto glede na preglednico 1; merilo 5 mm); (foto: Fariba Sharifnia, 1–10) Cover: Fruit shape of the investigated pistachio cultivars (the letters indicate cultivars name according to Table 1, scale bar 5 mm); (photo: Fariba Sharifnia, 1–10) Acta agriculturae Slovenica Volume / Letnik 117 · Number / Številka 4 · 2021 Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers Analiza genetske raznolikosti izbranih akcesij sladkornega trsa (Saccharum officinarum L.) z uporabo označevalcev na osnovi enostavnih ponavljajočih se zaporedij (ISSR) Faith Ewewluegim EMEGHA, David Adedayo ANIMASAUN, Folusho BANKOLE, Gbadebo OLAOYE 1–10 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) The effect of season on in vitro degradability and fermentability of feeds in red deers’ (Cervus elaphus L.) rumen fluid Andrej LAVRENČIČ, Darko VETERNIK 1–11 Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics of ‘Saint-Pierre’ tomatoes grown in Algeria Vpliv ozonirane vode za namakanje na morfološke, bakteriološke in senzorične lastnosti paradižnika ‘Saint-Pierre’, rastočega v Alžiriji Fouzia BENALI, Nadia RAMDANI 1–10 Effects of soil nutrient amendments on growth and grain yield performances of quality protein maize grown under water deficit stress in Ibadan, Nigeria Učinki gnojenja na rast in pridelek zrnja na proteinih obogatene koruze v razmerah sušnega stresa, Ibadan, Nigeria Folake Bosede ANJORIN, Adeyinka ADEBAYO, Taiwo OMODELE, Adewale ADETAYO, James ADEDIRAN 1–14 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on se-lected weed species Fitotoksični učinki eteričnih olj iz dveh vrst mačje mete (Nepeta glocephalata Rech.f. in N. ispahanica Boiss.) na izbrane vrste plevelov Marjan DYANAT, Farzad ASGARI 1–12 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water for long periods of time through the CO2 efflux Ovrednotenje biokemičnih obravnavanj onesnaženih tal, ki so bila dalj časa namakana z vodo slabe kakovosti z meritvijo sproščanja CO2 Mohamed SABER, Alaa M ZAGHLOUL 1–12 Influence of plant growth regulators and salicylic acid on the production of some secondary metabolites in callus and cell suspension culture of Satureja sahendica Bornm. Vpliv rastlinskih rastnih regulatorjev in salicilne kisline na tvorbo nekaterih sekundarnih metabolitov v kalusu in suspenziji celične kulture vrste šetraja Satureja sahendica Bornm. Sarieh TARIGHOLIZADEH, Rouhollah MOTAFAKKERAZAD, Morteza KOSARI-NASAB, Ali MOVAFEGHI, Sakineh MOHAMMADI, Mohsen SABZI, Amir-Hossein TALEBPOUR 1–12 In vitro antifungal potential of surfactin isolated from rhizospheric Bacillus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe In vitro protiglivni potencial surfaktina, izoliranega iz bakterije Bacillus thuringiensis Berliner 1915 iz rizosfere koruze (Zea mays L.) proti patogeni glivi Fusarium graminearum Schwabe Muddasir KHAN, Muhammad SALMAN, Syed Hussain SHAH, Muhammad ISRAR 1–7 Expression of IRT1 gene in barley seedlings under zinc deficiency at optimal and low temperatures Izražanje IRT1 gena v sejankah ječmena ob pomanjkanju cinka pri optimalnih in nizkih temperaturah Natalia KAZNINA, Natalia REPKINA, Yulia BATOVA, Alexandr TITOV 1–5 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance applied at the beginning of vegetative and reproductive stages Ovrednotenje lastnosti korenin krušne pšenice (Triticum aestivum L.) povezanih s sušnim stresom v začetku vegetativne in reproduktivne faze razvoja Arman ESKANDARI SHAHRAKI, Mohammad-Mehdi SOHANI, Sadollah HOOSHMAND, Ali AALAMI, Habibullah SAMIZADEH 1–14 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria Razvrstitev odločitvenih dejavnikov povezanih s problemi namakanja zelenjave s faktorsko analizo na območju upravne enote Swaida, Sirija Maya AL-ABDALA, Afraa SALLOWM, Safwan ABOUASSAF 1–13 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes Uvajanje najboljših kriterijev za ovrednotenje tolerance na sušo pri genotipih sirka Leyla NAZARI, Ebrahim DEHGHANIAN, Afshar ESTAKHR, Azim KHAZAEI, Behzad SORKHILALEHLOO, Mohammad Reza ABBASI 1–13 Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard Vpliv obtežitve na pridelek in kakovost grozdja sort Merlot in Vranac (Vitis vinifera L.) v vinogradu v Trebinju Mersija DELIĆ, Fikreta BEHMEN, Saša MATIJAŠEVIĆ, Šaćira MANDAL, Saud HAMIDOVIĆ, Senad MURTIĆ 1–7 Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross Višina rastlin, masa slame in površina lista zastavičarja so od fiziologov prepoznane morfološke lastnosti, ki nakazujejo toleranco na sušni stres Manel SALMI, Zine El Abidine FELLAHI, Abdelkader BENBELKACEM, Amar BENMAHAMMED, Hamenna BOUZERZOUR 1–10 Review Article / Pregledni znanstveni članek Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline Endophytic fungi as biological control agents and their indirect effects on plants Primož ŽIGON, Franci Aco CELAR 1–12 Short Scientific Article / Kratki znanstveni prispevek Development of efficient integrated management package against sweet potato weevil (Cylas formicarius [Fabricius, 1798]) Razvoj učinkovitega integriranega načina zatiranja hrošča Cylas formicarius (Fabricius, 1798) na sladkem krompirju Mohammad Monirul Hasan TIPU, Raunak JAHAN, Jubaidur RAHMAN, Md Mizanur RAHMAN, Md Ariful ISLAM, Md Mosiur Rahman Bhuyin APU 1–4 Acta agriculturae Slovenica, 117/4, 1–10, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2015 Original research article / izvirni znanstveni članek Analysis of genetic diversity in selected sugarcane (Saccharum officinar- um L.) accessions using inter simple sequence repeat (ISSR) markers Faith Eweluegim EMEGHA 1, 2, David Adedayo ANIMASAUN 3, Folusho BANKOLE 1, Gbadebo OLAOYE 1 Received December 24, 2020; accepted August 19, 2021. Delo je prispelo 24. decembra 2020, sprejeto 19. avgusta 2021 1 Department of Agronomy, Faculty of Agriculture, University of Ilorin, Kwara State, Nigeria 2 Corresponding author, e-mail: faith.emegha84@gmail.com 3 Department of Plant Biology, Faculty of Life Sciences, University of Ilorin, Kwara State, Nigeria Analysis of genetic diversity in selected sugarcane (Saccha- rum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers Abstract: Genetic diversity information among a popu- lation is important in exploiting heterozygosity for the im- provement of crop species through breeding programmes. This study was therefore, conducted to assess genetic diversity and establish molecular relationships among 20 selected exotic sug- arcane accessions from the Unilorin Sugar Research Institute germplasm using Inter Simple Sequence Repeat (ISSR) molecu- lar markers. Genomic DNA was extracted from the sugarcane leaf. Fragments amplification was then performed by poly- merase chain reaction (PCR) with ISSR markers and the data obtained were analyzed using MEGA 4 software. Analysis of the electropherogram showed a total of 39 loci consisting of 369 bands, out of which 95.8% were polymorphic. The biplot analy- sis showed all the markers contributed to the observed diversity with the least achieved with ISSR6. The principal co-ordinate analysis grouped the accessions into four clusters, comprising mixtures of all the six collection sites. The polymorphism ob- tained in the present study showed that the ISSR markers are effective for assessment of genetic diversity of the sugarcane ac- cessions as it reveals the genetic similarity or divergence of the accessions regardless their place of origin or cultivation. Key words: Dendrogram; genetic diversity; germplasm resources; ISSR marker; sugarcane Analiza genetske raznolikosti izbranih akcesij sladkornega trsa (Saccharum officinarum L.) z uporabo označevalcev na osnovi enostavnih ponavljajočih se zaporedij (ISSR) Izvleček: Informacija o genetski raznolikosti znotraj po- pulacij je pomembna za uporabo heterozigotičnosti za izbolj- šanje gojenih rastlin v žlahtniteljskih programih. Ta raziskava je bila narejena za oceno genetske raznolikosti in vzpostavitev molekularnih povezav med 20 izbranimi ekzotičnimi akce- sijami sladkornega trsa na Inštitutu za preučevanje genetskih resursov sladkornega trsa v Unilorinu (Sugar Research Insti- tute germplasm) z uporabo molekularnih markerjev na osnovi enostavno ponavljajočih se zaporedij. Genomska DNK je bila ekstrahirana iz listov sladkornega trsa s pomočjo mini kita (DNeasy Mini Kit,Qiagen). Namnožitev fragmentov je bila iz- vedena s polimerazno verižno reakcijo (PCR) z ISSR označeval- ci, pridobljeni podatki so bili analizirani s programom MEGA 4. Analiza elektroferogramov je pokazala, da je celokupno šte- vilo 39 lokusov sestavljalo 369 trakov, od katerih je bilo 95,8 % polimorfnih. Biplot analiza je pokazala, da so vsi označevalci prispevali k opaženi raznolikosti, z najmanjšim deležem ozna- čevalca ISSR6. Analiza glavnih komponent je združila akcesije v štiri skupine, ki so bile mešanica vzorcev iz vseh šestih vzor- čenih mest. Polimorfizem, ugotovljen v tej raziskavi je pokazal, da so vsi ISSR označevalci učinkoviti za ugotavljanje genetske raznolikosti akcesij sladkornega trsa ne glede na njihovo mesto izvora in načina gojenja. Ključne besede: dendrogram; genetska raznolikost; ge- netski viri; ISSR označevalci; sladkorni trs Acta agriculturae Slovenica, 117/4 – 20212 F. E. EMEGHA et al. 1 INTRODUCTION Sugarcane (Saccharum officinarum L.) is one of the most important economic crops. In Nigeria, sugarcane is mainly grown for its sugar juice, as a raw material for manufacturing sugar (Wayagari et al., 2003a,b), molasses and bagass, and recently ethanol and renewable energy. As an industrial crop, sugarcane production has con- tributed to the nation’s GDP and provided the opportu- nities for job creation. Other use of sugarcane include; fertilizer, bio-plastics, paper, sugarcane wax (Khushk and Pathan, 2006). Commercial cultivation is mainly through plant- ing of vegetative cutting (setts) of mature stalks. During sugarcane breeding programs, exchange and shipment of elite clones and breeding lines in the form of stalk cut- tings across different test locations occur regularly for the purpose of verifying parental source or desired use of a clone in an experiment (Pan, 2010). To increase sugar yield within the Nigerian sugar industry, it is important to optimize varietal trials and breeding activities of sug- arcane (NSDC, 2015). In Sugarcane breeding, mislabeling errors could oc- cur during the process of planting and selection, due to the use of large number of accessions in the varietal de- velopment program, which can only be revealed later in the selection program. Mislabeling could alter breeding goals by using the wrong variety for breeding activities. Molecular tools ensure that breeders have the correct clones involved in their crosses as well as varietal trials (Pan, 2010). Diversity analysis based on morphological attributes may not be sufficient or may be inflated due to environmental influences, particularly for new va- rieties or those that are new to a region (Animasaun et al., 2015). In these situations, a more accurate and clear means of identification is required to avoid downstream consequences such as risk of disease outbreak and/or poor productivity. The use of molecular marker techniques such as RFLP (Restriction fragment length polymorphism), SSLP (Simple sequence length polymorphism), AFLP (Amplified fragment length polymorphism), RAPD (Random amplification of polymorphic DNA), ISSR (Inter simple sequence repeat), SSR Microsatellite poly- morphism (Simple sequence repeat), SNP (Single nu- cleotide polymorphism), RAD markers (Restriction site associated DNA makers) etc, in the analysis of genetic variation among genetic materials, has facilitated correct determination of the nature of association among eco- nomic traits. This is because the techniques ensure devel- opment of accurate genetic maps since they are devoid of environmental influences, thereby helping to achieve set objectives within breeding programs leading to the achievement of significant yield increases in breeding programmes (Dilon et al., 2007). The use of Inter Simple Sequence Repeat (ISSR) marker in crops plant diversity study and fingerprinting is advantageous (Animasaun et al., 2015). The markers can detect a range of loci and allelic diversity among the genetic materials (Ajibade et al., 2000, Pfeiffer et al., 2011) as well as provide information on their unique identity that deserves conservation attention and improvement programmes (Da Costa et al., 2011, Animasaun et al., 2021). Furthermore, molecular-based information ob- tained would be a reliable basis for developing a template and workable strategy for germplasm conservation and future improvement through the selection of appropri- ate parents to maximize yield, establishment of proper identity of the genotypes and maintain genetic diversity. The approach also has the capacity to provide useful in- formation on the extent of genetic diversity among the germplasm accessions, and prevent possible misidentifi- cation, which may render the work previously done dur- ing selection unreliable. The ultimate goal in sugarcane breeding is to devel- op genetically improved varieties with high sugar yield (cane yield and sucrose content) that is economically sustained over several ratoon crops. Therefore, germ- plasm materials are usually assessed for their breeding behaviour with the objective of utilizing them either for direct cultivation on the sugar estates or as parents in hy- bridization for evolving new and superior progenies in- tended as replacement to the existing cultivars (Kwajaffa & Olaoye, 2014). In an effort to identify productive sug- arcane varieties for the rainforest and savanna ecologies of Nigeria, a detailed evaluation of 40 selected sugarcane varieties from six (6) breeding stations was conducted under the auspices of the West Africa sugarcane Devel- opment (WASD) Project (Olaoye et al. 2017), which pro- vided some information on morphology, cane yield and yield components. However, in order to have a proper diagnostic as- sessment of the genetic attributes of these varieties, twenty accessions were further selected for genetic di- versity and allelic polymorphism assessment using the molecular approach. The objectives were to characterize the selected sugarcane accessions using ISSR marker and provide detailed information on the nature of genetic diversity among them for further varietal improvement activities. Acta agriculturae Slovenica, 117/4 – 2021 3 Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL Twenty (20) exotic sugarcane accessions were se- lected from the pool of germplasm materials from six sugarcane breeding stations currently being maintained at the Unilorin Sugar Research Institute (USRI) Farm. The accessions were selected base on the morphological superiority reported elsewhere (Olaoye et al., 2017). De- tails of their origin, parentage (where available) and yield attributes are contained in Table 1. 2.2 DNA ISOLATION Genomic DNA (gDNA) was isolated from young unfolding leaf tissues. About 1 g of fresh leaf tissue was grounded into a fine powder in prechilled mortar and genomic DNA from individual accession was extracted using, DNAeasy Plant Mini Kit (QIAGEN, USA). The DNA extraction was performed in accordance with the manufacturer’s instruction. DNA concentration were determined comparatively by electrophoresis at current of 100 amps, 80 volts, for 40 minutes using agarose gel (0.8  %) electrophoresis, by applying 5  μl gDNA loaded after mixing with 3 μl 6X loading dye (Promega, USA) to check the quality of the DNA by comparing the inten- sity of the bands with a 1kb standard (Thermo Scientific, USA). The gel was visualized under a UV transillumina- tor and the gel was imaged with a gel documentation sys- tem (Ingenius-3, Syngene, USA) to confirm the quality of the genomic DNA. Parental identification S/N Variety Point of collection oBrix content (°Bx): Cane yield (t ha-1) Female Male 1 B97375 Barbados NA NA 2 B96812 Barbados NA NA 3 B96723 Barbados NA NA 4 B93757 Barbados 23.71 58.52 5 B47419+ Barbados 20.13 72.89 6 DB8134 Demarara NA NA 7 M1176/77 Mauritius 22.31 81.91 N55805 CP5530 8 M1246/84 Mauritius 21.2 93.40 M555/60 R570 9 M1334/84 Mauritius 19.58 95.55 M555/60 10 M1954/91 Mauritius 21.89 69.38 M2077/78 M1030/71 11 RB72/454 Brazil 21.26 58.89 CP53-76 12 RB86/3129 Brazil 21.63 608.09 RB763411 13 SP81-3250 Brazil 20.53 72.09 CP70-1547 SP71-1279 14 RB94/2991 Brazil 22.89 66.42 15 Co88025 Coimbatore 20.92 59.00 16 Co91017 Coimbatore 21.27 64.64 17 Co997+ Coimbatore 21.48 79.29 18 CoC671 Coimbatore 22.63 72.80 19 KNB9288 Sudan NA NA B871245 POLY 20 KNB9253 Sudan NA NA Table 1: List of selected 20 Sugarcane varieties and their attributes Brix content (°Bx): This is the proportion of sucrose in a solution, therefore its correlates with density of liquid. One degree brix is 1 g of sucrose in 100 g of solution Sources: G. Olaoye, Y. A Abayomi and F.O .Takim (2017). NSDC 2015 NA: Information not available Acta agriculturae Slovenica, 117/4 – 20214 F. E. EMEGHA et al. 2.3 ISSR PRIMER SELECTION AND PCR CONDI- TION Eight reproducible and informative ISSR primers of 14-19 bp were selected from a total of 10 tested ISSR primers. The primers were selected based on published experimental results on sugarcane and related Saccha- rum species (Da Costa et al., 2011). The selected primers synthesized by a commercial molecular biology company (Inqaba Biotec West Africa Ltd. Ibadan, Nigeria) were used for the Polymerase Chain Reaction (PCR) proce- dure. The primers were optimized and PCR conditions for experiment were set-up. For an efficient molecular characterization of sugarcane genotypes, initially six in- dividuals were randomly selected to screen the primers for their polymorphism and reproducibility at 52, 54, 57, 61, 62 and 65 °C annealing temperature (Table 2). Band intensity and reproducibility of all conditions were com- pared and optimized. The selection includes a spectrum of primers with different repeat motifs. The PCR reaction was carried out with 20  μl fi- nal reaction volume in 200  μl thin wall PCR tube in a Thermocycler (Applied Biosystems, Foster city, USA), containing 10.5 μl reaction mixture, 1.5 μl of 10 pmole primer 1 μl of 50 ng genomic DNA and 7 μl of ultra-pure nuclease free water (Ambion, USA). The PCR condition was an initial denaturation at 94  °C for 5 minutes, fol- lowed by 35 cycles of denaturation at 94 °C for 1 sec, an- nealing at 59 °C (annealing temperature was different for each primers) for 30 sec and extension at 72 °C for 1 min followed by a single cycle of final extension at 72 °C for 10 min. The reaction was put on hold at 4 °C. The PCR products were visualized to confirm amplification by the method of Animasaun et al., 2018. The targeted PCR amplification was confirmed by mixing 5 μl of PCR product with 2 μl  of 6X gel 6X gel loading dye (Promega, USA) and electrophoresed on 1.5  % agarose gel stained with 0.75  µl EN-Vision blue eye DNA dye for 40 min at 100 volts in 1X TBE buffer. Fragment sizes of the amplicons were determined from the gel by comparison with standard molecular weight marker ladder-low range Generuler1  Kb DNA Ladder (Thermofisher, USA). The amplified loci were visualized and photographed in a gel documentation system (Inge- nius-3, Syngene, USA). 2.4 BAND SCORING AND DATA ANALYSIS The PCR fragments were scored for the presence (1) or absence (0) of equally sized bands and two matrices of the different ISSR phenotypes were assembled and used in the statistical analysis. The fragments were only con- sidered on ability to detect clearly resolved and polymor- phic amplified loci among the populations studied for the eight ISSR primers selected for analysis. The data were entered in to binary matrix for analysis. Module analysis was performed with NTSYS-pc (Numerical Taxonomy and Multivariate Analysis System) and Cluster Analysis was performed using the unweighted pair group method with arithmetic averages (UPGMA), genetic differentia- tion and Shannon’s index (I) was determined using PAST software. In addition, to compare genotypes and evaluate pat- terns of genotype clustering, neighbour-joining (NJ) was used with Free Tree 0.9.1.50 (Saitou & Nei, 1987; Pavliček et al., 1999). To further examine patterns of genetic re- lationship among individual genotypes principal coor- dinate analysis (PCoA) was performed using agglom- erative technique using the Un-weighted Pair Group Method with Arithmetic Mean (UPGMA) Method and dendrogram was constructed as the output for the ge- netic relationship. S/N Oligo ID Sequence (5’ – 3’) *Tm (o C) No of base pairs GC- contents (%) 1 ISSR 1 GAGAGAGAGAGACC 52.61 14 57.14 2 ISSR 2 CTCTCTCTCTCTCTCTAC 57.62 18 50.00 3 ISSR 3 CACACACACACAAG 49.69 14 50.00 4 ISSR 4 CAGCACACACACACACA 60.16 19 52.63 5 ISSR 5 GTGTGTGTGTGTCC 52.61 14 57.14 6 ISSR 6 CTCTCTCTCTCTCTCTGC 59.9 18 55.56 7 ISSR 8 AGCACGAGCAGCAGCGG 64.43 17 70.59 8 ISSR 10 AGCACGAGCAGCAGCGT 62.02 17 64.71 Table 2: List and properties of ISSR primers selected for the molecular study of the molecular characterization of 20 exotic sugar- cane varieties (Ilorin, Nigeria) *Tm: melting temperature of the primers Acta agriculturae Slovenica, 117/4 – 2021 5 Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers 3 RESULT AND DISCUSSION Genetic diversity in sugarcane provides breeders with the necessary materials and opportunity to develop improved and new varieties possessing desirable char- acteristics (Govindaraj et al., 2015). Molecular analysis is important to protect genetic identity/purity because, morphological traits could be environmentally influ- enced (Animasaun et al., 2018). This is also necessary for authentication of accession prior to multiplication of setts for planting. Data obtained from molecular tech- niques can be analysed prior to application in diversity studies by analyzing the genetic relationship among sam- ples (Govindaraj et al., 2015). 3.1 LEVEL OF POLYMORPHISM The ISSR analysis, carried out on 20 varieties pro- duced 369 bands/alleles with an average of 46.13 alleles per primer. Eight primers produced distinct and repro- ducible bands among the primers tested and the ampli- fied PCR products showed arrays of monomorphic and polymorphic bands, 349 were polymorphic alleles and 20  monomorphic alleles with an average of 43.25 ISSR polymorphic alleles per primer. In other words, the ISSR markers detected high level of polymorphism. A total of 39  loci were amplified by eight ISSR primers, out of which 38 (91.66  %) were polymorphic. Loci amplifica- tion per primer ranged from 3 to 7 with an average of 4.88 loci per primer, and a mean allelic richness of 46.1 alleles/primers (Table 3), number of polymorphic alleles ranged from 2 (ISSR 6) to 7 (ISSR8). The code, sequence and other properties of the primers used are presented in Table 2. Though, seven out of eight of the primers were highly polymorphic, ISSR6 resulted in the least polymor- phic loci with 33.3  % polymorphism, ability of primer ISSR 8 and ISSR 10 to produce higher allele frequencies and polymorphic loci in this study indicated that the two primers are most informative and suitable for diversity study in sugarcane accessions. ISSR markers have been showed to possess high resolution ability in sugarcane fingerprinting and diver- sity analysis (Da Costa et al., 2011), they are effective and efficient in the identification of polymorphisms within and among populations and/or species. This current study is the first investigation to assess molecular genetic diversity within and among introduced sugarcane acces- sions in the USRI germplasm. Again, since polymorphic information is related to expected heterozygosity and is usually determined from allele frequency (Animasaun et al., 2015), the existing variation in the studied accession could be selected for the crop improvement. The present study reveals the existence of high level of genetic diversity and relatedness among and within the investigated sugarcane accessions, which were intro- duced into Nigeria as part of University of Ilorin Sugar Research Institute (USRI) germplasm. Similar findings were reported for some sugarcane accessions by Srivas- tava and Guota (2008) who recorded 78.48 % polymor- phism in a diversity screening among sugarcane varieties in India using ISSR markers. Smiullah et al. (2013) also detected 85.25 % polymorphism with ISSR markers on Sugarcane accessions from Pakistan. Thus, the studied genotypes showed considerable heterologous amplifica- tion of the alleles, whereby 91.66  % were polymorphic and only 8.34  % were monomorphic. High polymor- phism and higher number of alleles are very important for correct estimation of genetic diversity of a germplasm (Animasaun et al., 2015). The degree of polymorphism S/N Marker Code TNA TNL NML NPL P % M % 1 ISSR1 32 5 0 5 100 - 2 ISSR 2 34 5 0 5 100 - 3 ISSR 3 16 4 0 4 100 - 4 ISSR4 50 5 0 5 100 - 5 ISSR 5 49 4 0 4 100 - 6 ISSR 6 30 3 1 2 33.3 66.6 7 ISSR 8 81 7 0 7 100 - 8 ISSR 10 77 6 0 6 100 - TOTAL Average 369 46.1 39 4.88 1 38 91.66 % 8.3% Table 3: Amplification information of 8 ISSR markers used in the diversity study of the 20 exotic sugarcane accessions in the germplasm of USRI TNA: Total number of alleles; TNL: total number of loci; NML: Number of monomorphic loci; NPL: Number of polymorphic loci; P %: percentage polymorphism; M %: Percentage monomorphism Acta agriculturae Slovenica, 117/4 – 20216 F. E. EMEGHA et al. showed the extent of diversity and effectiveness of the markers (Pfeifer et al., 2011) and allele phenotype are use as reference to interpret microsatellite profile in diversity studies (Esselink et al., 2004). 3.2 GENETIC SIMILARITY AND DISTANCE Estimate of similarity coefficient was determined using Jaccard similarity coefficient-based pairwise com- parisons, based on the DNA amplification of the 20 ac- cessions of sugarcane (supplementary table 1), similarity ranged from 0.78 to 0.13 with mean of 0.455. Accessions DB8134 and M1246/84 showed the highest genetic simi- larity having similarity coefficient of 0.78 and were ad- judged to be closely related. However, the maximum ge- netic distance was observed between accessions DB8134 and SP81-3250 with 0.13, M1246/84 and SP81-3250, B74541 and SP81-3250 had a genetic similarity coeffi- cient of 0.136 each, followed by M1334/4 and SP81-3250 with genetic distance of 0.148. 3.3 BIPLOT ANALYSIS The distribution of the accessions into different spatial plane and co-ordinates by biplot analysis (Fig 1) showed the involvement of markers in separating ac- cessions into quadrants. Co-occurrence of B74541 and M1246/84 in quadrant three and B80689 and Co88025 in quadrant four on the same plane suggested a common ancestor. The overlapping of B85266 and B93757, which are close to RB72/454, indicated the accessions are ge- netically similar. Samples in quadrant I, III and IV are more closely related, except for M1954/91, while those in quadrant II are diffused i.e. they are relations separated by geographical isolation for a long time (Animasaun et al., 2015). The spatial closeness of the accessions in biplot analysis indicate their genetic similarity. In addition, the dispersion of the markers from the centroid reflects their effectiveness in delimiting the accessions (Animasaun et al., 2015). However, the distant location of M1954/91, SP81-3250 and DB8134 in quadrant I, II and IV implies the existence of genetic distance. This may be due to ac- cumulation of some genes through selection resulting from domestication of the accessions by local farmers (Animasaun et al., 2015). Meanwhile the obtained mark- er efficiency as revealed by biplot analysis supported ISSR makers as a useful tool for the initial assessment of intra- specific genetic variation (Devarumath et al., 2012). Figure 1: Bootstrapped Biplot of twenty accessions of sugarcane accessions characterized by eight ISSR primers for diversity and genotyping analysis (at p < 0.05) Acta agriculturae Slovenica, 117/4 – 2021 7 Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers 3.4 CLUSTER ANALYSIS Neighbor Joining-based and UPGMA tree con- struction methods on the basis of Jaccard’s similarity co- efficient was used to construct dendrogram to examine the relationship among sugarcane accessions based on 369 ISSR bands amplified by eight primers. The dendro- gram derived from UPGMA-based cluster analysis of the whole ISSR data with 20 sugarcane accessions shows all the accessions in the first cluster were clustered closely together which shows high genetic similarity consisting of nine (9) accessions (group 2). Cluster analysis by den- drogram depicting the genetic relationship classified the accessions into to group (1 and 2). On the other hand, the second group (group 2) sub-divided into 2a and 2b (Fig 3). The 2b group split into two clusters, 2b(i) and 2b(ii) (Fig 3). Cluster 2a is comprised of four genotypes, cluster 2b(i) and 2b(ii) is made up of three and four ac- cessions respectively. The different accessions formed clusters irrespective of their different geographical ori- gins. The neighbor joining (Fig 2) showed that B97375 is a distance neighbors from the other accessions. In ad- dition, RB86/3129 and Co997+ were joined together as neighbours, M134/84 was a close neighbor to Co88025. Also, B96723 was the closest neighbour to RB72/454. Clustering parameters such as, Principal coor- dinate analysis, UPGMA, Neighbour joining showed close clustering of individuals and intermixing in clus- ters irrespective of their origin. Interestingly, according to Ullah et al. (2013) accessions grouped in same clus- ter are more similar to each other but less similar to the accessions in other clusters. This means that accessions in the same cluster are genetically similar or related. This knowledge is important because, based on their ge- netic relationship, recognizing and classifying different individuals in homogeneous groups will help breeders select parents and improve efficiency in preparing cross- es for breeding programs (Zeni Neto et al., 2020). Figure 2: Neighbor joining diagram of genotypes based on ISSR marker analysis. 1 = B97375, 2 = B96812, 3 = B96723, 4 = B93757, 5 = B47419+, 6 = DB8134, 7 = M1176/77, 8 = M1246/84, 9 = M1334/84, 10 = M1954/91, 11 = RB72/454, 12 = RB86/3129, 13 = SP81-3250, 14 = RB94/2991 15 = Co88025, 16 = Co91017, 17 = Co997+, 18 = CoC671, 19 = KNB9288, 20 = KNB9253 Figure 3: Genetic relatedness of the sugarcane genotypes based on UPGMA cluster analysis Acta agriculturae Slovenica, 117/4 – 20218 F. E. EMEGHA et al. 3.5 PRINCIPAL COORDINATE ANALYSIS Pattern of variation among the sugarcane acces- sions was also observed using Principal coordinate analysis based on Jaccard’s similarity coefficient using PAST Statistical software package. The sugarcane ac- cessions clustered in the four quadrants irrespective of their place of origin. The ordination of the accessions on principal component axes PCo1 versus PCo2 based on cluster analysis of ISSR allelic data (Fig 4). It provided distinct, groupings that established sub-groups within the quadrants, which illustrate the degree of relatedness and diversity within the quadrants. In quadrant 1 acces- sions B96723, B93757, RB72/454 appeared to be closely related, but formed a sub group with accessions Co997+ and M1954/91 located a far distance away from the other accessions in quadrant 1. The occurrence of accessions B96723 and B93757 and the closeness of RB72/454 in quadrant 1 showed that they are closely related and in- dicates a common ancestor. In quadrant II, accessions SP81-3250, KNB9253, CoC671 formed a sub group while KNB9288 stands alone located close to the centroid. Also clustering of accessions SP81-3250, CoC671, KNB-9288, SP81-3250, KNB-9253 are far apart in quadrant II sug- gesting different origin. The diffuse pattern shows genetic divergence among the populations with accession SP81- 3250 being at the farther end of the quadrant revealing utmost genetic variation and distance from other popu- lations. In quadrant III, two subgroups were formed one with accessions M1176/77 and RB94/2991 and another with B97375 and M1246/84 showing similarity. Quad- rant IV had the highest number of accessions (7) among other quadrants. Accessions RB86/3129, M1334/84, B47419+ formed a sub-group within the quadrant. The clustering of accessions B97375, B96812 and Co88025 in quadrant IV indicates a common ancestor, while acces- sion DB8134 is far away from other accessions in quad- rant IV. Principal coordinate analysis revealed the impor- tant components contributing to the observed variation among the 20 sugarcane accessions. This form of ad- mixture may result from the participation of sugarcane genotypes in breeding programs to enhance some of the characteristics of commercially exploited varieties, so that parental breeding lines may be exchanged across the world’s sugarcane growing regions to achieve these goals (Tazeb et al., 2017). Another explanation for the high levels of similarity between subgroups and groups is Figure 4: The ordination of twenty Sugarcane (Saccharum officinarum) accessions on principal component axes PCo 1 versus PCo 2 based on cluster analysis of ISSR allelic data. 1 = B97375, 2 = B96812, 3 = B96723, 4 = B93757, 5 = B47419+, 6 = DB8134, 7 = M1176/77, 8 = M1246/84, 9 = M1334/84, 10 = M1954/91, 11 = RB72/454, 12 = RB86/3129, 13 = SP81-3250, 14 = RB94/2991, 15 = Co88025, 16 = Co91017, 17 = Co997+, 18 = CoC671, 19 = KNB9288, 20 = KNB9253 Acta agriculturae Slovenica, 117/4 – 2021 9 Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers that during the sugarcane breeding program, the lineages were exposed to a higher degree of intercultivar gene flow (Rodriguez et al., 2005). Comprehensively, it is possible for geographically different population to form a cluster with other population, because the majority of commer- cial sugar cane cultivars bred after the turn of the 20th century are interspecific hybrids between Saccharrum of- ficinarum and Saccharrum spontaneum L. (D’Hont et al., 1996). Thus, the cross progeny may have clustered from other regions with their progenitors or parents or the parents may have clustered from distantly related popu- lations with their cross progeny (Tazeb et al., 2017). Clus- tering of all sugarcane accessions together irrespective of their sources showed their remarkable genetic similarity and reinforced the postulate of a common progenitor for the accessions (Jauhar & Hanna, 1998). 4 CONCLUSION Adequate genetic information is prerequisite to identify potential parental combinations required in hybridization programme aimed to create segregating progenies with maximum genetic variability for further selection. The information from this study could provide accurate information to sugarcane breeding in Nigeria for strategic conservation of the germplasm resources and future improvement work of the sugarcane by select- ing suitable parents for breeding programs aimed at op- timizing sugar yield, establishing the proper identity of the accessions and preventing duplication of accessions. In addition, ISSR markers showed reliability and ef- ficiency in detecting polymorphisms within and among the sugarcane accessions studied. It has also been shown that ISSR markers have high-resolution ability in sug- arcane fingerprinting and diversity analysis and are therefore effective and efficient in the identification of polymorphisms within and among populations and/or species. However, seven out of the eight primers were polymorphic, ability of primer ISSR 8 and ISSR 10 to produce higher allele frequencies and polymorphic loci in this study indicated that the two primers are the most informative and suitable for diversity study in sugarcane. It is, therefore recommended that molecular marker ap- proach be deployed in investigating the remaining germ- plasm for diversity and breeding programs. 5 ACKNOWLEDGEMENTS Part of the fund used for this study was sourced from the grant received from the National Sugar Devel- opment Council (NSDC), Abuja on morphological char- acterization of the new exotic sugarcane varieties in the Unilorin Sugar Research Institute (USRI), Ilorin. We are grateful to the Management of the two organizations for their support. 6 REFERENCES Ajibade, S. R., Weeden, N. F. & Michite, S. (2000). In- ter simple repeat analysis of genetic relationship in the genus Vigna. Euphytica, 111, 47-55. https://doi. org/10.1023/A:1003763328768 Animasaun, D. A., Adikwu, V. O., Alex, G. A., Akinsunlola, T. P., Adekola, O. F. & Krishnamurthy, R. (2021). Morho- agronomic traits variability, allelic polymorphism and di- versity analysis of African yam bean: towards improving utilisatilization and germplasm conservation. Plant Ge- netic Resources, 19(3), 216-228. https://doi.org/10.1017/ S1479262121000253 Animasaun, D. A., Awujoola, K. F., Oyedeji, S., Morakinyo, J. A. & Krishnamurthy, R. (2018). Diversity level of genomic mi- crosatellite among cultivated genotypes of Digitaria species in Nigeria. African Crop Science Journal, 26(2), 303 – 311. https://doi.org/10.4314/acsj.v26i2.11 Animasaun, D. A., Morakinyo, J. A., Mustapha, O. T., and Krishnamurthy, R. (2015). Assessment of genetic diversi- ty in accessions of pearl millet (Pennisetum glaucum) and napier grass (Pennisetum purpureum) using microsatel- lite (ISSR) markers. Iranian Journal of Genetics and Plant Breeding, 4(1), 25-35. http://ijgpb.journals.ikiu.ac.ir/arti- cle_840.html Da Costa, MLM, Amorim, LLB, Onofre, AVC, De Melo, LJOT, De Oliveira, MBM, De Carvalho, R. and Benko-Iseppon, AM (2011). Assessment of genetic diversity in contrast- ing sugarcane varieties using inter-simple sequence repeat (ISSR) markers. American Journal of Plant Sciences, 02(03), 425-432. https://doi.org/10.4236/ajps.2011.23048 Devarumath, R., Kalwade, S., Kawar, P. and Sushir, K. (2012). Assessment of genetic diversity in sugarcane germplasm using ISSR and SSR markers. Sugar Tech, 14(4), 334-344. https://doi.org/10.1007/s12355-012-0168-7 D’Hont A., Rao PS., Feldmann P., Grivet L., Islam-Faridi N., Taylor P., Gaszmann JC (1995). Identification and char- acterization of sugarcane intergeneric hybrids, Saccharum officinarum and Erianthus arundinaceus, with molecular marker and DNA in situ hybridization. Theory Applied Ge- netics, 91, 320-326. https://doi.org/10.1007/BF00220894 Dillon, S., Shapter, F., Henry, R., Cordeiro, G., Izquierdo, L. and Lee, L. (2007). Domestication to crop improvement: Genet- ic resources for Sorghum and Saccharum (Andropogoneae). Annals of Botany, 100(5), 975-989. https://doi.org/10.1093/ aob/mcm192 Esselink, G.D., Nybom, H. and Vosman, B. (2004). Assignment of allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting—peak ratios) method. Theoretical and Applied Genetics, 109(2), pp.402-408. htt- ps://doi.org/10.1007/s00122-004-1645-5 Govindaraj M., Vetriventhan M. and Srinivasan M (2015). Im- Acta agriculturae Slovenica, 117/4 – 202110 F. E. EMEGHA et al. portance of genetic diversity assessment in crop plants and its recent advances. An overview of its analytical perspec- tives. Genetics Research International, 2015, 1-16. https:// doi.org/10.1155/2015/431487 Jauhar, P.P and Hanna, W.W. (1998). Cytogenetics and genetics of pearl millet. Advances in Agronomy, 64(1), 2-22. https:// doi.org/10.1016/s0065-2113(08)60501-5 Khushk, M. and Pathan, A (2006). Sugarcane and its by-prod- ucts. [online] Pakissan.com. Available at: https://www.pa- kissan.com/2017/06/15/sugarcane-and-its-by-products/ [Accessed 15 Feb. 2019] Kwajaffa, A. and Olaoye, G. (2014). Flowering behaviour, pol- len fertility and relationship of flowering with cane yield and sucrose accumulation among sugarcane germplasm accessions in a savanna ecology of Nigeria. International Journal of Current Agricultural Research, 3(12), 104-108 National Sugar Development Council Document (NSDC). (2015). Sugarcane Variety Importation at National Sugar Development Council (NSDC), Abuja, Nigeria; p. 10 Olaoye G., Abayomi, A. and Takim, O. (2017). Advancing sugar development in Nigeria: A Report of the West Africa Sugar Development Project on Varietal Identification for Rain For- est Ecologies of Nigeria (2010-2016). Project implementa- tion Agency, (p. 15) Ilorin: University of Ilorin, Unilorin Sugar Research Institute. Pan Yong-Bao , (2010). Databasing molecular identities of sug- arcane (Saccharum spp.) clones constructed with micros- atellite (SSR) DNA markers. American Journal of plant sci- ence, 1(2), 87 -94. https://doi.org/10.4236/ajps.2010.12011 Pavliček, A., Hrda, S. and Flegr, J. (1999). Free tree: Freeware program for construction of phylogenetic trees on the ba- sis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD Analysis of genus Frenkelia. Folia Biologica (Praha), 45(3), 97-99. Pfeiffer, T., Roschanski, A., Pannell, J., Korbecka, G. and Schnit- tler, M. (2011). Characterization of microsatellite loci and reliable genotyping in a polyploid plant, Mercurialis peren- nis (Euphorbiaceae). Journal of Heredity, 102(4), 479-488. https://doi.org/10.1093/jhered/esr024 Rodriguez AMH., Marco A., Cassillo C., Ericka P., Flores B. (2005). Genetic diversity of the most important sugarcane cultivars in Mexico. E-Gnosis , 3(1), 1-10. https://www.re- dalyc.org/articulo.oa?id=73000301 Saitou, N. and Nei, M. (1987). The neighbor-join methoding: a new method for reconstructing phylogenetic trees. Mo- lecular Biology and Evolution, 4(4), 406-425. https://doi. org/10.1093/oxfordjournals.molbev.a040454 Smiullah, F., Usman, K. and Ijaz, A. (2013). Genetic vari- ability of different morphological and yield contributing traits in different accession of Saccharum officinarum L. Universal Journal of Plant Science, 1(2), 43-48. https://doi. org/10.13189/ujps.2013.010203 Srivastava S and Gupta O.S. (2008) “Inter simple sequence repeat profile as a genetic marker system in sugar-cane,” Sugar Technology, 10(1), 48-52. https://doi.org/10.1007/ s12355-008-0008-y Tazeb A., Haileselassie T., Tesfaye K., (2017). Molecular charac- terization of introduced sugarcane genotype in Ethiopia us- ing inter simple sequence repeat (ISSR) molecular markers. African Journal of Biotechnology, 16(10), 434 – 449. Ullah. S., Farooq A. K., Abdullah A. A., Rameez I & Usman I. (2013). Genetic diversity assessment among sugarcane ac- cessions by means microsatellite (SSR) markers. Journal of Plant Breeding and Crop Science, 5(10), 214-219. https://doi. org/10.5897/JPBCS2012.024 Wayagari, J.W., Ayool .G.B., Imolehin, E. D. and Misari, S. M. (2003b): Economic evaluation of chewing sugarcane production in the central zone of Nigeria. Short Com- munication of Sugar Technology. Society of Sugar Research and Promotion, 5(1&2), 81-84. https://doi.org/10.1007/ BF02943771 Wayagari, J.W., Ayoola, G.B., Imolehin, E.D. and Misari, S.M.(2003b): Economic evaluation of chewing sugarcane production in the central zone of Nigeria. Sugar technology, 5(1 and 2), 81-84. https://doi.org/10.1007/BF02943771 Zeni Neto H., Borsuk, L. G. M., Santos, L. R. F. D., Angeli, H. S., Berton, G. S & Sousa, L. L. (2020). Genetic diversity and population structure of sugarcane (Saccharum spp.) acces- sions by means of microsatellites markers. Acta Scientiar- um. Agronomy, 42, e4508. Epub May 11 2020. https://doi. org/10.4025/actasciagron.v42i1.4508 Acta agriculturae Slovenica, 117/4, 1–11, Ljubljana 2021 doi:10.14720/aas.2021.117.4.987 Original research article / izvirni znanstveni članek Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) Andrej LAVRENČIČ 1, 2, Darko VETERNIK 3 Delo je prispelo 10. decembra 2018, sprejeto 25. oktobra 2021 Received December 10, 2018; accepted October 25, 2021. 1 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za zootehniko, Ljubljana, Slovenija 2 Korespondenčni avtor, e-naslov: andrej.lavrencic@bf.uni-lj.si 3 LPN Kozorog Kamnik, Kamnik, Slovenija Vpliv sezone na in vitro razgradljivost in fermentabilnost kr- mil v vampovem soku navadnega jelena (Cervus elaphus L.) Izvleček: Prehod iz poletja v zimo povzroči spremem- be v fiziologiji prebavnega trakta navadnega jelena in v tam prisotnih prebavnih procesih. Zato je bil namen raziskave ugotoviti, kako sezona vpliva na in vitro navidezno (ivNRSS) in pravo razgradljivost suhe snovi (ivPRSS), na kazalnike in vitro tvorbe plina in na sproščanje hlapnih maščobnih kislin (HMK) iz enajstih krmil, ki jih zauživajo ali dokrmljujemo košutam navadnega jelena v Sloveniji (plodovi kostanja in gradna ter želod in dva vzorca sveže trave, dva vzorca mrve in travne silaže, jabolčne tropine in koreni sladkorne pese). IvNRSS in ivPRSS, določeni z inkubacijo krmil v puferiranem vampovem soku, se med sezonama nista razlikovali, prav tako tudi nismo ugotovili velikih razlik pri večini kazalnikov produkcije plina. Le kazalnik »C« (specifična hitrost tvorbe plina) je bil večji (p < 0,05) v zimski sezoni. Tudi količine HMK so bile med sezonama zelo podobne. Vendar pa je bil delež ocetne kisline pogosto nekoliko večji pozimi kot jese- ni (0,05 < p < 0,10), medtem ko sta bila deleža propionske in maslene kisline pri krmilih, ki so vsebovala več vlaknine, večja (0,05 < p < 0,10) jeseni kot pozimi. Nasprotno pa smo ob inkubaciji krmil, ki vsebujejo veliko škroba (želod in ko- stanj), določili večji (p < 0,05) delež propionske kisline po- zimi, medtem ko je bil pri teh krmilih delež maslene kisline večji (p < 0,05) jeseni. Čeprav sta bila tako število uporablje- nih substratov (n = 11) kot število živali (n = 6), darovalk vampovega soka, majhna, pa ti rezultati kažejo na spremembo presnove vampovih mikroorganizmov med jesenjo in zimo. Ključne besede: navadni jelen; Cervus elaphus L.; pre- hrana živali; sezona; krmila; in vitro prebavljivost; in vitro produkcija plina; hlapne maščobne kisline; vamp The effect of season on in vitro degradability and ferment- ability of feeds in red deers’ (Cervus elaphus L.) rumen fluid Abstract: Transition from summer to winter changes red deer digestive tract physiology and digestive processes. The objective of the trial was to determine the effects of sea- son on in vitro apparent (ivADMD) and true dry matter (ivT- DMD) digestibility, in vitro gas production parameters and short-chain fatty acid synthesis (SCFA) in red deer hinds of eleven substrates naturally occurring in Slovenia (chestnut fruits, acorns of common and sessile oak, two fresh grasses) and those frequently used in supplemental red deer feeding (two grass hays and two grass silages, apple pomace and sugar beet roots). There were no differences in ivADMD, ivTDMD, determined by incubation of feeds in buffered rumen fluid, as there were no differences in majority of gas production parameters between autumn and winter season. Only the parameter “C” (specific gas production rate) was frequently higher (p < 0.05) in winter season than in autumn season. The amounts of SCFA were similar between two seasons. Howev- er, the proportion of acetic acid tended to be higher in winter, while the proportions of propionic and butyric acid tended to be higher in autumn than in winter especially in high fibre feeds. On contrary, in high starch feeds such as oak acorns and chestnut fruits, the proportion of propionic acid was higher (p < 0.05) in winter, while of butyric acid in autumn (p < 0.05). Despite the fact that the number of used substrates (n = 11) and animal rumen fluid donors (n = 6) were small, these results indicate a shift in rumen microbial metabolism between autumn and winter season. Key words: red deer; Cervus elaphus L.; animal nutri- tion; season; feed; in vitro digestibility; in vitro gas produc- tion; short-chain fatty acids; rumen Acta agriculturae Slovenica, 117/4 – 20212 A. LAVRENČIČ in d. VETERNIK 1 UVod Uspešno upravljanje s populacijo navadnega jele- na (Cervus elaphus L.) in s tem povezano preprečevanje škod na gozdnem drevju, še posebej na območjih, kjer je populacija jelenov zelo številčna, je v veliki meri od- visno od poznavanja navad jelenov in od njihove učin- kovitosti izkoriščanja naravnih virov. Pri tem pogosto navajamo razlike v obnašanju jelenov med hranjenjem, ki so povezane s prisotnostjo plenilcev in obstojem ka- kovostnega kritja, ter razlike, povezane z oblikovanjem skupin, ki pa so odvisne od socialnih povezav, gostote živali na posameznem območju in stopnje vznemirja- nja živali (Hafner in Černe, 2015). Podobno kot druge živali so tudi prežvekovalci podvrženi naravnim ritmom pridobivanja in izgublja- nja telesne mase, zauživanja krme, porabe energije, pre- snove in razmnoževanja. Dva pomembna mehanizma, ki vplivata na navedene kazalnike, sta razpoložljivost prehranskih virov in dolžina dneva (Clauss in sod., 2010), za katera pa ni povsem jasno, ali delujeta povsem ločeno ali v različnih kombinacijah. Zauživanje krme in prirast telesne mase sta najmanjša v zimski in največja v poletni sezoni (Milne in sod., 1978, Stevens in sod., 2003; Arnold in sod., 2004). Sibbald in Milne (1993), Ar- nold in sod. (2015) ter Weckerly in sod. (2018) navajajo, da se v poletni sezoni pri jelenih povečata tudi napol- njenost in masa prebavil (vamp in siriščnik). Weckerly in sod. (2018) so pri belorepem jelenu (Odocoileus vir- ginianus) ugotovili, da je masa prebavil povezana pred- vsem z napolnjenostjo prebavil (digesta load) in ne s hranilno vrednostjo krme. Ker je napolnjenost prebavil dobro povezana s prostornino vampa, je logično, da je v zimski sezoni manjša tudi prostornina predželodcev in drugih jelenovih prebavnih organov (Hofmann, 1985; Sibbald in Milne, 1993; Arnold in sod., 2015). Z manjšo prostornino predželodcev pa je povezana tudi dnevno zaužita količina energije, ki je v pozni jeseni in zgodnji zimi najmanjša, svoj vrh pa doseže med junijem in ok- tobrom (Arnold in sod., 2015). S prehranskega vidika so v prehrani navadnega je- lena pomembni predvsem obnašanje med zauživanjem krme, sposobnost jelenov za prebavo večjih količin krme, učinkovito izkoriščanje hranljivih snovi in konč- nih produktov prebave ter zmožnost živali, da zadostijo potrebam po hranljivih snoveh (Milne in sod., 1978). V splošnem velja, da so razlike v fermentaciji in razgra- dnji krme in hranljivih snovi v predželodcih povezane z razlikami v kemični sestavi zaužitih obrokov. Palmer in sod. (1976) navajajo, da lahko pričakujemo veliko variabilnost v aktivnosti vampovega soka med jeleni iz naravnega okolja, saj ti jeleni zauživajo zelo raznoliko krmo, od dobro prebavljivih sadežev in semen do sla- bo prebavljive voluminozne krme. Prav tako pa lahko pričakujemo veliko variabilnost v aktivnosti vampove- ga soka med sezonami, saj se razpoložljivost naravnih prehranskih virov med sezonami zelo spreminja, spre- minja pa se lahko tudi zaradi dokrmljevanja jelenov v obdobju pomanjkanja naravnih virov krme. Namen naše raziskave je bil ugotoviti, ali sezona vpliva na aktivnost vampovega soka navadnega jelena. Kriteriji, ki smo jim sledili, so bili in vitro navidezna in prava razgradljivost suhe snovi, kazalniki in vitro tvor- be plina in količina in vitro tvorjenih hlapnih maščob- nih kislin ter razmerja med njimi. Pri tem smo upora- bili krmo, ki jo jeleni običajno najdejo v naravi ali pa so sestavni del obroka pri zimskem dokrmljevanju. 2 MAterIAL In Metode V študiji smo uporabili 11 krmil, ki jih pogosto najdemo bodisi v naravnem okolju bodisi jih pogosto uporabljamo pri zimskem dokrmljevanju navadnega jelena. Vsa ta krmila smo uporabili že v prejšnjih po- skusih (Lavrenčič in Veternik, 2018a in b). Krmila iz naravnega okolja jelena so bila: dva vzorca sveže tra- ve-paše (z območja Jelendola in Kokre), plodovi div- jega kostanja (Aesculus hippocastanum) ter želod doba (Quercus robur) in gradna (Quercus petraea). Vzor- ci krmil, ki so bila namenjena dokrmljevanju, so bili: dva vzorca mrve in travne silaže (z območja Jelendo- la in Kokre), koreni sladkorne pese in jabolčne tropi- ne. Vzorce paše, korenov sladkorne pese in jabolčnih tropin smo pred kemijskimi analizami in inkubacijo v vampovem soku v 48 urah posušili pri 60 °C. Suhe vzor- ce smo zmleli skozi 1 mm sito in analizirali na vsebnost surovih beljakovin (SB), surovega pepela (SP), surovih maščob (SM) in surove vlaknine (SV) z metodami po Neumann in Bassler (1986), medtem ko smo vsebnosti v nevtralnem detergentu netopne vlakinen analizirali z aparatom ANKOM220 Fibre Analyser (Ankom Techno- logy, Macedon, NY) z metodo po avtorjih Goering in Van Soest (1970), pri čemer pa smo uporabili natrijev sulfit. Kemična sestava uporabljenih krmil je podana v preglednici 1. Za primerjavo značilnosti vampovega soka smo izbrali dve sezoni. Za jesensko sezono smo vzeli me- sece september, oktober in november, medtem ko so zimsko sezono predstavljali meseci december, janu- ar in februar. V lovni sezoni smo uplenili šest košut, od tega štiri jeseni, med sredino septembra in sredino oktobra 2011, dve pa pozimi, prvo v sredini decembra 2011, drugo pa v januarju 2012, za kar smo dobili po- sebno dovoljenje Ministrstva za kmetijstvo, gozdarstvo in prehrano (št. 341-1/2012/6 z dne 18. 1. 2012). Da- Acta agriculturae Slovenica, 117/4 – 2021 3 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) tumi uplenitev so bili v skladu z začetkom zimskega dokrmljevanja jelenov, ki ga v Lovišču s posebnim na- menom (LPN) Kozorog Kamnik začnejo izvajati konec novembra ne glede na vremenske in snežne razmere. V celotni zimi 2011-2012 je bilo jelenjadi ponujeno sku- pno približno 30 ton sena, travne silaže in sladkorne pese, vendar pa živali krmišča niso redno obiskovale. Ocenjujemo, da je bila številčnost jelenjadi na krmiščih le okoli 30 % v primerjavi s številčnostjo v zimah, ki so bile bogate s snežno odejo. Košute smo v tem LPN uplenili tako na območju Jelendola (46°24’27.79” N in 14°24’30.65” E; 850 m nadmorske višine; občina Tržič) kot na območju lovskega revirja Kokra (46°21’57.37” N in 14°25’25.36” E; 925 m nadmorske višine; občina Preddvor). Takoj po uplenitvi smo iz živali odstranili vamp in ga v največ 45 minutah v zaprti, toplotno izo- lirani posodi prepeljali v laboratorij. Vsako inkubacijo, ki je predstavljala eno serijo, smo opravili za vsako žival posebej. In vitro navidezno in pravo razgradljivost suhe snovi (ivNRSS in ivPRSS) smo določili po postopkih, ki sta jih opisala Lavrenčič in Veternik (2018a). Iz vsebine vampa vsake košute posebej smo pripravili inokulum tako, da smo vampovo vsebino ročno oželi skozi štiri pasti bombažne gaze in jo razredčili z raztopino pufra v razmerju 1 : 2 (v/v). Okoli 450 mg zmletega krmila smo zatehtali v filtrske vrečke ANKOM F57 (ANKOM Tech- nology, Macedon, NY, USA) in jih toplotno zavarili. Za vsako krmilo smo pripravili štiri vrečke in jih po 2 vsta- vili v 2 inkubacijski posodi (2 vrečki/posodo). V vsako posodo smo vstavili 24 vrečk F57 (2 vrečki/krmilo + dve prazni vrečki (slepi vzorec)) in dodali dva litra pu- feriranega vampovega soka, ki smo ga predhodno pre- pihovali z ogljikovim dioksidom. Posode smo vstavili v inkubator na 39 °C za 24 h. V času inkubacije je bilo zagotovljeno mešanje puferiranega vampovega soka v inkubacijskih posodah. Po končani inkubaciji smo vrečke intenzivno sprali pod tekočo vodo, posušili in stehtali ter izračunali ivNRSS. Vrečke smo v nadaljeva- nju tretirali še 1 uro v raztopini nevtralnega detergenta (ND) pri 100 °C v aparaturi ANKOM220 fibre analyser (ANKOM Technology, Macedon, NY, USA), jih sprali v vodi, posušili in stehtali ter izračunali in vitro pravo razgradljivost suhe snovi (ivPRSS). Obe, tako ivNRSS kot ivPRSS, sta bili izračunani kot delež med razliko zatehte suhe snovi vzorca in ostankom suhe snovi v vrečki po inkubaciji oz. obdelavi vzorca z ND. Dobljene ivNRSS in ivPRSS smo nato korigirali na izgubo mase slepega vzorca (prazne vrečke) med inkubacijo oz. ob- delavo z ND (količnik med maso oprane in posušene prazne vrečke po inkubaciji oz. obdelavi z ND in maso neinkubirane oz. z ND neobdelane prazne vrečke). Inokulum za izvedbo plinskega testa smo pripra- vi na enak način kot inokulum za določanje ivNRSS (Menke in Steingass, 1988; Lavrenčič in Veternik, 2018b). Okoli 200 mg zmletega krmila smo inkubirali v anaerobnih pogojih pri 39 °C v 100 ml steklenih bri- zgalkah, ki so vsebovale 30 ml puferiranega vampove- ga soka (inokuluma). Količino sproščenega plina smo odčitali po 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 in 96 urah. Po 24 urah smo tekočino dveh od štirih brizgalk prene- sli v 50 ml centrifugirne epruvete in jih do analize na vsebnost hlapnih maščobnih kislin (HMK) zamrznili na −20 °C. V vsaki seriji smo inkubirali tri brizgalke s slepim vzorcem (vsebovale so samo inokulum brez substrata) in tri brizgalke s standardnim vzorcem. Vse postopke od uplenitve živali do začetka inku- bacije smo izvedli v manj kot 2 urah, v času, ki ga še kot primernega navajata tudi Schwartz and Nagy (1972). Ekstrakte za analizo HMK smo pripravili iz pu- feriranega vampovega soka po 24 urah inkubacije po modificirani metodi Holdeman in sod. (1977). HMK smo določili s plinskim kromatografom Hewlett Pac- kard 5890 A (Hewlett Packard, Bellefonte, Pennsylva- nia, USA), opremljenim s split/splitless injektorjem in FID detektorjem. Za ločevanje HMK smo uporabili 30 m NUKOL TM, FUSED SILICA kapilarno kolono (SUPELCO, Bellefonte, Pennsylvania, USA). Dobljene količine plina smo korigirali na 1 g suhe snovi krmila in tudi na količino plina, sproščenega iz slepega vzorca. Kazalnike plinskega testa smo za vsak substrat ocenili s pomočjo Gompertzove enačbe (La- vrenčič in sod., 1997): Yt = B × exp(-C × exp(-D × t)), kjer je Yt količina sproščenega plina (ml/g DM) v času ‘t’, ‘B’ največja količina sproščenega plina (skupna po- tencialna količina plina; (ml/g SS)); ‘C’ specifična hi- trost sproščanja plina, na katero vpliva konstantni faktor ‘D’, s katerim opisujemo zmanjševanje specifične hitrosti tvorbe plina (ki je posledica zmanjševanja hi- trosti rasti mikroorganizmov in zmanjševanja količine fermentabilnega substrata) in ‘t’ čas v urah. Poleg kazal- nikov tvorbe plina smo izračunali tudi količino plina, sproščenega po 24 urah inkubacije (GAS24; ml/g suhe snovi), s pomočjo prvega in drugega odvoda Gompert- zove enačbe po času pa še največjo hitrost tvorbe plina (MFR) in čas, ko je bila dosežena največja hitrost tvor- be plina (TMFR; Lavrenčič in sod., 1997). Neto količino HMK, sproščeno v 24 urah inkuba- cije, smo izračunali tako, da smo od bruto količine po- sameznih HMK odšteli posamezne HMK, ki so se spro- stile v tem času v slepem vzorcu. Dobljene količine smo nato korigirali na 1 g inkubirane suhe snovi krmila. Rezultate smo nato analizirali z enofaktor- sko (one-way analysis of variance) analizo varian- ce s proceduro splošnega linearnega modela (GLM) statističnega paketa SAS/STAT version 9.4 (SAS, Acta agriculturae Slovenica, 117/4 – 20214 A. LAVRENČIČ in d. VETERNIK 2015). Primerjali smo kazalnike med sezonama (jesen vs zima) za vsako krmilo posebej. Rezultate pred- stavljamo kot povprečne razlike, dobljene s testom naj- manjših kvadratov (least square means). Kot statistično značilne smo sprejeli razlike pri p ≤ 0,05, medtem ko smo kot trende obravnavali razlike pri 0,05 < p < 0,10. 3 rezULtAtI Vzorce, katerih sestavo navajamo v preglednici 1, sta v svoji raziskavi uporabila že Lavrenčič in Veternik (2018a). Med njimi je obstajala velika raznolikost v ke- mični sestavi in bi jih lahko v grobem razdelili v dve skupini: na voluminozno krmo, kamor uvrščamo svežo travo-pašo, mrvo in travne silaže ter glede na vsebnost surove vlaknine in NDV tudi jabolčne tropine, ki pa jih lahko, zaradi fizikalne strukture, uvrščamo tudi v sku- pino močne krme, za katero je značilna majhna vseb- nost vlaknine (tako surove vlaknine kot NDV) in rela- tivno velika vsebnost BDI in NVOH (preglednica 1). Podatke o in vitro navidezni (ivNRSS) in pravi razgradljivosti suhe snovi (ivPRSS) obravnavanih kr- mil pri košutah v jesenski in zimski sezoni prikazujemo v preglednici 2. Tako ivNRSS in ivPRSS sta bili številč- no večinoma vedno večji v jesenski sezoni kot pozimi, a smo le za svežo travo iz Jelendola in za korene sladkor- ne pese ugotovili trende (0,05 < p < 0,10) pri ivNRSS. Nasprotno pa so imeli plodovi divjega kostanja ter že- lod doba večje ivNRSS in ivPRSS pozimi kot jeseni, a je bila le ivPRSS želoda doba statistično večja (p < 0,05). Kazalnike plinskega testa, ocenjene s pomočjo Gompertzovega modela, prikazujemo v preglednici 3. Skupna potencialna tvorba plina (kazalnik “B”) je bila večinoma večja v zimski sezoni, čeprav so bile razlike statistično značilne (p < 0,05) le pri sveži travi iz Jelen- dola, jabolčnih tropinah, korenih sladkorne pese ter želodu gradna in doba. Velik vpliv sezone pa smo ugo- Krmilo / Feed SS DM SB CP SM EE SV CF SP Ash BDI NFE NDV NDF NVOH NFC (g/kg) (g/kg SS – g/kg DM) Sveža trava (Jelendol) Fresh grass (Jelendol) 202 203 29 197 64 506 440 171 Sveža trava (Kokra) Fresh grass (Kokra) 282 134 24 278 70 494 600 91 Travna silaža (Jelendol) Grass silage (Jelendol) 387 128 31 340 97 404 640 42 Travna silaža (Kokra) Grass silage (Kokra) 586 194 28 237 107 434 517 72 Mrva (Jelendol) Grass hay (Jelendol) 867 107 18 304 97 472 543 178 Mrva (Kokra) Grass hay (Kokra) 843 93 19 282 70 537 563 192 Jabolčne tropine Apple pomace 153 58 28 218 27 669 482 338 Koreni sladkorne pese Sugarbeet roots 202 68 5 61 27 840 141 695 Plodovi divjega kostanja Chestnut fruits 372 85 16 143 25 730 389 422 Želod gradna Sessile oak acorns 579 52 36 130 22 760 278 542 Želod doba Common oak acorns 508 53 37 134 22 753 291 532 Preglednica 1: Kemična sestava krmil, uporabljenih v poskusu table 1: Chemical composition of feeds used in the experiment SS = suha snov – DM = dry matter; SB = surove beljakovine – CP = crude protein; SM = surove maščobe – EE = ether extract; SV = surova vlaknina – CF = crude fiber; SP = surovi pepel – Ash = crude ash; BDI = brezdušični izvleček (BDI = SS − (SP + SB + SM + SV)) – NFE = nitrogen- free extract (NFE = DM − (Ash + CP + EE + CF)); NDV = v nevtralnem detergentu netopna vlaknina – NDF = neutral detergent fiber, NVOH = nevlakninasti ogljikovi hidrati (NVOH = SS − (SP + SB + SM + NDV)) – NFC = non-fiber carbohydrates (NFC = DM − (Ash + CP + EE + NDF)) Acta agriculturae Slovenica, 117/4 – 2021 5 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) nih v zimski sezoni, pri tem pa so bile razlike statistič- no značilne (p < 0,05) le za svežo travo iz Jelendola, za mrvo iz Kokre in korene sladkorne pese, medtem ko smo za mrvo iz Jelendola ugotovili le trend. Čas, ko je bila hitrost tvorbe plina največja (TMFR), je bil številč- no večinoma daljši pri inkubaciji vzorcev v inokulu- mu, pripravljenim iz vampove vsebine košut, uplenje- nih pozimi. Vendar pa smo statistično značilne razlike (p < 0,05) izračunali samo za nekatera krmila, pri in- kubaciji sveže trave iz Jelendola, jabolčnih tropin ter za želode gradna in doba, medtem ko smo pri inkubaciji sveže trave iz Kokre, korenov sladkorne pese in plodov divjega kostanja izračunali zgolj trend (0,05 < p < 0,10). Tudi največja hitrost tvorbe plina (MFR) je bila večino- ma večja ob inkubaciji vzorcev v inokulumu, pripra- tovili pri kazalniku »C« (specifični hitrosti razgradnje). Ta je bil večji (p < 0,05) pri večini krmil, inkubiranih v vampovem soku košut, uplenjenih v zimski sezoni. Izjema je bila samo mrva iz Jelendola, medtem ko smo pri korenih sladkorne pese in želodu gradna ugotovi- li le trend (0,05 < p < 0,10). Nasprotno pa se kazalnik »D« pri posameznih substratih med sezonama ni raz- likoval (p > 0,05), čeprav je bil pri številnih substratih številčno manjši v jesenski sezoni. Statistično značilno (p < 0,05) različna kazalnika »D« smo dobili pri inku- baciji mrve iz Kokre in pri inkubaciji želoda gradna. Pri primerjavi izračunanih kazalnikov tvorbe pli- na (preglednica 4) smo ugotovili, da se je v 24 urah sprostilo več plina (GAS24) ob fermentaciji substratov, ki smo jih inkubirali v vampovem soku košut, uplenje- Krmilo Feed ivNRSS ivADMD ivPRSS ivTDMD jesen autumn zima winter SED jesen autumn zima winter SED Sveža trava (Jelendol) Fresh grass (Jelendol) 646* 564* 44,8 801 750 34,4 Sveža trava (Kokra) Fresh grass (Kokra) 454 437 33,1 611 595 25,2 Travna silaža (Jelendol) Grass silage (Jelendol) 491 460 41,8 606 587 34,3 Travna silaža (Kokra) Grass silage (Kokra) 560 526 68,5 742 702 32,0 Mrva (Jelendol) Grass hay (Jelendol) 540 517 32,7 670 652 37,2 Mrva (Kokra) Grass hay (Kokra) 465 423 32,8 624 586 28,4 Jabolčne tropine Apple pomace 497 489 16,8 704 699 11,5 Koreni sladkorne pese Sugarbeet roots 934* 881* 24,7 960 939 13,1 Plodovi divjega kostanja Chestnut fruits 408 436 25,4 725 738 12,7 Želod gradna Sessile oak acorns 523 517 20,3 767 775 7,7 Želod doba Common oak acorns 506 542 39,4 755b 791a 13,2 Preglednica 2: In vitro navidezna razgradljivost suhe snovi (ivNRSS; g/kg) in in vitro prava razgradljivost suhe snovi (ivPRSS) krmil, inkubiranih v inokulumih, pripravljenih iz vampove vsebine jeseni in pozimi uplenjenih košut table 2: In vitro apparent dry matter degradability (ivADMD; g/kg) and in vitro true dry matter digestibility (ivTDMD; g/kg) of feeds, incubated in inocula, prepared from rumen contents of hinds shot in autumn and winter ivNRSS = in vitro navidezna razgradljivost suhe snovi (mg/g SS) – ivADMD = in vitro apparent dry matter degradability (mg/g DM); ivPRSS = in vitro prava prebavljivost suhe snovi (mg/g SS) – ivTDMD = in vitro true dry matter digestibility (mg/g DM); SED = standardna napaka razlike – SED = standard error of the differenc a, b povprečja, označena z različnimi črkami znotraj parametra se statistično razlikujejo pri p < 0,05 – a, b means with different superscripts within parameter differ significantly (p < 0.05) * povprečja znotraj parametra kažejo trende (0,05 < p < 0,10) – means within the parameter show trends (0.05 < p < 0.10) Acta agriculturae Slovenica, 117/4 – 20216 A. LAVRENČIČ in d. VETERNIK vljenem iz vampove vsebine košut, uplenjenih v zimski sezoni, pri čemer pa smo statistično značilne razlike (p < 0,05) izračunali pri obeh mrvah (iz Jelendola in iz Kokre), pri korenih iz sladkorne pese in želodu gradna, medtem ko smo trend (0,05 < p < 0,10) izračunali le pri inkubaciji travne silaže iz Kokre. V preglednici 5 prikazujemo podatke o količini sproščenih hlapnih maščobnih kislin (HMK) in njiho- vih deležih. Ob inkubaciji vzorcev v inokulumu, pripra- vljenem iz vampovega soka košut, uplenjenih pozimi, se je pri večini vzorcev sprostilo več HMK kot iz tiste- ga, pripravljenega iz vampovega soka jeseni uplenjenih košut. Kljub temu pa smo statistično značilne razlike (p < 0,05) izračunali le pri mrvi iz Jelendola, medtem ko smo pri jabolčnih tropinah in želodu gradna izra- čunali le trend (0,05 < p < 0,10). Nasprotno pa smo pri inkubaciji sveže trave iz Kokre, travne silaže iz Jelendo- la in želoda doba ugotovili, da je bila produkcija HMK večja, če smo te vzorce inkubirali v inokulumu pripra- vljenem iz vampa jeseni uplenjenih košut, pri tem pa je bila razlika statistično značilna (p < 0,05) le pri in- kubaciji sveže trave iz Kokre. Pri 24-urni inkubaciji v vampovem soku košut, uplenjenih pozimi, smo v vseh vzorcih določili številčno večji delež ocetne kisline, čeprav smo statistično značilno odstopanje (p < 0,05) zabeležili samo pri korenih sladkorne pese, trende (0,05 < p < 0,10) pa pri sveži travi iz Kokre, travni si- laži iz Kokre, obeh vzorcih mrve in plodovih divjega kostanja. Nasprotno pa so bili deleži maslene kisline pri vseh vzorcih številčno večji v vampovem soku ko- šut, uplenjenih jeseni, a smo statistično značilne razlike (p < 0,05) izračunali le pri vzorcih, uvrščenih v skupino Krmilo Feed B (ml/g SS – ml/g DM) C D jesen autumn zima winter SED jesen autumn zima winter SED jesen autumn zima winter SED Sveža trava (Jelendol) Fresh grass (Jelendol) 260a 277b 5,4 2,00b 2,23a 0,052 0,159 0,162 0,0064 Sveža trava (Kokra) Fresh grass (Kokra) 222 230 8,3 2,01b 2,22a 0,071 0,092 0,091 0,0087 Travna silaža (Jelendol) Grass silage (Jelendol) 226 225 10,1 2,51b 2,79a 0,149 0,109 0,113 0,0431 Travna silaža (Kokra) Grass silage (Kokra) 223 223 8,0 2,24b 2,51a 0,118 0,111 0,127 0,0151 Mrva (Jelendol) Grass hay (Jelendol) 229 235 7,8 2,04 2,24 0,152 0,132 0,150 0,0100 Mrva (Kokra) Grass hay (Kokra) 219 216 6,3 1,81b 2,12a 0,091 0,096b 0,124a 0,0107 Jabolčne tropine Apple pomace 307b 342a 18,1 2,32b 2,57a 0,111 0,216 0,170 0,2107 Koreni slad. pese Sugarbeet roots 339b 380a 6,6 2,15* 2,43* 0,144 0,305 0,293 0,0176 Plodovi div. kostanja Chestnut fruits 260 264 28,2 2,16b 2,59a 0,108 0,120 0,120 0,0125 Želod gradna Sessile oak acorns 278b 293a 5,7 2,48* 2,75* 0,135 0,160a 0,131b 0,0056 Želod doba Common oak acorns 279b 294a 5,4 2,51b 3,09a 0,106 0,131 0,126 0,0067 Preglednica 3: Ocenjeni kazalniki in vitro tvorbe plina krmil, inkubiranih v inokulumih, pripravljenih iz vampove vsebine jeseni in pozimi uplenjenih košut table 3: Estimated in vitro gas production parameters of feeds, incubated in inocula, prepared from rumen contents of hinds shot in autumn and winter B = skupna potencialna tvorba plina – B = total potential gas production; C = relativna hitrost tvorbe plina, na katero vpliva konstantni kazalnik mikrobne učinkovitosti D – C = relative gas production rate as affected by a constant factor of microbial efficiency D SED = standardna napaka razlike – SED = standard error of the difference a,b povprečja, označena z različnimi črkami znotraj parametra se statistično razlikujejo pri p < 0,05 – a,b means with different superscripts within parameter differ significantly (p < 0.05) * povprečja znotraj parametra kažejo trende (0,05 < p < 0,10) – means within the parameter show trends (0.05 < p < 0.10) Acta agriculturae Slovenica, 117/4 – 2021 7 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) močnih krmil (koreni sladkorne pese, plodovi divjega kostanja ter želod doba in gradna), pri voluminozni krmi pa samo pri inkubaciji sveže trave iz Kokre. Ob inkubaciji vzorcev voluminozne krme in korenov slad- korne pese v vampovem soku košut, uplenjenih jeseni, smo zabeležili tudi številčno večji delež propionske ki- sline, medtem ko je bil pri inkubaciji plodov divjega kostanja ter želodu doba in gradna, delež propionske kisline večji, če smo jih inkubirali v vampovem soku košut, uplenjenih pozimi. Pri tem smo statistično zna- čilne razlike določili pri plodovih divjega kostanja in želodu gradna. Pri mrvi iz Kokre, jabolčnih tropinah, korenih sladkorne pese in želodu doba pa smo ugoto- vili samo trend (0,05 < p < 0,10). 4 rAzPrAVA Vpliv sezone na prebavljivost, razgradljivost in fer- mentabilnost hranljivih snovi je le redko obravnavan pri jelenih. V redkih virih avtorji (npr. Milne in sod., 1978; Sibbald in Milne, 1993; Freudenberger in sod., 1994; Domingue in sod., 1991) ugotavljajo, da v in vivo prebavljivosti organske, suhe snovi oz. dušika pri jele- nih ni bilo velikih razlik med poletno in zimsko sezo- no. Nasprotno pa sta Jiang in Hudson (1996) ugotovila, da je bila in vivo prebavljivost paše veliko večja poleti kot pozimi, kar je povsem razumljivo, saj sta slednja v poskusu uporabila svežo travo oz. pašo, katere hranil- na vrednost se je znotraj in med sezonami neprestano spreminjala, medtem ko so prej našteti avtorji vedno Krmilo Feed GAS24 (ml/g SS – ml/g DM) TMFR (h) MFR (ml/h) jesen autumn zima winter SED jesen autumn zima winter SED jesen autumn zima winter SED Sveža trava (Jelendol) Fresh grass (Jelendol) 249a 264b 4,7 4,4b 5,0a 0,17 15,2 16,3 0,53 Sveža trava (Kokra) Fresh grass (Kokra) 174 177 2,9 7,3* 8,9* 0,57 7,3 7,6 0,45 Travna silaža (Jelendol) Grass silage (Jelendol) 183 186 10,6 8,4 9,1 1,38 10,7 9,3 3,33 Travna silaža (Kokra) Grass silage (Kokra) 190 197 8,1 7,1 7,3 0,69 9,5* 10,3* 1,14 Mrva (Jelendol) Grass hay (Jelendol) 209* 220* 5,4 5,3 5,4 0,40 11,1b 13,0a 0,69 Mrva (Kokra) Grass hay (Kokra) 179b 192a 4,1 6,4 6,2 0,50 7,5b 9,7a 0,66 Jabolčne tropine Apple pomace 301 327 16,9 3,3b 5,6a 0,97 23,7 21,4 10,76 Koreni slad. pese Sugarbeet roots 338b 379a 6,5 2,5* 3,0* 0,25 37,8b 40,9a 1,92 Plodovi div. kostanja Chestnut fruits 226 226 5,1 6,6* 8,0* 0,71 11,3 11,6 0,67 Želod gradna Sessile oak acorns 263 258 5,1 5,7b 7,9a 0,24 16,1a 13,7b 0,64 Želod doba Common oak acorns 249 252 4,0 7,0b 9,0a 0,22 13,4 13,6 0,59 Preglednica 4: Ocenjeni kazalniki in vitro tvorbe plina krmil, inkubiranih v inokulumih, pripravljenih iz vampove vsebine jeseni in pozimi uplenjenih košut table 4: Calculated in vitro gas production parameters of feeds, incubated in inocula, prepared from rumen contents of hinds shot in autumn and winter GAS24 = prostornina plina, proizvedena v 24 urah inkubacije – Gas24 = gas volumen produced in 24 hours of incubation; MFR= največja hi- trost tvorbe plina – MFR = maximum fermentation rate; TMFR = čas, ko je dosežena največja hitrost tvorbe plina – TMFR = time of maximum fermentation rate; SED = standardna napaka razlike – SED = standard error of the difference a,b povprečja, označena z različnimi črkami znotraj parametra se statistično razlikujejo pri p < 0,05 – a,b means with different superscripts within parameter differ significantly (p < 0.05) * povprečja znotraj parametra kažejo trende (0,05 < p < 0,10) – means within the parameter show trends (0.05 < p < 0.10) Acta agriculturae Slovenica, 117/4 – 20218 A. LAVRENČIČ in d. VETERNIK Pr eg le dn ic a 5: K ol ič in e in d el ež i h la pn ih m aš čo bn ih k isl in , k i s o se sp ro st ile iz k rm il, in ku bi ra ni h v in ok ul um ih , p rip ra vl je ni h iz v am po ve v se bi ne v je se ni in p oz im i u pl en je ni h ko šu t ta bl e 5: T ot al a m ou nt o f s ho rt -c ha in fa tty a ci ds a nd m ol ar p ro po rt io ns o f a ce ta te , p ro pi on at e an d bu ty ra te re le as ed fr om fe ed s, in cu ba te d in in oc ul a, pr ep ar ed fr om ru m en c on te nt s of h in ds sh ot in a ut um n an d w in te r K rm ilo Fe ed H M K – S C FA (m m ol /g S S – m m ol /g D M ) A c (g /g H M K – g /g S C FA ) Pr (g /g H M K – g /g S C FA ) Bu (g /g H M K – g /g S C FA ) je se n au tu m n zi m a w in te r SE D je se n au tu m n zi m a w in te r SE D je se n au tu m n zi m a w in te r SE D je se n au tu m n zi m a w in te r SE D Sv ež a tr av a (J el en do l) Fr es h gr as s ( Je le nd ol ) 6, 76 6, 84 1, 26 0 0, 61 8 0, 67 6 0, 06 80 0, 26 9 0, 23 7 0, 04 84 0, 11 3 0, 08 7 0, 02 05 Sv ež a tr av a (K ok ra ) Fr es h gr as s ( K ok ra ) 5, 05 a 3, 83 b 0, 51 3 0, 66 9* 0, 72 9* 0, 02 87 0, 22 2 0, 19 2 0, 02 27 0, 10 9a 0, 07 9b 0, 01 21 Tr av na si la ža (J el en do l) G ra ss si la ge (J el en do l) 4, 79 6, 13 1, 62 2 0, 59 3 0, 62 8 0, 04 75 0, 30 0 0, 26 4 0, 04 10 0, 10 7 0, 10 8 0, 01 31 Tr av na si la ža (K ok ra ) G ra ss si la ge (K ok ra ) 5, 84 4, 74 1, 38 7 0, 65 0* 0, 71 2* 0, 01 39 0, 27 0 0, 22 0 0, 04 10 0, 07 9 0, 06 7 0, 00 95 M rv a (J el en do l) G ra ss h ay (J el en do l) 5, 86 b 9, 49 a 1, 31 0 0, 65 4* 0, 71 6* 0, 03 98 0, 24 1 0, 20 3 0, 03 02 0, 10 5 0, 08 1 0, 01 47 M rv a (K ok ra ) G ra ss h ay (K ok ra ) 4, 44 4, 83 0, 86 1 0, 66 9* 0, 74 4* 0, 04 18 0, 24 0* 0, 18 8* 0, 03 24 0, 09 1* 0, 06 8* 0, 01 64 Ja bo lč ne tr op in e A pp le p om ac e 7, 31 * 11 ,2 4* 1, 78 2 0, 66 7 0, 72 1 0, 02 43 0, 20 4* 0, 18 0* 0, 01 27 0, 12 9 0, 09 9 0, 03 41 K or en i s la d. p es e Su ga rb ee t r oo ts 10 ,5 1 13 ,1 9 1, 81 6 0, 52 6b 0, 61 8a 0, 03 84 0, 32 0* 0, 26 2* 0, 31 00 0, 15 4a 0, 12 0b 0, 13 05 Pl od ov i d iv . k os ta nj a C he st nu t f ru its 6, 40 7, 67 1, 94 7 0, 61 2* 0, 66 3* 0, 02 30 0, 13 5b 0, 16 3a 0, 00 89 0, 25 3a 0, 17 4b 0, 03 50 Ž el od g ra dn a Se ss ile o ak a co rn s 7, 15 * 9, 62 * 1, 11 1 0, 66 1 0, 69 2 0, 02 10 0, 10 6b 0, 15 7a 0, 01 76 0, 23 4a 0, 15 1b 0, 02 71 Ž el od d ob a C om m on o ak a co rn s 7, 77 7, 61 1, 40 1 0, 61 0 0, 64 6 0, 03 90 0, 14 9* 0, 19 8* 0, 02 71 0, 24 1a 0, 15 6b 0, 02 17 H M K = h la pn e m aš čo bn e ki sli ne – S C FA = sh or t- ch ai n fa tt y ac id s; A c = oc et na k is lin a – A c = ac et ic a ci d; P r = pr op io ns ka k is lin a – Pr = p ro pi on ic a ci d; B u = m as le na k is lin a – Bu = b ut yr ic a ci d SE D = st an da rd na n ap ak a ra zl ik e – SE D = st an da rd e rr or o f t he d iff er en ce a, b p ov pr eč ja , o zn ač en a z ra zl ič ni m i č rk am i z no tr aj p ar am et ra s e st at is tič no r az lik uj ej o pr i p < 0 ,0 5 – a, b m ea ns w ith d iff er en t s up er sc ri pt s w ith in p ar am et er d iff er si gn ifi ca nt ly (p < 0 .0 5) * po vp re čj a zn ot ra j p ar am et ra k až ej o tr en de (0 ,0 5 < p < 0, 10 ) – m ea ns w ith in th e pa ra m et er sh ow tr en ds (0 .0 5 < p < 0. 10 ) Acta agriculturae Slovenica, 117/4 – 2021 9 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) uporabili predhodno konzervirano voluminozno krmo (mrvo) in krmila. Za razliko od in vitro pogojev, ki smo jih imeli v našem poskusu, so vsi zgoraj omenjeni avtorji ugota- vljali prebavljivost oz. razgradljivost z in vivo metoda- mi. V in vivo pogojih sta prebavljivost in razgradljivost hranljivih snovi pogojena s trajanjem zadrževanja krme v prebavilih, predvsem v predželodcih (MRT). Milne in sod. (1978), Domingue in sod. (1991), Sibbald in Milne (1993) in Freudenberger in sod. (1994) so ugotovili, da jeleni poleti zaužijejo več krme kot pozimi, kar bi ob nespremenjeni prebavljivosti hranljivih snovi pomeni- lo, da je poleti MRT krajši kot pozimi, kar je skladno z rezultati Domingue in sod. (1991), ki so ugotovili, da se poleti poveča iztok tekoče faze. Nasprotno pa Freuden- berger in sod. (1994) ugotavljajo, da se poleti, kljub ne- spremenjenem zauživanju krme, upočasni iztok lignina (marker) iz predželodcev, kar nakazuje na podaljšan MRT pri jelenih v poletni sezoni. Slednje je lahko po- sledica visokih temperatur okolja, povsem možno pa je tudi, da je to posledica večje prostornine (in mase) predželodcev pri jelenih v poletni sezoni (Sibbald in Milne, 1993; Arnold in sod., 2015). Na ivNRSS in ivPRSS posameznih krmil vpliva tudi obrok, ki ga košute zauživajo (Gordon in sod., 2002). V pričujočem poskusu nismo mogli spremljati količin in vrste sestavin, ki so jih košute dejansko za- užile z obroki. Menimo, da so bili obroki po sestavi, kakovosti in hranilni vrednosti v jeseni in pozimi zelo podobne, saj košute poselijo predvsem območja, na ka- terih rastejo rastline z večjo hranilno vrednostjo (Bar- boza and Bowyer, 2000) in da košute izbirajo obroke tako, da je v njih najmanj 30 % trav (Adamič, 1990, cit. po Jerina, 2007). V sezoni 2011–2012 so imele košute jeseni na voljo tudi velike količine plodov kostanja, žira in želoda, ki so bili takrat dostopni v velikih količinah. Kljub temu, da so imele košute pozimi poleg plodov plodonosnega drevja na voljo tudi mrvo, travno silažo, jabolčne tropine in korene sladkorne pese, s katerimi delavci LPN redno dokrmljujejo jelenjad na obeh ob- močjih (D. Veternik, ustni vir, 25. januar 2012), slednjih niso zauživale v velikih količinah. Zato predvidevamo, da so bili jesenski in zimski obroki po svojih fizikalnih in kemijskih lastnostih zelo podobni. V nam dostopni literaturi nismo uspeli dobiti no- benih podatkov o vplivu sezone na kazalnike tvorbe plina pri navadnem jelenu. Dobljeni kazalniki tvorbe plina, predvsem skupna potencialna tvorba plina in produkcija plina v prvih 24 urah inkubacije, se niso razlikovali od tistih, ki sta jih Lavrenčič in Veternik (2018b) določila ob fermentaciji teh krmil v puferira- nem vampovem soku ovac. Skupna potencialna tvorba plina v zimski sezoni je bila večja predvsem pri vzorcih močne krme, medtem ko pri inkubaciji vzorcev volu- minozne krme nismo zaznali večjih razlik. Prav tako večjih razlik nismo zaznali pri kazalniku D, medtem ko je bil kazalnik C v zimski sezoni pri večini krmil ve- čji kot jeseni (preglednica 3). Posledica teh sprememb v kazalnikih tvorbe plina je podaljšan čas, v katerem tvorba plina doseže največjo hitrost, kar je še posebej izrazito pri močni krmi (preglednica 4). Vendar pa se prostornina v 24 urah proizvedenega plina ni bistve- no razlikovala med sezonama, prav tako pa med sezo- nama ni bilo bistvenih razlik v ivPRSS. Tudi količine sproščenih hlapnih maščobnih kislin se med sezonama niso razlikovale, čeprav smo nekoliko večje količine HMK določili pri skoraj vseh substratih v zimski sezo- ni. Tudi Domingue in sod. (1991) pri navadnem jele- nu niso ugotovili statistično značilnih razlik v količini sproščenih HMK, medtem ko Freunderberger in sod. (1994) in DeLiberto in sod. (1989) pri belorepem jelenu (Odocoileus virginnianus) navajajo večjo sintezo HMK v poletni kot v zimski sezoni, kar utemeljujejo z večjo prostornino predželodcev v poletni sezoni, zaradi česar se MRT podaljša, s tem pa se podaljšata tako čas delova- nja kot aktivnost mikroorganizmov. Nasprotno pa Ar- nold in sod. (2015) navajajo, da je učinkovitost prebave največja pozimi, saj se zaradi podaljšanega MRT, ki je posledica manjšega zauživanja krme in manjše veliko- sti prebavnih organov, poveča sinteza HMK. Vendar pa DeLiberto in sod. (1989) niso ugotovili nobenih razlik v sproščanju HMK med jesensko in zimsko sezono. So se pa v preučevanih sezonah razlikovali deleži tvorjenih posameznih HMK. Tako se je pri fermentaciji v predželodcih košut jeseni tvoril manjši delež ocetne kisline (trend) kot pozimi, medtem ko sta bila jeseni deleža propionske in maslene kisline običajno večja kot pozimi. Le deleži propionske kisline, ki so nastale ob fermentaciji plodov kostanja in želoda, so bili večji po- zimi kot jeseni. Razlike v deležih propionske in maslene kisline med preučevanima sezonama so posebej izra- zite pri krmi z majhno vsebnostjo vlaknine, ne pa tudi pri voluminozni krmi. Ozka razmerja med ocetno in propionsko kislino so značilna za obroke, v katerih pre- vladuje močna krma in vsebujejo veliko fermentabilnih ogljikovih hidratov. V našem primeru je takšne obro- ke jelenjad zauživala v obeh sezonah, saj je bil takrat obrod plodonosnih vrst dreves, ko sta hrast in bukev, izjemno velik, obenem pa jelenjad ni množično obisko- vala njej namenjenih krmišč. Vendar pa smo pri večini krmil izračunali (podatki niso prikazani) nekoliko širše razmerje med ocetno in propionsko kislino ob njihovi fermentaciji v inokulumu, pripravljenem iz vampove vsebine košut, uplenjenih pozimi. Samo pri krmilih, ki vsebujejo veliko škroba (plodovi kostanja in želod obeh hrastov), so bila razmerja med ocetno in propionsko Acta agriculturae Slovenica, 117/4 – 202110 A. LAVRENČIČ in d. VETERNIK kislino širša, ko smo jih določili v inokulumu, pripra- vljenim z vampovim sokom košut, uplenjenih jeseni. Iz propionske kisline se v procesu glukoneogeneze tvori glukoza, zato povečan delež propionske kisline v jesen- ski sezoni pomeni tudi boljšo oskrbo živali z energijo, kar sovpada z jelenjim rukom in potrebami po energiji za uspešno osemenitev pri samicah. Ocetna kislina je prekurzor za sintezo telesnih maščob. Za nalaganje tele- snih maščob pa mora biti na razpolago dovolj NADPH, ki se tvori iz glukoze. Če glukoze oziroma NADPH pri- manjkuje, večina ocetne kisline oksidira, zaradi česar se poveča tvorba presnovne toplote (Domingue in sod., 1991), ki pa jelenjadi omogoča večje možnosti za preži- vetje v mrzlih zimah. 5 SKLePI Pri inkubaciji vzorcev krmil v inokulumu, pripra- vljenem iz vampovega soka košut, uplenjenih v jesenski in zimski sezoni, nismo ugotovili večjih razlik v in vitro navidezni razgradljivosti (ivNRSS) in pravi razgradlji- vosti (ivPRSS) suhe snovi, kakor tudi ne v kazalnikih in vitro tvorbe plina, razen v kazalniku »C« (specifična hitrost tvorbe plina), ki je bil v zimski sezoni večji kot jeseni. Tudi razlike v količini sproščenih hlapnih ma- ščobnih kislin (HMK) med sezonama niso bile velike. Zaradi velikega obroda plodonosnega drevja v jeseni 2011 in zaradi dokrmljevanja košut s travno silažo, mrvo, jabolčnimi tropinami in koreni sladkorne pese predvidevamo, da se aktivnost mikroorganizmov v vampu košut ni bistveno spremenila. Predvidevamo, da bi bili tudi rezultati in vivo podobni, saj se srednji čas zadrževanja krme v prebavilih med sezonami ne spre- minja bistveno, poleti zaradi večje prostornine preba- vil, pozimi pa zaradi manjše količine zaužitih sestavin obroka. Ugotovili pa smo, da so vampovi mikroorganiz- mi košut med sezonama nekoliko spremenili presnovo, kar se kaže v spremenjenih razmerjih med posamezni- mi HMK. Iz večje količine ocetne kisline lahko košute dobijo več toplote, kar je pomembno predvsem v hu- dih zimah, iz večje količine propionske kisline jeseni pa potrebno energijo za uspešno reprodukcijo. Tudi deleži maslene kisline so bili večji jeseni, torej v času intenziv- nega nalaganja telesnih rezerv. Te domneve bi morali še potrditi z dodatnimi in vitro raziskavami, predvsem pa bi morali v poskus vključiti večje število živali. Prav tako pa bi morali te domneve potrditi ali ovreči tudi z in vivo raziskavami na samih živalih ter v naravnem okolju. 6 VIrI Arnold, W., Ruf, T., Reimoser S., Tataruch, F., Onderscheka, K., Schober F. (2004). Nocturnal hypometabolism as an over- wintering strategy of red deer (Cervus elaphus). American Journal of Physiology – Regulatory, Integrative and Compar- ative Physiology, 286, R174–R181. https://doi.org/10.1152/ ajpregu.00593.2002 Arnold, W., Beiglböck, C., Burmeister, M., Guschlbauer, M., Lengauer, A., Schröder, B., … Breves, G. (2015). Con- trary seasonal changes of rates of nutrient uptake, organ mass, and voluntary food intake in red deer (Cervus ela- phus). American Journal of Physiology – Regulatory, Inte- grative and Comparative Physiology, 309(3), R277–R285. https://doi.org/10.1152/ajpregu.00084.2015 Barbosa, P., Bowyer, R. T. (2000). Sexual segregation in dimor- phic deer: a new gastrocentric hypothesis. Journal of Mam- malogy, 81(2), 473–489. https://doi.org/10.1644/1545- 1542(2000)081%3C0473:SSIDDA%3E2.0.CO;2 Clauss, M., Hume, D. I., Hummel, J., (2010). Evolutionary adaptations of ruminants and their potential relevance for modern production systems. Animal, 4(7), 979–992. https://doi.org/10.1017/S1751731110000388 DeLiberto, T. J., Pfister, J. A., Demarais, S., Van Vreede, G. (1989). Seasonal changes in physiological parameters of White-tailed deer in Oklahoma. Journal of Wildlife Man- agement, 53(3), 533–539. https://doi.org/10.2307/3809173 Domingue, B. M. F., Dellow, D. W. Wilson, P. R., Barry, T. N. (1991). Nitrogen metabolism, rumen fermentation, and water absorption in red deer, goats, and sheep. New Zea- land Journal of Agricultural Research, 34(4), 391–400. https://doi.org/10.1080/00288233.1991.10417682 Freudenberger, D. O., Tayakawa, K., Barry, T. N., Ball, A. J., Suttie, J. M. (1994). Seasonality in digestion and ru- men metabolism in red deer (Cervus elaphus) fed on a forage diet. British Journal of Nutrition, 71(4), 489–499. https://doi.org/10.1079/BJN19940157 Gordon, I. J., Pérez-Barbería, F. J., Cuartas, P. (2002). The influence of adaptation of rumen microflora on in vitro digestion of different forages by sheep and red deer. Canadian Journal of Zoology, 80(11), 1930–1937. https://doi.org/10.1139/z02-179 Goering, H. K., Van Soest, P. J. (1970). Forage fiber analyses (ap- paratus, reagents, procedures and some applications). Agri- culture handbook 379. Washington, DC, USA: ARS USDA. Hafner, M., Černe, B. (2015). Vplivi okoljskih dejavnikov na velikost skupin jelenjadi (Cervus elaphus L.) v vzhodnih Karavankah in Kamniško-Savinjskih Alpah. Gozdarski Ve- stnik, 73(3), 155–169. Hofmann, R. R. (1985). Digestive physiology of the deer – their morphophysiological specialisation and adaptation. V P. F. Fennesy in K. R. Drew (ur.), Biology of Deer Produc- tion (str. 393–407). Wellington: Bulletin 22 of The Royal Society of New Zealand. Holdeman, L. V., Cato, E. P., Moore, W. E. C. (1977). Ether extraction of volatile fatty acids. V: Anaerobe laboratory manual (str. 1–132). 4th edition. Virginia: Southern Print- ing Company. Acta agriculturae Slovenica, 117/4 – 2021 11 Vpliv sezone na in vitro razgradljivost in fermentabilnost krmil v vampovem soku navadnega jelena (Cervus elaphus L.) Jerina, K., 2007. The effects of habitat structure on red deer (Cervus elaphus) body mass. Zbornik Gozdarstva in Lesarstva, 82, 3–13. Jiang, Z., Hudson, R. J. (1996). Digestive responses of wa- piti Cervus elaphus Canadensis to seasonal changes. Acta Theriologica, 41(4), 415–423. https://doi.org/10.4098/ AT.arch.96-40 Lavrenčič, A., Stefanon, B., Susmel, P. (1997). An evaluation of the Gompertz model in degradability studies of for- age chemical components. Animal Science, 64(3), 423–431. https://doi.org/10.1017/S1357729800016027 Lavrenčič, A., Veternik, D. (2018a). Differences between sheep and red deer in in vitro apparent and true digestibility of commonly used red deer feeds. Acta Agriculturae Sloveni- ca, 112(1), 5–9. https://doi.org/10.14720/aas.2018.112.1.1 Lavrenčič, A., Veternik, D. (2018b). Gas and short-chain fatty acid production from feeds commonly fed to red deer (Cervus elaphus L.) and incubated with rumen in- oculum from red deer and sheep. Journal of Animal Physiology and Animal Nutrition, 102(5), 1146–1153. https://doi.org/10.1111/jpn.12943 Menke, K. H., Steingass, H. (1988). Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development, 28, 375–386. Milne, J. A., Macrae, J. C., Spence, A. M., Wilson, S. (1978). A comparison of the voluntary intake and digestion of a range of forages at different times of the year by the sheep and the red deer (Cervus elaphus). British Journal of Nutrition, 40(2), 347–357. https://doi.org/10.1079/ BJN19780131 Neumann, K., Bassler, R. (1976). Methodenbuch, Band III. Neudamm: Verlag J. Neumann. Palmer, W. L., Cowan, R. L., Amman, A. P. (1976). Ef- fect of inoculum source on in vitro digestion of deer foods. Journal of Wildlife Management, 40(2), 301–307. https://doi.org/10.2307/3800429 Sibbald, A. M., Milne, J. A. (1993). Physical characteristics of the alimentary tract in relation to seasonal changes in voluntary food intake by the red deer (Cervus elaphus). Journal of Agricultural Science, Cambridge, 120(1), 99–102. https://doi.org/10.1017/S0021859600073640 Statistical Analysis Systems Institute (2015). SAS/STAT user’s guide: Statistics (release 9.4). Cary, NC: SAS Institute. Schwartz, C. C., Nagy, J. G. (1972). Maintaining deer rumen flu- id for in vitro digestion studies. Journal of Wildlife Manage- ment, 36(4), 1341–1343. https://doi.org/10.2307/3799281 Stevens, D. R., Webster, J. R., Corson, I. D. (2003). Effects of seasonality and feed quality on the feed requirements and live weight gain of young deer – a review. V M. J. Casey (ed), The nutrition and Management of Deer on Grazing Systems (str. 17–23). https://doi.org/10.33584/ rps.9.2002.3418 Tilley, J. M. A, Terry, R. A. (1963). A two-stage tech- nique for the in vitro digestion of forage crops. Jour- nal of the British Grassland Society, 18(2), 104–111. https://doi.org/10.1111/j.1365-2494.1963.tb00335.x Weckerly, F. W., Bhaskar, G., Duarte, A., Luna, R. S., Starns, H. D. (2018). Heavier rumen-reticulum organs in white tailed deer is consistent with dietary bulk not quality. Canadian Journal of Zoology, 96(7), 748–752. https://doi.org/10.1139/cjz-2017-0036 Acta agriculturae Slovenica, 117/4, 1–10, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2256 Original research article / izvirni znanstveni članek Influence of ozonised irrigation water on the morphological, bacterio- logical and sensory characteristics of ‘Saint-Pierre’ tomatoes grown in Algeria Fouzia BENALI 1, 2, Nadia RAMDANI 1 Received June 19, 2021; accepted September 25, 2021. Delo je prispelo 19. junija 2021, sprejeto 25. septembra 2021 1 Djillali Liabès University, Faculty of Exact Sciences, Department of Chemistry, Sidi Bel-Abbès, Algeria 2 Corresponding author, e-mail: fouzia.benali@univ-sba.dz Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics of ‘Saint-Pierre’ tomatoes grown in Algeria Abstract: This article focuses on the study of the influ- ence of ozonised water irrigation on the morphological, bacte- riological and sensory characteristics of ‘Saint-Pierre’ tomatoes grown in Algeria. The results were compared with those irri- gated with non-ozonised tap water called control of the same varietal type and grown under the same conditions. The work was carried out on seedlings of tomatoes grown and irrigated with ozonised water at different ozonisation times: 10- seconds, 20- seconds and 30-seconds, corresponding to lot I (tomato at 10-s), lot II (tomato at 20-s) and lot III (tomato at 30-s), respec- tively. Irrigation with ozonised water does not cause defects in shape, skin or colour of the fruits. They are, distinguished by a round shape, very red in colour, consistent and slightly acidic in taste. With good microbiological stability in accordance with the standard and good organoleptic quality except for the taste character of tomatoes at 30-seconds where a majority of evalu- ators estimated that ‘they were bland and no big difference for the other criteria analysed. In general, our results showed that the ozonisation of irrigation water improves the growth, devel- opment, vigour and yield of tomato plants without altering the marketability of the fruits. This process encourages the use of ozonised water in agriculture since it has a high added value from an environmental and economic point of view and it can be generalized to other crops. Key words: ozonised water; irrigation; ‘Saint-Pierre’ to- matoes; morphological; bacteriological and sensory character- istics Vpliv ozonirane vode za namakanje na morfološke, bakterio- loške in senzorične lastnosti paradižnika ‘Saint-Pierre’, rasto- čega v Alžiriji Izvleček: Članek se osredotoča na vpliv ozoniranja vode za namakanje na morfološke, bakteriološke in senzorične lastnosti paradižnika ‘Saint-Pierre’ rastočega v Alžiriji. Rezulta- ti so primerjani s tistimi, kjer je bila voda za namakanje iz vodo- voda in ni bila ozonirana, kar je služilo kot kontrola, pri isti sor- ti paradižnika, gojenega v enakih razmerah. Sadike paradižnika za bile zalivane z vodo, ozonirano različno dolgo in sicer: 10 s, 20 s in 30 s, kar je ustrezalo naborom paradižnikov v poskusu: I (paradižnik pri 10 s), II (paradižnik pri 20 s) III (paradižnik pri 30 s). Zalivanje z ozonirano vodo ni povzročilo poškodb v obliki, kožici in barvi plodov. Ti so bili značilno okrogle obli- ke, zelo rdeči, čvrsti in z rahlo kislim okusom. V primerjavi s standardom so imeli dobro mikrobiološko stabilnost in dobro organoleptično kakovost, razen okusa tistih, ki so bili zalivani z vodo ozonirano 30 s, za katere je večina ocenjevalcev oceni- la, da okus ni značilen, med ostalimi preučevanimi lastnostmi pa ni bilo velikih razlik. Na splošno so rezultati pokazali, da je ozoniranje vode za zalivanje izboljšalo rast, razvoj, vitalnost in pridelek paradižnika brez sprememb tržnih lastnosti plodov. Postopek vzpodbuja uporabo ozonirane vode v kmetijstvu, saj ima veliko dodano vrednost z vidika okolja in ekonomičnosti in bi se lahko splošno uporabljal tudi pri drugih kulturnih ra- stlinah. Ključne besede: ozonirana voda; zalivanje; ‘Saint-Pierre’ paradižnik; morfološke, bakteriološke in senzorične lastnosti Acta agriculturae Slovenica, 117/4 – 20212 F. BENALI and N. RAMDANI 1 INTRODUCTION Tomato (Solanum lycopersicum L.) is one of the most consumed market garden crops in the world after potatoes (FAOSTAT, s. d.), of variable shape (spherical, oblong, elongated), and various colours (white, pink, red, yellow, orange, green, purple and black) depending on the variety (Joseph et al., 2017; Renaud, 2003). In general, tomato occupies an important place in the human diet; where it is consumed fresh, whole, dried, paste, puree, juice, sauce or tomato powder (Bhat et al., 2020; Nethaji et al., 2020; Siti Fadlilah et al., 2020). It is low in calories and very rich in water, vitamins, antioxidants and macro- minerals such as iron, calcium, sulphur and potassium. It is rich in sugars (fructose and glucose), essential amino acids, organic acids and dietary fibres (Ali et al., 2020; García-Alonso et al., 2020). From the medicinal point of view, consumption of tomato fruits and their deriva- tive products has been associated with the prevention of cardiovascular disease (Cámara et al., 2020; Cheng et al., 2017; Saini et al., 2020), several types of cancer (Rowles et al., 2018; Wu et al., 2021; Yang et al., 2013), as well as the maintenance of bone health (Walallawita et al., 2020). Tomato production is increasing over the years, where its annual production in Algeria rises from 107 million tonnes in 2014 to 148 million tonnes in 2019 (FAOSTAT, s. d.), cultivated across various regions of the country, in particular, wilayas of Tlemcen, Mostaganem, Ain-Defla, Chlef, Tipaza, Jijel, Skikda, Guelma, Annaba, Adrar and Biskra (Algeria’s Ministry of Agriculture and Rural De- velopment, 2020). Plant diseases and pests affect tomato production, causing considerable yield drops and signifi- cant economic losses. Ozone has been applied for irrigation water disinfec- tion and to control nematodes that reduce crop yield of tomato (Guo et al., 2019; Landa Fernández et al., 2019). It is also used to residual pesticides decomposition follow- ing the overuse of chemicals in agricultural fields (Mit- sugi et al., 2017). It is a multifunctional reagent; it breaks down quickly into oxygen without leaving any chemical residues, In addition to its toxicity against a wide range of microorganisms (Pandiselvam et al., 2017) it is advanta- geous for many other applications, such as purification and disinfection treatment of waste and drinking water, preservation and extension of food shelf life, steriliza- tion of equipment, and elimination of unwanted aromas produced by bacteria during storage and shipping. It is also used to inactivate microorganisms on fresh produce, such as fruits, vegetables, meat, poultry, fish and eggs, and dry produce, like cereals, pulses and spices. It is used in the gaseous form to disinfect the air in cold rooms by removing ethylene to slow down the ripening process of fruits and vegetables without altering the quality charac- teristics, and in aqueous form using the ozonized water for washing food and ensure product safety (Horvitz & Cantalejo, 2014; Pandiselvam et al., 2017). The goal of our research is to study the influence of irrigation using ozonised water on tomato seedlings by varying the ozonisation times of water: 10-seconds, 20-seconds and 30-seconds while comparing the results with the control (tomato seedlings irrigated with tap wa- ter without ozonisation), in order to assess the effects of irrigation with ozonised water on the morphological, bacteriological and sensory characteristics of ‘Saint- Pierre’ tomatoes grown in Algeria. To the best of our knowledge this study has not been reported before, and it offers high added value for environmental, scientific and economic research fields. 2 MATERIAL AND METHODS 2.1 EXPERIMENTAL DESIGN The study was carried out during the year 2020 in the north-western region of Algeria, exactly in the region of Oran, on tomato seedlings (seeds) of the same varietal type with the ‘Saint-Pierre’ appellation, of indeterminate growth. Sown in spring season in individual pots filled with potting soil, divided into four lots referenced as fol- lows: lot I (tomato at 10-s); lot II (tomato at 20-s); lot III (tomato at 30-s) and finally lot IV (control tomato), where each lot contains five pots. The irrigation water source used was tap water, then it was divided into four variants A, B, C and D. The first three (A, B and C) were exposed to constant flows of gas- eous ozone produced by ozone generator FM-C900 (BE- YOK ozone, China), as shown in Figure1, during 10, 20 and 30-seconds, respectively. Variant D consists only of non-ozonised tap water as a control. The plants of batch I, II and III were then irrigated, regularly and respective- ly, with variants A, B, C and subsequently compared with the control plants. Figure 1: Schematic of the experimental setup Acta agriculturae Slovenica, 117/4 – 2021 3 Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics ... After five weeks of cultivation, the seedlings are transplanted into large pots and staked, put outdoors, in a well-lit and sunny place. During the test period, the monthly average temperature was variable ranging from 20 °C to 30 °C, see Figure 2, with an average monthly hu- midity ranging from 76 % to 67 % (Nomades, s. d.). The irrigation was performed regularly twice a week during the grow cycle. 2.2 OZONE GENERATOR The cost, complexity, hazard potential, and genera- tion of residual ozone were arguments that prevented investment in ozonation of irrigation water. The recent ozone generators have been manufactured with automat- ed ozonation systems, more compact infrastructure, low cost and simplified maintenance which reduce invest- ment and operating costs and makes renovations more feasible. When operating an ozone water treatment system, the main concern in terms of failure is leakage in the ozone lines (feed gas and off gas). Serious ozone leaks represent a significant health hazard for farmers and crops, hence the integration of ozone gas sensors in all ozonation systems. A fail-safe system can represent a significant percentage of the overall cost but generally offers a moderate level of maintenance to ensure proper operation (Graham et al., 2011). We should note that, it will be better for our future research to integrate ozone gas sensors into the ozona- Figure 2: Temperature evolution during the test period of the year 2020 (Nomades, s. d.) tion system, in order to better control of any unexpected issues related to gas leakages. 2.3 QUALITY ANALYSIS Fruit quality analyses were carried out with the aim of determining the morphological, bacteriological and organoleptic characteristics of each batch of tomatoes (I, II, III and IV). These analyses were carried out on sam- ples of whole tomatoes, fresh, ripe, firm, healthy and of uniform red colour for the four batches, in order to eval- uate the effects of ozonised water on the quality of the tomatoes by comparing them with the control samples. 2.4 MORPHOLOGICAL ANALYSIS The analysis consists of determining the morpho- logical characteristics of tomatoes by calculating the shape coefficient (Cf), the number of cells (Nℓ), the mass volume (ρ) and the average fruit mass (Pm) (Agassounon Djikpo Tchibozo et al., 2012). The other physical parameters were determined us- ing a calliper such as height and diameter. These analyses make it possible to identify and characterize the variety of the tomato studied. The shape coefficient is given by the following equa- tion: Acta agriculturae Slovenica, 117/4 – 20214 F. BENALI and N. RAMDANI 2.7 UNCERTAINTIES AND SHORTCOMINGS It should be noted that our seedlings have been placed in outdoor conditions, while greenhouse and commercial conditions are to be expected in our future research in order to collect additional information such as gas exchange parameters and economic feasibility. Soil analyses before and after treatment with ozonised water will be recommended to assess any impact that could modify the quality of the latter. In addition, irrigation with aqueous ozone at 30-seconds gave a better yield but the tomatoes are of lower taste quality, this concentration should be taken into account and should be considered as a threshold value and study the possibility of improv- ing the sensory qualities of fruits. Finally, increasing the number of panel of tasters will lead to a better appraisal of the organoleptic char- acteristics. 3 RESULTS AND DISCUSSION 3.1 MORPHOLOGICAL CHARACTERISTICS OF THE TOMATOES STUDIED The four lots of tomatoes studied are distinguished by smooth-looking fruits, of a pronounced red colour, strongly lobed (6 to 8 lobes) and round (Cf  >  0.8). The width of the fruits for the four lots is slightly more ex- tended, which gives it the appearance of a large fruit, the height, the diameters and the average mass of the fruits are variable (Figure 3). The highest average mass was observed in the toma- to at 30-s, followed by the tomato at 20-s then the tomato at 10-s and finally the control tomato with 81 g, 77 g, 72 g and 63 g respectively; the same for the density which is 0.97 g cm-3, 0.94 g cm-3, 0.93 g cm-3 for the tomatoes at 30- s, 20-s and 10-s and 0.91 g cm-3 for the control tomatoes. (Table 2). The tomatoes responded differently despite the same growing conditions and the same varietal type, the highest number of tomatoes picked during the whole pe- riod of the trial was observed in the tomatoes at 30-s with 31.86 % followed by the tomatoes at 20-s with 28.32 % then the tomatoes at 10-s with 22.12  % and finally 17.70 % for the control tomatoes. The difference in the quantity of tomatoes picked for the three lots (I, II, III) is proportional to the concentra- tion of the ozonised water. In July, the batches (I, II, III) show an early harvest in relation to the control batch. (Figure 4). During the hottest months the four lots recorded a large number of tomatoes picked with different quanti- It allows varieties to be classified into three catego- ries: Cf  < 0.8: it is a flattened shape; Cf  > 1: it is an elongated shape; 0.8  < Cf <  1: it is a round shape. 2.5 MICROBIOLOGICAL ANALYSIS The purposes of these analyses were to assess the microbiological quality of the four batches of tomatoes (I, II, III and IV). They allow checking the possible pres- ence or the absence of microorganisms in the samples to be analysed which is summarized in the search and the enumeration of the total coliforms and the faecal coli- forms according to the standard ISO 7251:2005, as well as the determined yeasts and moulds by the ISO 21527- 1:2008 method. A quantity of 10 ± 2  g of each sample of tomatoes was weighed and added to 90 ml of Ringer’s solution, each placed in sterile sachets, crushed and ho- mogenized in the Stomacher, thus constituting the stock solution. The analysis was carried out by seeding the stock suspension of 1 ml aliquots and its 10-1, 10-2 and 10-3 decimal dilutions on suitable culture media and under aseptic conditions. Yeasts and moulds testify to the appearance of phe- nomena of deterioration of tomatoes, discoloration and modification of the flavour, on the other hand the pres- ence of total coliforms and faecal coliforms reflect the hy- gienic level of the fruits. Table 1 groups the culture media used. 2.6 APPRAISAL OF SENSORY CHARACTERISTICS The objective of this analysis is, first to evaluate the organoleptic characteristics of the experimental and con- trol tomatoes according the parameters of standard ISO 5492: 2008 which is summarized in: the appearance, col- our, consistency, smell, acidity, aroma and taste. Second, rank the tomato samples offered for tasting noting their acceptability or their preferences over each other. The evaluation was carried out on fresh, mature, healthy, firm and red tomatoes by a panel of tasters. To- mato samples and pre-established questionnaires were distributed to each taster who agreed to collaborate in the study and no information was provided on the differ- ent batches of tomatoes in order to objectively assess the perceived sensory characteristics. Acta agriculturae Slovenica, 117/4 – 2021 5 Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics ... ties and as the seasons progressed the quantities of fruit picked decreased. The largest quantities of the picked tomatoes were recorded during the month of August for the four lots. (Figure 4). Tomatoes at 30-s were among the best performing tomatoes compared to other lots in terms of production density, average mass and density (Figure 3) and (Table 2). No defect in development, shape, epidermis or col- ouring was observed in comparison with the control tomatoes, total absence on all the samples studied of Designation Culture centre Cultivation conditions Norm Total coliforms VRBL Agar (Violet Red Bile Lactose Agar). Incubation : 37 ºC for 24 ± 2 h ISO 7251:2005 Faecal coliforms VRBL Agar (Violet Red Bile Lactose Agar). Incubation : 44 ºC for 24/48 ± 2 h ISO 7251:2005  Yeasts and moulds DRBC Agar (Dichloran-rose bengalchloramphènicol) Incubation : 25 ºC ± 1 ºC for 5 days ISO 21527-1:2008  Table 1: The culture media used for the enumeration and the quantitative enumeration of the germs investigated in the tomatoes studied protuberance or crevice and no presence of corky bruis- ing of umbilical form or elongated epistolary scar. These fruits have good resistance to cracking, a very developed placenta and a large number of seeds. Table 2 shows the main morphological characteristics of the four batches of tomatoes at 10-s, 20-s, 30-s and control. 3.2 RESULTS OF MICROBIOLOGICAL ANALYSIS The results obtained for the enumeration of total and faecal coliforms as well as for the quantitative enumera- tion of yeasts and moulds, show a total absence of all the germs sought both for tomatoes irrigated with ozonised water as for control tomatoes, while the standard toler- ates 103 cfu g-1 for total coliforms, which means that the samples studied show stability and a good hygienic level. This coincides with the work of (Heß & Gallert, 2015; (Güzel-Seydim et al., 2004) where ozone has been used as an antimicrobial agent on a variety of pathogenic or- ganisms, such as strains Escherichia coli, Enterococcus, Staphylococcus, fungi and viruses on the other hand (Guo & Wang, 2017) reveal that E. coli treated with moderate concentrations of ozonised water (0.5 mg l-1 ozone at 28 °C) were completely inactivated. Irrigation with ozonised water has no harmful influence on the experimental to- matoes compared to the control tomatoes and the results obtained show compliance with the standard (Inter min- isterial decree, Official Journal No. 39, 2017). These to- matoes pose no health risk, and have good marketability. These results are shown in Table 3. Figure 3: The average values of mass, height and diameter of experimental and control tomatoes Designation Coefficient of form Cf Number of lobes Nℓ Average fruit mass Pm (g) Volumic mass in ρ (g cm-3) Protuberance crevasse, Green collar Seeds number Tomato at 10-s 0.83 6-8 72.27 0.93 absence > 200 Tomato at 20-s 0.84 6-8 77.18 0.94 absence > 200 Tomato at 30-s 0.85 6-8 80.87 0.97 absence > 200 Control tomato 0.81 6-8 63.22 0.91 absence > 200 Table 2: Morphological characteristics of experimental and control tomatoes Acta agriculturae Slovenica, 117/4 – 20216 F. BENALI and N. RAMDANI 3.3 SENSORY CHARACTERISTICS OF TOMATOES The test was carried out by a panel of tasters com- posed of twelve (12) people, men and women aged 27 to 58 years. It made it possible to evaluate the organoleptic qualities of the experimental tomatoes compared to the control tomatoes. The criteria used are colour (very red to red), taste (salty, sweet), acidity, consistency, texture (melting, crunchy and floury), aroma and odour. The sensory assessment was carried out on freshly harvested tomatoes for the four lots (I, II, III and IV). It emerges that twelve of the evaluators find that the three experimental batches are very red, identical to the con- trols. Recalling that colour of the fruit is linked to the abundant content of carotenoids in the peel and flesh of the fruit. As for the taste, the tomatoes at 10-s, 20-s and Figure 4: The number of tomatoes picked of the four lots during the test period the control tomatoes were judged to be lightly salted by eight of the tasters, while for the tomatoes at 30-s the assessment is in favour of bland with seven evaluators (Table 4b). On the other hand, eight people believed that the four batches of tomatoes were not very acidic while one taster did not find them at all acidic. The other most sought-after criterion is the scent character, nine of the evaluators believed that tomatoes had a strong odour, qualified as a strong tomato odour to little pronounced. The consistency of tomato fruits is also an important quality criterion as appreciated as the colour and the taste; it determines the resistance of the fruits to handling and their behaviour in the marketing circuit. The firm- ness was estimated manually, and ten evaluators consid- ered the four lots of tomatoes to be very firm to firm, while one taster felt they were not very firm (Table 4a). In order to assess (the salinity, acidity, sweetness, Designation E. coli Yeasts and moulds 10-1 10-2 10-3 10-1 10-2 10-3 Tomato at 10-s absence absence absence absence absence absence Tomato at 20-s absence absence absence absence absence absence Tomato at 30-s absence absence absence absence absence absence Control tomato absence absence absence absence absence absence Table 3: Bacteriological characteristics of tomatoes irrigated with ozonised water and controls Acta agriculturae Slovenica, 117/4 – 2021 7 Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics ... juiciness), and to translate the more or less intense char- acteristics of each of the criteria, we use the evaluation of the hedonic quality of the four batches (I, II, III, IV) tomatoes. The results show that all the lots show tomatoes of red colour, of round shapes, smooth and shiny, fleshy, tender, slightly acidic and less salty, of firm and tasty flesh, of easily removable cuticle. As a result, irrigating with ozonised water has no significant effect on the perception of the organoleptic characteristics of tomatoes compared to controls except for the taste character of tomatoes at 30-s where seven people of the panel estimated that ‘they were bland and there is no difference for the other criteria. The panellists’ results were reported in Table 4 (a,b). 3.4 COMPARATIVE ANALYSIS In our study, the agronomic characteristics of to- matoes irrigated with ozonised water such as seedling development, early germination, growth, vigour, plant size and yield were greater compared to control plants. Which is consistent with the work of Martínez-Sánchez & Aguayo(2019) where irrigation with aqueous ozone improved the development of greenhouse-grown pepper seedlings and the microbiological quality of the water. The plants developed a higher number of leaves and sec- ondary roots, which could improve the adaptability and yield of the seedlings when transplanted into the fields. Studies by Ohashi-Kaneko et al. (2009) reported that treatment with ozonised water improves root respiration and increases nutrient uptake and biomass production. The yield of the experimental tomatoes (10-s, 20-s and 30-s) was significantly higher than in the control plants. Guo & Wang (2017) reported that direct spray- ing of ozonised water on growing crops in fields increas- es their antioxidant content and photosynthetic activity, which enhances crop protection and prevents infection with plant pathogens. Plants subjected to ozonised water treatments below 10 mg l-1 had slightly increased plant diameter and height. The fresh mass of the leaf treated with 6 mg l-1 of ozonised water was remarkably increased by 40.6% compared to the control. Rozpądek et al. (2015) reported that plants subjected to aqueous ozone treat- ments during vegetation showed accelerated growth and reached marketable quality more quickly. The effects of irrigation of tomatoes with ozonised water at 10-s, 20-s and 30-s showed no morphological damage compared to the control, this is supported by the work of Guo & Wang (2017) reporting that no negative effects were observed after treatment with ozonised wa- ter spray at concentrations below 8 mg l-1 carried out on Chinese cabbage, on the other hand, visible damage to Sensory parameters of the four batches of tomatoes (I, II, III, IV) Colour Acidity Consistency Aroma and odour Level Tasters number Level Tasters number Level Tasters number Level Tasters number Very red 12 Very acidic 0 Very consistent 1 Very pronounced 0 Red 0 Acid 1 Consistent 9 Pronounced 3 Less red 0 Less acidic 8 Less consistent 1 Less pronounced 6 Not red 0 Not at all acidic 1 Not consistent 0 Not pronounced 1 Indifferent 0 Indifferent 2 Indifferent 1 Indifferent 2 Table 4a: Summarizes sensoria’s parameters of the four batches of the tomatoes as perceived by the tasters Other flavour Tomatoes at 10-s, 20-s and controls Other flavour for the tomatoes at 30-s Level Tasters number Level Tasters number Very salty 0 Very salty 0 Salty 1 Salty 1 Slightly salty 8 Slightly salty 1 Not at all salty 1 Not at all salty 2 Fade 0 Fade 7 Indifferent 2 Indifferent 1 Table 4b: Summarizes sensoria’s parameters of the four batches of the tomatoes as perceived by the tasters Acta agriculturae Slovenica, 117/4 – 20218 F. BENALI and N. RAMDANI the leaves was observed after the plants were exposed to 10 mg l-1 of ozonised water spray for 15 days during the reproductive phase of the plants. Aqueous ozone is a germination activator in li- mited quantities; excessive doses may affect the quality of the seeds. These results vary according to the varieties of species in question (Pandiselvam et al., 2020). An additional positive effect of ozonisation is the in- activation of pathogenic bacteria, such as Escherichia coli, Enterococcus and Staphylococcus strains (Heß & Gallert, 2015) which raise crop protection and prevents infection with plant pathogens, It is also used to disinfect irriga- tion water and as an alternative to pesticides (Guo et al., 2019; Landa Fernández et al., 2019). Finally, as future research, we can consider the ir- rigation with ozonised water at different concentrations at a large scale either in greenhouse or in open fields. Determining the threshold values of aqueous ozone con- centrations that can be applied into the different parts of the plants and at all growth stages, and duration and fre- quency of irrigation beyond which damage could occur affecting crops would be interesting to be investigated. The effect of ozonised water spraying onto the to- mato fruits and leaves could also be taken into account for the rest of our work. 4 CONCLUSION The objective of this work was the experimental study of the effect of irrigation with ozonised water on the morphological, bacteriological and sensory charac- teristics of ‘Saint-Pierre’ tomatoes grown in Algeria. On the basis of our study and taking into consideration all the observations on the experimental part, the results of the analysis reveal that the experimental fruits do not present any defect in development, shape, skin or colour- ing, all the batches show satisfactory firmness with good resistance to cracking. These characteristics constitute an advantage in the marketing of tomatoes; moreover, the latter have a good hygienic level, and present no health risk for consumers. The greater quantities of tomatoes picked as well as the size of the fruit is proportional to the concentration of the ozonized water. This observation proves the posi- tive influence of irrigation with ozonised water as well as the different concentrations of ozone on the tomato plants. Despite this, seven tasters felt that 30-s tomatoes tasted bland unlike other lot I and II. The yield of 20-s tomatoes was lower than that of tomatoes at 30-s but with better taste quality than the latter. Therefore, this concen- tration is highly recommended for cultivation under the climatic conditions of the study region. Irrigation with ozonised water can be used to in- crease the yield without negative impact on the environ- ment and the quality of the product, moreover the ozon- ised water generator does not require large investments, nor specific infrastructure of low energy consumption and maintenance, it is easy to use, which means that it can be implemented anywhere. However, studies must be continued for a better ap- plication of this technique in other growing conditions and on different varietal types or other market garden products. We have carried out this research on a small number of samples and on a single varietal type; we plan in the future to repeat this study on different plant species and to study them on a larger number of specimens. Our study will serve as a basis for introducing ozon- ised water into crop irrigation in the region of Oran (Western Mediterranean region) to increase yield, size and to accelerate germination, flowering and tomatoes fruit production. As perspectives, similar studies must be carried out in other regions of Algeria such as the Tellian, arid, semi-arid, Atlas and Saharan regions and determin- ing the impact of different concentrations of aqueous ozone on the characteristics agronomic, morphologi- cal, physicochemical, bacteriological, organoleptic and nutritional, for a better understanding of the climate ef- fect in parallel to the application of ozonised water at the large scale. A soil analysis would also be recommended for tomatoes produced with an environmentally friendly process. 5 REFERENCES Agassounon Djikpo Tchibozo, M., Gomez, S., Tchobo, F., Soumanou, M., & Toukourou, F. (2012). Essai de conser- vation de la tomate par la technique de la déshydratation imprégnation par immersion (DII). International Journal of Biological and Chemical Sciences, 6(2), 657-669. https://doi. org/10.4314/ijbcs.v6i2.10 Ali, M. Y., Sina, A. A. I., Khandker, S. S., Neesa, L., Tanvir, E. M., Kabir, A., Khalil, M. I., & Gan, S. H. (2020). Nutri- tional composition and bioactive compounds in tomatoes and their impact on human health and disease : A review. Foods, 10(1), 45. https://doi.org/10.3390/foods10010045 Bhat, N. A., Wani, I. A., & Hamdani, A. M. (2020). Tomato powder and crude lycopene as a source of natural antioxi- dants in whole wheat flour cookies. Heliyon, 6(1), e03042. https://doi.org/10.1016/j.heliyon.2019.e03042 Cámara, M., Fernández-Ruiz, V., Sánchez-Mata, M.-C., Díaz, L. D., Kardinaal, A., & Lieshout, M. van. (2020). Evidence of antiplatelet aggregation effects from the consumption of tomato products, according to EFSA health claim re- quirements. Critical Reviews in Food Science and Nutrition, 60(9), 1515-1522. https://doi.org/10.1080/10408398.2019.1 577215 Acta agriculturae Slovenica, 117/4 – 2021 9 Influence of ozonised irrigation water on the morphological, bacteriological and sensory characteristics ... Cheng, H. M., Koutsidis, G., Lodge, J. K., Ashor, A., Siervo, M., & Lara, J. (2017). Tomato and lycopene supplementation and cardiovascular risk factors : A systematic review and meta-analysis. Atherosclerosis, 257, 100-108. https://doi. org/10.1016/j.atherosclerosis.2017.01.009 FAOSTAT. (s. d.). Consulté 28 mai 2021, à l’adresse http://www. fao.org/faostat/fr/#compare García-Alonso, F.-J., García-Valverde, V., Navarro-González, I., Martín-Pozuelo, G., González-Barrio, R., & Periago, M. J. (2020). Tomato. In Nutritional Composition and Antioxi- dant Properties of Fruits and Vegetables (p. 255-271). Else- vier. https://doi.org/10.1016/B978-0-12-812780-3.00015-5 Graham, T., Zhang, P., Woyzbun, E., & Dixon, M. (2011). Re- sponse of hydroponic tomato to daily applications of aque- ous ozone via drip irrigation. Scientia Horticulturae, 129(3), 464-471. https://doi.org/10.1016/j.scienta.2011.04.019 Guo, Z., & Wang, Q. (2017). Efficacy of ozonated water against Erwinia carotovora subsp. carotovora in Brassica campestris ssp. chinensis. Ozone: Science & Engineering, 39(2), 127-136. https://doi.org/10.1080/01919512.2016.1270744 Guo, Z., Wang, Z., Li, Y., & Wang, Q. (2019). Effect of different concentrations of ozone on in vitro plant pathogens devel- opment, tomato yield and quality, photosynthetic activ- ity and enzymatic activities. Ozone: Science & Engineering, 41(6), 531-540. https://doi.org/10.1080/01919512.2019.159 1268 Güzel-Seydim, Z., Bever Jr, P. I., & Greene, A. K. (2004). Effica- cy of ozone to reduce bacterial populations in the presence of food components. Food Microbiology, 21(4), 475-479. https://doi.org/10.1016/j.fm.2003.10.001 Heß, S., & Gallert, C. (2015). Sensitivity of antibiotic resistant and antibiotic susceptible Escherichia coli, Enterococcus and Staphylococcus strains against ozone. Journal of wa- ter and health, 13(4), 1020-1028. https://doi.org/10.2166/ wh.2015.291 Horvitz, S., & Cantalejo, M. (2014). Application of ozone for the postharvest treatment of fruits and vegetables. Criti- cal reviews in food science and nutrition, 54(3), 312-339. https://doi.org/10.1080/10408398.2011.584353 Inter ministerial decree, Official Journal39. (2017, juillet 2). JOURNAL OFFICIEL DE LA REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE. https://www.joradp. dz/FTP/JO-FRANCAIS/2017/F2017007.pdf Joseph, H., Nink, E., McCarthy, A., Messer, E., & Cash, S. B. (2017). “The Heirloom tomato is ‘In’. Does it matter how it tastes?” Food, Culture & Society, 20(2), 257-280. https://doi. org/10.1080/15528014.2017.1305828 Landa Fernández, I. A., Monje-Ramirez, I., & Orta Ledesma de Velásquez, M. T. (2019). Tomato crop improvement using ozone disinfection of irrigation water. Ozone: Science & En- gineering, 41(5), 398-403. https://doi.org/10.1080/0191951 2.2018.1549474 Martínez-Sánchez, A., & Aguayo, E. (2019). Effect of irrigation with ozonated water on the quality of capsicum seedlings grown in the nursery. Agricultural Water Management, 221, 547-555. https://doi.org/10.1016/j.agwat.2019.05.027 Mitsugi, F., Abiru, T., Ikegami, T., Ebihara, K., & Nagahama, K. (2017). Treatment of nematode in soil using surface barrier discharge ozone generator. IEEE Transactions on Plasma Science, 45(12), 3076-3081. https://doi.org/10.1109/ TPS.2017.2708706 Nethaji, D. K., Suresh, S., Prasanna, J. E. H., Vijayagopal, V., & Ramesh, G. (2020). Development of mango and tomato paste and it’s physico-chemical characterization. Interna- tional Journal of Scientific Research in Science, Engineer- ing and Technology, 165-171. https://doi.org/10.32628/ IJSRST207537 Nomades, D. C. (s.  d.). Météo en Algérie en 2020. Historique Météo. Consulté 19 juin 2021, à l’adresse https://www.his- torique-meteo.net/afrique/algerie/2020/ Ohashi-Kaneko, K., Yoshii, M., Isobe, T., Park, J.-S., Kurata, K., & Fujiwara, K. (2009). Nutrient solution prepared with ozonated water does not damage early growth of hydropon- ically grown tomatoes. Ozone: Science & Engineering, 31(1), 21-27. https://doi.org/10.1080/01919510802587523 Pandiselvam, R., Mayookha, V. P., Kothakota, A., Sharmila, L., Ramesh, S. V., Bharathi, C. P., Gomathy, K., & Srikanth, V. (2020). Impact of ozone treatment on seed germination – A systematic review. Ozone: Science & Engineering, 42(4), 331-346. https://doi.org/10.1080/01919512.2019.1673697 Pandiselvam, R., Sunoj, S., Manikantan, M. R., Kothakota, A., & Hebbar, K. B. (2017). Application and kinetics of ozone in food preservation. Ozone: Science & Engineering, 39(2), 115-126. https://doi.org/10.1080/01919512.2016.1268947 Renaud, V. (2003). Tomate. Tous les legumes courants, rares ou méconnus cultivables sous nos climats. Ulmer. Paris, Ulmer, 135-137. Rowles, J. L., Ranard, K. M., Applegate, C. C., Jeon, S., An, R., & Erdman, J. W. (2018). Processed and raw tomato consump- tion and risk of prostate cancer : A systematic review and dose–response meta-analysis. Prostate Cancer and Prostatic Diseases, 21(3), 319-336. https://doi.org/10.1038/s41391- 017-0005-x Rozpądek, P., Nosek, M., Ślesak, I., Kunicki, E., Dziurka, M., & Miszalski, Z. (2015). Ozone fumigation increases the abun- dance of nutrients in Brassica vegetables : Broccoli (Bras- sica oleracea var. italica) and Chinese cabbage (Brassica pe- kinensis). European Food Research and Technology, 240(2), 459-462. https://doi.org/10.1007/s00217-014-2372-z Saini, R. K., Rengasamy, K. R. R., Mahomoodally, F. M., & Keum, Y.-S. (2020). Protective effects of lycopene in cancer, cardiovascular, and neurodegenerative diseases : An update on epidemiological and mechanistic perspectives. Pharma- cological Research, 155, 104730. https://doi.org/10.1016/j. phrs.2020.104730 Siti Fadlilah, Adi Sucipto, & Mohamad Judha. (2020). Cucum- ber (Cucumis sativus) and tomato (Solanum lycopersicum) juice effective to reduce blood pressure. GSC Biological and Pharmaceutical Sciences, 10(1), 001-007. https://doi. org/10.30574/gscbps.2020.10.1.0246 Walallawita, U. S., Wolber, F. M., Ziv-Gal, A., Kruger, M. C., & Heyes, J. A. (2020). Potential role of lycopene in the preven- tion of postmenopausal bone loss : Evidence from molecu- lar to clinical studies. International Journal of Molecular Sci- ences, 21(19), 7119. https://doi.org/10.3390/ijms21197119 Wu, H., Li, W., Wang, T., Rong, Y., He, Z., Huang, S., Zhang, L., Wu, Z., & Liu, C. (2021). α-tomatine, a novel early-stage au- tophagy inhibitor, inhibits autophagy to enhance apoptosis Acta agriculturae Slovenica, 117/4 – 202110 F. BENALI and N. RAMDANI via Beclin-1 in Skov3 cells. Fitoterapia, 152, 104911. https:// doi.org/10.1016/j.fitote.2021.104911 Yang, T., Yang, X., Wang, X., Wang, Y., & Song, Z. (2013). The role of tomato products and lycopene in the preven- tion of gastric cancer : A meta-analysis of epidemiologic studies. Medical Hypotheses, 80(4), 383-388. https://doi. org/10.1016/j.mehy.2013.01.005 Acta agriculturae Slovenica, 117/4, 1–14, Ljubljana 2021 doi:10.14720/aas.2021.117.4.1887 Original research article / izvirni znanstveni članek Effects of soil nutrient amendments on growth and grain yield perfor- mances of quality protein maize grown under water deficit stress in Ibadan, Nigeria Folake ANJORIN 1, 2, Adeyinka ADEBAYO 1, Taiwo OMODELE 1, and Adewale ADETAYO 1, James ADED- IRAN Received September 26, 2021; accepted November 15, 2021. Delo je prispelo 26. septembra 2021, sprejeto 15. november 2021 1 Institute of Agricultural Institute Research and Training, Obafemi Awolowo University, Ibadan, Nigeria 2 Corresponding author, e-mail: folakeawoeyo@yahoo.com Effects of soil nutrient amendments on growth and grain yield performances of quality protein maize grown under wa- ter deficit stress in Ibadan, Nigeria Abstract: Drought and poor soil fertility are major limita- tions to crop production, globally. To investigate the impacts of water deficit stress (WS) and soil nutrient amendment (SA) on growth and yield performances of maize. A two years factorial field study was carried out, using a quality protein maize (QPM) (ILE-1-OB) and a non QPM–drought tolerant check (TZPBSR- W) varieties in Ibadan. Treatments include; six fertilizer appli- cation rates; 50 and 100 (kg N ha-1) of NPK-20-10-10, 10.7 kg N ha-1of Tithonia Poultry Compost (TPC), 50 N + 10.7TPC and 100 N + 10.7TPC (kg N ha-1), three WS; the control (FW), WS at vegetative stage (STR1), and WS at reproductive stage (STR2). Leaf area (LA) and grain yield (GY) were measured using stan- dard procedures. From the results, across WS, LA ranged from STR1 (458.90 ± 12.4) to FW (598.81 ± 13.1 cm2), GY varied from STR2 (2.94 ± 0.2 t ha-1) to FW (6.59 ± 0.2 t ha-1), across fertilizers, LA varied from 0 N (397.65 cm2) to 100N + 10.7TPC (622.71 cm2) and 50 N + 10.7TPC (611.03 cm2), respectively. The GY varied from 0 N (2.37 t ha-1) to 100 N + 10.7TPC (5.82 t ha-1) and 50N + 10.7TPC (5.26 t ha-1). Drought stress reduced growth and GY performances of QPM, while SA with 50 kg N ha-1 of inorganic fertilizer and 10.7 kg N ha-1 of TPC enhanced growth and grain yield of maize under WS. Key words: fertilizer application rates; grain yield; growth and yield performances;quality protein maize; soil nutrient amendments; water deficit stress Učinki gnojenja na rast in pridelek zrnja na proteinih oboga- tene koruze v razmerah sušnega stresa, Ibadan, Nigeria Izvleček: Suša in slaba rodovitnost tal sta v globalnem obsegu glavna dejavnika, ki omejujeta produktivnost gojenih rastlin. Za preučevanje vpliva vodnega deficita (WS) in doda- janja hranil v tla (SA) na rast in pridelek koruze je bil izveden dvoletni faktorski poljski poskus na sorti ILE-1-OB, bogati na proteinih (QPM) in na sušo odporni sorti TZPBSR-W kot kon- troli, ki ni obogatena s proteini (non QPM), v Ibidanu, Nigerija. Obravnavanja so obsegala: šest načinov gnojenja (50 in 100 (kg N ha-1) z NPK-20-10-10, 10,7 kg N ha-1 komposta iz vrste Titho- nia pomešanega s kokošjim gnojem (TPC), 50 N + 10,7 TPC in 100 N + 10,7 TPC (kg N ha-1), tri stopnje vodnega deficita (WS) v vegetativni (STR1) in reproduktivni fazi (STR2) in kontrolo s polnim namakanjem. Listna površina (LA) in pridelek zrnja (GY) sta bila izmerjena s standardnimi metodami. Listna povr- šina je v vegetativni fazi ob pomanjkanju vode znašala 458,90 ± 12,4 cm2, ob polnem zalivanju pa 598,81 ± 13,1 cm2. Pridelek zrnja je ob vodnem deficitu v reproduktivni fazi znašal 2,94 ± 0,2 t ha-1, pri polnem zalivanju pa 6,59 ± 0,2 t ha-1. Listna povr- šina je bila glede na načine gnojenja sledeča: 0 N (397,65 cm2), 100 N + 10,7 TPC (622,71 cm2) in 50 N + 10,7 TPC (611,03 cm2). Pridelek zrnja je glede na načine gnojenja dosegel nasle- dnje vrednosti: 0 N (2,37 t ha-1), 100 N + 10,7 TPC (5,82 t ha-1) in 50 N + 10,7 TPC (5,26 t ha-1). Sušni stres je zmanjšal rast in pridelek sorte QPM, gnojenje s 50 kg N ha-1 kot anorganskim gnojilom dopolnjeno z 10,7 kg N ha-1 v organski obliki je pospe- šilo rast in pridelek zrnja koruze v razmerah vodnega deficita. Ključne besede: na proteinih obogatena koruza; odmerki in vrste gnojil; rast; pridelek zrnja; sušni stress 309. Acta agriculturae Slovenica, 117/4 – 20212 F. ANJORIN et al. 1 INTRODUCTION Maize is an important cereal crop with wide range of utilization in several countries of the world. Apart from been a major staple crop, maize is an important ingredi- ent in livestock feed formulation for the rising poultry business in the sub Saharan Africa. Maize has remained a unique crop with great potentials to survive across dif- ferent agro-ecology and vegetations, globally. However, the detrimental impacts of drought and poor soil fertility on profitable maize production in the tropics cannot be overemphasized (Goldblatt, 2010; Ammani et al., 2012). Unpredictable weather conditions, erratic rainfall pat- terns, and incidences of occasional pockets of drought even at the peak of rains are characteristics attributes of Nigeria’s climate, lately. The consequences of climate change are gradually having its turn on the nation’s veg- etation and cropping system. An estimated value of about 15  % reductions in global maize production has been attributed to drought alone (Edmeades, 2013). Inadequate water availability af- fects virtually all physiological and metabolic processes in maize development. Processes such as germination, seedling growth, leaf formation, stem elongation, and overall crop development (Anjorin et al., 2017; Anjor- in et al., 2018). The severity of damage resulting from drought stress depends on the duration of drought and the phenological stage of plant development as at time of stress (Chaves et al., 2002; Jongdee et al., 2002). The reproductive developmental stage has been shown to be the most critical stage for maize sensitivity to drought. Monneveeux et al. (2006) in a similar view, reported that grain yield in maize could be drastically reduced by drought prolonged beyond 12 days during grain filling and flowering stages. Apart from drought, uncontrolled soil nutrient min- ing due to continuous cropping without supplementary replacement has been a common and regular practice in most countries of sub-Saharan Africa (Ngetich et al., 2012). An estimated average annual nutrient depletion ranged from 20 kg to 50 kg NPK ha-1yr-1 in majority of developing countries to more than 100 kg NPK ha-1yr-1 in the least developed countries of Africa (Tan et al., 2005). Crops appear more devastated especially when both drought and nutrient stresses occur simultaneously. However, the use of drought tolerant crop genotypes and fertilizers has the potentials to enhance crop growth and yield in the face of prevailing climatic challenges. Over time, several integrated soil fertility management strat- egies (ISFM) that could enhance soil fertility potentials and productivity in Africa had been advocated (Scoones & Toulmin, 1998). These include the use of fertilizers, organic inputs and improved germplasm in addition to the technicalities of adapting these practices to local en- vironments (Vanlauwe et al., 2010; Sanginga & Woomer, 2009). Therefore, there is a need for a balance in moisture and nutrient availability in the crop environment with regards to stages of plant development for optimum crop yield. As at present much work has not been carried out in this part of the world on soil fertility management strategies with regards to occurrences of drought dur- ing various phenological growth stages in maize. Hence, this study aimed at assessing the impact of inorganic and organic fertilizers (using Tithonia poultry compost) soil amendment interventions at ameliorating the impact of water deficit stress (drought) on maize phenology. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE, LOCATION AND DE- SIGN The study was conducted on the research field (Lon- gitude 3o50’56.1”E and latitude 7o22’ 20” N) during the dry seasons between Decembers – March in 2014/ 2015 and 2015/2016 at the Institute of Agricultural Research and Training (I.A.R&T), Moor Plantation in Ibadan. The I.A.R&T is located in the derived savanna agro ecology of Nigeria (Figure 1). 2.2 TREATMENTS 2.2.1 Water Deficit Stress (i) No water stress (FW): plots receive water up to field capacity till plant maturity (ii) Water stress for 14 days (withdrawn of watering) at three weeks after seedling emergence, while normal watering resumed till plant maturity (STR1) (iii)Water stressed imposed in maize plots by water withdrawer for 14 days at 6 weeks after seedling emer- gence after which normal watering resumed till plant maturity (STR2). 2.2.2 Fertilizer rates (i) Three rates of N fertilizer (NPK-20-10-10); 0 N, 50 N, 100 N (kg ha-1) (ii) One rate of Tithonia - Poultry Compost (TPC): 10 TPC (t ha-1) (10.7 kg N ha-1), Acta agriculturae Slovenica, 117/4 – 2021 3 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria (iii) Two rates of N fertilizer and TPC combinations; 50 N + 10 TPC and 100 N +10 TPC. 2.2.3 Varieties Two maize varieties consisting of one quality pro- tein maize variety (ILE-1-OB) and a drought tolerant maize (TZPBSR-W) (Smale et al., 2011) are both open pollinated (intermediate maturing) high yielding charac- terized by flint texture and white colour seeds, were col- lected from the seed store of I.A.R & T, Ibadan. 2.2.4 Experimental design The maize field was planted in 3 x 6 x 2 factorial ar- rangements using randomized complete block design (r = 3). Each of the three main plots was 27.5 m by 14 m in size were separated by 5 m apart to prevent water seep- age across the main plot during irrigation processes, the sub-plot was 4 m x 7.5 m while the sub-sub plot was 4 m x 3.75 m. There were thirty - six plots in the each main plot, each of the sub - sub plot consisted of six (6) rows of two plants per hill at a planting distance of 75 cm x 50 cm inter rows and intra rows spacing, respectively. 2.3 LAND PREPARATION, PLANTING AND CROP MANAGEMENT The pre crop for both first and second year is maize. The land was prepared mechanically by ploughing and harrowing. Initial wetting was done before each of the operations to ease the operations because the land was very dry and compacted as expected during the dry sea- son. After land preparations, maize seeds were sown at three seeds per hill. The young maize seedlings down to two vigorous healthy seedlings per stand. Pre emergence herbicides (Atrazin® 4 kg ha-1 and Glyphosate) were ap- plied to control weeds, while subsequent weeding was done with local hoes. 2.4 COMPOST PREPARATION AND FERTILIZER APPLICATION The compost was prepared from fresh cuttings of Mexican sunflower (Tithonia diversifolia (Hemsl.) us- Figure 1: Map showing the experimental plots and location of the experiment at the Institute of Agricultural Research and Train- ing in Ibadan, Oyo state, Nigeria Acta agriculturae Slovenica, 117/4 – 20214 F. ANJORIN et al. ing the heap method described by Fernhill, (2011). Nine (9) kilogram of Mexican sun flower (Tithonia diversi- folia) plant cuttings of about 10 centimeters long were weighed, chopped and spread on the earth surface. The spread plant cuttings were alternated in layers with the spreading of three kilogram (3 kg) of cured fecal poultry droppings to form heap of 1.3 m height. Several heaps made were sprinkled with water before covering with black polythene sheet to increase temperature, moisture maintenance and escape of gases. The heaps were over turned fortnightly with the aid of long garden fork and moisturized adequately to enhance effective microbial growth and activities. Adequate aeration was achieved using 1 m diameter pipes inserted vertically and horizon- tally into the heaps to ensure adequate ventilation. The pH and temperature were monitored until the compost matured (AAFRD, 2005). The compost heaps were al- lowed to stay for a period of 21/2 months after which the compost materials were ready for use. The compost ma- terial was spread thinly on a drying surface under shade and allowed to dry very well before storing in bags. Sam- ple of the matured compost were analysed for chemical properties (Anjorin, 2018). Compost was applied a-week before planting to each of the designated plots to initi- ate early mineralization of nutrients. Inorganic fertilizer (urea) was applied to the designated plots in splits at two weeks and five weeks after emergence based on the pre - determined rate. 2.5 IRRIGATION Irrigation was done using sprinklers while tensiom- eter (Eijkelkamp.co) was used to monitor the soil water potential. 2.6 DATA COLLECTION - Plant height (using meter rule and measured in centimeter from the base of the plant to the base of the last emerged leaf). - Leaf area (obtained by measuring in cm2 using the meter rule to measure the length of a fully expanded tagged leaf and the breadth at mid leaf. The product of the length and the width was multiplied by 0.75 which is the calibration factor for maize leaf (Francis et al., 1969). - Number of ears per plant (by visual counting) - Number of rows per cob, number of kernels per row (by visual counting ), number of kernels per cob (obtained by multiplying the number of kernels per rows with number of rows per cob), cob length (measuring the length of a cob using the meter rule) - Grain yield was taken from total ear harvest per plot. - Mass of 1000 grains and total grain yield (after Figure 2: Mean monthly temperature (oC), humidity and precipitation (mm) during 2014, 2015 and 2016 planting seasons. Source: Nigerian Meteorological Agency, Ibadan (NIMET) Acta agriculturae Slovenica, 117/4 – 2021 5 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria adjusting to 12 % moisture content) using weighing bal- ance. 2.7 DATA ANALYSIS The data collected were pooled across the two years and subjected to analysis of variance (ANOVA) for split - split - split plot in RCBD using Statistical Tool For Agri- cultural Research (STAR, version 2.0.1 2014). Significant means were separated using Tukey Honest Significant Difference at 5% probability level. 3 RESULTS 3.1 WEATHER INFORMATION The mean monthly temperature (oC), humidity and precipitation (mm) during the experimental studies were shown in Figure 2. No rainfall was recorded for the months of December, January, February (actual periods when water deficit stress was imposed). About 3.43 and 3.49 mm total number of rainfall were recorded in March in 2015 and 2016, respectively. Maximum temperatures were recorded in February while relative humidity values were significantly reduced in January and February of the years of the trials. 3.2 SOIL PHYSICO-CHEMICAL PROPERTIES The soil obtained from the experimental field was a loamy-sandy soil of classification series “Typic Kan- haplustalf”. Result of the chemical analyses showed that there were slight variations in the soil chemical proper- ties in the two years of the experimental studies (Table 1). The pH value of the soil samples appeared slightly acidic in 2015 (6.00) and slightly basic in 2016 (7.25). Soil total nitrogen (0.06  %, 0.05  %), available phosphorus (13.16 mg kg-1, 6.84 mg kg-1), organic carbon (0.44 %, 0.86 %), potassium and the micronutrients were very low in 2015 and 2016 compared with recommended soil requirement for Nigerian soils. 3.3 CHEMICAL PROPERTIES OF COMPOST USED The compost was slightly basic with pH value of 8.30, total nitrogen content was 0.70  %, while the val- ues of phosphorus and potassium were 0.91 mg kg-1 and 0.61 cmol kg-1, respectively (Table 2). The compost had high carbon to nitrogen ratio value (7.47), and very high micronutrients (Iron (9587), Zinc (436) and manganese (597) mg kg-1). 3.4 PLANT HEIGHT (PHT) Water deficit stress significantly influenced plant height and fertilizer application rates (p < 0.001) (Table 3). Significant reduction in plant heights were observed in maize subjected to water deficit stress at three weeks after emergence compared with maize grown under FW and STR2, plant heights ranged from 119.79 cm (STR1) to 150.76 cm (FW) (Table 4). Across the fertilizer rates, maize heights ranged between 116.88 cm (0 N) to 141.18 cm (100 N + 10 TPC), there was no significant difference in the plant heights observed across the fertilizer appli- cation rates, except for the control which had relatively shorter plants. Maize variety TZPBSR-W (136.43 cm) are taller than ILE-1-OB (132.01 cm). 2015 2016 Parameter pH (H20) 6.00 7.25 Organic carbon (%) 0.44 0.86 Total nitrogen (%) 0.06 0.05 Available P (mg kg-1) 13.16 11.84 Bulk density (Mg m-3) 1.31 1.31 ECEC(cmol) 7.11 5.56 Base saturation (%) 99.02 99.28 Exchangeable cation (cmol kg-1) K 0.22 0.37 Na 0.39 0.63 Ca 5.53 3.80 Al+H 0.07 0.04 Exchangeable micronutrient (mg kg-1) Fe 7.10 0.06 Zn 3.60 0.65 Cu 1.10 0.15 Mn 22.8 44.10 Soil particle analysis Sand g kg-1 854 842 Silt g kg-1 82 86 Clay g kg-1 64 72 Textural class loamy -Sandy loamy -Sandy Table 1: Pre-planting physico - chemical properties of soil used for the experiments Acta agriculturae Slovenica, 117/4 – 20216 F. ANJORIN et al. Water deficit stress and fertilizer interaction (WS x F) effect on plant height was significant (p < 0.05). Plant height ranged from 0 N (99.99 cm) (STR1) to 160.40 cm 10 TPC (FW) (Figure 3a). Plant heights at STR1 across the various fertilizers application rates were not signifi- cantly different but higher than 0N (99.99 cm) (p < 0.05). Highest plant height was observed at 10 TPC (160.40 cm) (FW) but lowest at 0 N (STR1). Water deficit stress and variety interaction (WS x V) interaction effect on plant heights was significant (p < 0.05) (Table 5). Maize variety TZPBSR-W (160.26 cm) had taller stems under full watering than ILE-1-OB (149.18 cm), however no differences observed in the heights at STR1 and STR2, respectively. 3.5 LEAF AREA (LA) Leaf area differed significantly across water deficit stress and fertilizer application rates (p < 0.001) (Ta- ble 3). The leaf areas varied from 458.90 cm2 (STR1) to 598.81 cm2 (FW) (Table 4). Across F rates, the largest leaf area size was observed when 100 N + 10 TPC was ap- plied (622.71 cm2), this LA value was however not sig- nificantly different from LA’s obtained when 50 N + 10 TPC (611.03 cm2) and 10 TPC (581.57 cm2) were applied, while the control (0 N) had least LA size of 397.65 cm2. The leaf areas of the two maize varieties were not signifi- cantly different. Water deficit stress and fertilizer interaction (WS x F) effect on LA was significant (p < 0.001) (Figure 3b). Large leaf area (LA) sizes of maize plant were observed at 100 N + 10 TPC (645.31 cm2) and 50 N + 10 TPC (647.47 cm2) under FW. The leaf areas obtained were not significantly different from LA’s obtained under 50 N and 100 N and 10 TPC fertilizer applications rates except 0 N (465.11 cm2). Similar trend was observed in STR2 across the fertilizers application rates. Significant reduction in leaf sizes were observed in STR1 across the fertilizer rates, however considerably larger leaf area sizes were ob- served with applications of 100 N + 10 TPC (541.47 cm2) and 50 N + 10 TPC (528.19 cm2), respectively. 3.6 NUMBER OF EAR PER PLANT (E/P) The number of ear per plant was not significantly influenced by WS (p < 0.05) (Table 3), however the E/P varied significantly across fertilizer application rates (p < 0.01). Applications of 100 N + 10 TPC (1.57) and 50 N + 10 TPC (1.60) produced more ear per plant than other F-application rates and the control which had the least value of 1.27 of ear per plant. Fertilizer and variety interaction (F x V) effects on number of ear per plant of two maize was significant (p < 0.05). Maize variety ILE-1-OB (1.39) had fewer numbers of ears than TZPBSR-W (1.15) under the control, maize variety TZPBSR-W (1.55) had more ears than ILE-1-OB (1.35) under 100 N (Table 6). 3.7 COB LENGTH (CBT) The cob length was significantly influenced by water deficit stress and fertilizer application rates (p < 0.001) (Table 3), across WS, the cob length ranged from 12.46 cm (STR2) to 18.02 cm (FW) (Table 4). Cobs length ranged between 0 N (12.02 cm) to 100 N + 10 TPC (16.44 cm). However, no significant difference was observed between cob lengths of 100 N (15.45 cm) and 10 TPC (15.28 cm). 3.8 NUMBERS OF ROWS PER COB (R/C) Water deficit stress and fertilizer significantly in- fluenced number of rows per cob (p < 0.001) (Table 3). The effect of WS on R/C, varied between STR2 (11.83) to FW (13.72), while applications of 100 N + 10 TPC and 50 N + 10 TPC and 100 N had the highest number of rows per cob compared with R/C of other fertilizer ap- plications rates but lowest in the control (11.03) (Table Parameter Values pH (H20) 8.30 Organic carbon (%) 5.25 Total nitrogen (%) 0.70 Available P (mg kg-1) 0.91 C/N ratio 7.47 Exchangeable cation (cmol kg-1) K 0.61 Na 0.62 Ca 4.95 Mg 0.92 Exchangeable micronutrient(mg kg-1) Fe 9587 Zn 436 Cu 31.0 Mn 597 Table 2: Chemical properties of the Tithonia poultry compost used as soil amendment Acta agriculturae Slovenica, 117/4 – 2021 7 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria So ur ce o v ar ia tio n D .F PH T (c m ) LA (c m 2 ) E/ P C BT (c m ) R/ C K /R K /C 10 00 -K M (g ) G Y (t h a-1 ) Re p 2 20 .7 6 n s 10 56 .6 8n s 0. 02 ns 0. 92 ns 0. 28 ns 29 .1 3* 52 25 .0 6* 33 .6 8n s 0. 88 ns W at er D efi ci t S tr es s ( W S) 2 87 51 .4 6* ** 18 99 71 .9 8* ** 0. 09 ns 27 8. 99 ** * 32 .9 3* ** 14 08 .2 5* ** 38 30 53 .6 2* ** 76 71 .4 7* * 12 8. 15 ** * Er ro r( a) 4 30 .4 8 11 93 .0 8 0. 18 2. 26 0. 39 2. 20 47 2. 02 96 .6 8 0. 66 Fe rt ili ze r ( F) 5 15 49 .1 0* ** 12 94 68 .9 5* ** 0. 25 ** 53 .8 8* ** 16 .0 9* ** 43 5. 31 ** * 11 52 00 .0 2* ** 43 36 .7 2* ** 26 .1 6* ** W S x F 10 17 0. 81 * 74 90 .9 9* ** 0. 04 ns 2. 83 ns 0. 99 ns 16 .6 5* * 43 02 .0 1* * 53 8. 55 * 1. 50 ** Er ro r ( b) 30 73 .0 9 1 49 2. 64 0. 05 1. 55 0. 50 3. 44 10 41 .1 2 2 01 .5 7 0 .4 0 Va rie ty (V ) 1 52 4. 92 ** 45 72 .7 6n s 0. 00 ns 1. 45 ns 1. 05 ns 3. 35 ns 14 93 .1 6n s 25 5. 38 ns 0. 41 ns W S x V 2 21 7. 86 * 51 50 .8 0n s 0. 01 ns 1. 34 ns 0. 38 ns 6. 39 ns 14 12 .4 4n s 11 5. 18 ns 0. 51 ns F x V 5 30 .3 9n s 11 31 .8 6 n s 0. 08 * 1. 76 ns 0. 39 ns 4. 91 ns 10 65 .8 1n s 10 7. 53 ns 0. 57 n s W S x F x V 10 30 .2 3n s 13 37 .7 8n s 0. 02 1n s 0. 67 ns 0. 74 ns 4. 14 ns 11 36 .8 9n s 55 .2 1n s 0. 37 n s Er ro r ( c) 36 54 .6 1 20 72 .0 1 0. 02 5 1 .3 0 0. 79 4. 19 13 15 .1 6 11 5. 31 0. 34 To ta l 10 7 Ta bl e 3: M ea n sq ua re o f A N O VA o f t he e ffe ct s o f w at er , f er til iz er , v ar ie ty a nd re su lt of f- in te ra ct io n on g ro w th a nd y ie ld co m po ne nt s o f t w o m ai ze v ar ie tie s e va lu at ed in Ib ad an * ** * ** S ig ni fic an t a t p < 0 .0 5, 0 .0 1, 0 .0 01 , n s = n ot si gn ifi ca nt . D .F = D eg re e of fr ee do m † M ea ns n ot fo llo w ed b y th e sa m e. Le tte rs w ith in a co lu m n ar e s ig ni fic an tly d iff er en t a t P = 0 .0 5 ac co rd in g to T uk ey H SD . P H T = Pl an t h ei gh t, LA = L ea f a re a, E /P = E ar p er p la nt , C BT = C ob le ng th R /C = R ow p er co b, K /R = K er ne l p er ro w, K /C = K er ne l p er co b, 1 00 0- K M = M as s o f 1 00 0 ke rn el s a nd G Y = G ra in y ie ld Acta agriculturae Slovenica, 117/4 – 20218 F. ANJORIN et al. PH T (c m ) LA (c m 2 ) E/ P C BT (c m ) R/ C K /R K /C 10 00 -K M (g ) G Y (t h a-1 ) W at er re gi m e FW 15 0. 76 a 59 8. 81 a 1. 54 a 18 .0 2a 13 .7 2a 30 .3 4a 41 9. 68 a 24 4. 73 a 6. 59 a ST R1 11 9. 79 c 45 8. 90 c 1. 46 a 14 .9 0b 12 .5 0b 23 .0 5b 29 2. 98 b 23 1. 43 b 3. 92 b ST R2 13 2. 11 b 56 2. 77 b 1. 44 a 12 .4 6c 11 .8 3c 17 .9 0c 21 5. 33 c 21 5. 57 c 2. 94 c F.r at e (F ) 0 N 11 6. 88 b 39 7. 65 c 1. 27 b 12 .0 2d 11 .0 3c 15 .5 7c 17 7. 34 c 20 3. 78 c 2. 37 c 50 N 13 2. 58 a 49 4. 84 b 1. 51 ab 14 .6 4c 12 .1 8b 21 .5 8b 26 6. 72 b 22 2. 23 ab 3. 98 b 10 0 N 13 3. 75 a 53 3. 15 ab 1. 45 ab 15 .4 5b c 13 .1 1a 25 .5 7a 33 7. 58 ab 23 3. 16 ab 4. 85 ab 10 T PC 14 0. 13 a 58 1. 57 a 1. 49 ab 15 .2 8b c 12 .8 2a b 23 .3 3a b 30 6. 96 ab 23 6. 01 a 4. 61 ab 50 N + 1 0 TP C 14 0. 79 a 61 1. 03 a 1. 60 a 16 .4 4a b 13 .5 0a 26 .6 9a 36 2. 46 ab 24 6. 68 a 5. 26 a 10 0 N + 10 T PC 14 1. 18 a 62 2. 71 a 1. 57 a 16 .9 1a 13 .4 6a 29 .8 4a 40 4. 92 a 24 1. 60 a 5. 82 a Va rie ty 15 .0 1 67 .8 5 0. 27 1. 26 0. 72 3. 26 56 .6 6 15 .9 5 0. 78 TZ PB SR -W 13 6. 43 a 54 6. 18 a 1. 48 a 15 .0 1a 13 .1 4a 23 .5 8a 30 5. 61 a 23 2. 13 a 4. 58 a IL E- 1- O B 13 2. 01 b 53 3. 65 a 1. 48 a 15 .2 4a 12 .7 7a 23 .9 4a 31 3. 31 a 22 9. 05 a 4. 51 a s e 1. 69 10 .4 5 0. 02 0. 29 0. 14 0. 69 11 .3 8 2. 17 0. 19 M ea n 13 4. 22 54 0. 16 1. 48 15 .1 2 12 .6 8 23 .7 6 30 9. 33 23 0. 58 4. 48 Ta bl e 4: M ai n eff ec t o f W at er D efi ci t S tr es s, fe rt ili ze r a nd v ar ie ty e ffe ct o n gr ow th a nd y ie ld co m po ne nt s o f t w o m ai ze v ar ie tie s e va lu at ed in Ib ad an † M ea ns n ot fo llo w ed b y th e sa m e le tte r w ith in a co lu m n ar e sig ni fic an tly d iff er en t a t p < 0 .0 5 ac co rd in g to T uk ey H on es t S ig ni fic an t D iff er en ce . S TR 1 = W at er st re ss at v eg et at iv e gr ow th st ag e, ST R 2 = W at er st re ss a t r ep ro du ct iv e gr ow th st ag e an d FW = F ul l w at er in g, P H T = Pl an t h ei gh t, LA = L ea f a re a, E /P = E ar p er p la nt , C BT = C ob le ng th R /C = R ow p er c ob , K /R = K er ne l p er ro w, K /C = K er ne l pe r c ob , 1 00 0 K M = M as s o f 1 00 0- ke rn el s a nd G Y = G ra in y ie ld Acta agriculturae Slovenica, 117/4 – 2021 9 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria 4). Interaction effects on number of rows per cob were not significant 3.9 NUMBERS OF KERNELS PER ROW (K/R) Number of kernels per row varied across the repli- cates (p < 0.05), WS and F (p < 0.001) and WS x F (0.01) (Table 3). Effect of WS on K/R was the lowest at STR2 (17.90) but the highest at FW (30.34), across fertilizer ap- plication rates (Table 4). Across SA rates, the number of kernel per row also ranged between 0 N (15.57) to 100 N + 10 TPC (29.84), though number of K/R at 100 N + 10 TPC was not significantly different from K/R recorded for 50 N + 10 TPC (26.69) and 100 N (25.57). The num- ber of kernels per row ranged from 0 N (22.64) to 100 N + 10 TPC (34.37) under FW, while no significant dif- ference among K/R formed by the applications of 100 N + 10 TPC (34.37), 50 N + 10 TPC (28.79) and 10 TPC (32.99) (Figure 3c). High significant reductions in num- ber K/R was observed at STR2 and K/R ranged from 0 N (9.76) to 100 N + 10 TPC (24.37) followed by 50 N + 10 TPC (21.59). 3.10 NUMBERS OF KERNELS PER COB (K/C) The numbers of kernels per cob varied significantly across the replicates (p < 0.05), WS and F (p < 0.001) and WS x F (p < 0.01) (Table 3). Effect of WS on K/C varied from STR2 (215.33) to FW (419.68) (Table 4). Across F- rates, the highest number of kernel per cob was recorded at 100 N + 10 TPC (404.92) and the lowest in the control (177.34), number of kernels per cob at 100 N, 10 TPC and 50 N + 10 TPC were not significantly different. Water deficit stress and fertilizer interaction effect was signifi- cant on K/C, least number of K/C was obtained at STR2 (98.86) while application of 100 N + 10 TPC (514.94) gave highest number of K/C at FW. 3.11 MASS OF1000-KERNELS (1000-KM) The mass of 1000-kernels was significantly influ- enced by WS (p < 0.01), F (p < 0.001) and WS x F (p < 0.05) (Table 3). The effect of WS on 1000-kernel mass varied from STR2 (215.57 g) to FW (244.73 g) (Table 4). Across the fertilizer application rates, the mass of 1000-kernel was the highest at 50 N + 10 TPC (246.68 g), though not significantly different from 1000-KM of 100 N+ 10 TPC (241.60 g) and 10 TPC (236.01 g), while the control had the least value of 203.78 g. Water deficit stress, fertilizer interaction effect shows that the highest 1000-kernel mass was obtained at 50 N + 10 TPC under FW (266.13 g), this value was not significantly different from 1000-kernel mass observed at 100 N + 10 TPC (261.38 g), 10 TPC (257.78 g) and 10 TPC (257.50 g), while the smallest value of 1000-kernel mass was observed under STR2 at 0 N (179.10 g). Across STR1, the mass of 1000-kernels were not significantly different. 3.12 GRAIN YIELD (GY) Grain yield varied significantly at WS, F (p < 0.001) and WS x F (p < 0.01) (Table 3). The effect of WS on GY ranged between STR2 (2.94 t ha-1) and FW (6.59 t ha-1) (Table 4). Across F- applications, 100 N + 10 TPC (5.82 t ha-1) and 50 N + 10 TPC (5.26 t ha-1) produced the high- est GY, while the control showed the least GY (2.37 t ha- 1). Application of 8.33 t ha-1 (100 N + 10 TPC) under FW Water stress Variety Plant height FW ILE-1-OB TZPBSR-W 149.18 ± 5.18 160.26 ± 4.06 STR1 ILE-1-OB TZPBSR-W 126.03 ± 3.27 128.90 ± 2.54 STR2 ILE-1-OB TZPBSR-W 141.80 ± 4.96 139.76 ± 4.42 Table 5: Water deficit stress and Variety interaction effect on plant heights of two maize varieties in Ibadan STR 1 = Water stress at vegetative growth stage, STR 2 = Water stress at reproductive growth stage and FW = Full watering Fertilizer Variety Number of earper plant 0 N ILE-1-OB 1.39 ± 0.08 TZPBSR-W 1.15 ± 0.06 50 N ILE-1-OB 1.52 ± 0.06 TZPBSR-W 1.49 ± 0.11 100 N ILE-1-OB 1.35 ± 0.05 TZPBSR-W 1.55 ± 0.06 10 TPC ILE-1-OB 1.43 ± 0.07 TZPBSR-W 1.54 ± 0.08 50 N + 10 TPC ILE-1-OB 1.64 ± 0.11 TZPBSR-W 1.55 ± 0.07 100 N + 10 TPC ILE-1-OB 1.58 ± 0.06 TZPBSR-W 1.57 ± 0.07 Table 6: Variety and fertilizer application rates interaction effects on number of ear per plant of two maize varieties in Ibadan Acta agriculturae Slovenica, 117/4 – 202110 F. ANJORIN et al. produced the highest GY, while GY was the lowest at 0 N under STR2 (0.88 t ha-1) (Figure 3f). The GY of the two maize varieties were not significantly different (p < 0.05). 4 DISCUSSION Drought and low soil fertility are major abiotic fac- tors militating against profitable maize production in the tropics. The use of drought tolerant crop genotypes and soil amendment has potential to enhance growth and yield performances of crops grown under drought condi- tion. To investigate the role of soil nutrient amendment on the growth and yield responses of crop to water deficit stress, field experiment was established in Ibadan, Nige- ria. Results obtained show that 14 days withdrawal of watering during the vegetative growth stage (STR1) re- sulted in maize plants with reduced heights and leaf ar- eas. The reduction in leaf area as a result of water deficit stress may be attributed to decrease in rate of leaf ini- tiation and expansion and or accelerated rate of leaf se- nescence and leaf shedding which consequently reduce grain yield compared with grain yield obtained under well watered condition (Bolaños & Edmeades, 1996; Nam et al., 1998; Anjum et al., 2011). As leaves with reduced leaf area do not fully intercept solar radiation which in turn strikes the ground, and consequently increased the evaporation - transpiration ratio (Araus, 2002). Reduc- tion in plant height from water deficit stress interferes with over all crop photosynthetic efficiency (Imadi et al., 2016). Hence, plants with greater heights are often larger in overall plant size, intercept more light and use water faster by transpiration. In this study, water deficit stress at vegetative stage (STR1) accounted for 41 % loss in grain yield, this find- ing agreed with the report of Rufino et al. (2018). Water deficit stress affects all the various metabolic processes and yield components in plant and in turn reduced crop yield potential. Borra’s et al. (2003), inferred that the overall indirect impact of water stress during vegeta- tive stage on grain yield is source limiting as water stress decreased the source potential and available assimilates level and decreases grain weight. For instance, the kernel rows in maize are determined between V7 to V8 maize growth phase, while the number of kernels on each ear and size of ear in maize is determined at V12 of the maize growth stage (Ritchie & Hanway, 1993; Annony- mous, 2013). Therefore, occurrence of water deficit stress during vegetative growth phases becomes detrimental to Figure 3: Water deficit stress and fertilizer interaction effects on (a) PHT (Plant Height) (b) LA (Leaf Area) (c) Number of K/R (Kernels/Row), (d) K/C (Kernels/cob), (e) 1000-Kernel weight and (f) GY(Grain Yield) of two maize varieties planted under three water deficit stress and six fertilizer application rates in Ibadan. FW = Full watering, STR1 = Water stress at vegetative growth stage STR 2 = Water stress at reproductive growth stage Acta agriculturae Slovenica, 117/4 – 2021 11 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria the final crop grain yield (Ritchie & Hanway, 1993). This is because, water deficit stress during vegetative growth stage decreases plant source potential and assimilates lev- el thereby decreasing grain weights (Borra’s et al., 2003; Fatemi et al., 2006 and Khalili et al. 2010) The impact of preanthesis water deficit stress (STR2) in this study resulted in 55.37 % loss in grain yield, this finding agreed with the reports of Denmead & Shaw, (1960) and Sah et al. (2020). Farre & Faci, (2009), and Mansouri, et al. (2010), which earlier inferred that grain yield of maize is highly determined by the amount of ir- rigation water. The number of ear formed per plant were not significantly different across the water deficit stress regime, but ears obtained from plants subjected to prean- thesis water deficit stress (STR2) were smaller in size with few grains while some were even barren. The significant reduction in the number of grain per row and 1000-ker- nel weight under the water deficit stress observed in this study agreed with the earlier reports of Carpici (2009) and Kuscu (2010). In the view of Grant et al. (1989) and Hargurdeep & Westgate (2010) water deficit stress dur- ing pre anthesis stage of maize development could be im- plicated for abnormal development of embryo sac, grain sterility and decreased fertile grain number. While im- position of water deficit stress during preanthesis growth stage resulted in reduced number of kernels per cob, ker- nel set per row and the total grain yield (sink limiting). Increased fertilizer applications significantly en- hance R/C, K/R, K/C, Weight of 1000-kernels and GY across the water deficit stress regime in this study. De- ficiencies in N supply have been reported to impair pollination synchronization, increased kernel abortion (Uribelarrea et al., 2002; Uhart & Andrade, 1995), re- sulting in reduced kernel number per plant and decrease grain yield observed in the fertilizer control (Carcova et al., 2000; Paponov et al., 2005). Apart from water, soil nutrient especially nitrogen also had significant impact on the yield components and grain yield of maize in this study. Increased maize growth and yield responses were obtained under increased fertilizer application rates es- pecially when 10 t ha-1 of compost was added to each in- organic fertilizer rates of 50 and 100 kg ha-1 respectively. Application of inorganic fertilizer with compost to crop has been reported to have the advantage of providing nutrients to meet crop nutrition requirements and main- tain soil health (Abedi et al., 2010; Kazemeini et al., 2010; Efthimiadou et al., 2010). High level of micronutrient in the compost (Table 2) may have helped to improve gen- eral plant performance. Apart from water, soil nutrient especially nitrogen also had significant impact on the growth and yield components of maize in this study. Application of nitrogen fertilizer have been shown to increased the uptake of other nutrients, this is because nitrogen enhances growth and development of small roots and root hairs which in turn facilitate the absorb- ing ability per unit of dry weight (Gheysari et al., 2009; Hammad et al., 2011). Nitrogen is also needed to estab- lish and maintain the enzymatic processes essential for carbon utilization and growth, and is also a major con- stituent of endosperm storage protein (Cazetta et al., 1999; Duvnjak et al., 2021). The use of 10 t ha-1 of Titho- nia poultry compost in combination to each of 100 kg N ha-1 and 50 kg N ha-1 of nitrogen fertilizer significantly enhanced grain yield than sole applications of each of in- organic fertilizer rate in this study. The compost (Table 2) has a very high carbon to nitrogen ratio, also very rich in essential micronutrients needed for maize production. Application of inorganic fertilizer with compost to crop has been reported to have the advantage of providing nutrients to meet crop nutrition demands and maintain soil health (Efthimiadou et al., 2010). Compost had been reported to improve soil water holding capacity as well as buffering rapid changes in soil pH (Tambone et al., 2007; Zemánek, 2011). The significant water regime by fertilizer interac- tion effects on the various growth and yield components in this study indicated that growth and yield increased resulting from fertilizer application depended on the availability of water (Pandey et al., 2000). Hence, ade- quate moisture availability is vital to nutrient mineraliza- tion, growth and grain yield of maize (Hokmalipour et al., 2010). Water deficit stress at the vegetative stage of growth not only deprived the plant of adequate mois- tures supply needed for cellular meristematic activities but also hinder nutrient supply which are needed for the development of yield component potential. Despite the impact of the water stress on the various yield com- ponents of maize, increased application of fertilizer was seen to enhanced grain yield of the two maize varieties. Increased nitrogen application has been shown to have the capability of improving drought tolerance and en- hancing grain yield in maize (Boutras, 2001; Xu et al., 2005). Variety TZPBSR-W appeared to performed better than ILE-1-OB most especially under well watered con- dition but such superiority could not be maintain under the first and second water stress conditions as observed in the number of kernels per row and number of kernels per cob. The effect of water stress on seed formation, ker- nel set and grain yield was most severe during the repro- ductive growth stage and under reduced nutrient avail- ability. Water stress and low nutrient availability might have reduced the sink strength and capacity of the maize plants which are determined by genetic and environmen- tal factors (Alvarez Prado et al., 2014). Moisture availability and nutrient availability to a large extent, determines seed formation, kernel set, and Acta agriculturae Slovenica, 117/4 – 202112 F. ANJORIN et al. the final grain yield in this study. Nitrogen fertilizer ef- fect on the various yield components and grain yield improved as the N application increases. Maize plant performed best when inorganic fertilizer was used along with organic fertilizer than when organic or inorganic fer- tilizer was applied alone. The result of the present finding on water regime nitrogen interaction also revealed that growth and yield components and grain yield performed better under adequate moisture availability. Nitrogen had been reported to improve water use efficiency in maize (Ogola et al., 2002). Growth and yield components were improved with increase N application even under water stress conditions. Therefore, optimization of N and water management could be an efficient way to attain sustain- able agriculture. The two maize varieties were similar in yield responses to the varying stress periods and ferti- lizer application rates in the two years of the experimen- tal studies. Similar report of variability in crop genotypic response under water stress had earlier been reported by Hufsteler et al., (2007); Abayomi & Abidoye, (2009). Application of 10 t ha-1 of Tithonia poultry compost alone to the maize field produced taller maize plants and broader leaves better than maize plants obtained when 100 kg N ha-1 inorganic fertilizer were applied, but this alone could not sustain the plant adequately beyond the pollination process. The evidence of this was the rapid appearance of yellow lower leaves in treatment with 10 t ha-1 (Tithonia poultry compost alone). Explanations for this could be that the N supply by the compost alone at the transition stage from vegetative to reproductive was not adequate enough for N demand for post pollination activities. Hence the need for remobilisation of N from the lower leaves for grain filling was inevitable. 5 CONCLUSION Climate change and its associated attributes have impacted negatively on general crop development across the world. Drought emanating from erratic rainfall pat- tern has constituted serious menace to profitable maize production in the sub Saharan Africa. From this study it was obvious that water deficit stress reduced growth and yield performances of the two maize varieties result- ing into grain yield losses of 41.0 % and 55.37 % under vegetative and reproductive stages water deficit stresses, respectively. However, this study has been able to explore soil fertility management at enhancing growth and yield performances of maize subjected to water deficit stress. Different rates of nitrogen fertilizer from inorganic, or- ganic sources and their combinations were applied to the two maize varieties at different phenological growth –water deficit stages. From the result, it is obvious that 50 kg N of inorganic fertilizer and 10.7 kg N of Titho- nia Poultry Compost significantly enhance growth and yield performances of the two maize varieties across water stresses in this study. The 50 kg N of inorganic fertilizer represents half dose of recommended 100 kg N of nitrogen fertilizer (inorganic) application rate for the agro ecological zone of the country. Minimal use of inorganic fertilizer rate will help reduce environmental issues associated with the increase use of chemical ferti- lizers and cost of production. The maize varieties grown under 50 kg N ha-1 NPK-20-10-10 and 10.7 kg N ha-1 TPC subjected to water deficit stress must have benefited immensely from fast release of plant nutrient (inorganic fertilizer) with high; micronutrients, organic carbon con- tent and moisture retention of compost. Augmenting re- duced rate of inorganic fertilizer with Tithonia compost is hereby recommended for profitable maize production in derived savanna ecology of Nigeria. In spite of the nu- merous benefits associated with the use of compost, the bulkiness and availability of enough quantities for large scale maize production remains a great challenge. Farm- ers should be adequately trained on compost preparation techniques and the importance of combine use of inor- ganic and organic fertilizers to boost maize production in the face of the prevailing climate change. Government should support and empower unemployed youth to em- brace commercial compost production so as to cater for the anticipated high compost demand by commercial farmers. More funding should be made available for soil fertility management and climate change adaptability studies. Funding: This research was supported by Research grant From the Federal Ministry of Agriculture and Nat- ural Resources and the Institute of Agricultural Research and Training, Moor Plantation, Ibadan, Nigeria. 6 REFERENCES Abayomi, Y. A., & Abidoye, T.O. (2009). Evaluation of cowpea genotypes for soil moisture stress tolerance under screen house conditions. African Journal of Plant Science, 3(10), 229-237. Online at http://www.academicjournals.org/AJPS Abedi, T., Alemzadeh, A., & Kazemeni, S.A. (2010). Effect of organic and inorganic fertilizer on grain yield and protein banding pattern of wheat. Australian Journal of Crop Sci- ence, 4, 384-389. Alvarez Prado, S., Sadras, V.O., & Borras, L. (2014). Independ- ent genetic control of maize (Zea mays) kernel weight de- termination and its phenotypic plasticity. Journal of Experi- mental Botany, 65, 4479–4487. https://doi.org/10.1093/jxb/ eru215 Ammani, A.A., Ja’afaru, A.K., Aliyu, J.A., & Arab, A.I. (2012). Climate change and maize production: Empirical evidence from Kaduna State. Journal of Agricultural Extension Acta agriculturae Slovenica, 117/4 – 2021 13 Effects of soil nutrient amendments on growth and grain yield performances ... under water deficit stress in Ibadan, Nigeria Nigeria, 16(1), 1–9. https://doi.org/10.4314/jae.v16i1.1 Anjorin, F.B. (2018). Effects of water deficit stress, inorganic and organic fertilizers on growth and yield performance of qual- ity Protein maize. Ph.D dissertation University of Ibadan, Ibadan. Anjorin, F.B., Adejumo, S.A., Are, K.S., & Ogunniyan, D. J. (2017). Seedling establishment, biomass yield and water use efficiencies of four maize varieties as influenced by wa- ter deficit stress. Cercetări Agronomice în Moldova, 50(2), 21–34. https://doi.org/10.1515/cerce-2017-0012 Anjum S. A., Xie, X., Wang, L., Saleem, M. F., Man, C., & Wang. L. (2011). Morphological, physiological and biochemical responses of plants to drought stress. African Journal of Ag- ricultural Research, 6(9), 2026-2032. Annonymous, (2013).Corn Growth Stages Channel.com Tech- nology Development & Agronomy, Musanto Araus, J. L., Slafer, G.A., Royo, C., & Serret, M.D. (2008). Breed- ing for yield potential and stress adaptation in cereals. Criti- cal Reviews in Plant Sciences, 27(6), 377–412. https://doi. org/10.1080/07352680802467736 Bolaños, J., & Edmeades, G.O. (1996). The importance of the anthesis-silking interval in breeding for drought tolerance in tropical maize. Field Crops Research, 48, 65-80. https:// doi.org/10.1016/0378-4290(96)00036-6 Borra’s, L., Westgate, M.E., & Otegui, M. E. (2003). Control of kernel weight and kernel water relation by post-flowering source-sink ratio in maize. Annals of Botany, 91, 857-867. https://doi.org/10.1093/aob/mcg090 Carpici, E.B. (2009). Evaluation of the effects of plant densities and nitrogen rates on stress physiology traits in silage corn (Zea mays L.) production. PhD thesis, Field Crops, Gradu- ate School of Natural and Applied Sciences. Uludag Univer- sity, Bursa, Turkey Cazetta, J.O., Seebauer, J. R., & Below, F.E. (1999). Sucrose and nitrogen supplies regulate growth of maize kernels. Annals of Botany, 84, 747-754. Article No. anbo.1999.0976, avail- able online at http:}}www.idealibrary.com on. https://doi. org/10.1006/anbo.1999.0976 Chaves, M.M., Pereira, J.S., Maroco, J., Rodriques, M.L., Ri- cardo, M.L., Osorio, M.L., Carvatho, I., Faria, T., & Pinheiro, C. (2002). How plants cope with water stress in the field photosynthesis and growth? Annals of Botany, 89, 907–916. https://doi.org/10.1093/aob/mcf105 Denmead, O.T., & Shaw, R.H. (1960). The effects of soil mois- ture stress at different stages of growth on the development and yield of corn. Agronomy Journal, 52, 272–277. https:// doi.org/10.2134/agronj1960.00021962005200050010x Edmeades, G.O. (2013). Progress in achieving and delivering tol- erance in maize. An update, SAAA Ithaca, NY. Global Sta- tus of Commercialized Biotech/GM Crops: 1- 4. Duvnjak, M., Kljak, K., & Grbeša, D. (2021). Nitrogen Stor- age in Crops: Case Study of Zeins in Maize. DOI: 10.5772/intechopen. 95380. https://doi.org/10.5772/in- techopen.95380 Edmeades, G.O., J. Bolaños, S.C. Chapman, H.R. Lafitte., & Bänziger, M. (1999). Selection for drought tolerance increases maize yields across a range of nitrogen levels. Crop Science, 39(4), 1306–1315. https://doi.org/10.2135/ cropsci1999.3951306x Efthimiadou, A., Bilalis, D., Karkanis, A., & Froud-Williams, B. (2010). Combined organic/inorganic fertilization enhance soil quality and increased yield, photosynthesis and sustainability of sweet maize crop. Australian Journal of Crop Science, 4(9), 722-729. Eghball, B., & Maranville, J.W. (1991). Interactive effects of wa- ter and nitrogen stresses on nitrogen utilization efficiency, leaf water status and yield of corn genotypes. Communi- cations in Soil Science and Plant Analysis, 22, 1367-1382. https://doi.org/10.1080/00103629109368498 Farré, I., & Faci, J.M. (2009). Deficit irrigation in maize for reducing agricultural water use in a Mediterranean envi- ronment. Agricultural Water Management, 96(3), 383-394. https://doi.org/10.1016/j.agwat.2008.07.002 Fatemi, R., Kahraryan, B., Ghanbary, A., & Valizadeh, M. (2006). The evaluation of different irrigation regimes and water requirement on yield and yield components of corn. Journal of Agronomy and Crop Science, 12(1), 133-141. Fernhill, (2011). Difference between compost and fertilizer. Re- trieved online fernhillcompost.com, 3(15), 427-2821. Francis, C.A., Rutger, J. N., & Palmer, A. F. E. (1969). Rapid method for plant leaf area estimation in maize (Zea mays L.) Crop science, 9(5), 537-539. https://doi.org/10.2135/cro psci1969.0011183X000900050005x Gheysari, M., Mirlatifi, S.M., Bannayan, M., Homaee, M., & Hoogenboomb, G. (2009). Interaction of water and nitrogen on maize grown for silage. Agricultural Water Management, 96, 809-82. https://doi.org/10.1016/j.agwat.2008.11.003 Goldblatt, A. (2010). Agriculture: Facts and Trends, South Af- rica. Retrieved from http://aWSRssets.wwf.org.za/down- loads/facts_brochure on 12/06/2017, 1-32. Grant, R.F., Jackson, B.C., Kiniry, J.R., & Arkin, G.F. (1989). Water deficit timing effects on yield components in maize. Agronomy Journal, 81, 61-65. https://doi.org/10.2134/agron j1989.00021962008100010011x Hammad, H.M., Ahmad, A.A., Wajid, A., & Akhter, J. (2011). Maize response to time and rate of nitrogen application. Pa- kistan Journal of Botany, 43(4), 1935-1942. Hargurdeep, S. S., & Westgate, M. E. (2000). Reproductive development in grain crops during drought. Advances in Agronomy, 68(1), 59-96. https://doi.org/10.1016/S0065- 2113(08)60843-3 Hokmalipour, S., Shiri-e-Janagard, M., Darbandi, M.H., Pe- yghami-e-Ashenaee, F., Hasanzadeh, M., Seiedi, M.N., & Shabani, R. (2010). Comparison of agronomical nitro- gen use efficiency in three cultivar of corn as af- fected by nitrogen fertilizer levels. World Applied Science Journal, 8(10), 1168-1174. http://www.redorbit.com/news/ science/1070340/the_effects_of_shortterm_compost Hufsteler, E.V., Boerma, H.R., Carter, T.E., & Earl, H.J. (2007). Genotypic variation for three physiological traits affecting drought tolerance in soybean. Crop Science, 47, 25-35. htt- ps://doi.org/10.2135/cropsci2006.04.0243 Imadi, S.R., Gul, A., Dikilitas, M., Karakas, S., Sharma, I., & Ahmad P. (2016). Water stress: types, causes, and impact on plant growth and development. In: Ahmad P, ed. Water Stress and Crop Plants. Chichester, UK: John Wiley & Sons, Ltd; 343-355. doi: 10.1002/9781119054450.ch21. https://doi.org/10.1002/9781119054450.ch21 Acta agriculturae Slovenica, 117/4 – 202114 F. ANJORIN et al. Jongdee, B., Fukai, S., & Cooper, M. (2002). Leaf water potential and osmotic adjustment as physiological traits to improve drought tolerance in rice. Field Crops Research, 76, 153-163. https://doi.org/10.1016/S0378-4290(02)00036-9 Khalili, M., Moghaddam, M., Kazemi Arbat, H., Shakiba, M.R., Kanooni, H., & Choukan, R. (2010). Effect of drought stress on different corn genotypes. Journal of Agricultural Science, 2(20), 67-84. Kuscu, H. (2010). Effects of deficit irrigation on yield and yield components of maize grown under Bursa conditions. PhD thesis, Irrigation and Agricultural Structures, Graduate School of Natural and Applied Sciences, Uludag University, Bursa, Turkey. Mansouri-Far, C.S.A., Sanavy, M.M., & Saberali, S.F. (2010). Maize yield response to deficit irrigation during low sensi- tive growth stages and nitrogen rate under semi-arid cli- matic conditions. Agricultural Water Management, 97(1), 12-22. https://doi.org/10.1016/j.agwat.2009.08.003 Monneveux, P.C., Sánchez, D., Beck, C., & Edmeades, G.O. (2006). Drought improvement in maize source population: evidence of progress. Crop Science, 41, 180-191. https://doi. org/10.2135/cropsci2005.04-0034 Ngetich, F.K., Shisanya, C.A., Mugwe, J., Mucheru-Muna, M., & Mugendi, D. (2012). The Potential of Organic and Inor- ganic Nutrient Sources in Sub-Saharan African Crop Farm- ing Systems - A Global Perspective, Dr. Joann Whalen (Ed.), ISBN: 978-953-307-945-5, In Tech, Available from: http:// www.intechopen.com/books/soil-fertility-improvement-and integrated-nutrient-management-a-global-perspective on 04/06/2016,Pp 1 -27. Ogola, J.B.O., Wheeler, T.R., & Harris, P.M. (2002). Effects of nitrogen and irrigation on water use of maize crops. Field Crops Research, 78, 105-117. www.elselvier.com. htt- ps://doi.org/10.1016/S0378-4290(02)00116-8 Pandey, R.K., Marienville, J.W., & Adum, A. (2000). Deficit ir- rigation and nitrogen effect on maize in a Sahelian envi- ronment. I .Grain yield components. Agricultural Water Management, 46, 1-13. https://doi.org/10.1016/S0378- 3774(00)00073-1 Paponov, I.A., Sambo, P., Erley, G.S.A., Presterl, T., Geiger, H.H., & Engels, C. (2005). Kernel set in maize genotypes differing in nitrogen use efficiency in response to resource availability around flowering. Plant and Soil, 272, 101–110. https://doi.org/10.1007/s11104-004-4210-8 Ritchie, S.W., Hanway, J.J., & Benson, G.O. (1993). How a Corn Plant Develops. Iowa State Univ. p. Rpt. No. 48. Available online at http://maize.agron.iastate.edu/corngrows.html Rufino, C.A., Fernandes-Vieira, J., Martín-Gil, J., Júnior, J.S.A., Tavares, L.C., Fernandes-Correa, M., & Martín-Ramos, P. (2018). Water stress influence on the vegetative period yield components of different maize genotypes. Agronomy, 8(8), 151. https://doi.org/10.3390/agronomy8080151 Sah, R.P., Chakraborty, M., Prasad, K. et al. (2020). Impact of water deficit stress in maize: Phenology and yield compo- nents.  Scientific Report, 10, 2944. https://doi.org/10.1038/ s41598-020-59689-7 Sanginga, N., & Woomer, P. L. (2009). Integrated soil fertility management in Africa: principles, practices and develop- mental process. Tropical Soil Biology and Fertility Insti- tute of the International Centre for Tropical Agriculture, Nairobi, 1-263. Scoones, I., & Toulmin, C. (1998). Soil nutrient balances: What use for policy? Agriculture, Ecosystems & Environment, 71, 255-267. https://doi.org/10.1016/S0167-8809(98)00145-5 Smale, M., Byerlee, D., & Jayne, T.S. (2011). Maize Revolutions In Sub-Saharan Africa. World Bank Policy Research Working Paper, 5659, 1-47. https://doi.org/10.1596/1813-9450-5659 STAR, (2014). Statistical Tool For Agricultural Research Version version 2.0.1 Biometrics and Breeding Informatics, PBGB Division, International Rice Research Institute, Los Baños,Laguna. Tambone, F., Genevini, P.D., &’Imporzano, G.A. (2007). The effects of short-term compost application on soil chemical properties and on nutritional status of maize plant. Com- post Science and Utilization, 15(3), 176-183. https://doi.org/ 10.1080/1065657X.2007.10702330 Tan, Z. X., Lal, R., & Wiebe, K. D. (2005). Global soil nutri- ent depletion and yield reduction. Journal of Sustainable Agriculture, 26(1), 123-146. Available online at http://www. haworthpress.com/web/JSA. https://doi.org/10.1300/ J064v26n01_10 Uhart, S.A., & Andrade, F.H. (1995). Nitrogen deficiency in maize. II.Carbon–nitrogen interaction effects on kernel number and grain yield. Crop Science, 35, 1384–1389. htt- ps://doi.org/10.2135/cropsci1995.0011183X00350005002 1x Uribelarrea, M., Carcova, J., Otegui, M.E., & Westgate, M.E. (2002). Pollen production, pollination dynamics, and ker- nel set in maize. Crop Science, 42, 1910–1918. https://doi. org/10.2135/cropsci2002.1910 Vanlauwe, B., Descheemaeke, K., & Giller, K.E et al. (2015). Integrated soil fertility management in sub-Saharan Af- rica: unravelling local adaptation. Soil, 1, 1239–1286. htt- ps://doi.org/10.5194/soil-1-491-2015 Xu, Z.Z., Yu, Z.W., Wang, D., & Zhang, Y.L. (2005). Water use water Kisintili applications to yield. Turkish nitrogen accu- mulation and translocation for winter. Journal of Agricul- ture and Forestry, 23, 233-241. Zemánek, P. (2011). Evaluation of compost influence on soil water retention. Acta Universitatis Agriculturae et Silvicul- turae Mendelianae Brunensis, 54(3), 227–232. https://doi. org/10.11118/actaun201159030227 Acta agriculturae Slovenica, 117/4, 1–12, Ljubljana 2021 doi:10.14720/aas.2021.117.4.1520 Original research article / izvirni znanstveni članek Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species Marjan DYANAT 1, 2, Farzad ASGARI 1 Received February 15, 2020; accepted October 16, 2021. Delo je prispelo 15. februarja 2020, sprejeto 16. oktobra 2021 1 Department of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Corresponding author, e-mail: Ma_dyanat@yahoo.com Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species Abstract: In the present study the bioherbicidal activity of essential oils hydrodistilled from Nepeta glocephalata Rech.f and N. ispahanica Boiss were investigated on four weed spe- cies (barnyard grass (Echinochloa crus-galli (L.) Beauv), redroot pigweed (Amaranthus retroflexus L.), lambsquarters (Chenopo- dium album L.) and canary grass (Phalaris canariensis L.)). A total of 37 components were identified from the essential oils of N. glocephalata and N. ispahanica constituting approximately 98.61 % and 96.1 % of the oils, respectively. In laboratory bio- assay different concentrations (0, 1, 2, 4 and 8 μl ml-1) of two Nepeta essential oils on germination, root and shoot length were studied. Results showed by increasing the concentration of oils, all studied traits of the weeds were decreased compared with control. In a glass house bioassay post-emergence appli- cation of Nepeta essential oils (1.25 %, 2.5 %, 5 % and 10 %, v/v) on 3-week-old weed plants caused visible injury (7-days after spray) ranging from chlorosis to necrosis of plant weeds. In foliar application under glasshouse conditions, both Nepeta essential oils reduced the seedling dry mass and concentra- tions of chlorophyll a chlorophyll b. The study concludes that Nepeta essential oils have phytotoxic effects and could be used as bioherbicides but the selectivity of these compounds should be considered also. Key words: Nepeta glocephalata Rech.f.; N. ispahanica Boiss.; bioherbicide; 1, 8-cineole; chlorophyll a; weed seed ger- mination; root length Fitotoksični učinki eteričnih olj iz dveh vrst mačje mete (Ne- peta glocephalata Rech.f. in N. ispahanica Boiss.) na izbrane vrste plevelov Izvleček: V raziskavi je bila preučevana bioherbicidna ak- tivnost vodnih destilatov eteričnih olj iz dveh vrst mačje mete (Nepeta glocephalata Rech.f in N. ispahanica Boiss. ) na štiri ple- velne vrste (navadna kostreba (Echinochloa crus-galli (L.) Be- auv), navadni (srhkodlakavi) ščir (Amaranthus retroflexus L.), bela metlika (Chenopodium album L.) in kanarska čužka (Pha- laris canariensis L.)). Celokupno je bilo v eteričnih oljih obeh vrstah določenih 37 sestavin, ki so predstavljale 98,61 % oziro- ma 96,1 % olja. V laboratorijskem poskusu so bili preučevani učinki različnih koncentracij (0, 1, 2, 4 in 8 μl ml-1) eteričnih olj iz obeh vrst mačje mete na kalitev, dolžino korenin in poganj- kov izbranih plevelov. Rezultati so pokazali, da so se vrednosti vseh merjenih parametrov plevelov zmanjševale s povečeva- njem koncentracije eteričnih olj. V poskusu v rastlinjaku so bile preučevane vidne poškodbe uporabe eteričnih olj iz obeh vrst mačje mete (1,25 %, 2,5 %, 5 % and 10 %, v/v) na tri tedne starih sejankah plevelov, sedem dni po škropljenju z eteričnimi olji, ki so se pojavile kot kloroze in nekroze. Pri foliarni uporabi eterič- nih olj obeh vrst mačje mete v rastlinjaku se je zmanjšala suha masa sejank plevelov, zmajšale so se tudi vsebnosti klorofila a in b. Na osnovi raziskave lahko zaključimo, da imajo eterična olja obeh vrst mačje mete fitotoksične učinke in bi lahko bile upo- rabljene kot bioherbicidi vendar je pri tem potrebno upoštevati selektivne učinke njihovih sestavin. Ključne besede: Nepeta glocephalata Rech. f.; N. ispaha- nica Boiss.; bioherbicid; 1, 8-cineol; klorofil a; kalitev semen plevelov; dolžina korenin Acta agriculturae Slovenica, 117/4 – 20212 M. DYANAT and F. ASGARI 1 INTRODUCTION Herbicide-resistant weeds and environmental con- cerns have led researchers to consider using alterna- tive ways to manage weeds (Vyvyan, 2002; Ashraf et al., 2017). Allelopathy is one of these ways (Weston, 1996). Allelopathic compounds can reduce the use of synthetic herbicides and thus reduce environmental pollution and lead to more safe crops (Singh et al., 2002, 2003, 2005a, b). Among the natural plant products, essential oils con- stitute an important group of that provide a characteristic odor to the aromatic plants (Singh et al., 2002). Earlier studies have documented that essential oils and their constituents inhibited seed germination and retard plant growth (Barney et al., 2005; Batish et al., 2006; Ens et al., 2009). The allelopathic activities of some essential oils and their monoterpenes on seeds germination or seed- ling growth at several species have been shown in pre- vious studies (Dudai et al., 1999; Abrahim et al., 2000; Tworkoski, 2002; Singh et al., 2004; Dudai et al., 2004; Armirante et al., 2006; Kordali et al., 2006; Kordali et al., 2007). Allelopathic properties of essential oils from dif- ferent aromatic plants belonging to Lamiaceae, Composi- tae, Myrtaceae, Cupressaceae, Rutaceae and Verbenaceae families have been reported (Dudai et al., 1999; Angelini et al., 2003; Kaur et al., 2010; Amri et al., 2013 ; Verde- guer et al., 2011). Also allelopathic potential of the es- sential oil of many plants from family Lamiaceae such as Salvia apiana Jeps. and Salvia leucophylla Greene (Muller et al., 1964), Satureja hortensis L. and Thymus vulgaris L. (Tworkoski 2002), Rosmarinus officinalis L., Satureja montana L. (Angelini et al., 2003), Lavandula spp. and peppermint (Mentha × piperita ‘Mitcham’) (Campiglia et al., 2007; Mahdavikia and Saharkhiz, 2015), Zataria mul- tiflora Boiss and its different chemotypes (Saharkhiz et al., 2010), Satureja khuzestanica Jamzad, Satureja bach- tiarica Bunge, Satureja rechingeri Jamzad and Satureja spicigera (K.Koch) Boiss. (Taban et al., 2013) have been previously reported. Genus Nepeta is one of the largest genera of the Lamiaceae family that comprises about 300 herbaceous perennial and annual species (Formisano et al., 2011). The greatest diversity and richness of species is found in Southwestern Asia, (especially Iran and Turkey), and the Western Himalayas. There are seventy-nine species of Ne- peta in Iran and about 39 of them are endemics (Jamzad, 2012). Much research was done on diversity, species rich- ness and chemical properties of Nepeta species. Most Ne- peta species are rich in essential oils. Diverse biological activities of Nepeta oil such as feline attractant, canine attractant, insect repellant, arthropod defense (Tucker and Tucker, 1988, Wagner and wolf, 1977), antibacterial, antifungal and antiviral activities (Tucker and Tucker, 1988) have been reported previously. There are several reports on the chemical composition of the essential oils of the genus Nepeta found in Iran (Sefidkon, 2004, 2005; Sajjadi, 2005; Sonboli et al., 2005; Jamzad, 2012). Allelo- pathic potential of this genus was revealed. Phytotoxicity of Nepeta essential oils has been mainly tested (Kobaisy et al., 2005, Eom et al., 2006, Mancini et al., 2009, Mutlu et al., 2011, Kekec et al., 2012, Bozari et al., 2013, Živković, 2013). Allelopathy of water extracts has been studied by Mutlu and Atici (2009) and Babaahmadi et al. (2013). No bioassays or field experiments had been done to study the allelopathic potential of Nepeta glocephalata Rech.f and N.ispahanica Boiss., Endemic plants of Iran. The aim of the present study was to study the essen- tial oil composition of N. glocephalata and N. ispahanica in order to know if these compositions have phytotoxic effects on germination, seedling growth injury and pho- tosynthesis of barnyard grass (Echinochloa crus-galli (L.) Beauv), a most important weed in rice (Oryza sativa L.), redroot pigweed (Amaranthus retroflexus L.) and lambs- quarters (Chenopodium album L.), annual plants serious- ly influencing summer crops and canary grass (Phalaris canariensis L.), serious weed of wheat (Triticum aestivum L.) fields in Iran. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL Above ground parts (leaves and flowers/inflores- cences) of N. glocephalata rech.f. were collected from natural sites of Kashan, Esfahan Province, at an altitude of 1600 m and the above ground parts of N. ispahanica Boiss. were collected from north-west of Tehran, at an altitude of 1800 m during the flowering period in July 2015 in Iran. The air-dried of the plant were powdered and hydrodistillated in a Clevenger-type apparatus for 3 h. The essential oils were dried over anhydrous sodium sulphate and stored at 3 °C in a dark before analysis. 2.2 GC AND GC/MS ANALYSES The oils were analyzed by GC and GC/MS. The GC analyses were performed using a Perkin-Elmer (UK) 8500 gas chromatograph equipped with Flame Ioniza- tion Detector (FID) and a DB-5 fused silica column ( 30 m × 0.25 mm, film thickness 0.25 μm .Oven temperature was held at 60 ºC for 3 min and programmed to 275 °C at a rate of 3 °C/min; injector temperature (split: 1: 25) 250 °C; detector temperature, 280 °C; carrier gas, N2 at 12 psi. Varian 3700 chromatography equipped with a CP- Acta agriculturae Slovenica, 117/4 – 2021 3 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species Sil5CB column (25 m×0.25 mm i.d., film thickness 0.39 μm) combined with a Varian MAT 44S, ionization ener- gy 70ev. The carrier gas was He and injector temperature was 270 ⁰C. Approximately, 0.1 μl of neat oil was injected under split condition (100:1) and the oven temperature was held at 60 ºC for 5min., programmed at 5 ºC min-1. to 220 C and then holds at this temperature for 20 min. 2.3 IDENTIFICATION OF COMPONENTS The compounds in the oil were identified by com- parison of their retention indices (RI, HP-5) with those reported in the literature as well as by comparing their mass spectra with the Wiley GC–MS Library, Adams Li- brary, Mass Finder 2.1 Library data, and published mass spectra data (McLafferty and Stauffer, 1989; Adams, 2007). 2.4 GERMINATION AND SEEDLING GROWTH BIOASSAY Seeds of two monocotyledon weeds (barnyard grass (Echinochloa crus-galli (L.) Beauv) and canary grass (Phalaris canariensis L.)) and two dicotyledon weeds (redroot pigweed (Amaranthus retroflexus L.) and lamb- squarters (Chenopodium album L.)) were collected from weeds growing in the summer crops. The germination tests were done in petri dishes (9 cm dia) in a germination chamber at 30 °C (day) and 20 °C (night) for barnyard grass, canary grass and redroot pigweed and at 20 °C and 10  °C for canary grass, respectively. For each essential oil, an oil-in-water emulsion was prepared at 1, 2, 4 and 8 μg ml-1 concentrations. Distillated water used as con- trol. Each Petri dish contained 25 weed seeds placed on two layers of filter paper (Whatman® No.5) wetted with 6 ml oil-in-water emulsion. To prevent evaporation, petri dishes were sealed with parafilm (16). After 14 days, all germinated seeds were counted. Seeds showing root emergence (2 mm) were recorded as germinated. After 14 days no seed germinations were observed. The ger- mination percentages were determined. Root and shoot length were measured by scientific ruler. 2.5 GLASS HOUSE STUDIES In another experiments, the effects of Nepeta species oils on 3-week-old weed plants raised under controlled conditions in experimental glass house were studied. Plants of the four weed species were raised from the seeds in plastic pots 12-cm in diameter. Pots were filled with 730 g garden soil (soil: sand: manure: 3:1:1, w/w) and ten seeds of each weed species were sown per pot. Pots were thinned to 5 equal-sized healthy plants per pot at one- week after sowing. Plants were watered every other day. Studied treatments in this experiment were 1.25, 2.5, 5 and 10 % (v/v) solution of essential oil or distilled water (control) at 3-week-old plants. A hand pressure sprayer filled with flooding nozzle was used for spraying at a rate of 400 l ha−1. The weed plants were examined for visible injury levels in terms of percent chlorotic and necrotic areas at 7-days after spray (DAS). Fresh leaves of all weed species (100 mg fresh leaf samples) were homogenized in 80 % aqueous acetone (5 ml). The homogenate was fil- tered through Whatman filter paper no. 1. The final vol- ume was adjusted to 5 ml by acetone (80 %). Chlorophyll a and chlorophyll b contents were determined spectro- photometrically using Unico 1200-Spectrophotometer at 663 nm for chlorophyll a and 647 nm for chlorophyll b. Calculations were completed using Lichtenthaler’s equa- tion (Lichtenthaler, 1987) and expressed as mg g-1 dry mass. Also dry mass of plant were measured after were oven-drying at 750 ⁰C for 48 h. 2.6 STATISTICAL ANALYSIS All the experiments were repeated and the present- ed data are average of the two experiments. The experi- mental design used for both experiments was completely randomized in a 5 x 2 factorial scheme (5 concentrations × 2 Nepeta essential oils), with four replications. ANO- VA was used to test for significant differences between the means of each Nepeta species and each essential oil concentration. For all statistical analysis, the SAS ver 9.1 program was used. The means were compared by Tukey’s HSD post hoc test (p < 0.05). 3 RESULTS 3.1 CHEMICAL COMPOSITIONS OF THE EXAM- INED ESSENTIAL The chemical compositions of the two Nepeta es- sential oils compounds were listed in Table 1. Total of 35 compounds were identified in N. ispahanica and N. glocephalata essential oils by GC/MS analysis. Eight- een components were identified, representing more than 96.1  % of the total oil components of N. ispa- hanica essential oil detected. The major components of N. ispahanica oil were 1,8-cineole (66.4  %), β-pinene (10.7  %) and α-pinene (3.1  %). Twenty-nine com- pounds reached 98.6  % of the total N. glocephalata es- Acta agriculturae Slovenica, 117/4 – 20214 M. DYANAT and F. ASGARI sential oils. The main components of N. glocephalata oil were 1,8-cineole (34.1  %), β-pinene (21.5  %), α-pinene (8.1  %) sabinene (7.8  %), (Z)-β-ocimene (7.6  %) and (E)-β-ocimene (6.9 %). Other components were pre- sent in amounts less than 3 %. No Compound IR % N. ispahanica N. glocephalata 1 α-Thujene 935 - 0.8 2 α-Pinene 940 3.1 8.1 3 Camphene 954 - 0.2 4 Sabinene 981 1.9 6.6 5 β-Pinene 986 10.7 21.5 6 Myrcene 998 - 1.7 7 δ-3-Carene 1011 - 0.5 8 α-Terpinene 1024 - 0.2 9 p-Cymene 1034 - 0.8 10 1,8-Cineole 1041 66.4 34.1 11 (Z)-β-Ocimene 1046 - 7.1 12 (E)-β-Ocimene 1056 - 6.5 13 γ-Terpinene 1066 - 0.3 14 trans-Sabinene-hydrate 1075 0.4 0.8 15 cis-Sabinene hydrate 1088 0.4 - 16 Tepinolene 1095 - 0.3 17 Linalool 1107 - 0.4 18 trans-Pinocarveole 1129 1.1 - 19 cis-p-menth-2-en-1-ol 1131 - 0.2 20 Verbenol 1134 0.6 - 21 Allo-ocimene 1137 - 0.2 22 trans-Sabinole 1149 - 0.5 23 Pinovarvone 1172 0.9 0.2 24 Myrtenal 1175 1.0 - 25 δ-Terpineole 1177 1.1 0.5 26 Myrtenol 1184 1.0 - 27 Terpinen-4-ol 1187 1.0 1.8 28 Cryptone 1196 - 0.2 29 α-Terpineole 1200 2.0 2.9 30 Myrthanol 1207 - 0.5 31 4aα,7α,7aα-Nepetalactone 1422 0.1 - 32 β-caryophyllene 1434 0.2 0.1 33 Germacrene D 1496 - 1.2 34 Bicyclogermacrene 1512 - 0.3 35 4aβ,7α,7aα-Nepetalactone 1575 2.1 - 36 β-caryophyllene oxide 1585 2.1 - 37 Spathulenole 1595 - 0.1 Total - 96.1 98.6 Table 1: Percentage composition of the essential oils of Nepeta species Retention Indices (The retention indices were determined on CPSil5CB column) Acta agriculturae Slovenica, 117/4 – 2021 5 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species and N. glocephalata essential oils, respectively. The re- gression lines between seed germination and essential oil concentrations confirm the different susceptibility of weed species (Fig. 1). There were significant differences among control and all concentrations, and between two Nepeta species essential oils for each weed species. The regression analysis between oil concentrations and root length showed that increasing concentration of essential oil increased the inhibitory effects on weed root length till a lethal dose (Fig. 2). When the root length of redroot pigweed and lambsqaurters was completely inhibited by essential oils of N. ispahanica at 4 μg ml-1, the root length was reduced respectively to 70 % for barnyard grass and to 73 % for canary grass, which explain that monocots weeds were more resistant than dicots. No significant differences among control and all concentrations, and between two Nepeta essential oils observed for shoot length of barnyard grass and canary grass. The shoot re- duction of barnyard grass compared with control was the 29 % and 28 % with N. glocephalata and N. ispahanica essential oils at the highest concentration, respectively. Canary grass showed a reduction of the 28 % with N. glo- cephalata and 25 % with N. ispahanica essential oils at the same concentration. Shoot length of redroot pigweed and lambsqaurters reached to zero at 4 and 8 μg ml-1 of N. ispahanica at 8 μg ml-1 of N. glocephalata. 3.2 GERMINATION AND SEEEDLING GROWTH BIOASSAY The effect of Nepeta species essential oils against seed germination, root length and shoot length of barnyard grass, canary grass, redroot pigweed and lambsquarters is shown in Figs. 1–3. Significant differences were found among control and all concentrations of Nepeta species essential oil tested. The essential oils of two Nepeta species reduced the germination of all studied weeds. Further- more the difference between the control and the lowest concentration was significant for all weed species. At 1 μg ml-1 of N. ispahanica germination reduction compare to control was 25 %, 11 % and 44 % and 49 % for barnyard grass, canary grass, redroot pigweed and lambsqaurters, respectively. Also germination reduction of barnyard grass, canary grass, redroot pigweed and lambsqaurters was 21.5 %, 8 % and 29 % and 44.75 % at 1 μg ml-1 of N. glocephalata, respectively (Fig. 1). Redroot pigweed and lambsquarters were most sensitive to N. ispahanica es- sential oil, their germination was completely inhibited by it at concentration 4 μg ml-1. At highest concentration 8 μg ml-1 germination percentages were 6.5 % and 10.20 % for canary grass by N. ispahanica and N. glocepha- lata, respectively. Barnyard grass seeds germinated 3 % and 11.75 % at highest concentration of N. ispahanica Figure 1: Effect of N. ispahanica and N. glocephalata essential oils on (a) barnyard grass, canary grass (b) redroot pigweed (c) and lambsqaurters (d) germination measured after 2 weeks. Vertical bars along each data point represent the standard error Acta agriculturae Slovenica, 117/4 – 20216 M. DYANAT and F. ASGARI Figure 2: Effect of N. ispahanica and N. glocephalata essential oils on (a) barnyard grass, canary grass (b) redroot pigweed (c) and lambsqaurters (d) root length measured after 2weeks. Vertical bars along each data point represent the standard error Figure 3: Effect of N. ispahanica and N. glocephalata essential oils on (a) barnyard grass, canary grass (b) redroot pigweed (c) and lambsqaurters (d) shoot length measured after 2 weeks. Vertical bars along each data point represent the standard error Acta agriculturae Slovenica, 117/4 – 2021 7 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species 3.3 GLASS HOUSE STUDIES For more investigation of herbicidal activity of Nepeta essential oils, an experiment was done on 3-week- old weeds. The mature plants of test weeds were dam- aged upon spray of Nepeta essential oils and showed vis- ible injury ranging from chlorosis to necrosis of plants. In general, the visible injury symptoms observed 7 days after spraying increased with increasing concentrations of both Nepeta essential oils (Table 2). At the lowest con- centration 1.25  % of both Nepeta essential oils, all the test weeds showed sign of injury. At the highest concen- tration (10 v/v) visible injury by N. ispahanica essential oil were 44 % 45.5 % 59.25 % and 51.62 % for barnyard grass, canary grass, redroot pigweed and lambsquarters, respectively. While visible injury of barnyard grass, ca- nary grass, redroot pigweed and lambsquarters caused by N. glocephalata essential oil were 39 % 36.62 % 44 % and 41.5 %, respectively at 7 days after spraying that did not have significant difference each other’s (Table 2). Increasing Nepeta essential oils concentration de- creased dry mass of all weed species. The inhibition rates of barnyard grass dry mass ranged from 24.89  % to 75.21 %, and from 16.75 to 61.17 % in N. ispahanica and N. glocephalata, respectively. In canary grass the in- hibition rates of dry mass, ranged from 22.49 to 63.26 %, and from 13.95 to 56.56 % at concentrations for N. ispa- hanica and N. glocephalata, respectively. Essential oil of N. ispahanica caused dry mass inhibition of redroot pigweed from 37.5 % to 90 % while N. glocephalata re- duced it from 23.75 % to 81.5 %. The inhibition rates of lambsquarters dry mass ranged from 19.75  % to 86  %, and from 14.75 to 76 % in N. ispahanica and N. glocepha- lata, respectively. There was a significant difference in the inhibition of dry mass among concentrations and the the highest inhibition of dry mass caused by N. ispahanica at concentration of 10 v/v that was significantly different from other treatments. Increasing essential oil concentration decreased chlorophyll a and chlorophyll b at all studied weeds. For example contents of chlorophyll a in barnyard grass were reduced 12.77  %, 25.40  %, 44.38  % and 49.62  % by N. ispahanica at concentrations of 1.25 %, 2.5 %, 5 % and 10 % v/v, respectively. N. glocephalata concentrations of 1.25 %, 2.5 %, 5 % and 10 % inhibited contents chloro- phyll a in barnyard grass, 11.65 %, 15.56 %, 29.1 % and 43.56 %, respectively that the difference between the con- centrations of 1.25 v/v and 2.5 v/v was not significant. The highest inhibition of chlorophyll a in canary grass was caused by N. ispahanica at concentration of 10 v/v which was not significantly different from N. glocepha- lata. In redroot pigweed, N. ispahanica at the concentra- tion of 1.25 v/v decreased chlorophyll a by 22.05 % and decreased further by increasing concentration. In lambs- quarters, no significant difference was observed between N. ispahanica and N. glocephalata at a concentration of 2.5 v/v (Table 4). N. ispahanica essential oil inhibited chlorophyll b of barnyard grass by 11.30  %, 21.59  %, 29.13  % and 36.79 % at concentrations of 1.25 %, 2.5 %, 5 % and 10 %, respectively. At concentrations of 1.25 %, 2.5 %, 5 % and 10 % N. glocephalata essential oil reduced chlorophyll b by 7.86  %, 12.09  %, 22.21  % and 33.35  %, respectively in barnyard grass. The highest of inhibition of chloro- phyll b in canary grass and redroot pigweed was caused by N. ispahanica at a concentration of 10 v/v which was not significantly different from N. glocephalata in redroot pigweed. In lambsqarters, there was no significant differ- ence between N. ispahanica and N. glocephalata at a con- centration of 1.25 v/v, but at the highest concentration, a significant difference was observed between these two species (Table 5). In this studies chlorophyll a decreased more than chlorophyll b in all species weeds (Tables 4 and 5). 4 DISCUSSION Many researchers reported the presence of nepeta- lactones in several Nepeta species in relatively high con- centrations (Sefidkon and Shaabani, 2004; Rustaiyan et al., 2000, Rustaiyan and Nadji, 1999, Sajjadi and Khatam- saz, 2000) but no nepetalactones were detected in N. glo- cephalata essential oil. 1, 8-cineole, which was the first major component of the studied oils, has been reported in the oil of some Nepeta species from Iran (Rustaiyan et al., 2000; Rustaiyan and Nadji, 1999; Sajjadi and Khatam- saz, 2000). 1, 8-Cineole was also reported previously to be the main compound of N. ispahanica oil (Sefidkon et al., 2005). β-pinene has also been found in the oils of some Nepeta species (Thappa et al., 2001; Baser et al., 2000; Rustaiyan et al., 2000; Rustaiyan and Nadji, 1999; Sefid- kon et al., 2002) but the concentrations of it found in this study was the most in comparison with previous studies. β-pinene and α-pinene are typical in most Nepeta species (Gkinis et al., 2003; Thappa et al., 2001, Baser et al., 2000; Rustaiyan and Nadji, 1999; Sefidkon et al., 2002). The herbicidal activity of both Nepeta essential oils were due to the high percentage of 1,8-Cineole. This is in agreement with Zunino and Zygadlo (2004) who re- ported that monoterpenes such as 1,8- cineole, thymol, geraniol and camphor have been reported to inhibit root growth in maize (Zea mays L.). In a study with 27 monoterpenes, against seed germination and primary root growth of radish (Raphanus sativus L.) and garden cress (Lepidium sativum L.), only 1, 8-cineole, inhibited Acta agriculturae Slovenica, 117/4 – 20218 M. DYANAT and F. ASGARI C on ce nt ra tio n (v /v ) Ba rn ya rd g ra ss C an ar y gr as s R ed ro ot p ig w ee d L am bs qu ar te rs N . i sp ah an ica N . g lo ce ph al at a N . i sp ah an ica N . g lo ce ph al at a N . i sp ah an ica N . g lo ce ph al at a N . i sp ah an ica N . g lo ce ph al at a 1. 25 19 ± 1 .6 3 d 13 .7 5 ± 1. 89 e 17 .8 7 ± 2. 01 d 10 .9 7 ± 0. 41 e 21 .5 ± 3 .7 6 e 11 .1 25 ± 1 .1 0 f 20 .2 5 ± 2. 28 d 12 .4 3 ± 0. 47 e 2. 5 32 ± 2 .1 6 c 19 .5 ± 0 .5 7 d 28 .0 1 ± 1. 31 c 20 .0 1 ± 1. 07 d 31 .5 ± 1 .0 8 cd 25 .8 75 ± 2 .7 8 de 31 .7 5 ± 1. 48 c 22 .6 8 ± 1. 21 d 5 40 .7 5 ± 0. 9 ab 29 .2 5 ± 0. 95 c 37 .2 8 ± 0. 4 b 29 .0 1 ± 0. 81 c 43 .7 5 ± 1. 04 b 36 .5 ± 1 .2 2 c 42 .2 5 ± 0. 45 b 32 .8 7 ± 0. 93 c 10 44 ± 3 .3 6 a 39 ± 1 .8 2 b 45 .5 5 ± 2. 65 a 36 .6 ± 2 .4 7 b 59 .2 5 ± 6. 7 a 44 ± 2 .6 1 b 51 .6 2 ± 3. 01 a 41 .5 ± 0 .5 4 b Ta bl e 2: E ffe ct s o f N ep et a es se nt ia l o ils o n vi sib le in ju ry o f b ar ny ar d gr as s, ca na ry g ra ss , r ed ro ot p ig w ee d an d la m bs qu ar te rs at 7 d ay s a fte r s pr ay in g Va lu es a re m ea ns ± st an da rd e rr or o f f ou r r ep lic at es . W ith in e ac h sp ec ie s, di ffe re nt le tte rs in di ca te th at m ea ns a re d iff er en t a t t he 9 5  % le ve l o f p ro ba bi lit y (T uk ey ’s H SD p os t h oc te st )s C on ce nt ra tio n (v /v ) B ar ny ar d gr as s C an ar y gr as s R ed ro ot p ig w ee d L am bs qu ar te rs N .is pa ha ni ca N .g lo ce ph al at a N . i sp ah an ica N .g lo ce ph al at a N .is pa ha ni ca N .g lo ce ph al at a N .is pa ha ni ca N .g lo ce ph al at a 1. 25 24 .8 9 ± 1. 67 g 16 .7 4 ± 0. 56 h 22 .4 9 ± 4. 53 f 13 .9 5 ± 2. 18 g 37 .5 ± 1 .7 3 f 23 .7 5 ± 2 .2 1 g 19 .7 5 ± 2. 5 f 14 .7 5 ± 2. 21 f 2. 5 39 .5 5 ± 1. 84 e 28 .5 8 ± 0. 96 f 32 .5 5 ± 2. 23 d 22 .1 5 ± 0. 80 e 53 ± 2 .1 6 e 39 .2 5 ± 0. 95 f 41 ± 2 .5 8 d 29 .5 ± 2 .6 4 e 5 69 .1 3 ± 1. 70 c 51 .7 4 ± 1. 84 d 52 .6 0 ± 7. 06 b 43 .9 5 ± 3. 30 c 77 .5 ± 2 .0 8 c 63 ± 2 .4 4 d 61 .5 ± 3 c 59 ± 4 .6 c 10 75 .2 1 ± 0. 61 a 67 .1 7 ± 0. 24 b 63 .2 6 ± 4. 52 a 56 .5 6 ± 2. 8 ab 90 ± 01 a 81 .5 ± 1 .2 9 b 86 ± 1 .4 1 a 76 ± 1 .8 2 b Ta bl e 3: E ffe ct s o f N ep et a es se nt ia l o ils o n dr y w ei gh ts in hi bi tio n % o f b ar ny ar d gr as s, ca na ry g ra ss , r ed ro ot p ig w ee d an d la m bs qu ar te rs at 7 d ay s a fte r s pr ay in g Va lu es a re m ea ns ± st an da rd e rr or o f f ou r r ep lic at es . W ith in e ac h sp ec ie s, di ffe re nt le tte rs in di ca te th at m ea ns a re d iff er en t a t t he 9 5  % le ve l o f p ro ba bi lit y (T uk ey ’s H SD p os t h oc te st ) C on ce nt ra tio n (v /v ) Ba rn ya rd g ra ss C an ar y gr as s Re dr oo t p ig w ee d L am bs qu ar te rs N . i sp ah an ica N .g lo ce ph al at a N . i sp ah an ica N . g lo ce ph al at a N . i sp ah an ica N . g lo ce ph al at a N . i sp ah an ica N .g lo ce ph al at a 1. 25 12 .7 7 ± 6. 79 c 11 .6 5 ± 0. 55 c 13 .8 0 ± 3g 8. 71 ± 1 .3 6g 22 .0 5 ± 1. 02 d 14 .4 7 ± 1. 45 f 20 .2 7 ± 5. 85 cd 11 .6 5 ± 0. 51 e 2. 5 25 .4 0 ± 1. 31 b 15 .5 6 ± 3. 57 c 31 .5 9 ± 5. 92 ef 19 .0 9 ± 2. 43 e 37 .8 0 ± 1. 97 d 26 .4 7 ± 5. 13 e 32 .9 0 ± 5. 74 c 27 .5 6 ± 0. 69 c 5 44 .3 8 ± 5. 0a 29 .1 ± 5 .7 4b 39 .1 3 ± 4. 41 bc 32 .2 1 ± 1. 33 cd 52 .4 7 ± 2. 17 b 44 .6 9 ± 3. 78 c 54 .3 8 ± 5. 72 a 41 .6 ± 4 .5 3 b 10 49 .6 2 ± 2. 5a 43 .5 6 ± 2. 43 a 46 .7 9 ± 3. 57 a 41 .6 0 ± 1. 77 ab 59 .3 2 ± 1. 0a 53 .0 7 ± 1. 85 b 59 .6 2 ± 2. 54 a 54 .8 1 ± 0. 56 a Ta bl e 4: E ffe ct s o f N ep et a es se nt ia l o ils o n C hl or op hy ll a in hi bi tio n% o f b ar ny ar d gr as s, ca na ry g ra ss , r ed ro ot p ig w ee d an d la m bs qu ar te rs at 7 d ay s a fte r s pr ay in gg Va lu es a re m ea ns ± st an da rd e rr or o f f ou r r ep lic at es . W ith in e ac h sp ec ie s, di ffe re nt le tte rs in di ca te th at m ea ns a re d iff er en t a t t he 9 5  % le ve l o f p ro ba bi lit y (T uk ey ’s H SD p os t h oc te st ) C on ce nt ra tio n (v /v ) B ar ny ar d gr as s C an ar y gr as s R ed ro ot p ig w ee d La m bs qu ar te rs N . i sp ah an ica N .g lo ce ph al at a N . i sp ah an ica N . l oc ep ha la ta N . i sp ah an ica N . g lo ce ph al at a N .is pa ha ni ca N .g lo ce ph al at a 1. 25 11 .3 0 ± 2. 20 c 7. 86 ± 2 .1 2c d 17 ± 2 .3 7d 8. 93 7 ± 1. 46 e 19 .5 4 ± 3. 42 ef 15 .1 1 ± 4. 87 f 15 .4 3 ± 1. 25 d 11 .0 4 ± 1. 20 e 2. 5 21 .5 9 ± 5. 93 b 12 .0 9 ± 5. 99 c 28 .7 5 ± 1. 49 c 20 .6 8 ± 1. 91 d 29 .6 3 ± 2. 71 cd 23 .5 2 ± 2. 52 de 25 .8 0 ± 1. 38 c 18 .9 1 ± 3. 19 d 5 29 .1 3 ± 4. 41 ab 22 .2 1 ± 1. 34 b 39 .2 5 ± 0. 46 ab 25 .8 7 ± 2. 13 c 38 .2 7 ± 2. 99 ab 33 .1 8 ± 1. 11 bc 32 .9 0 ± 1. 73 b 26 .2 1 ± 0. 48 c 10 36 .7 9 ± 3. 58 a 33 .3 5 ± 2. 88 a 42 .1 2 ± 2. 21 a 37 ± 3 .3 6b 45 .1 1 ± 4. 2a 40 ± 2 .3 7a b 40 .5 0 ± 2. 60 a 37 .3 4 ± 2. 35 a Ta bl e 5: E ffe ct s o f N ep et a es se nt ia l o ils o n C hl or op hy ll b in hi bi tio n % o f b ar ny ar d gr as s, ca na ry g ra ss , r ed ro ot p ig w ee d an d la m bs qu ar te rs at 7 d ay s a fte r s pr ay in g Va lu es a re m ea ns ± st an da rd e rr or o f f ou r r ep lic at es . W ith in e ac h sp ec ie s, di ffe re nt le tte rs in di ca te th at m ea ns a re d iff er en t a t t he 9 5  % le ve l o f p ro ba bi lit y (T uk ey ’s H SD p os t h oc te st ) Acta agriculturae Slovenica, 117/4 – 2021 9 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species their root elongation at the lowest concentrations (10−5 M, 10−6 M) applied (De Martino et al., 2010). Romagni et al. (2000) have shown that 1, 8-cineole, and its natural analogue 1, 4-cineole, both suppress the growth of sev- eral weeds. 1, 8-cineole inhibits the germination, speed of germination, seedling growth, chlorophyll content and respiratory activity of Ageratum conyzoides L. 1753 not Hieron. 1895 nor Sieber ex Steud. 1840. Singh et al. (2002) and De Feo et al. (2002) have investigated the herbicidal activity of 10 volatiles compounds from Ruta graveolens L. essential oils and showed that 1,8-cineole significantly inhibits the germination and radical elonga- tion of radish. The effects of the allelochemicals in studied traits directly dependent on the concentration and Nepeta spe- cies. The germination and root length decreased with increasing concentrations of essential oils. These results are in agreement with that of Ibáñez and Blázquez (2017) who reported that there are significant effects in shoot and/or shoot + root length of weeds depending on the weed and dose. N. ispahanica oil exerted the more in- hibitory effect than N. glocephalata for all weed species. It can be due to higher concentration of 1,8-cineole in N. ispahanica. While the inhibition is not similar between the Nepeta species oils, weed species differed in their response to the toxic effect of each oil. It was reported that the degree of allelopathic interference can even vary within species (Li et al. 2009). My observations in glass house indicated that both Nepeta oils can act as contact herbicides. These observa- tions are in agreement with previous studies showing that volatile oils and even their monoterpenes exhibit herbicidal activity (Tworkoski, 2002; Singh et al., 2005, 2006). Batish et al. (2004, 2007) concluded that the 5 % essential oil from E. citriodora caused 50–80 % visible in- jury in A. viridis, P. minor and E. crus-galli. In addition Poonpaiboonpipat et al. (2013) reported that the essen- tial oil lemon grass (Cymbopogon citratus (DC ex Nees) Stopf) applied on barnyardgrass in greenhouse caused leaf wilting. The reduction in seedling dry mass, chloro- phyll a and chlorophyll b content observed in my study is in agreement with previous reports indicating that the monoterpenes had a potential to reduce chlorophyll con- tent (Chowhan et al. 2011; Kaur et al. 2010; Gouda et al., 2016). It may be due to inhibition of biosynthesis of chlo- rophyll and/or degradation of chlorophyll. 5 CONCLUSION From the present study, it could be concluded that Nepeta essential oils strongly inhibited the germination and root length of all weeds. Dicot weeds (lambsquarters and redroot pigweed) were significantly more sensitive than monocot weeds (barnyard grass and canary grass). Indeed, at the dose of 4  μg ml-1, germination of lamb- squarters and redroot pigweed was totally inhibited by N. ispahanica essential oil. Further studies are required to investigate the herbicidal potential of Nepeta essential oils under field conditions and determine the effects on crop species and other weed species. This study is con- sidered the first study regarding of herbicidal effects of N. glocephalata and N. ispahanica but the selectivity of these compounds should be considered. 6 REFERENCES Abrahim, D., Braguini, W.L., Kelmer-Bracht, A.M. and Ishii- Iwamoto, E.L. (2000). Effects of four monoterpenes on ger- mination, primary root growth, and mitochondrial respi- ration of maize. Journal of Chemical Ecology, 26, 611-624. https://doi.org/10.1023/A:1005467903297 Adams, R.P. (2007). Identification of Essential Oil Components by GasChromatography/mass Spectrometry, 4th ed. Allured Publishing Corporation, Carol Stream, USA. Amri, I., Hamrouni, L., Hanana, M. and Jamoussi, B. (2013). Reviews on phytotoxic effects of essential oils and their individual components: news approach for weeds manage- ment. International Journal of Applied Biology and Pharma- ceutical Technology, 4, 96-114. Angelini, L.G., Carpanese, G., Cioni, P.L., Morelli, I., Macchia, M. and Flamini, G. (2003). Essential oils from Mediterra- nean Lamiaceae as weed germination inhibitors. Journal of Agricultural and Food Chemistry, 51, 6158-6164. https:// doi.org/10.1021/jf0210728 Armirante, F., De Falco, E., De Feo, V., De Martino, L., Mancini, E. and Quaranta, E. (2006). Allelopathic activity of essential oils from Mediterranean Labiatae. Acta Horticulture, 723, 347–352. https://doi.org/10.17660/ActaHortic.2006.723.47 Ashraf R., Sultanal B., Yaqoob S., Iqbal, M. 2017. Allelochemi- cals and crop management, CSP, 3, 1-13. Babaahmadi, H., Ghanbari, A., Asadi, G. and Emami, M.K. (2013). Allelopathic effect from some medicinal plants on germination of Alyssum hirsutum and Amaranthus retro- flexus. International Journal of Agronomy and Plant Protec- tion, 4, 3344-3347. Barney, J.N., Hay, A.G. and Weston, L.A. (2005). Isolation and characterisation of allelopathic volatiles from mugwort (Artemisia vulgaris). Journal of Chemical Ecology, 31, 247– 265. https://doi.org/10.1007/s10886-005-1339-8 Baser K.H.C., Kirimer N., Kurkcuoglu M. and Demirci, B. (2000). Essential oils of Nepeta species growing in Turkey. Chemical Natural Compounds, 36, 356–359. https://doi. org/10.1023/A:1002832628159 Batish, D.R., Setia, N., Singh, H.P. and Kohli, R.K. (2004). Phy- totoxicity of lemon-scented eucalypt oil and its potential use as a bioherbicide. Crop Protection, 23, 1209–1214. htt- ps://doi.org/10.1016/j.cropro.2004.05.009 Batish, D.R., Singh, H.P., Setia, N., Kaur, S. and Kohli, R.K. Acta agriculturae Slovenica, 117/4 – 202110 M. DYANAT and F. ASGARI (2006). Chemical composition and inhibitory activity of essential oil from decaying leaves of Eucalyptus citriodo- ra. Zeitschrift für Naturforschung, 61, 52–56. https://doi. org/10.1515/znc-2006-1-210 Batish, D.R., Singh, H.P., Setia, N., Kohli, R.K., Kaur, S. and Yadav, S.S. (2007). Alternative control of littleseed canary grass using eucalypt oil. Agronomy Sustainable Develop- ment, 27, 171–177. https://doi.org/10.1051/agro:2007008 Bozari, S., Agar, G., Aksakal, O., Erturk, F.A. and Yanmis, D. (2013). Determination of chemical composition and geno- toxic effects of essential oil obtained from Nepeta nuda on Zea mays seedlings. Toxicological Industrial Health, 29, 339- 48. https://doi.org/10.1177/0748233711433939 Campiglia, E., Mancinelli, R., Cavalieri, A. and Caporali, F. (2007). Use of essential oils of cinnamon (Cinnamomum zeylanicum L.), lavender (Lavandula spp.) and peppermint (Mentha x piperita L.) for weed control. Italian Journal of Agronomy, 2, 171-175. https://doi.org/10.4081/ija.2007.171 Chowhan, N., Singh, H.P., Batish, D.R. and Kohli, R.K. (2011). Phytotoxic effects of b-pinene on early growth and associ- ated biochemical changes in rice. Acta Physiologiae Plan- tarum, 33, 2369–2376. https://doi.org/10.1007/s11738-011- 0777-x De Feo V., Simone F.D., Senatore F. 2002. Potential allelo- chemicals from the essential oil of Ruta graveolens. Phyto- chemistry, 61, 573-578. https://doi.org/10.1016/S0031- 9422(02)00284-4 De Martino, L., Mancini, E., Almeida, L. F. R. and De Feo, V. (2010). The antigerminative activity of twenty-seven monoterpenes. Molecules, 15, 6630–6637. https://doi. org/10.3390/molecules15096630 Dudai, N., Ben-Ami, M., Chaimovich, R. and Chaimovitsh, D. (2004). Essential oils as allelopathic agents: bioconversion of monoterpenes by germinating wheat seeds. Acta Horti- culture, 629, 505–508. https://doi.org/10.17660/ActaHor- tic.2004.629.65 Dudai, N., Poljakoff-Mayber, A., Mayer, A.M., Putievs- ky, E. and Lerner, H.R. (1999). Essential oils as alle- lochemicals and their potential use as bioherbicides. Journal of Chemical Ecology, 25, 1079–1089. https://doi. org/10.1023/A:1020881825669 Ens, E.J., Bremner, J.B., French, K. and Korth, J. (2009). Iden- tification of volatile compounds released by roots of an in- vasive plant, bitou bush (Chrysanthemoides monilifera spp. rotundata), and their inhibition of native seedling growth. Biological Invasion, 11, 275–287. https://doi.org/10.1007/ s10530-008-9232-3 Eom, S.H.,Yang, H.S. and Weston, L.A. (2006). An evaluation of the allelopathic potential of selected perennial groundcov- ers: foliar volatiles of catmint (Nepeta × faassenii) inhibit seedling growth. Journal of Chemical Ecology, 32, 1835-48. https://doi.org/10.1007/s10886-006-9112-1 Formisano C., Rigano D. and Senatore F. (2011). Chemical con- stituents and biological activities of Nepeta species. Chemi- cal Biodiversity, 8, 1783–1818. https://doi.org/10.1002/ cbdv.201000191 Gkinis, G., Tzakou, O., Iliopoulou, D. and Roussis, V. (2003). Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones. Z. Naturforsch. Canadian Journal of Bioscience, 58, 681–686. https://doi. org/10.1515/znc-2003-9-1015 Gouda, N. A. A., Saad, M. M. G. and Abdelgaleil, S. A. M. (2016). PRE and POST Herbicidal Activity of Monoterpe- nes against Barnyard Grass (Echinochloa crus-galli).Weed Science, 64,191-200. https://doi.org/10.1614/WS-D-15- 00045.1 Ibáñez M. D. and Blázquez. M. A. (2017). Herbicidal value of essential oils from oregano-like flavor species. Food Agri- cultural Immunology, 28, 1168–1180. https://doi.org/10.10 80/09540105.2017.1332010 Jamzad, Z. (2012). Flora of Iran Lamiaceae, 76, 577-580. Kaur, S., Singh, H. P., Mittal, S., Batish, D. R. and Kohli, R. K. (2010). Phytotoxic effects of volatile oil from Artemisia scoparia against weeds and its possible use as a bioherbi- cide. Indian Crops and Production, 32, 54-61. https://doi. org/10.1016/j.indcrop.2010.03.007 Kekec, G., Mutlu, S., Alpsoy, L., Sakcali, M S. and Atici, O. (2012). Genotoxic effects of catmint (Nepeta meyeri Benth.) essential oils on some weed and crop plants. Toxi- cological and Industrial Health, 29, 504-513. https://doi. org/10.1177/0748233712440135 Kobaisy, M., Tellez, M. R., Dayan, F. E., Mamonov, L. K., Mu- kanova, G. S., Sitpaeva, G. T. andGemejieva, N. G. (2005). Composition and phytotoxic activity of Nepeta pannonica L. essential oil. Journal of Essential Oil Research, 17, 704. https://doi.org/10.1080/10412905.2005.9699037 Kordali, S., Aslan, I., Calmasur, O. and Cakir, A. (2006). Tox- icity of essential oils isolated from three Artemisia species and some of their major components to granary weevil, Sit- ophilus granarius (L.) (Coleoptera: Curculinonidae). Indian Crops Production, 23, 162–170. https://doi.org/10.1016/j. indcrop.2005.05.005 Kordali, S., Cakir, A. and Sutay, S. (2007). Inhibitory effects of monoterpenes on seed germination and seedling growth. Zeitschrift für Naturforschung, 62, 207–214. https://doi. org/10.1515/znc-2007-3-409 Li, H., Pan, K., Liu, Q. and Wang, J. (2009). Effect of enhanced ultraviolet-B on allelopathic potential of Zanthoxylum bungeanum. Scientia Horticulturae, 119, 310–314. https:// doi.org/10.1016/j.scienta.2008.08.010 Lichtenthaler, H.K. (1987).Chlorophylls and carotenoids: pig- ments of photosynthetic biomembranes. Methods En- zymology, 148, 350–382. https://doi.org/10.1016/0076- 6879(87)48036-1 McLafferty, F.W. and Stauffer, D.B. (1989). The Wiley/NBS Reg- istry of Mass Spectral Data. Wiley and Sons, New York. Mancini, E., Arnold, N. A., De Feo, V., Formisano, C., Riga- no, D., Piozzi, F. and Senatore, F. (2009). Phytotoxic ef- fects of essential oils of Nepeta curviflora Boiss. and Nepeta nuda L. subsp. albiflora growing wild in Leba- non. Journal of Plant Interaction, 4, 253-259. https://doi. org/10.1080/17429140903225507 Mahdavikia F. and Saharkhiz M. J. (2015). Phytotoxic activity of essential oil and water extract of peppermint (Mentha × piperita ‘Mitcham’). Journal of Applied Research Medicinal Aromatic Plants, 2, 146–153. https://doi.org/10.1016/j.jar- map.2015.09.003 Muller, C.H., Muller, W.H., Haines, B.L. (1964). Volatile growth Acta agriculturae Slovenica, 117/4 – 2021 11 Phytotoxic effects of essential oils from Nepeta glocephalata Rech.f. and N. ispahanica Boiss. on selected weed species inhibitors produced by aromatic shrubs. Science, 143, 471– 473. https://doi.org/10.1126/science.143.3605.471 Mutlu, S. and Atici, O. (2009). Allelopathic effect of Nepeta meyeri Benth. extracts on seed germination and seedling growth of some crop plants. Acta Physiologiae Plantarum, 31, 89-93. https://doi.org/10.1007/s11738-008-0204-0 Mutlu, S., Atici, O, Esim, N. and Mete, E. (20110. Essential oils of catmint (Nepeta meyeri Benth.) induce oxidative stress in early seedlings of various weed species. Acta Physiologiae Plantarum, 943-951. https://doi.org/10.1007/s11738-010- 0626-3 Pal Singh, H., Kaur, S., Mittal, S., Batish, D. R. and Kohli, R. K. (2008). Phytotoxicity of major constituents of the volatile oil from leaves of Artemisia scoparia Waldst. & Kit. Zeitschrift für Naturforschung, 63, 663-666. https://doi.org/10.1515/ znc-2008-9-1009 Poonpaiboonpipat, T., Pangnakorn, U., Suvunnamek, U., Tee- rarak, M., Charoenying, P. and Laosinwattana, C. 2013. Phytotoxic effects of essential oil from Cymbopogon citratus and its physiological mechanisms on barnyardgrass (Echi- nochloa crus-galli). Indian Crops Production, 41, 403– 407. https://doi.org/10.1016/j.indcrop.2012.04.057 Romagni, J.G., Allen, S.N. and Dayan, F.E. (2000). Allelo- pathic effects of volatile cineoles on two weedy plant spe- cies. Journal of Chemical Ecology, 26, 303-313. https://doi. org/10.1023/A:1005414216848 Rustaiyan, A. and Nadji, K. (1999). Composition of the es- sential oils of Nepeta ispahanica Boiss. and Nepeta binaludensis Jamzad from Iran. Flavour Fragrance Journal, 14, 35–37. https://doi.org/10.1002/(SICI)1099- 1026(199901/02)14:1<35::AID-FFJ776>3.0.CO;2-N Rustaiyan, A., Komeilizadeh, H., Monfared, A., Nadji, K., Ma- soudi, S. and Yari, M. (2000). Volatile constituents of Nepeta denudata Benth. and N. cephalotes Boiss. from Iran. Journal of Essential Oil Research, 12, 459–461. https://doi.org/10.10 80/10412905.2000.9699565 Saharkhiz, M.J., Esmaeili, S. and Merikhi, M. (2010). Es- sential oil analysis and phytotoxic activity of two ecotypes of Zataria multiflora Boiss. growing in Iran. Natural Product Research, 24, 1598–1609. https://doi. org/10.1080/14786411003754280 Sajjadi. S.E. and Khatamsaz, M. (2000).Volatile constituents of Nepeta heliotropifolia Lam. Journal of Essential Oil Research, 13, 204–205. https://doi.org/10.1080/10412905.2001.96996 65 Sajjadi, S. E. (2005). Analysis of the essential oil of Nepeta sin- tenisii Bornm. from Iran. DARU. Journal of Pharmaceutical Sciences, 13, 61-4. Sefidkon, F., Dabiri, M. and Alamshahi, A. (2002). Analysis of the essential oil of Nepeta fissa CA Mey from Iran. Fla- vor Fragrance Journal, 17, 89–90.https://doi.org/10.1002/ ffj.1045 Sefidkon, F. and Shaabani, A. (2004). Essential oil composition of Nepeta meyeri Benth. from Iran. Flavour Fragrance Jour- nal, 19, 236–238.https://doi.org/10.1002/ffj.1294 Sefidkon, F., Jamzad, Z. and Mirza. M. (2005). Chemical com- position of the essential oil of five Iranian Nepeta species (N. crispa, N. mahanensis, N. ispahanica, N. eremophila and N. rivularis). Flavor Fragrance Journal, 21, 764–767. https://doi.org/10.1002/ffj.1668 Singh H.P., Batish D.R., Kaur S., Ramezani H. and Kohli R.K. (2002). Comparative phytotoxicity of four monoterpe- nes against Cassia occidentalis. Annals of Applied Biology, 141,111- 116. https://doi.org/10.1111/j.1744-7348.2002. tb00202.x Singh, H.P., Batish, D.R. and Kohli, R.K. (2003). Allelopathic interactions and allelochemicals: newpossibilities for sus- tainable weed management. Critical Review Plant Science, 22, 239-311. https://doi.org/10.1080/713610858 Singh, HP, Batish, D.R, Kaur, S., Vaid, S. and Kohl, I. R. (2004). Weed suppressing ability of some monoterpenes. Journal of Plant Diseases Protection, 111, 821-828. Singh, H.P., Batish, D.R., Setia, N. and Kohli, R.K. (2005a). Herbicidal activity of volatile essential oils from Eucalyp- tus citriodora against Parthenium hysterophorus. Annals of Applied Biology, 146, 89–94. https://doi.org/10.1111/j.1744- 7348.2005.04018.x Singh, H.P., Batish, D.R., Setia, N. and Kohli, R.K. (2005b). Herbicidal activity of volatile oils from Eucalyptus ci- triodora against Parthenium hysterophorus. Annals of Ap- plied Biology, 146, 89–94. https://doi.org/10.1111/j.1744- 7348.2005.04018.x Singh, H.P., Batish, D.R., Kaur, S., Kohli, R.K. and Arora, K. (2006). Phytotoxicity of volatile monoterpene citronel- lal against some weeds. Zeitschrift für Naturforschung, 61, 334–340. https://doi.org/10.1515/znc-2006-5-606 Sonboli ,A., Salehi, P. and Allahyari, L. (2005). Essential oilcom- position of Nepeta involucrate from Iran. Chemical Natural Compounds, 41, 683-5. https://doi.org/10.1007/s10600- 006-0011-z Thappa R.K., Agarwal S.G., Srivastava T.N. and Kapahi, B.K. (2001). Essential oils of four Himalayan Nepeta species. Journal of Essential Oil Research, 13, 189–191. https://doi.or g/10.1080/10412905.2001.9699658 Tucker A.O. and Tucker S.S. (1988). Catnip and the catnip response. Economic Botany, 42, 214–231. https://doi. org/10.1007/BF02858923 Tworkoski, T. (2002). Herbicide effects of essential oils. Weed Science, 50, 25–431. https://doi.org/10.1614/0043- 1745(2002)050[0425:HEOEO]2.0.CO;2 Verdeguer, M., García-Rellán, D., Boira, H., Pérez, E., Gan- dolfo, S. and Blázquez, M. A. (2011). Herbicidal activity of Peumus boldus and Drimys winterii essential oils from Chile. Molecules, 16, 403-411. https://doi.org/10.3390/mol- ecules16010403 Vyvyan, J.R. (2002). Allelochemicals as lead for new herbicides and agrochemicals. Tetrahedron, 58, 1631-1636. https://doi. org/10.1016/S0040-4020(02)00052-2 Wagner, H. and Wolf, P. (1977). New Natural Products and Plant Drugs with Pharmacological, Biological and Thera- peutical Activity. Springer Verlag, New York. https://doi. org/10.1007/978-3-642-66682-7 Weston, L.A. (1996). Utilization of allelopathy for weed man- agement in agroecosytems. Agronomy Journal, 88, 360–366. https://doi.org/10.2134/agronj1996.000219620036000600 04x Živković, J.M.N. (2013). Antioxidative, antimicrobial and allelo- pathic effects of three endemic Nepeta species (Lamiaceae). Acta agriculturae Slovenica, 117/4 – 202112 M. DYANAT and F. ASGARI Doctoral Dissertation. University of Belgrade, Faculty of biology. Zunino, M.P. and Zygadlo, J.A. (2004). Effect of monoterpenes 309. on lipid oxidation in maize. Planta, 219, 303- https://doi.org/10.1007/s00425-004-1216-7 Acta agriculturae Slovenica, 117/4, 1–12, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2111 Original research article / izvirni znanstveni članek Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water for long periods of time through the CO2 efflux Mohamed SABER 1, Alaa ZAGHLOUL 2, 3 Received February 21, 2021; accepted November 26, 2021. Delo je prispelo 21. februarja 2021, sprejeto 26. novembra 2021 1 Department of Agricultural Soil Microbiology, National Research Centre, Dokki, Cairo, Egypt 2 Soils and Water Use Department, National Research Centre, Dokki, Cairo, Egypt 3 Corresponding author, e-mail: alaazaghloul2008@gmail.com Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water for long periods of time through the CO2 efflux Abstract: To sightsee the bearings of the certain reme- diation amendments, usually applied in the bioremediation of soils irrigated with low quality water for extended periods on the indigenous microbial population, a greenhouse experiment was conducted at National Research Centre (NRC) where the soil ecosystem was supplied with varied mineral remediation amendments and the carbon dioxide (CO2) refluxes were fol- lowed up. In this study, microbial activity through CO2 efflux was taken as an indicator to evaluate the effectiveness of eight soil amendments in minimizing the hazards of inorganic pol- lutants in soil ecosystem irrigated with low quality water s for more than 40 years. Results showed that Ni and Zn were the most dominant contaminants that adversely influenced indig- enous microbial activities in untreated soil, while Cu was the most persuasive. All trailed remediation amendments signifi- cantly minimized the hazards of inorganic pollutants in treated soil ecosystems. In addition, modified bentonite (Probentonite) was the best persuasive one. Mechanisms take place between trailed remediation amendments and inorganic pollutants in the studied soil ecosystems were discussed. In conclusion ap- plication of certain raw or modified clay minerals especially Probentonite could be a good tool in decreasing the rate of the studied inorganic pollutants in a contaminated soil ecosystem irrigated with low quality water for extended periods. Key words: soil; low quality water; bioremediation; po- tential toxic elements; soil indigenous microbial Activities; modified clay minerals Ovrednotenje biokemičnih obravnavanj onesnaženih tal, ki so bila dalj časa namakana z vodo slabe kakovosti z meritvijo sproščanja CO2 Izvleček: Za prepoznavanje obremenitev, ki jih povzroča- jo nekateri remediacijski dodatki, ki se navadno uporabljajo pri bioremediaciji tal namakanih dalj časa z vodo slabe kakovosti na samoniklo mikrobno populacijo, je bil izveden poskus v ra- stlinjaku v nacionalnem raziskovalnem centru (NRC). Tlem so dodajali različne mineralne remediacijske dodatke in spremlja- li sproščanje ogljikovega dioksida (CO2). Mikrobna aktivnost, izražena kot iztok CO2, je služila kot indikator ovrednotenja učinkovitosti osmih talnih dodatkov, ki naj bi zmanjšali škodo, ki jo v talnem ekosistemu povzročajo anorganska onesnaže- vala iz vode za namakanje slabe kakovosti v obdobju več kot 40 let. Rezultati so pokazali, da sta bila Ni in Zn dominantna kontaminanta, ki sta negativno vplivala na aktivnost samoni- klih mikrobov v obravnavanih tleh medtem, ko je bil učinek Cu največji. Vsi poskusi dodatkov v remediaciji so značilno zmanjšali tveganja poškodbe tal zaradi anorganskih polutantov v obravnavanih talnih ekosistemih. Pri tem je bil spremenjeni bentonit (Probentonite) najučinkovitejši. V raziskavi so inter- pretirani mehanizmi, ki potekajo med dodatki v remediaciji in anorganskimi onesnaževali v preučevanih talnih ekosistemih. Zaključek je, da je dodatek nekaterih osnovnih ali spremenje- nih glinenih mineralov, še posebej probentonita lahko dobro sredstvo za zmanševanje onesnaženja z nekaterimi anorganski- mi onesnaževali v onesnaženih ekosistemih, ki so bili namakani z vodo slabe kakovosti v daljšem obdobju. Ključne besede: tla; voda slabe kakovosti; bioremediacija; potencialno toksični elementi; aktivnost samoniklih talnih mi- krobov; spremenjeni glineni minerali Acta agriculturae Slovenica, 117/4 – 20212 M. SABER and A. ZAGHLOUL 1 INTRODUCTION Through low quality water farming varied organic and inorganic contaminates reach the soil together with enteric pathogens, and cause vital adverse agricultural, environmental and public health troubles. Biological pollutants  include bacteria, viruses, and parasites that are responsible for waterborne diseases, such as typhoid fever, cholera, dysentery, polio, hepatitis, and schistoso- miasis. The presence of coliform bacteria is indicator of recent fecal pollution, this type of contamination is ex- clusively attributed to human and animal waste. Also, in these materials inorganic pollutants include cations and anions, most of them naturally occurring in soils, sedi- ments, and rocks. Cations include heavy metals, such as cadmium (Cd), chromium (Cr VI), lead (Pb), manganese (Mn), mercury (Hg), and nickel (Ni). These highly toxic chemicals may reach soil after mineral dissolution with low quality waters applied or from industrial activities or after industrial emissions (Saber et al., 2015).Although low quality water farming was always associated with po- tential benefits as well as with problems, yet appropriate practices to ensure its safe and effective sustainable use are not well developed (Doaa Ali et al, 2020). The role of microorganism on the health of soil ecosystem is incon- testable. Since 1930 huge amounts of agrochemicals were applied to soil ecosystems and adversely impacted their biome activities, Khan and Scullion (2000) recorded a shift in bacterial to fungal population in soil ecosystems as a result of contamination with inorganic pollutants. Recently, one of the narrative concepts of soil health is bi- oremediation of contaminated soil ecosystems using var- ied remediation amendments, some of which with nega- tive impacts on soil microbial activities (Kelly and Tate III, 1998). It is worthy to state that the influence of the inorganic pollutants on the respiration intensity in a soil ecosystem irrigated with low quality water for extended periods was somewhat inconsistent, Yonebayashi and Hattori (1989) and Doelman and Haanstra (1984) verified significant decreases in CO2 evolution from such soils, while Bardgett and Saggar (1994) and Welp (1999), on the other hand, recorded high increases. Such phenomenon is linked with varied interferences. The current study aimed at evaluating the delayed effect of a natural modified clay mineral fortified with certain remediation amendments on a soil irrigated with low quality water s for extended periods through their microbial activities represented by CO2 evolution. 2 MATERIAL AND METHODS 2.1 STUDY SITE A surface soil sample (0-30 cm), irrigated with low quality water for 40 years was collected from El-Rahawy farm, Giza governorate. The chemical characterization of the soil showed it to have pH, 6.83; EC, 0.2 dSm-1; OM (organic matter), 0.2 %; clay content, 4.4 % and with a sandy texture. Determined inorganic pollutants in the tested soil were 18 ppm Ni; 35.65 ppm Cu; 400.6 ppm Zn; 596 ppm Fe; 45.19 Mn 57.7ppm Pb and the Zn equivalent parameter was 633.9. 2.2 EXPERIMENTAL DESIGN In a completely randomized pot experiment with four replicates, single and combined mixtures of varied remediation inorganic amendments were trailed to re- tain PTE’s from a contaminated soil ecosystem irrigated with low quality water for extended periods. The soil was treated with either 2 % probentonite (T1), 2 % kaolinite (T2), 1 % probentonite + 1 % Kaolinite (T3), 1 % proben- tonite + 1 % rock phosphate (RP) (T4), 1 % kaolinite + 1 % RP (T5), 1 % Bentonite + 0.5 % kaolinite + 0.5 % RP (T6), 2 % iron oxide (T7) and 1 % iron oxide + 1 % RP (T8). Treated and untreated control soils were moistened to 60 % of the soil water holding capacity and incubated for 60 days at 25  oC. At the end of incubation time (60 days), a kinetic study was carried out on treated and un- treated soil ecosystems followed by a distribution study of inorganic pollutants studied. 2.3 INORGANIC POLLUTANTS INSTRUMENTA- TION AND ANALYSIS A Perkin–Elmer flame atomic absorption spectrom- eter (FAAS) and HACH DR890 colorimeter was used in inorganic pollutants instrumentation and analyses. Atomic absorption measurements were carried out using air: acetylene flame while HACH colorimeter measure- ment with the provided test kits. The operating param- eters for working elements were set of as recommended by the manufacturer. 2.4 POTENTIAL TOXIC ELEMENTS DISTRIBU- TION ANALYSIS Inorganic pollutants were fractionated to wa- ter soluble, exchangeable, (readily available form RA), Acta agriculturae Slovenica, 117/4 – 2021 3 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ... carbonate-bound, Fe-Mn oxides-bound ad organic- bound which was considered to be the residual fraction (Zaghloul, 2002). The soil quality criterion index (Zn equivalent model) was numerically expressed for the lev- els of PTE’s toxicity as described by Saber et al. (2012). A quality criterion index for zinc equivalent over 250 units indicated a risky situation. Kinetic studies were carried out using the Electrical Stirred Flow Unit (ESFU) meth- od. 2.5 CO2 EFFLUX AND KINETIC EQUATION CO2 evolved during the incubation was trapped in 1 M NaOH, and the excess NaOH was titrated with 0.1 M HCl after the addition of BaCl2. Total CO2 mineral- ized was calculated as cumulative CO2 evolution (Leifeld et al., 2002). The specific respiration activity (qCO2) was expressed as the production of CO2-C per unit biomass C and time. Kinetic equations: The following four kinetic equa- tions representing both empirical and theoretical equa- tions were used to test the conformity of the CO2 release data to each of them. 2.5.1 First order equation Log (qj - qt ) = Log q0 - k1 t where: qj = the maximum amount of CO2 release q0 = the initial amount of CO2 at the time of added the resin. qt = the amount of CO2 release at time t. t = time in minute k1 = the rate constant of reaction n in sec -1 2.5.2 Elovich équation q= (1/b) ln ( ab ) + (1/b) ln t where: q = amount of CO2 desorbed at time t a = constant in ppm CO2 min -1 b = constant in (ppm CO2) -1 t = time in minute 2.5.3 Modified Freundlich equation q = kd t b\ where: q = amount of CO2 desorbed in time t kd = desorption rate coefficient in mg CO2 kg -1 soil min-1 b\ = constant in mg CO2 kg -1 soil 2.6 SOIL MICROBIAL BIOMASS Soil microbial biomas was measured by the fumi- gation–extraction method after 24, 72, 96, 168, 240 and 336 hours. Three replicates of each treatment were fumi- gated with ethanol-free chloroform for 24 h at 25 ⁰C. The soil samples were then extracted with 0.5 M K2SO4 for 30 min. Three replicates of non-fumigated soil samples were extracted similarly. The extracted PTE’s were determined by dichromate oxidation at 100 ⁰C (2 ml of extract, 1.5 ml of 15 M H2SO4 and 1.5‏ ml of saturated aqueous solu- tion of K2Cr2O7). The residual K2Cr2O7 was determined by photometrical at 565 nm (Kuzyakov, 1997). The cali- bration of the extracted C (carbon) measurements was carried using glucose. CO2 evolved during incubation was trapped in 1 M NaOH, and the excess NaOH was titrated with 0.1 M HCl after addition of BaCl2 (USEPA. 2001). Total mineralized C was calculated as cumulative CO2 evolution (Leifeld et al., 2002). The specific respira- tion activity (qCO2) was expressed as the production of CO2-C per unit biomass C and time (Anvar and Oliver Dilly, 2002). 2.7 STATISTICAL ANALYSIS Multiple linear regressions, discriminant analysis and the fitting of curves to the data were performed using separate two-way ANOVAs. The data of biomass inor- ganic pollutants and soil organic C were analyzed by dis- criminate analysis. Statistical analyses aimed to examine the succession of the applied remediation amendments in returning the studied contaminated soil ecosystem to its normal settings. SAS software was used to evaluate the kinetic models that describe CO2 efflux under the action of the used different remediation amendments. 3 RESULTS AND DISCUSSION 3.1 KINETICS OF CO2 EFFLUX IN BIO-REMEDI- ATED SOIL ECOSYSTEM Results drawn in Figure 1 demonstrate the kinetics of CO2 efflux from both contaminated and bio-remediat- ed soil ecosystems. All trailed remediation amendments Acta agriculturae Slovenica, 117/4 – 20214 M. SABER and A. ZAGHLOUL led to marked decreases in the rate of CO2 efflux com- pared to control contaminated soil ecosystem. In con- trol contaminated soil ecosystem, maximum CO2 efflux reached 14.11 mg kg-1 soil; while being 10.45 mg kg-1 soil in T3 (soil fortified with a mixture of bentonite + kaolin- ite) and decreased to 8.2 in T8 (iron oxide + RP). Other trailed remediation amendments, thereafter decreased CO2 efflux with values higher than in abovementioned treatments was recorded. Chander and Brookes (1993), stated that when Zn and Cu are present together in the soil ecosystem the in- crease in Zn and Cu bioavailability above 123 or 3.0 ppm causes marked decreases in the intensity of soil microbial biomass. Results given in Figure 2 show that the impacts of the trailed remediation amendments in minimizing the hazards of PTE’s varied according to their type. Modified bentonite, iron oxide, rock phosphate as well as the mix- ture of these remediation amendments decreased signifi- cantly the evolution of CO2 or restored the soil ecosystem to its normal conditions. Of all treatments T8, T3 and T7 were the best that condensed normal conditions, in some cases; however, certain remediation amendments increased the CO2 to a non-significant level compared to control especially when fortified with kaolinite (T5) even in the absence of RP. 3.2 RATE CONSTANTS OF BEST FITTED MODELS DESCRIBE CO2 EFFLUX AS AFFECTED BY REMEDIATION AMENDMENTS APPLIED TO SOIL ECOSYSTEM Kinetic approach was used to evaluate the effec- tiveness of the trailed remediation amendments on CO2 efflux that express the biomass activity as well as the depressing action of PTE’s associated with the trailed re- mediation amendments. As given in Tables 1-3, the rate constants of CO2 efflux from the soil irrigated with low quality water for extended periods, as an indicator for microbial activ- ity, varied according to type of the trailed remediation amendment. For MFE, the best fitted model, the appli- cation of kaolinite and RP (T5) decrease CO2 efflux to 0.71 mg g-1 soil, in control the value was 0.77 mg g-1 min- 1. Fortification of the soil ecosystem irrigated with low quality water with iron oxide also decreased CO2 efflux to 0.68 mg g-1 min-1, and hence might be used to cure such contaminated soil ecosystem. Also, using bentonite in T1 decreased the CO2 flux to 0.68 mg g -1 min-1. All other trailed remediation amendments significantly decreased CO2 efflux compared to control. The negative values re- corded for the capacity factor in the same model means returning the soil ecosystem to optimum conditions for microbial activity. 3.3 MICROBIAL BIOMASS IN PTE’S CONTAMI- NATED AND BIO-REMEDIATED SOIL ECO- SYSTEMS Results given in Table 4 show the amount of con- taminants absorbed by the microbial biomass in the soil ecosystem. In control microorganism’s absorbed signifi- cant amounts of inorganic pollutants reached 0.46 ppm Zn, 0.03 Cu and 0.10 Ni. The existence and uptake of all inorganic pollutants directly led to an increase in the microbial activity in the soil ecosystem. For example, in T1 only Ni was absorbed by microorganisms, meanwhile other studied inorganic pollutants did not presented in side microorganisms. The same trend was re-exhibited in T3, T5 and T7 treatments. Bentonite + kaolinite + rock phosphate (RP) treatment (T6) and iron oxide + RP treatment (T8) also increased the microbial activity through increasing CO2 efflux of rate constants of Elovich kinetic model (Table 3). Results in the same table point to that bentonite as remediation amendment decreased inorganic pollutants uptake at different degrees with some exception observed in case of Ni. Although the application of oxides as remediation amendment was extensively mentioned in literature, the application of iron oxides in this study enhanced Zn and Ni by microorganisms found in the soil ecosystem. It is worth to state that Cu was the only PTE that was de- pressed by the trailed remediation amendments though the increasing of non fumigated values compared to fu- migated ones. 3.4 CONTRIBUTION OF TRAILED REMEDIATION AMENDMENTS IN SOIL ECOSYSTEMS ON MINIMIZING THE HAZARDS OF PTE’S The effect of trailed remediation amendments on minimizing the hazards of Ni, Cu and Zn is drawn in Figure 3 that show all trailed treatments significantly decreased the available forms of the studied inorganic pollutants compared to control. The comparison be- tween the different treatments indicated that bentonite significantly decreased the available form of PTE’s. For instances, the application of modified bentonite to soil (T4) led to a decrease reaching 34 % of available of Cu, 79 % of available Zn and 77 % of available Ni. The mixture of bentonite and kaolinite decreased 68 % of available Cu, 66 % of available Zn and 59 % of Acta agriculturae Slovenica, 117/4 – 2021 5 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ... Fi gu re 1 : K in et ic s o f C O 2 e ffl ux fr om co nt am in at ed so il as a ffe ct ed b y re m ed ia tio n m at er ia ls co m pa re d to co nt ro l t re at m en t. W he re : ( T1 ) 2  % p ro be nt on ite , ( T2 ) 2  % k ao lin ite , (T 3) 1  % p ro be nt on ite + 1  % K ao lin ite , ( T4 ) 1  % p ro be nt on ite + 1  % ro ck p ho sp ha te (R P) , ( T5 ) 1  % ka ol in ite + 1  % R P, (T 6)  1  % b en to ni te + 0 .5  % k ao lin ite + 0 .5  % R P, (T 7) 2  % iro n ox id e an d (T 8) 1  % ir on o xi de + 1  % R Acta agriculturae Slovenica, 117/4 – 20216 M. SABER and A. ZAGHLOUL available Ni, this trend perhaps represents the selectivity of used clay minerals in retain PTE’s. Results pointed to that increasing of Cu and Zn was retained by kaolinite over bentonite, meanwhile a reverse trend was observed in Ni. The modification of kaolinite with RP increased the retention of Ni by 64 % in the soil ecosystem; meanwhile it did not exceed 18 % under sole kaolinite application. The same treatment, however, did not influence the retention level of Cu and Zn in the soil ecosystem. In contrast, fortification of the soil ecosystem with sole iron oxide decreased the retention of inorganic pollutants to 65, 72 and 45 % of Cu Zn and Ni respectively, while the mixture of PR with iron oxide decreased these values re- spectively to 50, 30 and 36 % of total form in soils. Results drawn in the same figure indicated that the mixture of all treatment (T6) did not exhibit the predicted trend since the decreasing orders of Inorganic pollutants did not ex- ceed 34, 62 and 55 % of total Cu, Zn and Ni compared to control in order to be a valued treatment but not the best 3.5 DISTRIBUTION OF THE STUDIED PTE’S IN REMEDIATED SOIL ECOSYSTEM Results drawn in Figure 4 exhibit the distribution of Ni, Cu and Zn in soil ecosystem irrigated with low qual- Figure 2: Map showing the experimental plots and location of the experiment at the Institute of Agricultural Research and Train- ing in Ibadan, Oyo state, Nigeria. (T1) 2 % probentonite, (T2) 2 % kaolinite, (T3) 1 % probentonite + 1 % kaolinite, (T4) 1 % probentonite + 1 % rock phosphate (RP), (T5) 1 % kaolinite + 1 % RP, (T6) 1 % bentonite + 0.5 % kaolinite + 0.5 % RP, (T7) 2 % iron oxide and (T8) 1 % iron oxide + 1 % RP Treatments a*10^4 b R2 SE cont -5.51 11.58 0.99** 0.21 T1 -5.96 10.95 0.99** 0.40 T2 -5.12 9.62 0.99** 0.35 T3 -7.71 12.53 0.97** 0.81 T4 -8.95 14.36 0.99** 0.80 T5 -6.43 11.33 0.96** 0.73 T6 -5.94 11.00 0.99** 0.43 T7 -3.88 7.40 0.99** 0.36 T8 -5.51 11.58 0.98** 0.21 Table 1: Rate constants of 1st order model describe CO2 efflux from contaminated soil as affected by remediation treatments Treatments a b R2 SE cont 0.77 2.29 0.99** 0.05 T1 0.68 -1.86 0.99** 0.04 T2 0.74 -2.17 0.99** 0.04 T3 0.72 -2.39 0.99** 0.05 T4 0.68 -2.56 0.99** 0.05 T5 0.71 -1.99 0.98** 0.07 T6 0.75 -2.29 0.99** 0.05 T7 0.68 -1.86 0.99** 0.04 T8 0.74 -2.17 0.99** 0.04 Table 2: Rate constants of MFE describe CO2 efflux from con- taminated soil as affected by remediation treatments Acta agriculturae Slovenica, 117/4 – 2021 7 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ... ity water for long periods as affected by the trailed re- mediation amendments applied to minimize the hazards of PTE’s and to optimize microbial activities through re- mediation of soil ecosystem. Generally, as shown in the Figure 4, trailing the different remediation amendments decreased the readily available form and increased the residual one with rates varied according to amendment used. Three main categories of the trailed remediation amendments are distinguished, the 1st category included the best ones, i.e., pro-bentonite (T4), pro-kaolinite (T5) and iron oxide (T7), which minimized the readily avail- able form to zero in Ni and from 98 to 100 % for Cu and Treatments a b R2 SE cont 3.85 -28.08 0.92** 1.77 T1 3.82 -27.09 0.95** 1.40 T2 3.34 -24.00 0.94** 1.25 T3 4.57 -33.12 0.95** 1.54 T4 5.24 -38.31 0.95** 1.76 T5 3.97 -28.46 0.94** 1.59 T6 3.82 -27.77 0.94** 1.44 T7 2.54 -18.10 0.93** 1.04 T8 3.85 -28.08 0.92** 1.77 Table 3: Rate constants of Elovich equation describe CO2 efflux from contaminated soil as affected by remediation treat- ments Zn, i.e., increased the residual form in these treatments. The 2nd category included the remediation amendments able to minimize the available form of one of the test- ed inorganic pollutants to zero such as T3 (the mixture between bentonite and kaolinite) in case of Cu. The 3rd category included the rest of treatments that significantly minimized inorganic pollutants at different rates accord- ing to the studied inorganic pollutants. It should be mention that all applied remediation amendments are locally available in Egypt and are con- sidered with economic low coast. Various methods such as hydrometallurgical technologies, ion exchange, electro dialysis, reverse osmosis, precipitation and adsorption had been trailed to remove inorganic pollutants from aqueous solution phase in aquatic ecosystems (La Grega et al., 1994). It is well known that the reduction in the readily available forms of inorganic pollutants’ in con- taminated soil ecosystems is a commonly technique used to reduce the negative impacts of inorganic pollutants on environment and improve the quality of contaminated soil ecosystems (Zaghloul, 2006). It is well known that clay minerals interact with almost all soil contaminants (Prost and Yaron, 2001). The adsorption of Ni, Cd, Zn, and Pb by the clay mineral montmorillonite was reported by Schulthess and Huang (1990). Sorption technique is one of the most efficient methods of cleaning the envi- ronment from Inorganic pollutants. In this study microbial activity through CO2 efflux was used as an indicator to evaluate the effectiveness of eight remediation amendments in minimizing the haz- ards of PTE’s in soil ecosystem. Bentonite is a well-known as one of the most effective remediation amendments Treatments NF Zn Fum. Zn NF Cu Fum Cu NF Ni Fum Ni ppm Control 2.65 3.11 1.24 1.27 1.80 10.9 T1 0.86 0.42 1.69 1.34 1.89 2.36 T2 2.00 0.99 0.82 0.71 2.3 1.61 T3 0.54 0.31 0.58 0.41 1.88 2.23 T4 6.53 4.85 1.21 1.08 2.04 2.22 T5 3.13 2.71 0.85 0.77 1.44 2.02 T6 0.3 0.44 0.96 0.52 1.66 2.36 T7 1.21 0.78 0.89 0.89 1.75 2.05 T8 3.29 4.39 0.88 0.78 1.89 2.71 Table 4: Microbial biomass for Ni, Cu and Zn in both contaminated and remediated soils NF: non-fumigated, Fum: Fumigated Acta agriculturae Slovenica, 117/4 – 20218 M. SABER and A. ZAGHLOUL Figure 3: Potential toxic elements availability in contaminated as affected by remediation materials compared to control treat- ments Acta agriculturae Slovenica, 117/4 – 2021 9 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ... Figure 4: Distribution of PTE’s in the studied low quality water soils as affcted by certain remediative amendments. (T1) 2 % probentonite, (T2) 2 % kaolinite, (T3) 1 % probentonite + 1 % kaolinite, (T4) 1 % probentonite + 1 % rock phosphate (RP), (T5) 1 % kaolinite + 1 % RP, (T6) 1 % bentonite + 0.5 % kaolinite + 0.5 % RP, (T7) 2 % iron oxide and (T8) 1 % iron oxide + 1 % RP Acta agriculturae Slovenica, 117/4 – 202110 M. SABER and A. ZAGHLOUL used in curing soil ecosystems contaminated with certain inorganic pollutants such as Ni, Cu, Zn and others (An- dini et al., 2006). In addition, bentonite has been shown to improve the overall soil quality (Phillips, 1998). Appli- cation of bentonite to soils irrigated with low quality wa- ter s significantly retained PTE’s in different mechanisms such as sorption and ion exchange mechanisms. The op- timum conditions of soil ecosystem to decrease the avail- ability of PTE’s might obviously monitored through the microbial activity of decreasing the CO2 efflux. Although both bentonite and pro-bentonite showed priority in minimizing the hazards of PTE’s in the stud- ied soil ecosystem, yet, kaolinite exhibited the least ex- changeability among bentonite clay mineral group, as several studies confirmed the potential of natural kaolin- ite in metal ion adsorption from solution. O’Day et al. (1994) mentioned that CO2 is always binding to kaolinite as co-complexes at both inner and outer sphere complex- es using X-ray absorption spectroscopy (XAS). Boron adsorption on kaolinite was studied by Singh and Mat- tigod (1992) using phenomenological equations and sur- face complication reactions. Samaneh  and  Jalali (2016) evidenced strong preference for the ion exchanged form of kaolinite for Cu ions. Exchange capacity of both cation as well as anion of kaolinite and their relation with homo ionic counterparts with Na+ was critically examined by Ferris and Jepson (1975). Results indicated that the succession of more than one model in describing the kinetic results having high R2 values ranged between 96-99** in used models. This means that the different mechanisms that took place in the sorption of studied PTE’s by the trailed remediation amendments improved CO2 efflux by decreasing the available form of PTE’s. The outer groups were situated along the unshared plane of the alumina hydroxyl sheet, while the inner groups were located along the plane that is shared with and borders on the silica oxide sheet. The movement of the inner hydroxyl plane is restricted as a result of chemi- cal bonding between the silica and alumina sheets. The pro- clay mineral treatment in all cases directly increased the retention of PTE’s, this trend decreased the microbial biomass of these treatment. This result might be due to the mode of phosphate reaction with all PTE’s in having complex compounds (Ma and Harris, 1997). Worth to mention that iron oxide exhibited the same trend when RP was applied to the soil ecosystem. Sorption and immobilization of inorganic pollut- ants in soil ecosystem is an effective detoxification pro- cess and thus it is an essential part of the buffering capac- ity of the soil ecosystem (Welp 1999). Immobilization of inorganic pollutants caused an increase in basal respiration rate, litter decomposition and microbial activity. There are several methods for immobilization of inorganic pollutants in soil, through either adding natural and synthetic chemical additives such as alkaline materials, phosphate minerals, iron and manganese oxides, alumino-silicates or coal fly ashes (Mench et al., 1998). Clay minerals are among the major materials that interact with almost all soil contaminants (Prost and Yaron, 2001). The adsorption of Ni, Cd, Zn, and Pb by montmorillonite was reported by Schulthess and Huang (1990). Immobilization of inorganic pollut- ants by natural zeolite (clinoptilolite) and six synthetic zeolites was studied by Oste et al. (2002), who found that the synthetic zeolites had an effect on immobilization of Cd and Zn. The improvement of the quality of the mi- crobe’s media through is mainly due to ameliorative ac- tion of PTE’s in soil ecosystem irrigated with low quality water for extended periods. 4 CONCLUSIONS AND RECOMMENDA- TIONS The application of microbial activity through CO2 flux in evaluation soil remediation technology(ies) can be a viable and innovative best way. In this work, the use of some clay minerals, crude or modified with some microbes, significantly reduced the content of harmful heavy metals in a soil ecosystem irrigated with low-qual- ity water for long periods. The treatments used led to a significant decrease in the available forms of the studied heavy elements in parallel with the increase in the resid- ual unavailable forms of pollutants. According to the ob- tained results, heavy metal ions showed a tendency to ac- cumulate on clay minerals, which shows the importance of this method of treatment and suitability for improving soil quality by restoring appropriate ecosystem condi- tions and flourishing microbial activity. This method is also characterized by low costs and an economical way that encourage the farmers to use it for having safe food. It is worth noting the importance and necessity of using this technique more in other studies with other polluted metals that did not fall within the scope of this research. 5 ABBREVIATIONS NRC: National Research Centre PR: Rock phosphate CO2: Carbon dioxide Cu: copper Ni: Nickel Zn: Zinc PR: Rock phosphate Acta agriculturae Slovenica, 117/4 – 2021 11 Evaluation of biochemical treatments applied in polluted soils irrigated with low quality water ... K2Cr2O7: Potassium dichromate ANOVA: Analysis of variance R2: Coefficient of determination SE: Standard Error NF: Non-fumigated Fum: Fumigated 6 ACKNOWLEDGMENT The authors would like to express their appreciation and gratitude to the Science, Technology & Innovation Funding authority (STDF) for financing the present work through the project number 41523 contracted with the National Research Center and extended till present. 7 REFERENCES Andini S., Cioffi F., Montagnaro F., and Santoro L. (2006). Si- multaneous adsorption of chlorophenol and heavy metal ions on organophilic bentonite. Applied Clay Science, 31, 126-133. https://doi.org/10.1016/j.clay.2005.09.004 Anvar S.H. and Oliver M. (2002). Soil microbial eco-physiol- ogy as affected by short-term variations in environmental conditions. Soil Biology and Biochemistry, 34, 1283–1290. https://doi.org/10.1016/S0038-0717(02)00071-8 Bardgett RD. and Saggar S. (1994). Effects of heavy metal con- tamination on the short-term decomposition of labelled 14C glucose in a pasture soil. Soil Biology and Biochemistry, 26, 727–733. https://doi.org/10.1016/0038-0717(94)90265- 8 Chander K and Brookes P (1993). Residual effects of zinc, cop- per and nickel in low quality water sludge on microbial biomass in a sandy loam. Soil Biology and Biochemistry, 25, 1231–1239. https://doi.org/10.1016/0038-0717(93)90219-2 Doaa Ali, Omaima Sharaf, Mohamed Saber, Essam Hoballah, Mohamed Khaled Ibrahim, Einas El- Shatoury and Alaa Zaghloul (2020). Bioremediation of potential toxic ele- ments in varied soil ecosystems (greenhouse scale). Plant Archives, 20, 9482-9490. Doelman P. and Haanstra L. (1984). Short-term and long term effects of cadmium, chromium, copper, nickel, lead and zinc on soil microbial respiration in relation to abiotic soil factors. Plant and Soil 79: 317-327. https://doi.org/10.1007/ BF02184325 Ferris A. and Jepson W. (1975). The exchange capacities of kaolinite and the preparation of homoionic clays. Jour- nal of Colloid Interface Science, 51, 245–259. https://doi. org/10.1016/0021-9797(75)90110-1 Khan M. and Scullion J. (1999). Microbial activity in grass- land soil amended with low quality water sludge contain- ing varying rates and combinations of Cu, Ni, Zn. Biology and Fertility of Soils, 30, 202–209. https://doi.org/10.1007/ s003740050609 Kelly J. and Tate R. (1998). Effects of heavy metals contami- nation and remediation on soil microbial communi- ties in the vicinity of a zinc smelter. Journal of Environ- mental Quality, 27, 609–617. https://doi.org/10.2134/ jeq1998.00472425002700030019x Kuzyakov Y. (1997). The role of amino acids and nucleic bases in turnover of nitrogen and carbon in soil humic fractions. European Journal of Soil Science, 48, 121–130. https://doi. org/10.1111/j.1365-2389.1997.tb00191.x La Grega M., Buckingham P. and Evans, J. (1994). Hazardous Waste Management, McGraw Hill Publication. Leifeld J., Siebert S. and Kogel-Knabner I. (2002). Biological ac- tivity and organic matter mineralization of soil amended with biowaste composts. Journal of Plant Nutrition and Soil Science, 165, 151–159. https://doi.org/10.1002/1522- 2624(200204)165:2<151::AID-JPLN151>3.0.CO;2-T Ma L., Tan F. and Harris W. (1997). Concentration and dis- tribution of eleven metals in Florida soils, Journal of En- vironmental Quality, 26, 769-775. https://doi.org/10.2134/ jeq1997.00472425002600030025x Mench M., Vangronsveld J., Lepp N. and Edwards R. (1998). Physio-chemical aspects and efficiency of trace element im- mobilisation by soil ammendments. In Metal contaminated soils: In situ inactivation and phytorestoration. Springer- Verlag and R.G. Edited by J. Vangronsveld and S.D. Cun- ningham. Landes Company, 151-182. Mohamed Saber, Hussein, F. Abuouziena, Esam Hoballah, Fat- ma Abd-Elzaher, Azza Tyrkey and Alaa Zaghloul (2015). Risk assessment and mitigation measures for certain amendments used in bioremediation of low quality waterd soils. International Journal of ChemTech Research CODEN (USA): IJPRIF (ISSN: 0974-4304), 8(6), 423-440. O’Day P., Parks G. and Brown G. (1994). Molecular structure and binding sites of Co2+ surface complexes on kaolinite from X-ray adsorption spectroscopy. Clays Clay Mineral, 42, 337–355. https://doi.org/10.1346/CCMN.1994.0420312 Oste L., Dolfing J., Ma W. and Lexmond T. (2001). Cd uptake by earthworms as related to the availability in the soil and the intestine. Environmental Toxicology and Chemistry, 20, 1785–1791. https://doi.org/10.1002/etc.5620200823 Phillips I.R. (1998) Phosphorus availability and sorption under alternating waterlogged and drying conditions. Commu- nication of Soil Science and Plant Analysis, 29, 3045–3059. https://doi.org/10.1080/00103629809370175 Prost R. and Yaron B. (2001). Use of modified clays for control- ling soil environmental quality. Soil Science,166, 880–895. https://doi.org/10.1097/00010694-200112000-00003 Saber M., Hobballa E., Soad El-Ashery and Zaghloul A. (2012). Decontamination of potential toxic elements in low quality water soils by inorganic amendments. Journal of Agricul- tural Science and Technology,A, 2, 1232-1244. Samaneh T. and Mohsen J. (2016). Sorption, desorption, and speciation of Cd, Ni, and Fe by four calcareous soils as affected by pH. Environmental Monitoring and Assessment, 6, 188-322. https://doi.org/10.1007/s10661-016-5313-4 SAS Institute (1985). SAS/STAT Guide for Personal Computers. 6th ed. SAS Ins., Cary, NC Schulthess C.P. and Huang C.P. (1990). Adsorption of heavy metals by silicon and aluminum oxide surfaces on clay min- erals. Soil Science Society American Journal, 54, 679-688. https://doi.org/10.2136/sssaj1990.03615995005400030008x Acta agriculturae Slovenica, 117/4 – 202112 M. SABER and A. ZAGHLOUL Singh S.P. and Mattigod S.V. (1992) Modeling boron adsorp- tion on kaolinite. Clays Clay Miner, 40, 192–205. https:// doi.org/10.1346/CCMN.1992.0400209 USEPA (2001). USEPA test methods. SW-846 manual. Available online at www.epa.gov/epaoswerlhazwaste/test/sw846.htm (verified11 Apr. 2003). USEPA, Washington, DC Welp G. (1999). Inhibitory effects of the total and water-soluble concentrations of nine different metals on the dehydroge- nase activity of a loess soil. Biology and Fertility of Soils, 30, 132-139. https://doi.org/10.1007/s003740050599 Yonebayashi K. and Hattori T. (1989). Chemical and biological studies on environmental humic acids: II. 1H–NMR and IR spectra of humic acids. Soil Science and Plant Nutrition, 35, 383–392. https://doi.org/10.1080/00380768.1989.10434771 Zaghloul A.M. (2002). Kinetics of potassium adsorption in some soils of Egypt using Electrical Stirred Flow unit (ESFU). Egyptian Journal of Soil Science, 42, 463–471. Zaghloul A.M., Camilia El-Dewany and Yousef R.A. (2006). Distribution of Pb and Zn in some Egyptian contaminated soils as affected by time of exposure and source of pollut- ants. Journal of Applied Science Research. 2, 284-289. Acta agriculturae Slovenica, 117/4, 1–12, Ljubljana 2021 doi:10.14720/aas.2021.117.4.773 Original research article / izvirni znanstveni članek Influence of plant growth regulators and salicylic acid on the production of some secondary metabolites in callus and cell suspension culture of Satureja sahendica Bornm. Sarieh TARIGHOLIZADEH 1, Rouhollah MOTAFAKKERAZAD 1, 2, Morteza KOSARI-NASAB 1, 3, Ali MOVAFEGHI 1, Sakineh MOHAMMADI 1, Mohsen SABZI 4, Amir-Hossein TALEBPOUR 5 Received April 23, 2018; accepted October 19, 2021. Delo je prispelo 23. aprila 2018, sprejeto 19. oktobra 2021 1 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran 2 Corresponding author, email: rmotafakker@tabrizu.ac.ir 3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 4 Ahar Faculty of Agriculture and Natural Resources, University of Tabriz, Iran 5 East Azerbaijan Research Center for Agriculture and Natural Resources, Tabriz, Iran Influence of plant growth regulators and salicylic acid on the production of some secondary metabolites in callus and cell suspension culture of Satureja sahendica Bornm. Abstract: The impact of combinations of plant growth regulators (PGRs) on callus culture of Satureja sahendica Bornm. was investigated. In nodal explants, the response of secondary metabolite production to different concentrations of PGRs was analyzed regarding the presence and absence of polyvinylpyrrolidone (PVP). The explants were cultured on MS media in presence of auxins (2,4-dichlorophenoxyacetic acid and naphthylacetic acid) and cytokinins (thidiazuron and kinetin); which were used in equal concentrations of 0.5, 1, and 2 mg l-1. The treatment of 2 mg l-1 2,4-D + 2 mg l-1 Kin (MD3) led to the highest production of total phenolics (4.303 ± 0.449 mg GAE g-1) and flavonoids (24.903 ± 7.016 mg QE g-1). Moreover, the effect of salicylic acid (SA) on the produc- tion of secondary metabolites in cell suspension culture of Satureja sahendica was evaluated. The cell suspension culture was established by culturing the nodal-derived friable callus in the liquid medium containing different concentrations of SA (0, 100, 150, 200 µM). An inverse relationship exists be- tween the fresh mass and secondary metabolites contents. In addition, there was a significant difference among concentra- tions of SA in the production of total phenolics and flavonoid compounds. SA enhances secondary metabolites production and decreases cell fresh mass. Key words: Satureja sahendica; callus induction; cell sus- pension culture; secondary metabolites; growth regulators; salicylic acid Vpliv rastlinskih rastnih regulatorjev in salicilne kisline na tvorbo nekaterih sekundarnih metabolitov v kalusu in su- spenziji celične kulture vrste šetraja Satureja sahendica Bor- nm. Izvleček: Preučevan je bil vpliv kombinacije rastlinskih rastnih regulatorjev (PGRs) na kulturo kalusa vrste šetraja Sa- tureja sahendica Bornm. V nodijskih izsečkih je bil preučevan odziv tvorbe sekundarnih metabolitov na različne koncentra- cije PGRs glede na prisotnost in odsotnost polivinilpirolidona (PVP). Izsečki so bili gojeni v MS gojišču v prisotnosti auksinov (2,4-diklorofenoksiocetna kislina in naftilocetna kislina) in ci- tokininov (tidiazuron in kinetin), ki so bili uporabljeni v enakih koncentracijah 0,5; 1, in 2 mg l-1. Obravnavanje 2 mg l-1 2,4-D + 2 mg l-1 Kin (MD3) je vodilo k največji tvorbi celokupnih fe- nolov (4,303 ± 0,449 mg GAE g-1) in flavonoidov (2,903 ± 7,016 mg QE g-1). Dodatno je bil ovrednoten učinek salicilne kisli- ne (SA) na tvorbo sekundarnih metabolitov v kulturi suspen- zije celic šetraja. Kultura suspenzije celic je bila vzpostavljena z gojenjem rahlega kalusa, pridobljenga iz nodijskih izsečkov v tekočem mediju, ki je vseboval različne koncentracije SA (0, 100, 150, 200 µM). Pojavilo se je obratno sorazmerje med svežo maso in vsebnostjo sekundarnih metabolitov. Med različnimi koncentracijami salicilne kisline ni bilo opaziti značilnih razlik na tvorbo celokupnih fenolov in flavonoidov. Salicilna kislina vzpodbuja tvorbo sekundarnih metabolitov in zmajšuje svežo maso celične kulture. Ključne besede: Satureja sahendica; indukcija kalusa; kultura suspenzije celic; sekundarni metaboliti; rastni regula- torji; salicilna kislina 2 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. 1 INTRODUCTION Satureja is an extra-large genus of Lamiaceae: Nepetoideae and comprises about 200 species of of- ten aromatic shrubs and herbs distributed in Asia, the Mediterranean region, and North America. The flora of Iran possesses 14 species of this genus, 9 of which are endemic. The members of this genus is widely found in mountainous areas in Iran (Rechinger, 1982; Jamzad, 1996; Mozaffarian, 1996). Many species of the genus are traditionally used for curing diseases such as wounds, muscle pains, diarrhea, nausea, infections, and gastro- enteritis (Hadian et al., 2012). Like many members of Lamiaceae, Satureja species are very rich in secondary metabolites such as flavonoids, phenolics diterpenes, and phenolic acids, and therefore they have gained at- tention from researchers for their applications  in  vari- ous fields (Ghotbabadi et al., 2012). Satureja sahendica Bornm., locally known as “Marze-Sahandi”, is an endemic species of Satureja in Iran and is distributed in Northwestern and West- ern regions in East Azerbaijan, Zanjan, and Kurdistan provinces (Mozaffarian, 1993). This plant is a peren- nial, branched, bushy, aromatic, and late flowering herb (Ghahreman, 1988, 1993). Literature reviews show that 39 components were identified in the oil of S. sahen- dica and the main constituents of the essential oils were thymol (19.6 - 41.7  %), p-cymene (32.5 - 54.9  %), and γ-terpinene (1.0 - 12.8  %) (Sefidkon et al., 2004; Has- sanpouraghdam et al., 2009). In recent years, the pres- ence of several antioxidant compounds in this plant was reported. Furthermore, some flavonoids such as derivatives of diosmetin, quercetin, luteolin, and api- genin were found in this species (Saeidnia et al., 2011; Hadjmohammadi et al., 2012). Although a significant amount of research has been done on the composition of the oil and the chem- ical and physical characteristics as well as medicinal properties of this species, tissue culture-related activi- ties have been limited to some species of Satureja. Actu- ally, major work has not been done to analyze Satureja sahendica metabolites. For instance, a study evaluated antimicrobial and antioxidant activities of the essential oil from aerial parts of Satureja hortensis L. via callus culture (Güllüce, 2003). Moreover, the production and optimization of rosmarinic acid (an important phe- nolic acid) were investigated in the callus culture of S. hortensis (Tepe & Sokmen, 2007). Development of appropriate conditions and tech- niques for the production of phenolic compounds is required due to their medical and commercial values including a range of biological activities such as anti- bacterial, anti-viral, and anti-cancer (Taveira et al., 2010). Previous researches illustrated that plant in vitro culture is a suitable technique for the production of valuable metabolites in most plants, and the stimulants could be applied as physical and chemical stress for the production of these metabolites as one of the most suc- cessful strategies (Ravishandera et al., 1999; Dörnen- burg & Knorr, 1995). The production and accumulation of secondary metabolites in plants is known as a part of the defense response against pathogenic attacks, which could be triggered and activated by growth regulators and elicitors (Zhong et al., 1996; Wang et al., 2004; Al- Sane et al., 2005; Shilpashree & Rai, 2009). Elicitors, ei- ther biological or non-biological compounds, increase the secondary metabolites production by activating the genes involved in the biosynthesis of these compounds (Neumann et al., 2009). Salicylic acid (SA) or 2-hy- droxybenzoic acid, a type of plant phenolics, is an effec- tive inducer of genes involved in plants’ defense system. Therefore, SA can effectively induce the enhancement of secondary metabolites production such as alkaloids, terpenoids, phenolics, and phytoalexins (Vlot, 2008). It has been reported that the production of taxol in the suspension culture of Taxus baccata L. was considerably increased by SA compared to the control (Khosrousha- hi, 2006). Salicylic acid caused also a significant increase in the production of alkaloids in the hairy root culture of Brugmansia x candida Pers. (Alvarez et al., 2000) and it likewise enhanced the synthesis of total flavonoids in the suspension culture of Andrographis paniculata (Burm.f.) Nees (Mendhulkar, 2013). The application of SA for the production of curcumin in the Catharanthus roseus (L.) G.D on cell culture has been also reported (Matkowski, 2008). Due to lack of a basic investigation on plant tissue culture in S. sahendica, and after our last research on the in vitro production of secondary metabolites of Lalle- mantia iberica (M.Bieb.) Fisch. & C.A.Mey. as a member of Lamiaceae (Pourebad et al., 2015), we became inter- ested to work on tissue and cell culture of this plant species. Correspondingly, based on our preliminary accomplished work (Tarigholizadeh et al., 2015), we aimed to study callus and cell suspension culture condi- tions and determination of total phenolic and flavonoid compounds in S. sahendica using different concentra- tions and combinations of growth regulators and SA. The present work focuses on the secondary metabolites production of S. sahendica via callus and cell suspen- sion cultures and highlights the potential of this plant species for further pharmaceutical researches. 3Acta agriculturae Slovenica, 117/4 – 2021 Influence of plant growth regulators and salicylic acid ... in callus and cell suspension culture of Satureja sahendica Bornm 2 MATERIALS AND METHOD 2.1 PLANT MATERIAL Seeds of Satureja sahendica Bornm. were obtained from the Botanical garden of East- Azerbaijan province in Iran, and were surface-sterilized with 70  % ethanol for 1 minute, rinsed once with water, followed by 10 minutes immersion in 1  % formaldehyde plus a few drops of 80  % Tween and interspersed with washings in sterile distilled water. After that, seeds were sterilized with commercial hypochlorite solution (20  %) for 10 minutes. After 3 rinses with sterile distilled water, the seeds were treated with giberellic acid (200 mg l-1 for 15 min) for the elimination of the dormancy problem (Tarigholizadeh et al., 2015). Then, the seeds were culti- vated on a hormone-free MS basal medium (Murashige & Skoog, 1962). 2.2 CALLUS INDUCTION Callus culture was started by nodal explants of in vitro germinated 30-days-old seedlings of S. sahendica and were cultured on MS medium supplemented with different combinations of 2,4-D (2,4-dichlorophenoxy- acetic acid) and kin (kinetin) as well as NAA (naphthyl- acetic acid) and TDZ (thidiazuron) in different concen- trations as presented in Table 1. In order to examine the effect of polyvinylpyrrolidone (PVP) on callus growth and secondary metabolites production, we aimed to ap- ply a distinct set of experiments with the same PGRs and their combinations mentioned above and 400 µl PVP. Therefore, there were two types and series of media (each medium: 250 ml) for callus induction: with the presence of PVP (PVP+) and absence of PVP (PVP-). The pH value of all media were adjusted at 5.6 - 5.8 with 1 N NaOH prior to adding of agar (8.0 g l–1) and subsequently they were autoclaved for 15 minutes (121  ˚C, 104 kPa) and dispensed into glass jars (each containing 250 ml). Three glass jars containing seven explants and each were cultured per treatment. For control groups, we used MS with and without PVP. All cultures were maintained in the growth room 16 h light (40 µmol m-2 s-1 white cool fluorescence) and 8 h dark at 23 ± 2˚C for four weeks. Then, callus samples were subcultured once and four weeks later, were collected for evaluation of the following parameters: callogenesis percentage, morphological traits and total phenolic and flavonoid contents. Growth regulators (mg l-1) Media (-PVP or + PVP) NAA 0.5 + TDZ 0.5 MN1 NAA 1.0 + TDZ 1.0 MN2 NAA 2.0 + TDZ 2.0 MN3 2,4-D 0.5 + Kin 0.5 MD1 2,4-D 1.0 + Kin 1.0 MD2 2,4-D 2.0 + Kin 2.0 MD3 Table 1: List of MS media supplemented with different growth regulators for callus induction 2.3 ESTABLISHMENT AND MAINTENANCE OF CELL SUSPENSION CULTURE In order to callogenesis for establishing cell sus- pension culture, callus induction was carried out in the same above-mentioned way. However, the exerted PGRs were only 2,4-D and Kin with two different con- centrations of each (0.5, 1 mg l-1). To establish suspen- sion culture, 0.5 g of healthy and two-month-age callus tissues were transferred to 50 ml of MS medium with mentioned PGRs compounds without agar and were kept on shakers at 110 rpm (revolutions per minute) and 25  ˚C in the dark. Cultured samples were subcul- tured once per three weeks to establish cell lines. In each subculture, 5 ml of medium containing cells was added to 45 ml of new medium with the same PGRs. Based on previous studies and our preliminary researches, differ- ent concentrations of SA were chosen and their effects on cultured cells and tissues were examined. For this propose, 5 ml of cell culture medium was transferred to 45 ml MS liquid medium containing 0 (control), 100, 150 and 200 µM concentrations of sterilized SA. Samples were then placed on a shaker (110 rpm, 25 ˚C) in darkness for three weeks. Finally, the samples were collected to examine the influence of different concen- trations of SA on growth and secondary metabolites production. 2.4 DETERMINATION OF TOTAL PHENOLIC AND FLAVONOID CONTENTS Callus cultures derived from nodal segments using the different combinations of growth regulators were dried in oven at 35  ˚C for 30 h. Then, 0.5 g of dried callus from each sample was mixed with methanol in a small glass tube and were put at 25 ˚C for 40 h. This procedure was done for each treatment and extracts and they were centrifuged at 16300 x g for 15 minutes. The supernatant was separated for measurement of 4 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. PVP). First callus tissues appeared on nodal explants after 10 days of culture. However, callogenesis in the combination of 2, 4-D + Kin occurred one week later. Callus tissues covered almost all the explants surface within 30 days. Green-compact and yellowish green- friable callus tissues were formed by NAA+TDZ and 2, 4-D + Kin groups, respectively. In treatments with 2, 4-D, and Kin, shoot formation was not observed and high concentrations of these PGRs resulted in a low cal- logenesis efficiency (Table 2). As expected, the absence of PGRs in MS and MS + PVP media (control groups) showed a strong shooting response without production of any callus tissues, and therefore this group was ex- cluded from further examination (data not shown). The PVP application with growth regulators induced a gen- eral increase in callogenesis (Table 2). Actually, the ad- dition of PVP to the medium was effective in overcom- ing the browning of the culture medium and promoted callogenesis. PVP hydrogen bonds are able to absorb phenolic substances, and thus reduce their amount in medium. These substances are released from tissues and cells into the surrounding medium and their ac- cumulation may result in decreased rates of growth and development of cultured materials (Saxena et al., 1986; Leyser et al., 2003). Similar effects of PVP were also re- ported by Saxena et al. (1999) and Ogita et al. (2001) in a bamboo (Dendrocalamus strictus (Roxb.) Nees) tissue culture. In the current study, S. sahendica nodal mate- rial was used to test PVP and PGRs effects on in vitro cultured tissues. As it is shown, NAA + TDZ promoted callus production with significant shoot formation. Previous reports revealed that TDZ alone and in com- bination with NAA strongly promoted compact-green nodular callus and shoot formation in shrubs (Tarig- holizadeh et al., 2015, Al-juboory et al., 1998; Murthy, 1998; Thiruvengadam & Chung, 2015). 3.2 THE EFFECT OF PGRS ON CALLUS TISSUES GROWTH Our experiments revealed that callus growth of S. sahendica was strongly affected by type, combination, and different concentrations of PGRs (Table 3). Statis- tically significant differences were found between 2,4- D + Kin and NAA + TDZ treatments. Moreover, cal- lus growth was also affected by the presence of PVP. PVP exerted its positive effects on both fresh and dry mass of callus tissues in PVP+ media (Fig. 1 and Table 3). As shown in Figure 1 and Table 3, we obtained the highest fresh mass from MN3 and MN1 in PVP- and PVP+ media, respectively. However, there were no sig- nificant differences among the different concentrations total phenolics and flavonoids content. In addition, in cell suspension culture, cell growth was determined by measuring the fresh mass (due to low dry mass in cell suspension culture) and total cell extract was prepared to measure total phenolics and flavonoids contents. For the determination of total phenolics content in callus and cell culture, Folin-Ciocalteu reagent was used (Singleton et al., 1999). In brief, 100 µl of each ex- tract was combined with 2.5 ml of distilled water, and then mixed thoroughly with 100 µl of Folin-Ciocalteu reagent. After 6 minutes, 150 µl of 20  % (w/v) sodium carbonate was added and the solution was left at room temperature for 30 minutes in the dark. The absorbance of the reaction mixtures was measured at 650 nm. The results were expressed in the form of gallic acid equiva- lents (GAE) per gram of dry mass. The total flavonoids content in callus and cell cul- ture was estimated by using the aluminum chloride colorimetric method (Chang et al., 2002). A quantity of 500 µl of each sample solution was combined with 2.5 ml of distilled water and subsequently with 150 µl of 5  % sodium nitrite (NaNO2) solution, after 6 min of mixing, 300 µl of 10  % aluminum chloride (AlCl3) was added and then allowed to stand 5 minutes, fol- lowed by adding 1000 µl of 1 M NaOH solution to the mixture. The obtained solution was thoroughly mixed, after which the absorbance was determined at 510 nm. The results were expressed as mg quercetin equivalents (QE) per gram of dry mass. 2.5 STATISTICAL ANALYSIS All experiments were performed in a completely randomized design. Each treatment was comprised of three replicates. A one-way analysis of variance (ANO- VA) was applied to statistically analyze the data that was obtained from callus tissues and the means were compared by Duncan’s Multiple Range Tests (DMRT). IBM SPSS statistic ver. 22 was used to determine the significance at p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 CALLUS INDUCTION AND MORPHOLOGY Table 2 shows the effects of PGRs on S. sahendica nodal explants cultured on MS medium, displaying the callogenetic and morphologic properties of callus tis- sues. Shoot formation was also observed during cal- lus growth, and therefore the percentage of produced organs by nodal explants was reported (in relation to 5Acta agriculturae Slovenica, 117/4 – 2021 Influence of plant growth regulators and salicylic acid ... in callus and cell suspension culture of Satureja sahendica Bornm Table 2: Effect of different combinations of PGRs on callus induction and morphology in presence (PVP+) or absence (PVP-) of polyvinylpyrrolidone Data within the two columns (PVP-/PVP+) of each growth parameter, followed by different letters are significantly different at p ≤ 0.05. The data are presented as means ± SE (n = 3) Media Callogenesis % Callus tissues morphology Shoot formation % PVP+ PVP- PVP± PVP+ PVP- MN1 100 ± 0.00 a 90.48 ± 9.52 ab Green, Compact 42.86 ± 0.00 b 76.19 ± 4.76 b MN2 95.24 ± 4.763 b 100 ± 0.00 a Green, Compact 71.43 ± 0.00 a 90.47 ± 4.76 a MN3 100 ± 0.00 a 100 ± 0.00 a Green, Compact 52.38 ± 12.60 b 71.43 ± 8.25 b MD1 100 ± 0.00 a 100 ± 0.00 a Yellowish-Green, Friable 0.00 ± 0.00 c 0.00 ± 0.00 c MD2 71.43 ± 0.00 c 90.48 ± 9.52 ab Yellowish-Green, Friable 0.00 ± 0.00 c 0.00 ± 0.00 c MD3 90.47 ± 4.763 b 66.67 ± 17.17 b Yellowish-Green, Friable 0.00 ± 0.00 c 0.00 ± 0.00 c Figure 1: S. sahendica callus tissues grown on MS medium in the presence (upper row) and in the absence (lower row) of PVP and with the addition of PGRs. a (MN1), b (MN2), c (MN3), d (MD1), e (MD2), f (MD3) Table 3: Effect of different combinations of PGRs on callus tissues growth in MS PVP- and PVP+ medium Different letters within two columns (PVP-/PVP+) of each growth parameter represent significant differences among treatments at p ≤ 0.05. The data are presented as means ± SE (n = 3) Media Fresh mass (mg) Dry mass (mg) PVP+ PVP- PVP+ PVP- MN1 1.969 ± 0.535 a 0.848 ± 0.162 ab 0.118 ± 0.023 a 0.066 ± 0.014 ab MN2 1.966 ± 0.393 a 1.354 ± 0.435 a 0.130 ± 0.022 a 0.096 ± 0.031 a MN3 1.460 ± 0.197 a 1.384 ± 0.155 a 0.099 ± 0.017 a 0.094 ± 0.011 a MD1 0.287 ± 0.028 b 0.477 ± 0.086 bc 0.026 ± 0.001 b 0.047 ± 0.014 abc MD2 0.334 ± 0.026 b 0.256 ± 0.056 bc 0.026 ± 0.002 b 0.022 ± 0.003 bc MD3 0.160 ± 0.02 b 0.140 ± 0.028 c 0.020 ± 0.004 b 0.011 ± 0.004 c of growth regulators within a group. The highest dry mass was achieved by MN2 in both PVP- and PVP+ media. Once again, there were no significant differences within a group. As an obtained result, the presence of NAA and TDZ in the medium improved the growth of callus tissues. These result regarding all other examined growth parameters reinforced the findings of our previ- ous work where the effects of the PGRs and PVP were observed on callus relative growth rate (RGR) (Tarig- holizadeh et al., 2015). Correspondingly, Ali et al. (2013) reported that NAA in combination with TDZ was more effective for callus formation of Artemisia absinthium L. than other combinations of PGR. They achieved the highest callogenesis frequency (83.3  %) and maximum 6 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. mg l-1 of 2,4-D and 0.1 mg l-1 Kin. Based on former re- ports, maximum production of phenolic compounds was obtained from the active growing cells (Fu et al., 2005; Antonigni et al., 2007). In most cases, secondary prod- ucts accumulation was promoted at the end of rapid cell division in the growth cycle. However, in some of the cell culture systems, the production of secondary metabolites did not follow a parallel way with the cell growth (James et al., 2008) and the production occurred along with low growth. In the present study, as shown in Figures 1 and 2 and Table 3, an opposite relationship between growth and secondary products formation was found in NAA + TDZ and 2,4-D + Kin groups. According to our results, it can be deduced that the type of PGRs was more effective than other parameters for the production of total pheno- callus biomass (FW: 132 gl-1) on MS medium supple- mented with 1 mg l-1 NAA + 1 mg l-1 TDZ. Similarly, the dry mass showed the highest amount in 1 mg l-1 of both NAA and TDZ in callus of S. sahendica, although varying this concentration resulted in the reduction of dry mass. Previously, decrease in callogenic responses or mass production has been observed using 2,4-D in combination with Kin (Jeong et al., 2007; Hakkim et al., 2007; Johnson et al., 2011; Walla Abdelazeez et al., 2017). Thus, combination of 2, 4-D and Kin is not appropriate for the induction of callus tissues in nodal explants of S. sahendica compared with NAA + TDZ group. 3.3 THE EFFECT OF PVP AND PGRS ON PHENO- LIC COMPOUNDS PRODUCTION IN CALLUS TISSUES The present study shows that total phenolics con- centration depends on the type and combination of PGRs in the culture medium. Accumulation of phenolic compounds indicated differences between two groups of growth regulators used in this study. As can be seen in Figures 2 and 3, media containing 2, 4-D + Kin not only stimulated a high accumulation of total phenolics, but also enhanced flavonoid contents. Based on the results, MD3 medium maximized the amount of these com- pounds in both PVP- (the highest amount for flavonoids: 24.903 ± 7.016 mg QE g-1) and PVP+ (the highest amount for total phenolics: 4.303 ± 0.449 mg GAE. g-1) media. It should be noted that along with PGRs, PVP may play a significant role in production of total phenolics. Based on the obtained results, PVP nearly improved the amounts of total phenolics and flavonoids, except for 2,4-D + Kin treatment, which accumulation of flavonoids decreased along with the presence of PVP (Fig. 2 and 3). Similarly, Rani and Nair (2006) reported that PVP has an effect on Vitix negundo L. callus organogenesis. They suggested that this might be due to PVP ability to bind phenolics and some toxic substances. In addition, high production of isoflavones and growth index were achieved in callus culture of Genista plants after addition of PVP to MS me- dium (Luczkeiwicz & Glod, 2003). Previously, enhanced phenolic compounds accumulation by the combination of 2, 4-D, and Kin was also observed in Ocimum sanctum L. (Hakkim et al., 2011). Furthermore, the highest accu- mulation of withanolide A was showed in MS medium containing 2,4-D (9.05 µM) and Kin (2.32 µM) in cell suspension cultures of Withania somnifera (L.) Dunal (Sivanandhan et al., 2013). Also, Han et al. (2012) have obtained the highest amounts of rutin and GABA via in- cubation of the immobilized Morus bombycis Koidzumi cells in a full-strength MS liquid medium containing 1 Figure 2: Content of total phenolics (mg GAE. g-1) in callus tissues of S. sahendica affected by different combinations of PGRs in MS medium (in absence and presence of PVP: PVP- and PVP +). The results are presented as means ± SE (n = 3) Figure 3: Content of flavonoids (mg QE. g-1) in callus tissues of S. sahendica affected by different combination of PGRs in MS medium (in absence and presence of PVP: PVP- and PVP +). The results are are presented as means ± SE (n = 3) 7Acta agriculturae Slovenica, 117/4 – 2021 Influence of plant growth regulators and salicylic acid ... in callus and cell suspension culture of Satureja sahendica Bornm lic compounds. Similarly, a reverse correlation between rosmarinic acid (RA) accumulation and callus growth was reported in the callus culture of Satureja hortensis L. (Tepe & Sokmen, 2007). Based on these results, RA ac- cumulation and growth relationship is anthocyanin type and phenolic compound accumulation enhanced in the stationary phase of cell growth. We suggest that the presence of 2,4-D in media is most likely more responsible for the above-mentioned opposite relationship. As it can be seen in Table 2, ap- plication of 0.5 mg l-1 of both 2,4-D and Kin in medium resulted in a 100 % callogenesis in presence and absence of PVP and callus formation percentage showed a sig- nificant reduction in high concentrations of these two PGRs. Actually, 2, 4-D as an auxin is commonly used as an herbicide, especially for broadleaf weeds control (WHO, 1989; US EPA, 2005b; Tomlin, 2006). It owns herbicidal and lethal effects at high concentrations, and this function probably causes the production of higher concentrations of secondary metabolites by explants in culture media. 3.4 THE EFFECT OF SA AND PGRS ON CELL GROWTH The mean comparison of cells’ fresh mass indicated a significant difference among the different concentra- tions of SA and control samples. Higher concentrations of SA reduced cell fresh mass. According to the data pre- sented in Table 4, the highest (304.67 ± 3.48a) and the lowest (148 ± 3.76c) amount of fresh mass in the liquid media were obtained by 100 µM (1 mg l-1 2,4-D + 1 mg l-1 Kin) and 200 µM of SA (0.5 mg l-1 2,4-D + 0.5 mg l-1 Kin), respectively. The treated cells with SA showed a higher amount of fresh mass in 1 mg l-1 2,4-D + 1 mg l-1 Kin compared to 0.5 mg l-1 2,4-D + 0.5 mg l-1 Kin (Table 4). It should be noted that due to proper and adequate growth of callus tissues derived from MS media containing 0.5 and 1 mg l-1 of 2,4-D and Kin; these treatments have been chosen for further examination. 3.5 THE EFFECT OF SA AND PGRS ON PHENO- LIC COMPOUNDS IN CELL SUSPENSION CULTURE According to the variance analysis and the results of phenolics and flavonoids assessment in control and treated samples with different concentrations of SA in cell suspension culture, there was a significant differ- ence among treatments at both concentrations of 2,4-D + Kin in nodal explants of S. sahendica. Total phenolics and flavonoids content was enhanced by increasing the concentrations of SA, but this increase was not linear and the amount of total phenolics was reduced by high concentrations of SA. The highest and the lowest total phenolics content were obtained by 150 µM SA (2.1 ± 0.22 mg GAE g-1) with 0.5 mg l-1 2,4-D + 0.5 mg l-1 Kin and control treatment (0.78 ± 0.007 mg GAE g-1) with 1 mg l-1 2,4-D + 1 mg l-1 Kin, respectively (Fig. 4 and 5). On the other hand, there was no significant difference among different concentrations of SA, except for the con- trol treatment (Fig. 4). According to Table 3, the highest (358.6 ± 0.00 mg QE g-1) and the lowest (70.88 ± 0.47 mg QE g-1) production of flavonoids were obtained by 150 Table 4: Effect of different combination of PGRs and SA on cell fresh mass in suspension cell culture of Satureja sahendica Different letters within the same column represent statistically significant differences among treatments at p ≤ 0.05 PGRs treatments Elicitor concentrations Fresh mass (mg)2,4-D + Kin (mg l-1) Salicylic acid (µM) 0.5 : 0.5 Control 175 ± 1.76b 100 266 ± 4.62a 150 186.67 ± 6.96b 200 148 ± 1.73c 1 : 1 Control 185.33 ± 2.96c 100 304.67 ± 3.48a 150 283.67 ± 3.18b 200 158 ± 4.04d 8 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. (Kovacik, 2009). They also reported that total phenolics content was increased by 50 mg l-1 SA. Due to an inverse relationship between growth and accumulation of sec- ondary metabolites, the inhibition of cell growth by SA might induce the synthesis of secondary metabolites. Because precursors of secondary metabolite biosynthe- sis originate from the primary metabolism, under severe stress the primary metabolism changes to the second- ary metabolism and the necessary resources are diverted from development to defense (Harfouche et al., 2008). Moreover, total phenolics and flavonoids contents were increased by different concentrations (0, 50, 100, 200, and 250 µM) of SA in Cynara scolymus L. (Samadi et al., 2014) and by the increasing concentration of SA up to 100 µM, total phenolics and flavonoids contents showed µM of SA and control treatment with 0.5 mg l-1 (2,4-D + Kin), respectively. No significant difference was found among different concentrations of SA in both concentra- tions (0.5 and 1 mg l-1) of 2,4-D + Kin (Fig. 6). Investigation of SA effects on total phenolics and flavonoids contents in suspension culture of nodal- derived callus tissues of S. sahendica showed that these parameters were increased by 100 to 150 µM of SA and decreased by enhancement in SA concentration to 200 µM. Presumably, reduction in total phenolics and fla- vonoids contents in 200 µM of SA can be caused by the limited ability of cells in response to stresses or reduced enzymes activity. The maximum amount of phenolics and flavonoids content was obtained by 150 µM of SA and the flavonoids and total phenolics were 2 and 5 times more than the control group, respectively (with 0.5 Kin + 0.5 2,4-D). Interestingly, with a further increase in the SA concentration, not only the increas- ing in accumulation of total phenolics and flavonoids was not achieved, but also the accumulation process of these parameters was declined. Similar to our results, Esmaeilzadeh Bahabadi and Rezaei (2014) using cell culture of Trigonella foenum- graecum L. showed a significant reduction in cell growth with the increasing concentrations of SA. Thus, higher concentrations of SA have a detrimental effect on plant’s oxidative condition and eventually cause plant death Figure 4: Comparison of total phenolics and flavonoids content after treatment with different concentrations of SA (0, 100, 150, and 200 µM, left to right) in cell suspension of S. sahendica. Upper row: Callus tissues grown on MS medium containing 1 mg l-1 2,4-D + 1 mg l-1 Kin. Lower row: callus tissues grown on MS medium containing 0.5 mg l-1 2,4-D + 0.5 mg l-1 Kin Figure 5: Content of total phenolics (mg GAE. g-1) in cell suspension culture of S. sahendica after treatment with differ- ent concentrations of SA in MS medium containing different combinations of 2, 4-D + Kin. The results are presented as means ± SE (n = 3) Figure 6: Content of flavonoids (mg QE. g-1) in cell suspen- sion culture of S. sahendica after treatment with different concentrations of SA in MS medium containing different combinations of 2, 4-D + Kin. The results are presented as means ± SE (n = 3) 9Acta agriculturae Slovenica, 117/4 – 2021 Influence of plant growth regulators and salicylic acid ... in callus and cell suspension culture of Satureja sahendica Bornm a significant increase. These results are in agreement with the findings of the present work. They also reported that the changes of phenylpropanoid compounds were positively correlated with phenylalanine ammonia lyase (PAL) activity, and total phenolics and flavonoids content was also increased by PAL increased activity (Samadi et al., 2014). The effects of different elicitors on polyphenols content and activity of other polyphenol-related enzymes were also studied. For instance, shikimate dehydroge- nase (SDH), tyrosine ammonia lyase TAL, cinnamate- 4-hydroxylase (C4H), 4-coumarate/coenzyme A ligase (4-CL), and dihydroflavonol 4-reductase (DFR) were activated by elicitors such as SA and methyl jasmonate (Ruiz-García and Encarna Gómez-Plaza, 2013; Kim et al., 2020). Interestingly, the activity of PAL was directly related to the concentration of SA, so that the PAL activ- ity changes were similar to total phenolics and flavonoids accumulation via increasing the amount of SA. The pre- vious reports have suggested that the amount of pheno- lics and flavonoids was strongly influenced by PAL en- zyme activity (Sun et al., 2012; Ruiz-García and Encarna Gómez-Plaza, 2013). SA, as a stress-inducing compound, activates the signaling pathway of PAL, subsequently, PAL results in activation of the phenylpropanoid pathway and increasing the production of phenolic compounds by increasing the transcription of specific mRNA, in which these compounds counteract the induced stress. Elicitors, such as SA and methyl jasmonate, play an im- portant role in the signaling process which induces the biosynthesis of phenolic compounds and expression of plant defense genes (Wen et al., 2004; Wang et al., 2009). Exposure to elicitors may lead to an increase in the con- tent of defense related compounds such as total pheno- lics, flavonoids and phytoalexins. This might be due to an increase in the expression levels of responsible genes for the biosynthesis of these metabolites. Similar to our results, Sadeghian et al. (2013) reported that in Satureja khuzistanica Jamzad polyphenol oxidase and superoxide dismutase enzymes activity as well as total protein con- tents of SA (0, 50, 100, 200, and 400 mg l-1). Moghadam et al. (2013) found that the application of SA (125, 250, 500 mmol) in suspension culture of Portulaca oleracea L. hairy roots increased dopamine production with the highest amount at the concentration of 250 mM. Moreo- ver, flavonoid content was enhanced by the application of SA (0.05, 0.5, 1, and 1.5 mM) in the suspension culture of Andrographis paniculata (Matkowski, 2008). The studies about the effect of SA concentration (1, 1.5, 2 mM) on stimulating of Cicer arietinum L. immune system showed that this plant quickly responds to 1.5 mM of SA, and polyphenol oxidase activity increases in this concentra- tion (Rajjou et al., 2006). Despite decreasing total phe- nolic contents at higher concentrations, increasing above mentioned concentration (1.5 mM) was reported. These results show that exposure to 1.5 mM of SA is harmless for plants and this concentration may induce the chemi- cal defense response. However, treatment of samples with 2 mM SA caused phytotoxicity, which in turn might lead to low production of phenolic compounds (War et al., 2011). These findings are in fair agreement with the findings of current work. Also, it has been reported that the high concentration of SA induces hypersensitivity response that leads to cell death, while low concentra- tions of SA induces immune response (Namadeo, 2007). Therefore, the increased production and accumulation of phenolic compounds and flavonoids which found in S. sahendica suspension culture can be attributed to the induceded defense responses with SA. 4 CONCLUSION In the present study, the culture medium for cal- lus induction from nodal explants of Satureja sahendica was optimized. Furthermore, the production of some secondary metabolites in the obtained callus tissues were determined. It was found that callus growth and secondary metabolites production of S. sahendica was strongly affected by type, combination, and different concentrations of PGRs and statistically significant dif- ferences were found between 2,4-D + Kin and NAA + TDZ treatments. Moreover, the media containing 2, 4-D + Kin stimulated the production of both total pheno- lics and flavonoid compounds. Along with PGRs, PVP was generally an effective component in improvement of the studied parameters. In addition, the impact of SA on the accumulation and biosynthesis of secondary metabolites by cell suspension cultures of S. sahendica was also investigated. Based on the obtained results, SA can be used as a stimulant to improve the amount of phenolic compounds in the cell suspension cultures of this plant under controlled conditions. 5 AUTHOR CONTRIBUTIONS R.M., M.K.-N. and A.M. planned the research. All authors have read and agreed to the published version of the manuscript. 6 ACKNOWLEDGMENTS The authors thank the University of Tabriz for sup- porting and making funds available for this work. 10 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. the essential oil and methanol extracts of herbal parts and callus cultures of Satureja hortensis L. Journal of Agricul- tural and Food Chemistry, 51(14), 3958-3965. https://doi. org/10.1021/jf0340308 Hadian, J., Akramian, M., Heydarian, H., Mumivan, H., As- ghari, B. (2012). Composition and in vitro antibacterial ac- tivity of essential oils from four Satureja species growing in Iran. Natural Product Research, 26(2), 98-108. https:// doi.org/10.1080/14786419.2010.534734 Hadjmohammadi, M. R., Soltani, M., Sharifi, V. (2012). Use of hollow fiber liquid phase microextraction and HPLC for extraction and determination of apigenin in human urine after consumption of Satureja sahendica Bornm. Journal of Chromatography B, 900, 85-88. https://doi.org/10.1016/j. jchromb.2012.05.022 Hakkim, F. L., Gowri-Shankar, C., Girija, S. (2007). Chemical composition and antioxidant property of holy basil (Oci- mum sanctum L.) leaves stems, and inflorescence and their in vitro callus cultures. Journal of Agricultural and Food Chemistry, 55(22), 9109-9117. https://doi.org/10.1021/ jf071509h Hakkim, F. L., Kalyani, S., Essa, M., Girija, S. Song, H. (2011). Production of rosmarinic acid in Ocimum sanctum (L.) cell suspension cultures by the influence of growth regu- lators. International Journal of Biological and Medical Re- search, 2(4), 1158-1161. Han, K.L., Lee, Y., Song, J. H., Hwang, Y. S., Lee, W. S., Kim, M. W., Kim, S. H. (2012). Enhanced production and secretion of rutin and GABA in immobilized cells of mulberry tree (Morus bombycis K.). Plant Cell, Tissue and Organ Culture, 108(3), 513-20. https://doi.org/10.1007/s11240-011-0028- 2 Harfouche, A. L., Rugini, E., Mencarelli, F., Botondi, R., Muleo, R. (2008). Salicylic acid induces H2O2 production and en- dochitinase gene expression but not ethylene biosynthesis in Castanea sativa in vitro model system. Journal of Plant Physiology, 165(7), 734-744. https://doi.org/10.1016/j. jplph.2007.03.010 Hassanpouraghdam, M. B., Safi-Shalamzari, M., Azami, M. A., Mohajjel-Shoja, A. (2009). γ-terpinene and carvacrol rich volatile oil of Satureja sahendica Bornm. from Maragheh district in Northwest Iran. Chemija, 20(3), 186-189. James, J. T., Meyer, R., Dubery, I. A. (2008). Characterization of two phenotypes of Centella asiatica in Southern Africa through the composition of four triterpenoids in callus, cell suspensions and leaves. Plant Cell, Tissue and Organ Culture, 94(1), 91-99. https://doi.org/10.1007/s11240-008- 9391-z Jamzad, Z. (1996). Satureja rechingeri (Labiatae): A new stream. Species from Iran. (pp. 75-7). Annalen des Naturhis- torischen Museums in Wien. Jeong, G. T., Woo, J. C., Park, D. H. (2007). Effect of plant growth regulators on growth and biosynthesis of phenolic com- pounds in genetically transformed hairy roots of Panax ginseng C. A. Meyer. Biotechnology and Bioprocess Engineer- ing, 12(2), 86-9. https://doi.org/10.1007/BF03028631 Johnson, M., Wesely, E. G., Kavitha, M. S., Uma, V. (2011). Antibacterial activity of leaves and inter-nodal callus ex- tracts of Mentha arvensis L. Asian Pacific Journal of Tropical 7 REFERENCES Ali, M., Abbasi, B. H., Ul-haq, I. (2013). Production of com- mercially important secondary metabolites and anti- oxidant activity in cell suspension cultures of Artemisia absinthium L. Industrial Crops and Products, 49, 400-406. https://doi.org/10.1016/j.indcrop.2013.05.033 Al-juboory, K. H., Skirvin, R.M., William, D.J. (1998). Callus induction and adventitious shoot regeneration of gar- denia (Gardenia jasminoides Ellis) leaf explants. Scientia Horticulturae, 72(3-4), 171-178. https://doi.org/10.1016/ S0304-4238(97)00060-5 Al-Sane, K. O., Shibli, R.A., Freihat, N.M., Hammouri, M.K. (2005). Cell suspension culture and secondary metabo- lites production in African violet (Saintpaulia ionantha Wendl.). Jordan Journal of Agricultural Sciences, 1(1), 84-92. Alvarez, P.S., Spollansky, T. C., Giulietti, A.M. (2000). The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme and Micro- bial Technology, 26(2-4), 254-258. https://doi.org/10.1016/ S0141-0229(99)00137-4 Antonigni, F., Zheng, S., Pagnucco, C., Baraldi, R., Poli, F., Biondi, S. (2007). Induction of flavonoid production by UV-B radiation in Passiflora quagrangularis callus cultures. Fitoterapia, 78(5), 345-352. https://doi.org/10.1016/j.fito- te.2007.02.001 Chang, C. C., Yang, M.Y., Wen. H. M., Chern, J. C. (2002). Es- timation of total flavonoid content in propolis by two com- plementary colorimetric methods. Journal of Food and Drug Analysis, 10(3), 178-182. https://doi.org/10.38212/2224- 6614.2748 Dörnenburg, H., Knorr, D. (1995). Strategies for the improve- ment of secondary metabolite production in plant cell cul- tures. Enzyme and Microbial Technology, 17(8), 674-684. https://doi.org/10.1016/0141-0229(94)00108-4 Esmaeilzadeh Bahabadi, S., Rezaei, A. (2014). Increased trigo- nelline production by salicylic acid in fenugreek (Trigo- nella foenum-graecum L.) cell culture. (in Persian). Journal of Cell and Tissue, 5, 165-172. Fu, C. X., Zhao, D.X., Huang, Y., Ma, F. S. (2005). Cellular ag- gregate size as the critical factor for flavonoid production by suspension cultures of Saussurea medusa. Biotechnology Letters, 27(2), 91-95. https://doi.org/10.1007/s10529-004- 6934-1 Ghahreman, A. (1988). Flora of Iran (in Persian). Research In- stitute of Forest and Rangeland, Tehran. Ghahreman, A. (1993). Plant Systematics: Cormophytes of Iran (in Persian). Iran University Press, Tehran. Ghotbabadi, F. S., Alizadeh, A., Zadehbagheri, M., Kamel- manesh, M. M., Shaabani, M. (2012). Phytochemical composition of the essential oil, total phenolic content, antioxidant and antimicrobial activity in Iranian Satureja sahendica Bornm. at different ontogenesis conditions. Jour- nal of Medicinal Plants Research, 6(19), 3525-3534. https:// doi.org/10.5897/JMPR11.374 Güllüce, M., Sokmen, M., Daferera, D., Ağar, G., Ӧzkan, H., Kartal, N., Polissiou, M., Sökmen, A., Sahin, F. (2003). In vitro antibacterial, antifungal, and antioxidant activities of 11Acta agriculturae Slovenica, 117/4 – 2021 Influence of plant growth regulators and salicylic acid ... in callus and cell suspension culture of Satureja sahendica Bornm Medicine, 4(3), 196-200. https://doi.org/10.1016/S1995- 7645(11)60068-0 Khosroushahi, A. Y., Valizadeh, M., Gasempour, A., Khos- roushahi, M., Naghdibadi, H., Dadpour, M. R., Omidi, Y. (2006). Improved taxol production by combination of inducing factor in suspension culture of Taxus bac- cata. Cell Biology International, 30(3), 262-269. https://doi. org/10.1016/j.cellbi.2005.11.004 Kim, N. S., Jung, D. H., Jung, C. R., Jeon, K. S., Park, H. W., & Park, S. U. (2020). Improvement of phenylpropanoid pro- duction with elicitor treatments in Pimpinella brachycarpa Nakai. Horticulturae, 6(4), 108. https://doi.org/10.3390/ horticulturae6040108 Kovacik, J., Backor, M., Strnad, M., Repcak, M. (2009). Salicylic acid-induced changes to growth and phenolic metabolism in Matricaria chamomilla plants. Plant Cell Reports, 28(1), 135-143. https://doi.org/10.1007/s00299-008-0627-5 Leyser, O., Day, S. (2003). Mechanism in plant development, Oxford, UK: Blackwell Science Ltd, ISBN: 978-0-86542- 742-6. Luczkeiwicz, M., Glod, D. (2003). Callus cultures of Geni- sta plants- in vitro material producing high amounts of isoflavones of phytoestrogenic activity. Plant Sci- ence, 165(5), 1101-1108. https://doi.org/10.1016/S0168- 9452(03)00305-4 Matkowski, A. (2008). Plant in vitro culture for the production of antioxidants — A review. Biotechnology Advances, 26(6), 548-560. https://doi.org/10.1016/j.biotechadv.2008.07.001 Mendhulkar, V. D., Moinuddin, M., Vakil, A. (2013). Elicitation of flavonoids by salicylic acid and Penicillium expansum in Andrographis paniculata (Burm. f.) Nees. cell culture. Re- search in Biotechnology, 4(2), 1-9. Moghadam, Y. A., Piri, K. H., Bahramnejad, B., & Habibi, P. (2013). Methyl jasmonate and salicylic acid effects on the dopamine production in hairy cultures of Portulaca olera- cea (purslan). Bulletin of Environment, Pharmacology and Life Sciences, 2(6), 89-94. Mozaffarian, V. (1993). A dictionary of Iranian plant names (pp. 482-483) Farhang Moaser Publication, Tehran, Iran. Mozaffarian, V. (1996). A dictionary of Iranian plant names. Farhang Moaser Publication, Tehran, Iran. Murashige, T., Skoog, E. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cul- tures. Physiologia Plantarum, 15(3), 473-497. https://doi. org/10.1111/j.1399-3054.1962.tb08052.x Murthy, B. N. S., Murch, S. J. Saxena, P. K. (1998). Thidiazuron: A potent regulator of in vitro plant morphogenesis. In Vitro Cellular & Developmental Biology-Plant, 34(4), 267-275. https://doi.org/10.1007/BF02822732 Namadeo, A. G. (2007). Plant cell elicitation for production of secondary metabolites— A review. Pharmacognosy Re- views, 1(1), 154-160. Neumann, K. H., Kumar, A., Imani, J. (2009). Plant cell and tissue culture. A tool in biotechnology (pp. 333). Springer- Verlag Berlin Heidelberg. Ogita, S., Sasamoto, H., Yeung, E. C., & Thorpe, T. A. (2001). The effects of glutamine of the maintenance of embryo- genic cultures of Cryptomeria japonica. In Vitro Cellular & Developmental Biology-Plant, 37(2), 268-273. https://doi. org/10.1007/s11627-001-0048-4 Pourebad, N., Motafakkerazad, R., Kosari-Nasab, M., Farsad- Akhtar, N., Movafeghi, A. (2015). The influence of TDZ concentrations on in vitro growth and production of sec- ondary metabolites by the shoot and callus culture of Lalle- mantia iberica. Plant Cell, Tissue and Organ Culture, 122(2), 331-339. https://doi.org/10.1007/s11240-015-0769-4 Rajjou, L., Belghazi, M., Huguet. R., Robin, C., Moreau, A., Job, C. (2006). Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Plant Physiology, 141(3), 910-23. https://doi.org/10.1104/pp.106.082057 Rani, D. N., Nair, G. M. (2006). Effects of plant growth regu- lators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, Nir- gundi (Vitix negundo L.). In Vitro Cellular and Developmen- tal Biology-Plant, 42(1), 69-73. https://doi.org/10.1079/ IVP2005727 Ravishandera, G. A., Bhyalakshmi, N., Ramachandra Rao, S. (1999). Production of food additive. Biotechnology: Second- ary metabolites (pp. 89-110). New Dehli: Oxford IBH. Rechinger, K. H. (1982). Satureja in Flora Iranica (pp. 495-504). Akademische Druck-u. Verlagsanstalt, Graz. Ruiz-García, Y., & Gómez-Plaza, E. (2013). Elicitors: a tool for improving fruit phenolic content. Agriculture, 3(1), 33-52. https://doi.org/10.3390/agriculture3010033 Sadeghian, F., Hadian, J., Hadavi, M., Mohamadi, A., Ghorban- pour, M., Ghafarzadegan, R. (2013). Effects of exogenous salicylic acid application on growth, metabolic activities and essential oil composition of Satureja khuzistanica Jamzad. Journal of Medicinal Plants, 3(47), 70-82. Saeidnia, S., Nourbakhsh, M. S., Gohari, A. R., Davood, A. (2011). Isolation and identification of the main com- pounds of Satureja sahendica Bornm. Australian Journal of Basic and Applied Sciences, 5(6), 1450-1453. https://doi. org/10.1021/jf303755w Samadi, S., Ghasemnezhad, A., Alizadeh, M. (2014). Investiga- tion on phenylalanine ammonia-lyase activity of artichoke (Cynara scolymus L.) affected by methyl jasmonate and salicylic acid in in-vitro conditions (in Persian). Journal of Plant Production Research, 21, 135-148. Saxena, P. K., & Gill, R. (1986). Removal of browning and growth enhancement by polyvinylpolypyrrolidone in protoplast cultures of Cyamopsis tetragonoloba L. Biologia Plantarum, 28(4), 313-315. https://doi.org/10.1007/BF02902302 Saxena, S., & Dhawan, V. (1999). Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis. Plant Cell Reports, 18(5), 438-443. https://doi.org/10.1007/s002990050600 Sefidkon, F., Jamzad, Z. Mirza, M. (2004). Chemical variation in the essential oil of Satureja sahendica from Iran. Food Chemistry, 88(3), 325-328. https://doi.org/10.1016/j.food- chem.2003.12.044 Shilpashree, H., Rai, R. (2009). Effects of growth regulators on in vitro plant regeneration and flavonoid production in Hy- pericum mysorense. International Journal of Integrative Biol- ogy, 8(1), 43-49. Singleton, V. L., Orthofer, R., Lamuela-Raventos, R. M. (1999). 12 Acta agriculturae Slovenica, 117/4 – 2021 S. TARIGHOLIZADEH et al. in Plant Biology, 11(4), 436-442. https://doi.org/10.1016/j. pbi.2008.05.003 Walla Abdelazeez, M. A., Khusnetdinova Landysh, Z., Timo- feeva Olga, A. (2017). Effect of different plant growth regu- lators on the in vitro induction and maintenance of callus from different explants of Hyoscyamus muticus L. Journal of Applied Environmental and Biological Sciences, 7(3), 27-35. https://doi.org/10.17816/snv201873101 Wang, K., Jin, P., Cao, S., Shang, H., Yang, Z., Zheng, Y. (2009). Methyl jasmonate reduces decay and enhances antioxi- dant capacity in Chinese bayberries. Journal of Agricul- tural and Food Chemistry, 57(13), 5809–5850. https://doi. org/10.1021/jf900914a Wang, Y., Yuan, Y., Wn, J. (2004). Induction studies of me- thyl jasmonate and salicylic acid on taxane production in suspension cultures of Taxus chinensis var. mairei. Bio- chemical Engineering Journal, 19(3), 259-265. https://doi. org/10.1016/j.bej.2004.02.006 War, A. R., Paulraj, M. G., War, M. Y., Ignacimuthu S. (2011). Jasmonic acid-mediated induced resistance in groundnut (Arachis hypogaea L.) against Helicoverpa armigera (Hub- ner) (Lepidoptera: Noctuidae). Journal of Plant Growth Regulation, 30(4), 512-523. https://doi.org/10.1007/s00344- 011-9213-0 Wen, P. F., Chen, J. Y., Kong, W. F., Pan, Q. H., Wan, S. B., Huang, W. D. (2004). Salicylic acid induced the expression of phenylalanine ammonia-lyase gene in grape berry. Plant Science, 169(5), 928-934. https://doi.org/10.1016/j.plants- ci.2005.06.011 World Health Organization (WHO). Environmental Health Cri- teria 84, Environmental aspects - 2,4-dichlorophenoxyacetic acid (2,4-D); International Programs on Chemical Safety. World Health Organization: Geneva, Switzerland; 1989. Zhong, J. J., Bai, Y., Wang, S. J. (1996). Effects of plant growth regulators on cell growth and ginsenoside saponin pro- duction by suspension cultures of Panax quinquefolium. Journal of Biotechnology, 45(3), 227-234. https://doi. org/10.1016/0168-1656(95)00170-0 Analysis of total phenols and other oxidation substrates and oxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152-178. Sivanandhan, G., Kapil Dev, G., Jeyaraj, M., Rajesh, M., Mu- thuselvam, M., Selvaraj, N., Manickavasagam, M., Gana- pathi, A. (2013). A promising approach on biomass accu- mulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma, 250(4), 885-898. https://doi.org/10.1007/s00709-012- 0471-x Sun, Z., Hou, S., Yang, W., & Han, Y. (2012). Exogenous ap- plication of salicylic acid enhanced the rutin accumulation and influenced the expression patterns of rutin biosynthesis related genes in Fagopyrum tartaricum Gaertn leaves. Plant Growth Regulation, 68(1), 9-15. https://doi.org/10.1007/ s10725-012-9688-0 Tarigholizadeh, S., Motafakkerazad, R., Kosari-Nasab, M., Movafeghi, A. (2015). Callus medium improvement for Sa- tureja sahendica as an important medicinal plant. Interna- tional Journal of Integrative Biology, 16(1), 13-18. Taveira, M., Ferreres, F., Pereira, M. D., Sousa, C., Andrade, B. P., Martins, A., Pereira, A. J., Valentao, P. (2010). In vitro culture of Brassica oleracea L.var. costata DC: potential plant bioreactor for antioxidant phenolic compounds. Journal of Agricultural and Food Chemistry, 57(4), 1247- 1252. https://doi.org/10.1021/jf803496x Tepe, B., Sokmen, A. (2007). Production and optimization of rosmarinic acid by Satureja hortensis L. callus cultures. Natural Product Research, 21(13), 1133-1144. https://doi. org/10.1080/14786410601130737 Thiruvengadam, M., Chung, I. M. (2015). Phenolic com- pound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L.). Elec- tronic Journal of Biotechnology, 18(4), 295-301. https://doi. org/10.1016/j.ejbt.2015.05.005 Tomlin, C. D. S. (2006). The Pesticide Manual: A World Com- pendium. 14th ed. British Crop Protection Council: Surrey, UK. United States Environmental Protection Agency (US EPA). Reregistration eligibility decision for 2,4-D. EPA 738-R-05- 002; June 2005b. Vlot, A. C., Klessig, D. F., Park, S. W. (2008). Systemic ac- quired resistance: the elusive signal(s). Current Opinion Acta agriculturae Slovenica, 117/4, 1–7, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2345 Original research article / izvirni znanstveni članek In vitro antifungal potential of surfactin isolated from rhizospheric Bacil- lus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phy- topathogen Fusarium graminearum Schwabe Muddasir KHAN 1, 2, Muhammad SALMAN 1, Abdullah 1, Syed Hussain SHAH 1; Muhammad ISRAR 3 Received September 16, 2021; accepted November 29, 2021. Delo je prispelo 16. septembra 2021, sprejeto 29. november 2021 1 Department of Health and Biological Sciences, Abasyn University Peshawar, Khyber Pakhtunkhwa, Pakistan 2 Corresponding author, e-mail: mk03025678947@gmail.com, muddasir.khan0302@gmail.com 3 Department of Microbiology, Abbottabad University of Science and Technology, Khyber Pakhtunkhwa, Pakistan In vitro antifungal potential of surfactin isolated from rhi- zospheric Bacillus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe Abstract: Fusarium graminearum fungus cause signifi- cant loss in maize (Zea mays L.) and other cereal crops all over the world. The usage of chemical agents cause severe envi- ronmental problems. Bacillus species and other plant growth- promoting bacteria (PGPR) play key role in biopesticide devel- opment. A wide range of environmentally safe antimicrobial agents are already being manufactured. The current investiga- tion was focused on exploring the antifungal activity of Bacillus thuringiensis lipopeptide surfactin against fungal phytopatho- gen Fusarium graminearum. B. thuringensis was isolated from the rhizosphere of maize crop and cultivated to produce lipo- peptides. Surfactin was identified by high-performance liquid chromatography (HPLC) from the extract at 210 nm, retention time 3-5 minutes and the obtained peaks area was 3.990. The growth of F. graminearum was successfully inhibited by surfac- tin at different concentrations. Among these, 80 % concentra- tion showed the highest zone of inhibition in comparison to 60 %, 40 % and 20 % concentrations (p < 0.005), respectively. The current study concludes B. thuringensis lipopeptide surfac- tin has a high potential to inhibit the growth of F. graminearum. Key words: surfactin; Bacillus; biological control; HPLC; Fusarium graminearum In vitro protiglivni potencial surfaktina, izoliranega iz bak- terije Bacillus thuringiensis Berliner 1915 iz rizosfere koruze (Zea mays L.) proti patogeni glivi Fusarium graminearum Schwabe Izvleček: Gliva Fusarium graminearum povzroča znantne izgube v pridelku koruze in drugih žit širom po svetu. Uporaba kemičnih sredstev za zatiranje povzroča resne okoljske proble- me. Vrste iz rodu Bacillus in druge rast vzpodbujajoče bakterije (PGPR) igrajo ključno vlogo pri razvoju biopesticidov. Proizve- den je bil že širok spekter okolju prijaznih antimikrobnih agen- sov. Raziskava se osredotoča na uporabo protiglivne aktivnosti lipopeptidnih surfaktinov iz bakterije Bacillus thuringiensis proti patogeni glivi Fusarium graminearum. Bakterija B. thu- ringensis je bila izolirana iz rizosfere posevka koruze in gojena za proizvodno lipopeptidov. Surfaktin je bil določen s tekočin- sko kromatografijo visoke ločljivosti (HPLC) iz izvlečka pri 210 nm, retencijskim časom 3-5 minut, dobljeni višek je bil 3.990. Rast patogene glive je bila uspešno zavrta pri različnih koncen- tracijah surfaktina. 80 % koncentracija surfaktina je pokazala največjo sposobnost zaviranja v primerjavi s koncentracijami 60 %, 40 % in 20 % (p < 0,005). Na osnovi te raziskave lahko zaključimo, da ima lipopeptidni surfaktin iz bakterije B. thu- ringensis velik potencial za zaviranje rasti glive F. graminearum. Ključne besede: surfaktin; Bacillus; biološka kontrola; HPLC; Fusarium graminearum Acta agriculturae Slovenica, 117/4 – 20212 M. KHAN et al. 1 INTRODUCTION Globally Bacillus thurenginisis is considered to be the most predominant soil-dwelling bacterium found in the plants rhizosphere known for their antimicrobial properties. Aforementioned, Bacillus strains are known as plant growth promoting rhizobacteria (PGPR) that are associated with plants tolerance against biotic, and abiotic stresses caused by certain fungal phytopathogens (Saxena et al., 2019). In this context the worldwide major loss of maize and other cereal crops is due to fungal phy- topathogens. The repertoire of fungal phytopathogens in- cluding Acremonium alternatum Link (Pal and Gardener, 2006), Ustilago maydis (DC.) Corda[ (Kwon et al., 2021), Aspergillus niger van Tieghem, Aspergillus flavus Link, Puccinia sorghi Schwein., Fusarium species (Rehman et al., 2021), Helminthosporium, Alternaria, Rhizopus, Peni- cillium, Drechslera (Snetselaar and McCann, 2017), Mac- rophomina phaseolina (Tassi) Goid., and Colletotrichum graminicola D.J. Politis (Saleem et al., 2012), cause varie- ties of disease in maize. Maize (Zea mays L.) is the most important cereal crop in the world, covering 75  % of the food require- ments all over the world (Hussain et al., 2013). In Pa- kistan, among the cereal crops, maize is the third most important crop, after wheat and rice. Among these, glob- ally the most important and significant phytopathogen is Fusarium graminearum, which causes significant loss of grain crops (Rauwane et al., 2020). The wide range of dis- eases caused by this plant pathogen includes; fruit rots, Fusarium head blight (FHB), wilts, and root rots (Kant et al., 2011). Chemical compounds have been used to manage these fungal phytopathogens for many decades. They have a potential to generate major environmental prob- lems. Alternative and less environmentally detrimental measures are required to control these plant diseases. Bacillus species and other PGPR play a key role among biopesticides. They produce various antimicrobial com- pounds such as enzymes, lipopeptides, and antibiotics that stimulate plant development while inhibiting patho- genic microbes (Shafi et al., 2017). For B. thurenginisis cyclic peptides including, surfactin, mycobacillin, myco- subtilin, subtilin, bacilysin, fengycin, bacillomycin, and iturin are reported that exhibit both antibacterial, and antifungal properties (Khan et al., 2021; Ntushelo et al., 2019). Surfactin is a lipopeptide composed of cyclic dep- sipeptides of β-hydoxy hepta with possible amino acid combinations of alanine, valine, leucine, or isoleucine at positions 2, 4, and 7 in the cyclic depsipeptide moiety and β-hydoxy fatty acid chain variants of C13 to C16 in the cyclic depsipeptide moiety and β-hydoxy fatty acid chain variant (Hue et al., 2001). According to the investigations surfactin has natural antifungal properties produced by Bacillus spp. that could inhibit the growth of certain fun- gal species including, F. graminearum (Khan et al., 2021), Fusarium oxysporum Schlecht. emend. Snyder & Hansen (Kim et al., 2010), Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc. (Snook et al., 2009), Fusarium verticillioides (Sacc.) Nirenberg (Dunlap et al., 2011), and Fusarium moniliforme (Sacc.) Nirenberg (Vitullo et al., 2012). Therefore, the current study was designed to iso- late and characterize B. thuringiensis lipopeptide from rhizospheric soil and also to assess its antifungal effica- cy against the fungal phytopathogen F. graminearum of maize. 2 MATERIALS AND METHODS 2.1 BACTERIAL AND FUNGAL ISOLATION A total of 20 maize rhizospheric soil samples were collected from various locations in Peshawar, Pakistan, for the isolation of B. thuringiensis (Figure 1). B. thur- ingiensis was identified using colony morphology, gram staining, and biochemical-tests such as citrate hydroly- sis, catalase, indole production, nitrate reduction, Vog- es-Proskauer (VP), motility, H2S production, and crys- tal formation (Amin et al., 2015). F. graminearum was isolated using a sample acquired from a diseased maize plant in Peshawar, Pakistan (Figure 1), and identified us- ing colony morphology and microscopic analysis (Uddin et al., 2019; John et al., 2006). 2.2 LIPOPEPTIDE EXTRACTION AND IDENTIFI- CATION In a shaking flask containing nutrient broth medi- um (Oxoid™), all morphological and biochemical based confirmed isolated colonies of B. thuringiensis were in- jected. The flask was incubated for 16 hours at 30 oC with shaking incubator at 200 rpm. Afterward the culture was transferred to an Erlenmeyer flask containing 99 ml of Tryptic Soy Broth (TSB) medium (Oxoid™) and incubat- ed overnight at 30 oC with shaking incubator at 200 rpm. The optical density (OD) of the B. thuringiensis growth curve was measured at 600 nm using a spectrophotom- eter (Shimadzu, UV-1800). After the decline phase of B. thuringiensis growth, the culture was removed and cen- trifuged at 6000 rpm for 30 minutes. The supernatant was filtered using a sterile 0.22 µm filter (Mater et al., 2009). The extract was then centrifuged for 10 minutes at 1000 rpm and 20 oC. The deposit was dissolved in a solution Acta agriculturae Slovenica, 117/4 – 2021 3 In vitro antifungal potential of surfactin ... against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe of methanol (Analytical grade, VWR Chemicals BDH®) and water (50:50, v/v) and filtered again using a 0.22 µm filter membrane. For purification, the sample was treated three times with 20 ml chloroform (VWR Chemicals BDH®). The bottom layer was collected and chloroform was evaporated at 50 oC temperature by using a hotplate stirrer. Methanol was used to dissolve the residue. Sur- factin from the extract were identified by introducing 50 µl of the extract into a Shimadzu 20A UV-Vis HPLC at a wavelength range of 200–250 nm. The isocratic HPLC method was employed, along with a 4.6 × 150 mm C-18 normal phase column (Mater et al., 2009). For the iden- tification of surfactin by HPLC experiment, acetonitrile was utilized as a mobile phase. Surfactin were discovered after comparing the observed peak to previously pub- lished data (Meena et al., 2014). 2.3 ANTIFUNGAL ACTIVITY OF LIPOPEPTIDE EXTRACT To test the antifungal activity of surfactin, four 5 mm wells were created on potato dextrose agar (PDA) (Oxoid™) using a sterilized cork borer. The methanol was used as a control and also used to create concentrations of the lipopeptide extract of 20 %, 40 %, 60 %, and 80 %, respectively. The wells were filled with 200 µl of methanol (control), 20 %, 40 %, 60 %, and 80 % concentrations of lipopeptide extract, respectively. A colony of active grow- ing F. graminearum was placed in the middle of media plates using sterile forceps and incubated at 30 oC for 3-7 days. Five repetitive antifungal analysis of the extracted lipopeptde was done by the same method described above. The inhibitory zones were measured and recorded (Mater et al., 2009). The obtained mean zone of inhibi- tions was analyzed using a one-way ANOVA test using the Statistical Packages for Social Sciences (SPSS) version 23.0 software and Microsoft Excel. 3 RESULTS AND DISCUSSION 3.1 BACTERIAL ISOLATE In 20 rhizospheric soil samples B. thuringiensis 12 isolates were confirmed by various criteria such as, col- ony morphology, gram staining, and biochemical assays (Table 1). Previous results revealed that Bacillus species are primarily found in rhizospheric soil and that their metabolites have antibiotic characteristics as they can inhibit or restrict the development of other microorgan- isms (Amin et al., 2015). 3.2 FUNGAL ISOLATE In context to this study, F. graminearum was isolated from infected maize plants and identified using colony morphology (white to pinkish), and microscopic assess- ment (Hyaline septate hyphae, two to multi-celled and Figure 1: (A) Sampling site of maize rhizospheric soil for the isolation of B. thuringiensis, (B) Diseased maize for the isolation of F. graminearum Acta agriculturae Slovenica, 117/4 – 20214 M. KHAN et al. sickle-shaped) in the current investigation. Fusarium head blight (FHB) disease is caused by F. graminearum in maize. This fungus exhibit certain sign of early bleaching during infection which could reduce grain production and quality (Ntushelo et al., 2019). 3.3 LIPOPEPTIDE IDENTIFICATION According to the current study findings, B. thuring- iensis was grown to produce lipopeptides, and the optical density (OD) of the growth curve was measured (Figure 2). Lipopeptides isolated from B. thuringiensis were ana- lyzed by HPLC using acetonitrile as the mobile phase. At 210 nm and retention period 3-5 minutes, the observed peak area was 3.990 (Figure 3), which is similar to the peaks found earlier in surfactin literature data (Mubarak et al., 2015). Previous studies are in agreement with our findings. According to the Deepak and Jayapradha (2015), they identified lipopeptide surfactin by HPLC which are produced by B. thuringiensis. In another study, the lipopeptde fengycin produced by B. thuringiensis was identified by HPLC techniques (Kim et al., 2004). 3.4 ANTIFUNGAL ACTIVITY OF LIPOPEPTIDE B. thuringiensis lipopeptide surfactin against the development of F. graminearum was investigated in this work. The surfactin lipopeptide efficiently suppressed the growth of F. graminearum (Figure 4). According to ear- lier research, isolated Bacillus spp. from the rhizosphere, particularly B. subtilis, reduced the growth of F. gramine- arum. Bacillus spp. is also effective in the prevention of Fusarium head blight (FHB) and root rot; they stimu- late plant development and inhibit the mycelial growth of fungal infections through antagonistic action (Herba et al., 2020; Madhi et al., 2020; Dukare et al., 2020). In this study, lipopeptide surfactin from B. thuringiensis was tested against F. graminearum at 20 %, 40 %, 60 %, and 80 % concentrations (Figure 4). The zone of inhibi- tion was the greatest at the 80 % concentration, followed by the 60 %, 40 %, and 20 % concentrations (p < 0.005), respectively. These findings are in agreement with previ- ous report, in which the surfactin action against F. ox- ysporum (Deepak and Jayapradha, 2015) was screened. According to a recent study, microorganisms were iso- lated from plant anthers and wheat kernels to test their antagonistic activity against F. graminearum, the causa- tive agent of Fusarium head blight (FHB). B. subtilis has a strong antifungal impact on F. graminearum mycelium, sporulation, and DON formation, with inhibition values of 87.9 %, 95.6 %, and 100 %, respectively (Zhao et al., 2014). 4 CONCLUSION Lipopeptides obtained from Bacillus species have less negative environmental effects as compared to chemical compounds. The current study concluded that B. thuringiensis isolated from the rhizosphere of maize crop may produce lipopeptide surfactin, which has a high potential to inhibit the growth of F. graminearum. The study is also emphasizing surfactin as potential bio- logical control agent with widespread usage. We are also encouraging other researchers to take advantage of newly invented techniques to explore mechanism of action of various Bacillus strains against phytopathogens. Tests Results Colony Morphology Circular, rough, opaque, fuzzy white or slightly yellow Gram Staining Gram Positive Shape Rod shaped Motility Positive Catalase Positive Indole production Negative Citrate utilization Positive H2S production Negative Crystals formation Positive Identified Strains B. thuringiensis Table 1: Morphological and Biochemical characteristics of B. thuringiensis Figure 2: Optical density (OD) of the growth curve of B. thur- ingiensis at 600 nm wavelength Acta agriculturae Slovenica, 117/4 – 2021 5 In vitro antifungal potential of surfactin ... against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe Figure 3: HPLC Chromatogram of B. thuringiensis lipopeptide surfactin obtained at 210nm, retention time between 3-5 minutes and peak area 3.990 Figure 4: B. thuringiensis lipopeptide surfactin zone of inhibition (mean) against F. graminearum at various concentrations (p < 0.005) Acta agriculturae Slovenica, 117/4 – 20216 M. KHAN et al. 5 REFERENCE Amin, M., Rakhisi Z., Ahmady A. Z. (2015). Isolation and identification of Bacillus Species from soil and evalua- tion of their antibacterial properties. Avicenna Journal of Clinical Microbiology and Infection, 2(1), e2323. https://doi. org/10.17795/ajcmi-23233 Deepak, R., Jayapradha, R. (2015). Lipopeptide biosurfactant from Bacillus thuringiensis pak 2310: a potential antago- nist against Fusarium oxysporum. Journal de Mycologie Medicale, 25(1), e15-e24. https://doi.org/10.1016/j.myc- med.2014.10.011 Dukare, A., Paul, S., Arambam, A. (2020). Isolation and efficacy of native chitinolytic rhizobacteria for biocontrol activities against fusarium wilt and plant growth promotion in pi- geon pea (Cajanus cajan L.). Egyptian Journal of Biological Pest Control, 30, 56. https://doi.org/10.1186/s41938-020- 00256-7 Dunlap, C. A., Schisler, D. A., Price, N. P., Vaughn, S. F. (2011). Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. Journal of Microbi- ology, 49, 603–609. https://doi.org/10.1007/s12275-011- 1044-y Hue, N., Serani, L., Laprevote, O. (2001). Structural investigation of cyclic peptidolipids from Bacillus subtilis by high energy tandem mass spectrometry. Rapid Communication. Mass Spectrometry, 15, 203–209. https://doi.org/10.1002/1097- 0231(20010215)15:3<203::AID-RCM212>3.0.CO;2-6 Hussain, N., Hussain, A., Ishtiaq, M., Azam, S., Hussain, T. (2013). Pathogenicity of two seed-borne fungi commonly involved in maize seeds of eight districts of Azad Jammu and Kashmir, Pakistan. African Journal of Biotechnology, 12(12), 1363-1370. John, F. L., Brett, A. S. (2016). The Fusarium laboratory manual, First edition, Blackwell Publishing, 2016. Kant, P., Reinprecht, Y., Martin, C. J., Islam, R., Pauls, K. P. (2011). Disease resistance / Pathology / Fusarium. Else- vier, 00263-4. https://doi.org/10.1016/B978-0-08-088504- 9.00263-4 Khan, M., Salman, M., Jan, S. A., Shinwari, Z. K. (2021). Biolog- ical control of fungal phytopathogens: A comprehensive re- view based on Bacillus species. MOJ Biology Medicine, 6(2), 90‒92. https://doi.org/10.15406/mojbm.2021.06.00137 Kim, P. I., Bai, H., Bai, D., Chae, H., Chung, S., Kim, Y., et al. (2004). Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. Journal of ap- plied microbiology, 97(5), 942-949. https://doi.org/10.1111/ j.1365-2672.2004.02356.x Kim, P. I., Ryu, J., Kim, Y. H., Chi, Y. T. (2010). Production of biosurfactant lipopeptides iturin A, fengycin and surfactin from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporides. Journal of Microbiology and Biotechnology, 20, 138–145. https://doi.org/10.4014/jmb.0905.05007 Kwon, S., Rupp, O., Brachmann, A., Blum, C. F., Kraege, A., Goesmann, A., Feldbrügge, M. (2021). mRNA inventory of extracellular vesicles from Ustilago maydis. Journal of Fungi, 7(7), 562. https://doi.org/10.3390/jof7070562 Madhi, Q. H., Jumaah, A. M. (2020). Affectivity evaluation of Bacillus subtilis in controlling eggplant root rot caused by Rhizoctonia solani and Fusarium solani. IOP Conference Se- ries: Earth and Environmental Science, 553, 012026. https:// doi.org/10.1088/1755-1315/553/1/012026 Mater, S. M., El-Kazzaz, S. A., Waigh, E. E., El-Diwany, A. I., Moustafa, H. E., Abo-Zaid, G. A., Elsalam, A. H. E., Hafez, E. E. (2009). Antagonistic and inhibitory effect of Bacillus subtilis against certain plant pathogenic fungi. Biotechnolo- gy, 8(1), 53-61. https://doi.org/10.3923/biotech.2009.53.61 Meena, K. R., Saha, D., Kumar, R. (2019). Isolation and partial characterization of iturin like lipopeptides (a bio-control agent) from a Bacillus subtilis strain. International Journal of Current Microbiology and Applied Sciences, 3(10), 121- 126.   Mubarak, M. Q. E., Hassan, A. R., Hamid, A. A., Khalil, S., Isa, M. H. M. (2015). A simple and effective isocratic HPLC method for fast identification and quantification of surfac- tin. Sains Malaysiana. https://doi.org/10.17576/jsm-2015- 4401-16 Ntushelo, K., Ledwaba, L. K., Rauwane, M. E., Adebo, O. A., Njobeh, P. B. (2019). The mode of action of Bacillus species against Fusarium graminearum, tools for investigation and future prospects. Toxins, 11, 606. https://doi.org/10.3390/ toxins11100606 Pal, K. K., Gardener, B. M. (2006). Biological control of plant pathogens. The Plant Health Instructor, 1-25. https://doi. org/10.1094/PHI-A-2006-1117-02 Rauwane, M. E., Ogugua, U. V., Kalu, C. M., Ledwaba, L. K., Woldesemayat, A. A., Ntushelo, K. (2020). Pathogenicity and virulence factors of Fusarium graminearum includ- ing factors discovered using next generation sequencing technologies and proteomics. Microorganisms, 8(2), 305. https://doi.org/10.3390/microorganisms8020305 Rehman, F., Adnan, M., Kalsoom, M., Naz, N., Husnain, M. G., Ilahi, H., Ilyas, M. A., Yousaf, G., Tahir, R., Ahmad, U. (2021). Seed-borne fungal diseases of maize (Zea mays L.): A review. Agrinula: Jurnal Agroteknologi Dan Perkebunan, 4(1), 43-60. https://doi.org/10.36490/agri.v4i1.123 Ren, J., Li, Z., Wu, P., Zhang, A., Liu, Y., Hu, G., et al. (2021). Ge- netic dissection of quantitative resistance to common rust (Puccinia sorghi) in tropical maize (Zea mays L.) by com- bined genome-wide association study, linkage mapping, and genomic prediction. Frontiers in plant science, 12, 1338. https://doi.org/10.3389/fpls.2021.692205 Saleem, M. J., Bajwa, R., Hannan, A., Qaiser, T. A. (2012). Maize seed storage mycoflora in Pakistan and its chemical control. Pakistan Journal of Botany, 44(2), 807-812. Saxena, A. K., Kumar, M., Chakdar, H., Anuroopa, N., Bagyaraj, D. J. (2020). Bacillus species in soil as a natural resource for plant health and nutrition. Journal of Applied Microbiology, 128(6), 1583-1594. https://doi.org/10.1111/jam.14506 Shafi, J., Tian, H., Ji, M. (2017). Bacillus species as versatile weapons for plant pathogens: a review. Biotechnology & Biotechnological Equipment, 31(3), 446–459. https://doi.or g/10.1080/13102818.2017.1286950 Snetselaar, K., McCann, M. (2017). Ustilago maydis, the corn smut fungus, has an unusual diploid mitotic stage. Myco- logia, 109(1), 140-152. https://doi.org/10.1080/00275514.2 016.1274597 Snook, M. E., Mitchell, T., Hinton, D. M., Bacon, C. W. (2009). Acta agriculturae Slovenica, 117/4 – 2021 7 In vitro antifungal potential of surfactin ... against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe Isolation and characterization of Leu7-surfactin from the endophytic bacterium Bacillus mojavensis RRC 101, a bio- control agent for Fusarium verticillioides, Journal of Agri- culture and Food Chemistry, 57, 4287–4292. https://doi. org/10.1021/jf900164h Uddin, M. N., Nasrullah, Khan, M. (2019). Isolation and Iden- tification of fungal pathogens associated with diseases of onion crop in district Swat, Pakistan. Abasyn Journal of Life Sciences, 2(2), 91-99. Vitullo, D., Di Pietro, A., Romano, A., Lanzotti, V., Lima, G. (2012). Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusar- ium oxysporum. Plant Pathology, 61, 689–699. https://doi. org/10.1111/j.1365-3059.2011.02561.x Zhao, Y., Selvaraj, J. N., Xing, F., Zhou, L., Wang, Y., Song, H., Tan, X., Sun, L., Sangare, L., Folly, Y. M. E. (2014). An- tagonistic action of Bacillus subtilis strain SG6 on Fusar- ium graminearum. PLoS ONE, 9, e92486. https://doi. org/10.1371/journal.pone.0092486 Acta agriculturae Slovenica, 117/4, 1–5, Ljubljana 2021 doi:10.14720/aas.2021.117.4.1998 Original research article / izvirni znanstveni članek Expression of IRT1 gene in barley seedlings under zinc deficiency at opti- mal and low temperatures Natalia KAZNINA 1, 2, Natalia REPKINA 1, Yulia BATOVA 1, Alexandr TITOV 1 Received December 17, 2020; accepted October 21, 2021. Delo je prispelo 17. decembra 2020, sprejeto 21. oktobra 2021 1 Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Petrozavodsk, Russia 2 Corresponding author, e-mail: kaznina@krc.karelia.ru Expression of IRT1 gene in barley seedlings under zinc defi- ciency at optimal and low temperatures Abstract: The deficiency or excess of zinc (Zn) cause neg- ative effect on plant metabolism and development. Therefore, plants have established a tightly controlled system, including protein transporters to balance the uptake and utilization of metal ions. In this study, the relative expression of HvIRT1 gene, encoding the transmembrane protein IRT1 was analyzed in shoots and roots of barley (Hordeum vulgare ‘Nur’) under zinc deficiency at optimal (22 °C) or low (4 °C) temperatures. The Zn deficiency (0 μmol) caused an increase in HvIRT1 gene ex- pression under both optimal temperature condition and cold. Although, the difference in mRNA content of HvIRT1 gene in roots of barley under optimal and low temperature was not ob- serve. However, the HvIRT1 expression in leaves was higher at optimal temperature compare with cold condition. Moreover, long-term (7 days) of low temperature influence along with zinc deficiency leads to a significant decrease in the amount of HvIRT1transcripts in leaves, that corresponds to a decrease of photosynthesis rate and biomass accumulation. Overall, these findings suggest that HvIRT1 gene play an important role in plant’s response to zinc deficiency under optimal temperatures condition as well as at cold. Key words: IRT1; Hordeum vulgare; zinc deficiency; low temperatures Izražanje IRT1 gena v sejankah ječmena ob pomanjkanju cin- ka pri optimalnih in nizkih temperaturah Izvleček: Pomanjkanje ali prebitek cinka (Zn) povzročata negativne učinke na presnovo in razvoj rasttlin. Zaradi tega so rastline razvile dobro nadzorovan sistem, vključno s proteinski- mi transporterji za uravnavanje privzema in porabe kovinskih ionov. V raziskavi je bilo analizirano izražanje HvIRT1 gena, ki kodira transmembranski protein IRT1 v poganjkih in koreni- nah ječmena (Hordeum vulgare ‘Nur’) ob pomankanju zinka pri optimalni (22 °C) in nizki (4 °C) temperaturi. Pomanjkanje cin- ka (0 μmol) je povzročilo povečano izražanje HvIRT1 gena pri optimalni kot pri nizki temperaturi. Razlika v vsebnosti mRNK HvIRT1 gena v koreninah ječmena v optimalnih razmerah in pri nizki temperaturi ni bila opažena, a kljub temu je bilo iz- ražanje gena HvIRT1 v listih večje pri optimalni temperaturi v primerjavi s hladnimi rastnimi razmerami. Daljša izpostavitev (7 dni) nizki temperature je ob pomanjkanju cinka povzročila značilno zmanšanje transkriptov HvIRT1 v listih, kar ustreza upadu fotosinteze in akumulacije biomase. Ta odkritja nakazu- jejo, da igra HvIRT1 gen pomembno vlogo pri odzivu rastlin na pomanjkanje cinka tako v optimalnih razmerah kot pri nizkih temperaturah. Ključne besede: IRT1; Hordeum vulgare; pomanjkanje cinka; nizke temperature Acta agriculturae Slovenica, 117/4 – 20212 N. KAZNINA et al. 1 INTRODUCTION Zn deficiency has been recognized as an impor- tant factor affecting crop production. Cell transmem- brane proteins from ZIP family (zinc-iron-regulated transporter) play an important role in providing plants of the necessary amount of zinc (Pedas et al., 2008; Lee and An, 2009; Yamunarani et al., 2013). The IRT1 (iron- regulated transporter1) proteins, belonging to the ZIP family, were firstly discovered in cereals. ZIP proteins are able to transport various divalent cations, such as Fe2+, Zn2+, Cu2+, Mn2+from the rhizosphere through the plas- ma membrane into the cytoplasm of root cells, as well as from xylem vessels in leaf mesophyll cells (Palmer and Guerinot, 2009). It has been reported that zinc deficiency leads to increase in the activity of IRT1 protein and IRT1 gene expression in parallel with high accumulation of Zn in roots and shoots of rice, maize and Arabidopsis (Ishi- maru, 2006; Pedas et al., 2008; Yamunarani et al., 2013; Kabir et al., 2017 etc.). Therefore, IRT1 protein play es- sential role in Zn uptake, translocation and storage of Zn in plant cells especially under Zn deficiency. Although, most of the evidence from these studies was performed on plants under optimal temperature conditions, howev- er, in nature plants are often exposed to low temperatures during the growing season, that caused in decrease in supply of nutrients to root cells, that result in their defi- ciency in plants (Hacisalihoglu et al., 2001). Perhaps this effect may be associated with a decrease in the activity of transport proteins (Guerinot, 2000; Hacisalihoglu et al., 2001). Despite this data, the effect of cold on IRT1 gene expression and IRT1 protein activity is still unclear. Some reports demonstrated that the IRT1 protein activity regu- lated at the both translation and transcription level (Shin et al., 2013; Brumbarova et al., 2015). According these findings, we studied the expression of the IRT1 gene in the roots and leaves of barley under zinc deficiency at op- timal and low temperatures. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL AND GROWTH CONDI- TIONS Seeds of barley (Hordeum vulgare ‘Nur’)were pur- chased from the Tula Research Institute of Agriculture, Tula, Russia. Seedlings were cultivated in a growth cham- ber with 14 h photoperiod, a photo-synthetic photon flux density of 180 μmol m-2 s-1, a temperature of 22 °C and a relative humidity of 60 - 70 % on Hoagland-Arnon nutri- ent solution (pH 6.2 to 6.4) with optimal (variant Zn 2 μmol + 22 °C) zinc content or its deficiency (variant Zn 0 μmol + 22 °C). Seven-day-old seedlings (initial level) were separated. One part of the plants of both variants was exposed to low temperature (4 ° С) (variants Zn 2 μmol + 4 °C and Zn 0 μmol + 4 °C), and the other part was left under the optimal temperature during 7 days. All parameters were measured at day 0 (initial level) and 1, 3, 7 days after treatments. 2.2 BIOMASS AND NET PHOTOSYNTHETIC RATE DETERMINATION For biomass determination plants were collected, their shoots and roots separated and dried in an oven at 85 °C for 24 h. The net photosynthetic rate (PN) was measured during a day using portable photosynthesis system HCM-1000 (Walz, Effeltrich, Germany). 2.3 GENE EXPRESSION The expression pattern of HvIRT1 gene in leaves and roots was monitored by real-time PCR. Frozen roots and leaf tissues were homogenized with liquid nitrogen. To- tal RNA was extracted using a TRizol reagent (Evrogen, Moscow, Russia) as instructed by them anufacturer. The total RNA was treated with RNase free DNase (Syntol, Moscow, Russia) to remove genomic DNA. The purity of RNA samples and their concentrations were determined spectrophotometrically (SmartSpecPlus, Bio-Rad, Hercu- les, USA): samples withA260/A280 ratios within 1.8 - 2.0 were used for further analysis. The total RNA (1 μg) was reverse-transcribed using a MMLV RT kit (Evrogen) fol- lowing the supplier’s recommendations. Real-time quan- titative PCR was performed using the iCycler iQ detec- tion system (Bio-Rad). Analyzes were performed using a SYBR Green PCR kit (Evrogen). The PCR conditions consisted of denaturation at 95 °С for 5 min followed by 45 cycles of denaturation at 95 °С for 15 s, annealing at 56 °С for 30s, and extension at 72 °С for 45 s. A dissocia- tion curve was generated at the end of each PCR cycle to verify that a single product was amplified using iCycler iQ. To minimize sample variations, mRNA expression of a target gene was normalized relative to the expression of a housekeeping gene actin. The mRNA content of target gene (HvIRT1) were quantified in comparison to the ac- tin by the ΔΔCt method (Livak and Schmittgen, 2001). Primers were designed (using the Primer Design pro- gram): HvActin (U21907) ATGTTTTTTTCCAGACG (direct) and ATCCAAGCCAACCCAAGT (reverse), HvIRT1 (EU54802) GTGCTTCCACCAGATGTTTGAG (direct) andGGATGCCGACGACGATGA (reverse). Acta agriculturae Slovenica, 117/4 – 2021 3 Expression of IRT1 gene in barley seedlings under zinc deficiency at optimal and low temperatures 2.4 STATISTICAL ANALYSIS All data are presented as means ± standard errors (SEs) from at least three independent replicates. Signifi- cant differences between variants and relative to the ini- tial level were calculated by two-way analysis of variance (ANOVA) using Microsoft Excel 2010. Student’s t-test was applied to compare statistical significance at level of p < 0.05. 3 RESULTS AND DISCUSSION Table 1 shows the effect of Zn deficiency under optimal and low temperatures on plant dry mass (DM) accumulation and net photosynthetic rate (PN). Un- der optimal temperature conditions Zn deficiency did not significant affect the DM and PN parameters com- pare with plants grown with optimal Zn concentration. However, after 7 days Zn deficiency (Zn 0 μmol + 22 °C) caused slightly reduction root DM compare with variant Zn 2 μmol + 22 °C. Despite the Zn concentration the low temperature leads to reduce DM accumulation and PN parameter. Al- though, after 7 days Zn deficiency in combination with low temperature (Zn 0 μmol + 4 °C) leads to significant decrease in root DM and photosynthesis activity com- pare with variant Zn 2 μmol + 4 °C. Under optimal growth conditions (variant Zn 2 μmol + 22 °C) the transcript level of HvIRT1 gene gradu- ally increase in roots and leaves of barley during 7 days (Fig.).While, at the initial level, the HvIRT1 gene expres- sion was 3-fold higher in roots seedlings grown with Zn deficiency (Zn 0 μmol + 22 °C). Further HvIRT1 gene mRNA content in variant Zn 0 μmol + 22°C slightly in- creased in compare with variant Zn 2 μmol+ 22 °C. At the initial level there was no significant difference in mRNA content of HvIRT1 gene in leaves between Zn 2 μmol + 22 °C and variant Zn 0 μmol + 22 °C variants (Fig.). However, leaves of barley exposed to Zn deficiency (Zn 0 μmol + 22 °C) showed higher the HvIRT1mRNA content within 1 day and slightly decrease on seventh day in compare with Zn 2 μmol + 22 °C variant. Low temperature caused an increase in HvIRT1 gene expression in roots along with time of exposure in both variants (Zn 2 μmol + 4 °C and Zn 0 μmol + 4 °C) (Fig.). However, on 7th day of experiment the amount of HvIRT1 gene transcripts in roots of barley variant Zn 0 μmol + 4 °C were greater than in variant Zn 2 μmol + 4 °C. Low temperature resulted in HvIRT1 gene transcript accumulation in leaves of barley variant Zn 2 μmol + 4 °C. After 1 day of cold impact the HvIRT1 gene expres- sion was 4-fold higher in variant Zn 2 μmol + 4 °С com- pare with initial level and 10-fold higher on seven day of experiment. In variant Zn 0 μmol + 4 °C the HvIRT1 gene mRNA content increased after 3 days of exposure to low temperature and significant decreased on seven day of experiment. In general, our results demonstrated a high toler- ance of barley ‘Nur’ to Zn deficiency. It was shown that under conditions of zinc deficiency, the accumulation of biomass and photosynthetic activity remained stable un- til the end of the experiment. Similar data were described previously (Hajiboland and Beiramzadeh. 2008; Kabir et al., 2017). The capability of plants to grow under Zn deficiency mostly depend on metal transporters activity, including protein IRT1 (Suzuki et al., 2012; Yamunarani et al., 2013; Kabir et al., 2017). Zn deficiency under op- timal temperature leads to increase in HvIRT1 transcript amount in roots and leaves of barley that resulted in ac- tivation of transport metal ions in cells and kept growth and photosynthetic activity. This also support the fact that after 7 days the gene expression of HvIRT1 decreased along with photosynthetic activity. The negative effect of Zn deficiency on photosynthesis process was described previously in Oryza sativa L. (Hajiboland and Beiramza- deh, 2008), Zea mays L. (Liu et al., 2016), Sorgum bicolor (L.) Moench (Li et al., 2013), however the transport pro- tein activity was not studied. There are fragmentary data about influence of low temperature on metal transporters activity. The negative effect of 0°С temperature on gene expression, encoding protein transporters ZIP1 and ZIP3 was described pre- viously (Grotz et al., 1998). We have also shown the in- crease in HvIRT1 gene expression in barley under chilling (Kaznina et al., 2019). It is supposed that due to negative influence of chilling on nutrient transport into cells the increase in HvIRT1 gene expression in roots that we re- ported can be a result of requirement of mesophyll cells of leaves in nutrients that are necessary for photosyn- thesis process. There are no data about activity of trans- porter proteins under chilling and Zn deficiency condi- tion. Thus considering the results described above for barley under low temperature and Zn deficiency during 7 days in leaves the HvIRT1 mRNA content significantly decreased. It seems to be a result of decrease in require- ment in nutrients caused by slowdown of photosynthetic activity and growth of seedlings under stress conditions. Additionally, it can be a result of interruption in signal transduction from leaves to roots that was demonstrated previously in plants under chilling and optimal nutrient level (Giehl et al., 2009; Romera et al., 2011). Acta agriculturae Slovenica, 117/4 – 20214 N. KAZNINA et al. Variant Time, days 0 (initial point) 1 3 7 Dry root biomass, mg Zn 2+22°C 5.51 ± 0.55 аA 6.72 ± 0.48 аA 7.29 ± 0.42 bA 8.21 ± 0.60 bA Zn 0 +22°C 5.10 ± 0.45 aA 6.51 ± 0.41 aA 6.92 ± 0.38 bA 7.96 ± 0.46 bA Zn 2+4°C 5.51 ± 0.55 аA 6.20 ± 0.47 aA 6.26 ± 0.48 aB 7.36 ± 0.54 bAB Zn 0+4°C 5.10 ± 0.45 aA 6.13 ± 0.29 aA 6.23 ± 0.35 aB 6.36 ± 0.46 aB Dry shoot biomass, mg Zn 2+ 22°C 20.13 ± 1.88 aA 20.89 ± 1.21 aA 27.21 ± 1.48 bA 31.32 ± 2.41 bA Zn 0 +22°C 18.99 ± 1.89 aA 19.51 ± 1.09 aA 24.06 ± 1.62 bA 32.13 ± 1.32 bA Zn2+4°C 20.13 ± 1.88 aA 20.00 ± 1.51 aA 20.87 ± 1.50 aB 24.77 ± 1.39 bB Zn 0 +4°C 18.99 ± 1.89 aA 19.63 ± 1.16 aB 18.42 ± 0.95 aC 19.28 ± 1.21 aC Net photosynthetic rate, μmol СО2 m 2·s-1 Zn 2+ 22°C 6.82 ± 0.16 aA 7.77 ± 0.17 bA 6.54 ± 0.50 aA 6.53 ± 0.10 aA Zn 0 +22°C 6.95 ± 0.14 aA 7.69 ± 0.14 bA 6.46 ± 0.49 aA 6.27 ± 0.12 aA Zn2+4°C 6.82 ± 0.16 aA 4.65 ± 0.20 bB 4.86 ± 0.12 bB 4.16 ± 0.09 cB Zn 0 +4°C 6.95 ± 0.14 aA 4.83 ± 0.12 bB 4.35 ± 0.15 bB 3.82 ± 0.17 cC Table 1: The effect of zinc deficiency on the root and shoot dry biomass and photosynthesis rate of barley plants ‘Nur’ at optimum (22 °C) and low (4 °C) temperatures Different lowercase letters indicate significant differences in columns (between variants), uppercase letters - in rows (relative to the initial level) (p < 0.05).Values perform mean ± SE (n = 10) Figure 1: The effect of zinc optimum (a, c) and zinc deficiency (b, d) on HvIRT1 gene transcription in the roots and leaves of bar- ley plants ‘Nur’ at 22 °C (a, b) and 4 °C (c, d). Different lowercase letters indicate significant differences relative to the initial level (p < 0.05) Acta agriculturae Slovenica, 117/4 – 2021 5 Expression of IRT1 gene in barley seedlings under zinc deficiency at optimal and low temperatures 4 CONCLUSIONS According to our results it was shown that Zn defi- ciency caused in increase in HvIRT1 gene expression in leaves of barley not only under optimal temperature as was shown in another reports but also under cold. For the first time we demonstrated that long-term exposure (7 days) to low temperature leads to significant decrease in HvIRT1 mRNA amount in leaves in parallel to slow down in photosynthetic activity and growth. Taken to- gether, the results presented here illustrate the participa- tion of the HvIRT1 gene in adaptation to Zn deficiency under optimal and low temperatures conditions. 5 ACKNOWLEDGEMENTS This research was carried out using the equipment of the Core Facility of the Karelian Research Centre of the Russian Academy of Sciences and under state order (No. 0218-2019-0074). 6 REFERENCES Brumbarova, T, Bauer, P, Ivanov, R (2015). Molecular mecha- nisms governing Arabidopsis iron uptake. Trends in Plant Science, 20(2), 124–133. https://doi.org/10.1016/j. tplants.2014.11.004 Giehl, R. F. H., Meda, A.R., von Wirén, N. (2009). Moving up, down, and everywhere: signaling of micronutrients in plants. Current Opinion in Plant Biology, 12, 320–327. htt- ps://doi.org/10.1016/j.pbi.2009.04.006 Grotz,N., Fox, T., Connolly, E., Park, W., Guerinot, M.L., Eide, D. (1998). Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proceedings of the National. Academy of Sciences USA, 95, 7220–7224. https://doi.org/10.1073/pnas.95.12.7220 Guerinot,M.L.(2000). The ZIP family of metal transporters. Biochimica et Biophysica Acta, 1465, 190–198. https://doi. org/10.1016/S0005-2736 (00)00138-3 Hacisalihoglu,G., Hart, J.J., Kochian, L.V. (2001). High- and low- affinity zinc transport systems and their possible role in zinc efficiency in bread wheat. Plant Physiology, 125, 456–463. https://doi.org/10.1104/pp.125.1.456 Hajiboland, R., Beiramzadeh, N. (2008). Growth, gas exchange and function of antioxidant defense system in two contrast- ing rice genotypes under Zn and Fe deficiency and hypoxia. Acta Bioogica. Szegediensis, 52(2), 283–294. http://www. sci.u-szeged.hu/ABS Ishimaru, Y., Suzuki, M., Tsukamoto, T., et al. (2006). Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. The Plant Journal, 45, 335–346. https://doi.org/10.1111/ j.1365-313X.2005.02624.x Kabir, A. H., Hossain, M.M., Khatun, M.A., Sarkar, M.R., Haid- er, S.A. (2017). Biochemical and molecular mechanisms associated with Zn deficiency tolerance and signaling in rice (Oryza sativa L.). Journal of Plant Interactions, 12(1), 447–456. https://doi.org/10.1080/17429145.2017.1392626 Kaznina, N.M., Titov, A.F., Repkina, N.S., Batova, Yu.V. (2019). Effect of zinc excess and low temperature on the IRT1 gene expression in the roots and leaves of barley. Dokla- dy Biochemistry and Biophysics, 48, 264–268. https://doi. org/10.1134/S1607672919040057 Lee, S., A., G. (2009). Over-expression of OsIRT1 leads to in- creased iron and zinc accumulations in rice. Plant, Cell and Environment, 32, 408–416. https://doi.org/10.1111/j.1365- 3040.2009.01935.x Li, Y., Zhang, Y., Shi, D., Kiu, X., Qin, J, Ge, Q. … Xu, J. (2013). Spatial-temporal analysis of zinc homeostasis reveals the response mechanisms to acute zinc deficiency in Sor- ghum bicolor. New Phytologist, 200, 1102–1115. https://doi. org/10.1111/nph.12434 Liu, H., Gan, W., Renge, Z., Zhao, P. (2016). Effects of zinc fertilizer rate and application method on photosynthetic characteristics and grain yield of summer maize. Journal of Soil Science and Plant Nutrition, 16(2), 550–662. https://doi. org/10.4067/S0718-95162016005000045 Livak, K.J., Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔct method. Methods, 25, 402–408. https://doi. org/10.1006/meth.2001.1262 Palmer, G.M., Guerinot, M.L. (2009). Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nature Chemical Biol- ogy, 5, 333–340. https://doi.org/10.1038/nchembio.166 Pedas, P., Ytting, C. K., Fuglsang, A. T., Jahn, T. P., Schioerring, J. K., Hasted, S. (2008). Manganese efficiency in barley: identification and characterization of the metal ion trans- porter HvIRT11[OA]. Plant Physiology, 148, 455–466. https:// doi.org/10.1104/pp.108.118851 Romera, F. J., García, M. J., Alcántara, E., Pérez-Vicente, R. (2011). Latest findings about the interplay or auxin, ethyl- ene and nitric oxide in the regulation of Fe deficiency re- sponses by strategy I plants. Plant Signaling and Behavior, 6, 167–170. https://doi.org/10.4161/psb.6.1.14111 Shin, L.-J., Lo, J.-C., Chen, G. H., Callis, J., Fu, H., Yeh, K-C. (2013). IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis. The Plant Cell, 25, 3039–3051. https://doi. org/10.1105/tpc.113.115212 Suzuki, M., Bashir, K., Inoue, H., Takahashi, M., Nakanishi, H., Nishizawa, N.K. (2012). Accumulation of starch in Zn- deficient rice. Rice, 59, 1–8. https://doi.org/10.1186/1939- 8433-5-9 Yamunarani, R., Ramegowda, V., Pavithra, J., Geetha, G., Rajashekar-Reddy, H., Udayakumar, M., Shankar, A. G. (2013). Expression of a rice Zn transporter, OsZIP1, in- creases Zn concentration in tobacco and finger millet transgenic plants. Plant Biotechnology Reports, 7, 309–319. https://doi.org/10.1007/s11816-012-0264-x Acta agriculturae Slovenica, 117/4, 1–14, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2166 Original research article / izvirni znanstveni članek Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance applied at the beginning of vegetative and reproductive stages Arman ESKANDARI SHAHRAKI 1, 2, Mohammad-Mehdi SOHANI 1, Sadollah HOOSHMAND 3, Ali AALAMI 1, Habibullah SAMIZADEH 1 Received April 10, 2021; accepted November 29, 2021. Delo je prispelo 10. aprila 2021, sprejeto 29. novembra 2021 1 Department of Agriculture and Plant Breeding, Guilan University, Rasht, Iran 2 Corresponding author, e-mail: ph.d.eskandari@gmail.com 3 Department of Agriculture and Plant Breeding, Shahrekord University, Shahr-e-kord, Iran Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance applied at the beginning of veg- etative and reproductive stages Abstract: Roots play an important role in wheat grain yield, especially under drought stress conditions. To investigate root characteristics under drought stress conditions in bread wheat, 90 lines F10 obtained from the crossing (‘Yecora Rojo’ × ‘Chinese Spring’) randomly with the parents of the population were examined. The study was conducted in the form of a split- plot design with a randomized complete block base in three conditions including: 1. no stress, 2. application of drought stress at the beginning of the vegetative stage, and 3. application of drought stress at the beginning of the reproductive stage. The results showed, interaction between genotype and condition of drought was significant for all root-related traits, except shallow root dry mass, at the level of 1 % probability. The response of root-related traits to different types of drought stress was very complex. The longest root length, decrease for 13.3 % was dur- ing stress at the beginning of the vegetative stage in compari- son to non-stress conditions, while the same trait increased for 4.9 % during stress at the beginning of the reproductive stage, comparison to non-stress conditions. The results of principal component analysis under non-stress conditions showed that by considering the distribution of genotypes compared to the first two components, genotypes can be identified that have more yield with the proper root condition and vice versa. Key words: deep root; drought tress; main components; shallow root; tolerance index Ovrednotenje lastnosti korenin krušne pšenice (Triticum aes- tivum L.) povezanih s sušnim stresom v začetku vegetativne in reproduktivne faze razvoja Izvleček: Korenine imajo pomembno vlogo za pridelek zrnja pšenice, še posebej v razmerah suše. Za preučevanje zna- čilnosti korenin krušne pšenice v razmerah sušnega stresa je bilo pridobljenih 90 linij F10 iz naključnih križanj med star- ševskima sortama Yecora Rojo in Chinese Spring. Raziskava je bila izvedena kot poskus z deljenkami kot popolni naključni bločni poskus v treh stresnih razmerah: 1. brez stresa, 2. sušni stres na začetku vegetativne faze razvoja in 3. sušni stres na za- četku reproduktivne faze razvoja. Rezultati so pokazali, da je bila interakcija med genotipom in razmerami stresa značilna za vse s koreninami povezane lastnosti na ravni 1 % verjetnosti, razen za suho maso plitvih korenin. Odziv s koreninami pove- zanih lastnosti na različne vrste sušnega stresa je bil zelo kom- pleksen. Zmanšanje dolžine najdaljših korenin za 13,3 % v pri- merjavi s kontrolo je bilo, ko je sušni stres nastopil na začetku vegetativne faze razvoja med tem, ko se je isti parameter pove- čal za 4,9 % ob nastopu sušnega stresa na začetku reproduktiv- ne faze razvoja v primerjavi z nestresnimi razmerami. Rezultati analize glavnih component iz poskusa v nestresnih razmerah so pokazali, da bi z upoštevanjem razvrstitve genotipov glede na dve prvi komponenti te lahko razdelili na tiste, ki imajo več pridelka in primeren koreninski sistem in obratno. Ključne besede: globoke korenine; sušni stres; glavne komponenete; plitve korenine; tolerančni indeks Acta agriculturae Slovenica, 117/4 – 20212 A. ESKANDARI SHAHRAKI et al. 1 INTRODUCTION Water scarcity is a serious challenge to survival, es- pecially in arid and semi-arid regions. Different climatic models predict that drought stress will increase in fre- quency and intensity and this will confirm the shortage of available water in the future (Nadeem, 2019). There- fore, it seems necessary to understand the consequences of these changes on the production of different types of crops (Shanker, 2014). To date, many traits such as: number of seeds per plant, plant biomass, thousand-seed mass (Ghassemi-Golezani, 2018), grain yield, harvest in- dex (Kadam, 2012), relative leaf water content, amount of tissue lost water, leaf wax content, leaf thickness, stomatal characteristics (Heidari, 2012), number of days to spike, number of days to physiological maturity, plant shading temperature, green content of the plant (Hasani, 2016), to study how crops react to drought stress in the field and laboratory has been studied. Most of the collected information about drought stress is related to traits that consider conditions above the soil surface and limited at- tention has been paid to plant root traits. Ignoring the selection of root traits in wheat is mainly due to problems in measuring the traits of the root system and how the roots are distributed (Richards, 2008). Under water re- striction conditions, plant growth is directly determined by the ability to absorb and convert water into plant biomass (Jin et al., 2018), therefore the ability of roots to grow under drought stress conditions is an adaptive feature for plants, especially in rainfed conditions and with limited irrigation (Dalal, 2018). Root architecture is one of the most promising features for drought stress and can be used positively in drought resistance breeding programs. This feature enables the plant to extract water more efficiently from deeper soil layers, under very dry environments (Nadeem, 2019). Therefore, genetic modi- fication of plants to have an effective root system, with all its problems, is very important for optimal production in rainfed conditions and with limited water (Kadam, 2012). Hammer et al. (2009) showed that root architecture and its associated water uptake are more important than canopy architecture and plant light uptake for biomass and plant performance in high-density vessels. Advances in water uptake from subsoil by rainfed wheat can make a significant contribution to its yield (Jin, 2015b). In or- der for wheat to be highly productive, it is necessary to remove barriers to plant growth by supporting the root system that is effective in absorbing water and nutrients (Jin, 2015a). Drought tolerance depends on the plant’s ability to avoid water leakage from plant tissues, which is affected by root architecture, including: increased root length, root density, and deep rooting, because the plant can much more than the soil to search for water absorp- tion (Sofi, 2018). Gao and Lynch (2016) reported that they are very effective in tolerating drought stress, increasing rooting depth, and subsequently improving water uptake from deeper soil levels. Also, traits such as root thickness, root dry mass, root volume and root density have high herit- ability that can have a positive effect on drought stress tolerance (Kadam, 2012). Axial roots are a key element in the plant root phenotype. Axial roots are the main structure of root biomass and form a framework for lat- eral root growth and therefore have a significant effect on lateral root penetration into deeper soil slopes (Gao and Lynch, 2016). On the other hand, the usefulness of a strong root system to increase yield in environments without water stress is much more effective than the same type of root system in drought stress conditions, because a strong root system may reduce the risk of depletion of soil water before completion, increase the grain filling stage (Sofi, 2018). In general, according to different reports for the root system, the root reaction under drought stress conditions is very complex and it is very difficult to maintain a bal- ance between traits to modify its characteristics. Howev- er, roots are a semi-latent plant that is difficult to ignore their importance in yield (Koolachart et al., 2013; Bardg- ett et al., 2014). At present, the study of root systems and their importance in water and nutrient uptake and their role in drought stress resistance has been considered by agricultural researchers. The study of plant root systems is very limited due to the difficulties associated with root studies, including observation, measurement of related traits and their manipulation in the field and the use of destructive methods (Sharma et al., 2011; Thangthong et al., 2016). Carrying out agronomic and physiological studies related to root systems in wheat and their results can be very useful in breeding programs to promote, adapt and stabilize grain yield of new wheat cultivars. As a result, identifying and understanding root characteristics for crop development is essential in stressful conditions (Jin et al., 2018). Due to the scarcity of water resources in the country and facing a drought crisis, it is necessary to pay attention to valuable gene resources for use in the wheat breeding program. Identifying beneficial gene sources and genes that control drought tolerance and improving some traits and creating ideal types will play an impor- tant role in the development of wheat breeding programs. Therefore, the purpose of this study was to investigate the important root-related traits in recombinant (RIL) layers of bread wheat in tolerance to drought stress applied at the beginning of the vegetative and reproductive stages. Acta agriculturae Slovenica, 117/4 – 2021 3 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... 2 MATERIALS AND METHODS From 156 recombinant F10 inbred lines from the cross between two Chinese Spring cultivars as female parents and ‘Yecora Rojo’ as male parents, 90 lines were randomly selected and named with RIL letters and geno- type number. This population was prepared by single seed selection. The two parents of the population differed sig- nificantly in some drought tolerance characteristics such as carbon isotope discrimination and other agronomic and morphological traits (Ehdaie and Waines, 1994). In addition to 90 recombinant inbred lines, the parents of the population and four cultivars named Sorkhatakhm, Pishtaz, Kalhidari and Aflak were also examined in this research. This study was conducted in the form of a split- plot design with a randomized complete block base in three conditions including: 1. no stress, 2. application of drought stress at the beginning of the vegetative stage (code 30 of Zadoks growth scale), and 3. application of drought stress at the beginning of the reproductive stage or boots swollen (code 45 of Zadoks growth scale). Since accurate evaluation of root characteristics in field condi- tions is almost impossible, an attempt was made to use near-field and controllable conditions in this field. First, a part of the field was selected, then the sur- face soil of that area was collected to a depth of 35 cm from the ground. In the same area, a canal 1 m deep, 2 m wide and 25 m long was dug to place the planting tubes. In order to implement the experimental design, plastic pipes with a diameter of 12 cm and a length of one meter were prepared and the pipes with a homogeneous com- bination of soil containing 35 % of arable soil collected from the field and 65 % of sand (in order to facilitate root tissue separation), was filled and used as an experimental unit. At the end of the plastic pipes, there were holes for water to drain. Also, the soil prepared for filling the cul- ture tubes was sampled and sent to the laboratory to de- termine the field capacity (FC), wilting point (PWP) and other characteristics, and the soil characteristics used are given in Table 1. In each experimental unit, three seeds of one geno- type were planted and after confirming the establishment of plants, one plant was maintained and the rest were re- moved. Drought stress was applied in two phenological stages including: beginning of the vegetative stage (code 30 of Zadoks growth scale), and beginning of the repro- ductive stage or boots swollen (code 45 of Zadoks growth scale) with complete cessation of irrigation, after each line reached the desired phenological stage. Under con- ditions without drought or normal stress, irrigation was done when the volume percentage of moisture reached about 70 % of the soil field capacity within the cultivation pipes. Volumetric moisture content was measured by PMS-714 humidity meter every three days in stress-free treatment. In order to properly feed the wheat, the ferti- lizer regime used for each cultivation tube, under stress and non-stress conditions, included 500 ml of Hoogland 50  % fertilizer solution, which was added to each tube in two stages and 250 ml in each stage, before reaching Added to the desired phenological stages for stress ap- plication and irrigation time. After physiological exami- nation of most recombinant inbred lines and before har- vest, the height of the last leaf from the soil surface was measured. To obtain healthy roots and prevent damage to them, the planting tubes were gently removed from the pit and placed on a horizontal platform, then the tubes were cut and the soil mass inside them was carefully re- moved. The tubes containing the remaining soil that had not been removed from the roots were then gently in- serted into the tub to remove the sand around the roots. The washed roots were placed on a plastic surface and the traits related to the roots included: the size of the longest root, the mass of the shallow roots (zero to 30 cm depth), the mass of the deep roots (deeper depth). (30 cm), root biomass, root to biomass ratio of total plant, root to shoot ratio, plant height, stem dry mass, panicle length, panicle mass, number of seeds per panicle, seed mass per plant, plant biomass, mass thousands of seeds and biological yield were measured. For more detailed information on the tolerance or susceptibility of genotypes to drought stress, stress tolerance indices, which in many studies had a significant correlation with yield, including mean productivity (MP) indices, geometric mean productivity (GMP) and stress tolerance index (STI) were calculated and evaluated. Yp and Ys yield genotypes under normal and stress conditions, respectively. Descriptive statistics, analysis of variance, evalua- tion of genotype response based on tolerance or drought sensitivity indices, ranking of genotypes based on toler- ance indices at all levels of stress, grouping of genotypes based on average rank index and checking the accuracy of grouping by independent comparison test, was done. Euclidean distance was used to measure the dissimilar- ity between genotypes by hierarchical clustering method (average linkage method). The main components for Sand (%) Silt (%) Clay (%) EC (ds.m-1) FC (%) P.W.P (%) pH 70 13.5 16.5 3.39 37.1 15.4 7.4 Table 1: The soil profile used to fill the planting tubes Acta agriculturae Slovenica, 117/4 – 20214 A. ESKANDARI SHAHRAKI et al. each of the stress-free and stress-free environments were analyzed. 3 RESULTS AND DISCUSSION The results of analysis of variance from the study of traits are presented in Table 2. According to the results, it was found that there is a significant difference between genotypes in terms of all root-related traits at the level of one percent probability. Also, the interaction of geno- type under environmental conditions was significant for all root traits, except shallow root dry mass, at the level of 1  % probability. Bardgett et al. (2014) reported that diversity in root traits not only exists between different species and cultivars, but also the diversity of root traits within the species is very high, and this has the potential for plants. It helps to show different abilities in absorbing water and nutrients, and there is a lot of evidence that some root-related traits respond quickly to environmen- tal changes. In another study of drought stress on wheat, the difference between maximum root length, total root biomass, root biomass up to a depth of 30 cm and root biomass greater than 30 cm depth, the difference have shown significance (Kadam et al., 2012). Also Sofi et al. (2018) showed that drought stress sig- nificantly affects root biomass, rooting depth, total root length, root volume. The results of analysis of variance of yield-related traits and yield components showed a sig- nificant difference in the level of 1 % probability between genotypes and also the existence of genotype interaction at drought stress levels for all these traits (Table 2). This indicates genetic diversity as well as different trends in the response of genotypes to drought stress conditions. Sinha et al. (2019) reported that there is an inverse relationship between drought stress levels and yield. Among the studied traits, the response of root-re- lated traits to different types of drought stress was of par- ticular importance (Table 3). The longest root decrease by 13.3  % compared to non-stress conditions during stress was determined at the beginning of the vegetative stage, while the same trait increased by 4.9 % compared to non-stress conditions during stress at the beginning of the reproductive stage. Root system-related studies that emphasize grain yield in wheat have reported a variety of positive, negative, and neutral relationships (Sofi et al., 2018). In 2018, study on wheat roots under drought stress, drought stress significantly increased the root length of some genotypes (Dalal et al., 2018). During the applica- tion of stress at the beginning of the vegetative stage, the dry mass of shallow roots (depth between 0 to 30 cm) decreased by 2.8  % compared to the non-stress condi- tions. The main feature of shallow roots, which are lo- cated at upper soil levels, is mainly the absorption of wa- ter-soluble nutrients, and the performance of roots with a penetration depth of more than 30 cm, in most cases water absorption from the deeper soil levels. (Ehdaie et al., 2016). Root biomass decreased by 13.1 % compared to non-stress conditions during stress application at the beginning of vegetative stage, while the same trait shows a 3.4 % increase compared to non-stress conditions dur- ing stress application at the beginning of reproductive stage (Table 3). Since root biomass was the result of the total dry mass of shallow and deep roots and the dry mass re- sponse of shallow roots to both drought stress conditions was decreasing, so the increase of root biomass during stress application at the beginning of reproductive stage. It can be caused by the increase in dry mass of deep roots. Biomass is a root trait that has been proposed as an im- portant feature in drought stress resistance and yield stability in bread wheat in places with variable moisture regimes (Ehdaie et al., 2012). In the ratio of root biomass to total biomass of wheat plant, a 51.9 % increase in this proportion was observed with the application of stress at the beginning of the vegetative stage and 29.6 % with the application of stress at the beginning of the reproductive stage compared to non-stress conditions (Table 3). The increase in the amount of this proportion during drought stress is due to the destructive effects of stress on plant biomass (biomass above the soil surface) which is part of the total plant biomass and is at the denominator of this proportion. The root-to-shoot ratio showed an in- crease of 94.7 % in terms of stress at the beginning of the vegetative stage and 60.5 % in terms of stress at the be- ginning of the reproductive stage compared to non-stress conditions. Stunting of plant shoots including shoots and stems is one of the primary effects of drought stress, which occurs indirectly through chemical signaling from root to stem (Jin et al., 2015a). Some reports suggest that the root-to-shoot ratio can be an important feature for drought tolerance, so that the higher the root-to-shoot ratio, the higher the plant’s tolerance to drought stress. A study on shoot and root characteristics of maize hybrids in drought tolerance showed that irrigated diets had less effect on root dry mass compared to stem dry mass (Jin et al., 2018). In the study of root-related traits, the response of the wheat plant was very different and even contradic- tory in some cases due to the stressful stage of growth. Acta agriculturae Slovenica, 117/4 – 2021 5 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... M ea n Sq ua re s S. O .V . D f Lo ng es t ro ot (c m ) Sh al lo w ro ot m as s ( g) Ro ot bi om as s (g ) Ra tio o f ro ot b io - m as s to T ot al pl an t bi om as s Ra tio o f ro ot to sh oo t Pl an t he ig ht (c m ) St em dr y m as s (g ) Sp ik e le ng th (c m ) Sp ik e m as s (g ) N um be r of g ra in pe r- sp ik e G ra in m as s pe r- pl an t (g ) To ta l pl an t b io - m as s ( g) Th ou sa nd gr ai n m as s ( g) Bi ol og ic al ye ild (g ) Bl oc k 2 10 60 .4 ns 6. 65 ** 38 .0 2* 0. 21 8n s 1. 67 ns 26 .3 ns 0. 14 ns 0. 46 ns 3. 75 ns 13 06 ns 1. 28 ns 63 .5 5* 62 .9 ns 4. 14 ns C on di tio n of D ro ug ht 2 23 22 .6 n s 0. 18 n s 12 .2 3 n s 1. 45 1 * * 9. 22 ** 92 3. 3 * * 1. 55 * 32 .3 ** 79 2. 29 ** 16 22 0* * 13 9. 49 ** 10 20 .9 ** 38 61 ** 86 3. 9* * Bl oc k × C on di tio n of D ro ug ht 2 52 1. 2 0. 23 5. 08 0. 04 7 0. 47 10 .5 0. 11 0. 19 1. 19 41 7 0. 29 5 4. 42 15 .8 0. 72 G en ot yp e 95 10 8. 9 * * 0. 12 ** 0. 94 ** 0. 03 0 * * 0. 27 ** 21 1. 7* * 0. 44 ** 4. 34 ** 7. 23 ** 30 80 ** 1. 24 1* * 11 .5 8* * 29 6* * 9. 55 ** C on di tio n of D ro ug ht × G en ot yp e 19 0 76 .9 ** 0. 07 n s 0. 82 ** 0. 00 8* * 0. 81 ** 73 ** 0. 18 ** 1. 55 ** 1. 76 ** 40 9* * 0. 27 9* * 3. 49 ** 32 .6 ** 2. 09 ** Su bs id ia ry er ro r 57 0 38 .5 4 0. 07 0. 49 0. 00 4 0. 03 16 .3 0. 07 0. 17 0. 38 11 6 0. 08 8 1. 25 7. 45 0. 51 C .V . ( % ) 21 .2 15 .5 30 .5 18 .1 29 .8 10 .4 14 .2 7. 3 23 .2 24 .4 27 .4 16 .2 11 .2 15 .5 Ta bl e 2: R es ul ts o f a na ly sis o f v ar ia nc e N s, * a nd ** : In di ca te s n o sig ni fic an t d iff er en ce , s ig ni fic an t d iff er en ce at 5  % a nd si gn ifi ca nt d iff er en ce at 1  % , r es pe ct iv el y Acta agriculturae Slovenica, 117/4 – 20216 A. ESKANDARI SHAHRAKI et al. C ha ra ct er ist ic s W ith ou t d ro ug ht st re ss St re ss at th e be gi nn in g of th e ve ge ta tiv e st ag e Pe rc en ta ge ch an ge co m pa re d to th e w ith ou t dr ou gh t s tr es s St re ss at th e be gi nn in g of th e re pr od uc tiv e st ag e Pe rc en ta ge ch an ge co m - pa re d to th e dr ou gh t s tr es s Lo ng es t r oo t ( cm ) 30 .1 7 26 .1 5 -1 3. 3 31 .6 4 +0 4. 9 Sh al lo w ro ot m as s ( g) 1. 77 1. 72 -0 2. 8 1. 75 -0 1. 1 Ro ot b io m as s ( g) 2. 37 2. 06 -1 3. 1 2. 45 +0 3. 4 Ra tio o f r oo t b io m as s t o To ta l p la nt b io m as s 0. 27 0. 41 +5 1. 9 0. 35 +2 9. 6 Ra tio o f r oo t t o sh oo t 0. 38 0. 74 +9 4. 7 0. 61 +6 0. 5 Pl an t h ei gh t ( cm ) 40 .5 0 37 .0 4 -0 8. 5 38 .3 0 -0 5. 4 St em d ry m as s ( g) 2. 04 1. 90 -0 6. 8 1. 96 -0 3. 9 Sp ik e le ng th (c m ) 6. 13 5. 46 -1 0. 9 5. 76 -0 6. 0 Sp ik e m as s ( g) 4. 38 1. 07 -7 5. 5 2. 52 -4 2. 4 N um be r o f g ra in p er -s pi ke 68 .5 9 20 .5 7 -7 0. 0 43 .1 8 -3 7. 0 N um be r o f s pi ke le ts p er -s pi ke 82 .2 0 27 .4 0 -6 6. 6 55 .2 0 -3 2. 8 G ra in m as s p er -p la nt (g ) 1. 80 0. 41 -7 7. 2 1. 02 -4 3. 3 To ta l p la nt b io m as s ( g) 8. 81 5. 04 -4 2. 8 6. 94 -2 1. 2 Th ou sa nd g ra in m as s ( g) 27 .3 2 20 .1 9 -2 6. 1 25 .2 1 -0 7. 7 Bi ol og ic al y ei ld (g ) 6. 43 2. 98 -5 3. 7 4. 48 -3 0. 3 Ta bl e 3: Th e av er ag e of tr ai ts in th re e co nd iti on s i nc lu di ng : 1 . n o st re ss , 2 . a pp lic at io n of d ro ug ht st re ss at th e be gi nn in g of th e ve ge ta tiv e st ag e, an d 3. ap pl ic at io n of d ro ug ht st re ss at th e be gi nn in g of th e re pr od uc tiv e st ag e.. Acta agriculturae Slovenica, 117/4 – 2021 7 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... In order to investigate the relationship between the measured traits, the correlation coefficient between the traits was calculated and interpreted (Table 4). Accord- ing to the results, a positive and very significant relation- ship at the level of 1 % probability was between the yield (grain mass per plant) with plant biomass (0.89**). Also, a very significant negative relationship was observed be- tween yield (grain mass per plant) with the ratio of root biomass to total plant biomass (0.71**) and ratio root to shoot biomass (0.65 **). Correlation table coefficients show a positive and very significant relationship between the longest root with root biomass (0.75**), total deep root length (0.55**) at the level of 1 % probability. Contrary to the results of this study, Kadam et al.(2012) showed that root biomass had a significant cor- relation with all measured traits except the longest root and no correlation was observed between root biomass and the longest root. In another study to locate quantita- tive traits related to root and shoot characteristics in a population of recombinant inbred lines of spring wheat, it was reported that between plant biomass and number of seeds per spikelet, dry mass of shallow roots, dry mass of deep roots and root biomass, there is a correlation at the level of 1 % probability (Ehdaie et al., 2016). Since grain yield in genotypes may be independ- ent of each other under normal and stress conditions, the tolerance or susceptibility index to stress was used to distinguish genotypes that responded better to stress conditions. The use of these indices was the identifica- tion and selection of genotypes that have relatively high yields under both normal and stress conditions. Studies show that the average productivity indices (MP), geo- metric mean productivity (GMP) and stress tolerance index (STI), due to high correlation with grain yield, are suitable for identifying high yield genotypes under nor- mal conditions, mild stress and severe drought stress (Ali and El-Sadek, 2016; Khosravi et al., 2020). In this study, the genotypes that showed the highest MP, GMP and STI and also had high yield, drought tolerant genotypes were considered and vice versa. After ranking the genotypes based on all three indices of tolerance or sensitivity to stress, the average ratings obtained for each genotype were also calculated as the mean rank index (RM). Cor- relation coefficients between the three indices of drought tolerance and yield under stress conditions at the begin- ning of the vegetative and non-stress stages showed that there is a positive and significant correlation at the level of one percent probability between all indices of stress tolerance and yield. Therefore, it seems that the indica- tors used in this study can be effective in identifying and differentiating high-yield genotypes under drought and non-stress conditions. Since the accuracy of independent comparison test is higher than independent comparison test and practical analysis test of observations or division of effects of treatments, so in order to group genotypes based on average rank index and measure the validity of grouping accuracy, the comparison test were used inde- pendently. First, the genotypes were ranked from 1 to 96 based on the mean RM (T) rank index. Then genotypes with ranks between one and 48 were placed in the first group and the rest of the genotypes were classified from 49 to 96 in the second group. For independent compari- son test, the first group was given a coefficient of +1 and the second group was given a coefficient of -1. Then, the independent comparison test between the first and sec- ond groups was performed according to the measured Characteristics Longest root (cm) Root biomass (g) Total deep roots length (cm) Ratio of root biomass to Total plant biomass Grain mass per- plant (gr) Total plant biomass (g) Ratio of root to shoot Longest root (cm) 1.00 0.75** 0.55** 0.29ns 0.19ns 0.49* 0.28ns Root biomass (g) 1.00 0.48* 0.47* 0.18ns 0.56** 0.47* Total deep roots length (cm) 1.00 0.13ns 0.03ns 0.17ns 0.12ns Ratio of root biomass to Total plant biomass 1.00 -0.71** -0.42* 0.96** Grain mass per-plant (g) 1.00 0.89** -0.65** Total plant biomass (g) 1.00 -0.37ns Ratio of root to shoot 1.00 Table 4: Correlation between the measured traits in all three stress conditions including: 1. no stress, 2. application of drought stress at the beginning of the vegetative stage, and 3. application of drought stress at the beginning of the reproductive stage Ns, * and **: Indicates no significant difference, significant difference at 5 % and significant difference at 1 %, respectively Acta agriculturae Slovenica, 117/4 – 20218 A. ESKANDARI SHAHRAKI et al. data related to grain yield in plant genotypes. The results of the independent comparison test between the two groups are given in Table 5. The results showed a signifi- cant difference in the level of 1 % probability between the two groups based on the mean rank index. Mohammadi and Abdullahi (2017) also reported the use of a stress tolerance index could not lead breed- ers to the best option and genotypes should be selected based on a combination of several tolerance indices or sensitivity to provide a more practical criterion for im- proving stress tolerance traits. Determining the size of the inbred lines and prox- imity to each other, as well as increasing productivity in effective parenting, can reduce breeding volume, costs, and time. To separate the genotypes, cluster analysis was used by Euclidean distance to measure the dissimilarity between genotypes by hierarchical clustering method (average linkage method), and finally the genotypes were divided into four main groups (Figure 1). The first group consists of two subgroups, which subgroup (a) includes eight genotypes with the names of RIL19, RIL46, RIL51, RIL62, RIL64, RIL89, RIL114 and RIL164 and subgroup (b) contains 63 genotypes. It seems that the reason for the divergence of the two subgroups from each other is how the genotypes in the group react to drought stress for grain yield per plant. So that the genotypes of sub- group (a) in all three conditions of the experiment were weak in terms of grain yield and most of them in terms of yield rank under non-stress conditions, drought stress at the beginning of vegetative stage and drought stress at the beginning of vegetative stage. Are at the bottom of the table. On the other hand, although in subgroup (b) grain yield per plant was very diverse and in some cases to some extent, but in general grain yield per plant geno- types in this subgroup, in all three conditions without stress and application of two The type of drought stress at the beginning of the vegetative and reproductive stages was favorable and was higher than the average popula- tion of the studied genotypes. The second group consists of eighteen genotypes named Aflak, RIL8, RIL9, RIL10, RIL16, RIL23, RIL48, RIL67, RIL76, RIL79, RIL82, RIL87, RIL112, RIL113, RIL122, RIL137, RIL150 and RIL151. The mean rank index of most genotypes in this group was in the upper half of the ranking table during the stress application conditions at the beginning of the vegetative stage and during the stress application condi- tions at the beginning of the reproductive stage. This indicates that the genotypes in this group did not respond well to drought stress in terms of tolerance or susceptibility to stress. The genotypes of this group in terms of the average of some measured traits such as: longest root size, root biomass and plant biomass, in all three conditions of the experiment, had higher ranks than the average of all studied genotypes. In other words, these genotypes had a favorable response to drought stress in terms of the average of the mentioned traits. In the third group, only one genotype named RIL35 was included. The fourth group consisted of six ruby egg genotypes, RIL117, RIL118, RIL141, RIL146 and RIL163. Seed yield per plant in this group was very low and weak compared to other genotypes under non-stress conditions, drought stress at the beginning of the veg- etative stage and drought stress at the beginning of the reproductive stage. However, there were good potentials among the genotypes of this group in terms of shallow root dry mass, root biomass, plant biomass and longest root. To determine the share of each measured trait in the variance of the study population, as well as reducing the number of studied traits by considering the correla- tion matrix between the measured traits and indices, the principal component analysis method was used. Under drought stress conditions at the beginning of the vegeta- tive stage, the results showed that the first four main com- ponents had specific values greater than one and together accounted for 86.19 % of the diversity in the study popu- lation (Table 6, Figure 2a). Based on the high and posi- tive specific values related to biological yield (BY), spike mass (SM), grain mass per plant (GMP) and total plant biomass (TPB) in the first component, the first compo- nent can be considered related to the yield of genotypes. In the second component, the highest eigenvalues were related to MP, GMP and STI indices, genotype yield un- der non-stress conditions (YP), 1000-grain mass (TGW) and genotype yield during drought stress conditions at the beginning of vegetative stage (YS1). The second com- ponent can be introduced as a component related to the response to sensitivity or tolerance to drought stress. The third component had the highest coefficients for deep root mass (DRM), total deep root length (TDRL), root biomass (RBio) and longest root (LR). According to these Independent comparison based on grain yield trait per-plant contrast Df Contrast ss Mean Square F value P r> F One 1 2369.8 2369.8 11.77 0.0007 Table 5: Independent comparison test between two groups of genotypes based on total rank of mean RM(T) Acta agriculturae Slovenica, 117/4 – 2021 9 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... Figure 1: Cluster analysis of 96 genotypes based on data obtained from measuring all traits Acta agriculturae Slovenica, 117/4 – 202110 A. ESKANDARI SHAHRAKI et al. Characteristics Abbreviation First component Second component Third component Fourth component Biological yeild (g) BY 0.96 -0.20 -0.10 Spike mass (g) SW 0.88 -0.21 -0.24 0.28 Grain mass per-plant (g) GWP 0.88 -0.24 -0.23 0.30 Total plant biomass (g) TPB 0.87 -0.36 0.25 0.10 Ratio of root biomass to total plant biomass RRT -0.74 0.59 0.21 Number of grain per-spike NGS 0.68 -0.58 -0.29 0.26 Ratio of root to shoot RRS -0.65 -0.10 0.64 0.26 Mean productivity MP(1) 0.37 0.87 0.24 0.10 Geometric mean productivity GMP(1) 0.41 0.86 0.23 0.14 Stress tolerance index STI(1) 0.44 0.85 0.20 0.15 Yield genotypes under normal conditions (g) YP 0.11 0.83 0.28 Thousand grain mass (g) TGW 0.54 0.74 0.15 0.22 Number of spikelets per-spike NSS 0.59 -0.60 -0.33 0.28 Deep root dry mass (g) DRW 0.15 -0.41 0.81 0.23 Total deep roots length (cm) TDRL 0.15 -0.39 0.80 0.15 Root biomass (g) Rbio 0.21 -0.44 0.80 0.24 longest root (cm) LR -0.29 0.71 Spike length (cm) SP 0.55 -0.11 0.31 -0.72 Yield genotypes under stress conditions at the beginning of the vegetative stage (g) YS(1) 0.54 0.74 0.15 0.22 Stem dry mass (g) SDW 0.56 0.30 -0.72 Plant height (cm) PH 0.55 0.33 -0.69 Shallow root mass (g) SRW 0.22 -0.22 0.23 0.12 Ratio of number of grain to number of spikelets per spike RNN 0.48 Eigen value 7.51 5.73 4.26 2.13 Variance (%) 32.69 24.95 18.54 10.00 Cumulative variance (%) 32.69 57.64 76.19 86.19 Table 6: Principal component analysis using evaluation traits under drought stress conditions at the beginning of the vegetative stage Characteristics Abbreviation First component Second component Third component Fourth component Biological yeild (g) BY 0.95 0.22 0.16 Spike mass (g) SW 0.91 0.18 0.33 Grain mass per-plant (g) GWP 0.91 0.19 0.33 Total plant biomass (g) TPB 0.86 0.21 0.39 0.18 Ratio of root biomass to total plant biomass RRT -0.81 -0.16 0.53 Number of grain per-spike NGS 0.89 -0.36 0.21 Table 7: Principal component analysis using evaluation traits under drought stress conditions at the beginning of the reproduc- tive stage Acta agriculturae Slovenica, 117/4 – 2021 11 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... Characteristics Abbreviation First component Second component Third component Fourth component Biological yeild (g) BY 0.93 -0.25 0.19 Spike mass (g) SW 0.87 -0.34 0.21 0.24 Grain mass per-plant (g) GWP 0.87 -0.33 0.22 0.24 Total plant biomass (g) TPB 0.96 0.15 0.13 Ratio of root biomass to Total plant biomass RRT -0.43 0.87 -0.14 Number of grain per-spike NGS 0.73 -0.32 -0.55 0.19 Ratio of root to shoot RRS -0.46 0.83 -0.13 Thousand grain mass (g) TGW 0.99 Number of spikelets per-spike NSS 0.70 -0.29 -0.58 0.14 Deep root dry mass (g) DRW 0.48 0.79 0.14 Total deep roots length (cm) TDRL 0.54 0.75 Root biomass (g) Rbio 0.51 0.82 0.16 longest root (cm) LR 0.54 0.70 0.12 Spike length (cm) SP 0.74 -0.54 Stem dry mass (g) SDW 0.72 0.25 -0.56 Plant height (cm) PH 0.72 0.11 -0.58 Table 8: Principal component analysis using evaluation traits under no drought stress conditions Continued Ratio of root to shoot RRS -0.79 -0.17 0.47 Mean productivity MP(2) -0.18 0.97 Geometric mean productivity GMP(2) -0.16 0.98 Stress tolerance index STI(2) -0.15 0.97 Yield genotypes under normal conditions (g) YP -0.25 0.94 Thousand grain mass (g) TGW -0.10 0.97 Number of spikelets per-spike NSS 0.80 -0.45 0.19 Deep root dry mass (g) DRW 0.95 0.17 Total deep roots length (cm) TDRL -0.11 0.11 0.89 0.14 Root biomass (g) Rbio 0.96 longest root (cm) LR 0.16 0.84 Spike length (cm) SP 0.71 0.18 0.22 -0.56 Yield genotypes under stress at the beginning of the reproductive stage (g) YS(2) -0.10 0.97 Stem dry mass (g) SDW 0.66 0.28 0.22 -0.62 Plant height (cm) PH 0.62 0.28 0.24 -0.63 Shallow root mass (g) SRW 0.12 0.25 -0.25 Ratio of number of grain to number of spikelets per spike RNN 0.53 0.20 -0.15 Eigen value 7.87 6.52 4.31 1.65 Variance (%) 34.24 28.35 18.74 07.20 Cumulative variance (%) 34.24 62.59 81.33 88.54 Acta agriculturae Slovenica, 117/4 – 202112 A. ESKANDARI SHAHRAKI et al. Continued Shallow rootmass(g) SRW 0.30 0.46 0.13 Ratio of number of grain to number of spikelets per spike RNN 0.37 -0.25 0.26 Eigen value 7.70 4.67 2.83 1.32 Variance (%) 40.57 24.59 14.93 06.94 Cumulative variance (%) 40.57 65.16 80.10 87.05 Fi gu re 2 : Th re e- di m en sio na l d ia gr am o f t he d ist rib ut io n of m ea su re d tr ai ts a nd in di ce s r el at iv e to th e pr in ci pa l c om po ne nt s a ) i n te rm s o f s tr es s at th e be gi nn in g of th e ve ge ta tiv e st ag e, b) in te rm s o f s tr es s a t t he b eg in ni ng o f t he re pr od uc tiv e st ag e, c) in co nd iti on s w ith ou t s tr es s Fi gu re 3 : T w o- di m en sio na l d ia gr am o f t he d ist rib ut io n of g en ot yp es re la tiv e to th e fir st o f t w o pr in ci pa l c om po ne nt s a ) u nd er d ro ug ht st re ss at th e be gi nn in g of th e ve ge ta tiv e st ag e, b) u nd er d ro ug ht st re ss at th e be gi nn in g of th e re pr od uc tiv e st ag e, c) w ith ou t s tr es s Acta agriculturae Slovenica, 117/4 – 2021 13 Evaluation of traits related to bread wheat (Triticum aestivum L.) root in drought tolerance ... results, the third component related to root traits is con- sidered. Based on the output of principal components dur- ing drought stress conditions at the beginning of the vegetative stage, it can be concluded that based on the first and second components and considering the distri- bution of genotypes relative to the first two components, genotypes that in terms of the first component, they have the highest value, have more performance during stress conditions, and also according to the second component, the reaction of these genotypes to drought stress at the beginning of the vegetative stage will be visible. Accord- ingly, the genotypes at the far right of the graph (Figure 3a) have a high yield and are more tolerant to drought stress at the beginning of the growing stage. These are low-yield, drought-sensitive genotypes when drought stress was applied at the beginning of the growing stage. The results of principal component analysis dur- ing drought stress conditions at the beginning of the reproductive stage are shown in Figures (2b) and (3b). As in the case of stress at the beginning of the vegeta- tive stage, here too the first four components had specific values greater than one, describing a total of 88.54 % of the diversity in the study population (Table 7). Due to the higher and positive eigenvalues for biological yield (BY), grain mass per plant (GMP), spike mass (SM), number of grains per spike (NGS) and total plant biomass (TPB) in the first component, can be The first component was related to the performance of the population. The highest eigenvalues in the second component are related to GMP, MP, STI, 1000-grain mass (TGM) indices, genotype yield during drought stress conditions at the beginning of reproductive stage (YS2) and genotype yield under non-stress conditions (YP). Conditions also, we define the second component as the component related to the response of susceptibility or tolerance of genotypes to drought stress at the beginning of the reproductive stage. In the third component, the highest coefficients were related to root biomass (RBio), deep root mass (DRM), total deep root length (TDRL) and longest root (LR). As a result, the third component related to the diversity of root traits was considered. Under stress-free conditions, the results of principal component analysis showed that the first four principal components with eigenvalues greater than one, in total, accounted for 87.05 % of the variance in the population of the recombinant inbred lines studied (Table 8). Ac- cordingly, the first component with total plant biomass (TPB), biological yield (BY), grain mass per plant (GMP) and spike mass (SM), justified 40.57 % of the diversity in the population (Figure 2c). Therefore, even in stress-free conditions, the first component can be introduced relat- ed to the performance of genotypes. The second compo- nent with a justification of 24.59 % of the diversity of the study population and the highest specific values related to the traits of root to plant biomass ratio (RRT), root to shoot ratio (RRS), root biomass (RBio), deep root mass (DRM) and the sum of deep root lengths (TDRL), could be introduced as a component of root traits. The result of principal component analysis output under stress-free conditions showed that based on the first and second components and considering the distri- bution of genotypes relative to the first two components, it is possible to identify genotypes that have the highest yield under conditions. No stress in terms of root traits had the best reaction and vice versa (Figure 3c). 4 CONCLUSION Despite the significant difference in the level of 1 % probability between genotypes and also the interaction of genotype with environmental conditions, for all root traits except shallow root dry mass, understanding the response of these traits to the types of drought stress was high complicated. For example, the longest root trait decreased by 13.3 % compared to stress-free conditions at the beginning of the vegetative stage, while the same trait increased by 4.9 % compared to non-stress condi- tions at the beginning of the reproductive stage. Also, in the case of root biomass trait during stress application at the beginning of the vegetative stage compared to the non-stress state decreased by 13.1 %, while the same trait increased by 3.4 % during stress application at the repro- ductive stage. However, finding a successful combination of shoot and root traits that can be used in breeding to improve further growth and productivity is a big chal- lenge, because in the present study, the response of the wheat plant to many stages It is different from stressful growth and even in some cases contradictory. 5 REFERENCES Ali, M. B. and A. N. El-Sadek. (2016). Evaluation of drought tolerance indices for wheat (Triticum aestivum L.) under irrigated and rainfed conditions. Communications in Biom- etry and Crop Science, 11(1), 77-89. Bardgett, R. D., L. Mommer and F. T. De-Vries. (2014). Going underground: root traits as drivers of ecosystem processes. Trends in Ecology & Evolution, 29(12), 692-699. https://doi. org/10.1016/j.tree.2014.10.006 Dalal, M., S. Sahu, S. Tiwari, A. R. Rao and K. Gaikwad. (2018). Transcriptome analysis reveals interplay between hor- mones, ROS metabolism and cell wall biosynthesis for drought-induced root growth in wheat. Plant Physiology Acta agriculturae Slovenica, 117/4 – 202114 A. ESKANDARI SHAHRAKI et al. and Biochemistry,130, 482-492. https://doi.org/10.1016/j. plaphy.2018.07.035 Ehdaie, B., A. P. Layne and J. G. Waines. (2012). Root system plasticity to drought influences grain yield in bread wheat. Euphytica, 186, 219-232. https://doi.org/10.1007/s10681- 011-0585-9 Ehdaie, B., S. A. Mohammadi and M. Nouraein. (2016). QTLs for root traits at mid-tillering and for root and shoot traits at maturity in a RIL population of spring bread wheat grown under well-watered conditions. Euphytica, 211(1), 17-38. https://doi.org/10.1007/s10681-016-1670-x Ehdaie, B. and J. G. Waines. (1994). Genetic analysis of car- bon isotope discrimination and agronomic characters in a bread wheat cross. Theoretical and Applied Genetics, 88(8), 1023-1028. https://doi.org/10.1007/BF00220811 Gao, Y. and J. P. Lynch. (2016). Reduced crown root num- ber improves water acquisition under water deficit stress in maize (Zea mays L.). Journal of Experimental Botany, 67(15), 4545-4557. https://doi.org/10.1093/jxb/erw243 Ghassemi-Golezani, K., S. Heydari and B. Dalil. (2018). Field performance of maize (Zea mays L.) cultivars under drought stress. Acta agriculturae Slovenica, 111(1), 25-32. https://doi.org/10.14720/aas.2018.111.1.03 Hammer, G. L., Z. Dong, G. McLean, A. Doherty, C. Messina, J. Schussler, C. Zinselmeier, S. Paszkiewicz and M. Cooper. (2009). Can changes in canopy and/or root system archi- tecture explain historical maize yield trends in the US corn belt. Crop Science, 49(1), 299-312. https://doi.org/10.2135/ cropsci2008.03.0152 Hassani, F. (2016). Evalution of terminal drought tolerance and validation of its related EST-SSRs in bread wheat. Thesis for the degree of Ph. D. in Plant Breeding, Shahre-kord univer- sity, Iran. (In Persian with English abstract). Heidari, Z. (2012). Determination of chromosomal position of genes controlling some physiological traits related to drought resistance in bread wheat (Triticum aestivum) and their rela- tionship with root traits, using selected alternative lines. The- sis for the degree of M.Sc. in Plant Breeding, Shahre-kord university, Iran. (In Persian with English abstract) Jin, K., J. Shen, R. W. Ashton, R. P. White, I. C. Dodd, M. A. Par- ry and W. R. Whalley. (2015a). Wheat root growth respons- es to horizontal stratification of fertiliser in a water-limited environment. Plant and Soil, 386(1-2), 77-88. https://doi. org/10.1007/s11104-014-2249-8 Jin, K., J. Shen, R. W. Ashton, R. P. White, I. C. Dodd, A. L. Phil- lips, M. A. Parry and W. R. Whalley. (2015b). The effect of impedance to root growth on plant architecture in wheat. Plant and Soil, 392(1-2), 323-332. https://doi.org/10.1007/ s11104-015-2462-0 Jin, Z., X. Qing-wu, K. E. Jessup, H. Xiao-bo, H. Bao-zhen, T. H. Marek, X. Wenwei, S. R. Evett, S. A. O’Shaughnessy and D. K. Brauer. (2018). Shoot and root traits in drought toler- ant maize (Zea mays L.) hybrids. Journal of Integrative Ag- riculture, 5(17), 1093-1105. https://doi.org/10.1016/S2095- 3119(17)61869-0 Kadam, S., K. Singh, S. Shukla, S. Goel, P. Vikram, V. Pawar, K. Gaikwad, R. Khanna-Chopra and N. Singh. (2012). Genomic associations for drought tolerance on the short arm of wheat chromosome 4B. Functional & Integrative Genomics, 12(3), 447-464. https://doi.org/10.1007/s10142- 012-0276-1 Khosravi, S., R. Azizinezhad, A. Baghizadeh and M. Maleki. (2020). Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid and hexa- ploid cultivars of wheat using stress tolerance indices. Acta agriculturae Slovenica, 115(1), 105-112. https://doi. org/10.14720/aas.2020.115.1.1336 Koolacharta, R., S. Jogloya, N. Vorasoota, S. Wongkaewb, C. C. Holbrookc, N. Jongrungklanga, T. Kesmalaa and A. Patanothaia. (2013). Rooting traits of peanut genotypes with different yield responses to terminal drought. Field Crops Research, 149, 366–378. https://doi.org/10.1016/j. fcr.2013.05.024 Mohammadi, R. and A. Abdulahi. (2017). Evaluation of durum wheat genotypes based on drought tolerance indices under different levels of drought stress. Journal of Agricultural Sciences, Belgrade, 62(1), 1-14. https://doi.org/10.2298/ JAS1701001M Nadeem, M., J. Li, M. Yahya, A. Sher, C. Ma, X. Wang and L. Qiu. (2019). Research progress and perspective on drought stress in legumes: a review. International Journal of Molecular Sciences, 20(10), 1-32. https://doi.org/10.3390/ ijms20102541 Richards, R. A. (2008). Genetic opportunities to improve cereal root systems for dryland agriculture. Plant Production Sci- ence, 11, 12–16. https://doi.org/10.1626/pps.11.12 Shanker, A. K., M. Maheswari, S. k. Yadav, S. Desai, D. Bhanu, N. B. Attal and B. Venkateswarlu (2014). Drought stress re- sponses in crops. Functional & Integrative Genomics, 14(1), 11-22. https://doi.org/10.1007/s10142-013-0356-x Harma, S., S. Z. Xu, B. Ehdaie, A. Hoops, T. J. Close, A. J. Lu- kaszewski and J. G. Waines. (2011). Dissection of QTL ef- fects for root traits using a chromosome arm-specific map- ping population in bread wheat. Theoretical and Applied Genetics, 122, 759–769. https://doi.org/10.1007/s00122- 010-1484-5 Sinha, R., V. Irulappan, B. Mohan-Raju, A. Suganthi and M. Senthil-Kumar. (2019). Impact of drought stress on simulta- neously occurring pathogen infection in field-grown chick- pea. ScientificReports, 9(1), 1-15. https://doi.org/10.1038/ s41598-019-41463-z Sofi, P. A., M. Djanaguiraman, K. H. M. Siddique and P. V. V. Prasad. (2018). Reproductive fitness in common bean (Pha- seolus vulgaris L.) under drought stress is associated with root length and volume. Indian Journal of Plant Physiology, 23(4), 796-809. https://doi.org/10.1007/s40502-018-0429-x Thangthonga N., S. Jogloya, V. Pensukb, T. Kesmalaa and N. Vorasoot. (2016). Distribution patterns of peanut roots under different durations ofearly season drought stress. Field Crops Research, 198, 40–49. https://doi.org/10.1016/j. fcr.2016.08.019 Acta agriculturae Slovenica, 117/4, 1–13, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2217 Original research article / izvirni znanstveni članek Classification of determinant factors of irrigated vegetable problems us- ing exploratory factor analysis in Swaida governorate, Syria Maya AL-ABDALA 1, 2, Afraa SALLOWM 3, Safwan ABOUASSAF 1 Received May 26, 2021; accepted October 26, 2021. Delo je prispelo 26. maja 2021, sprejeto 26. oktobra 2021 1 General Commission for Scientific Agricultural Research (GCSAR), Syria 2 Corresponding author, e-mail: mayaabdala6@gmail.com 3 Agricultural Economics, Faculty of Agriculture, University of Damascus, Syria Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida gover- norate, Syria Abstract: The objective of this research was to classify the determinant factors of irrigated vegetable problems and the amount of variance that is explained by each factor in Swaida Governorate/ Syria by using the Exploratory Factor Analysis. The research is based on the data which were collected through questionnaires that were obtained according to the opinions of farmers. It included questions about some of the social and eco- nomic characteristics of farmers, and the concerning problems related to irrigated agriculture by using multiple-choice ques- tions (on a 3-point scale) during the 2019-2020 Based on a sam- ple size of 92 farmers, representing 54.9 % of the studied sta- tistical community, and distributed randomly within the areas of spread of irrigated vegetable cultivation.. The results showed the success of using the exploratory factor analysis technique, using the Principal components methodology and Varimax in classifying six factors with an initial eigenvalues greater than one for each, and these factors are: agricultural technological progress, agricultural employment, sale outlets, natural condi- tions, prices, production requirements. These factors explained (13.21 %, 12.65 %, 12.55 %, 11.12 %, 10.94 %, and 9.85 %) of the total variance respectively, and together explained 70.33 %. Key words: exploratory factor analysis; principal compo- nent; factors; Varimax; irrigated vegetables Razvrstitev odločitvenih dejavnikov povezanih s problemi namakanja zelenjave s faktorsko analizo na območju upravne enote Swaida, Sirija Izvleček: Namen raziskave je bil s faktorsko analizo razvr- stiti odločitvene dejavnike, povezane s problemom namakanja zelenjave, in določiti vpliv posameznega dejavnika na območju upravne enote Swaida v Siriji. Raziskava temelji na mnenjih kmetov, ki so bila pridobljena s pomočjo anketnih vprašalni- kov. Ti so vključevali vprašanja o nekaterih socialnih in eko- nomskih značilnostih kmetov in problemih z namakanjem, s katerimi se srečujejo. Anketirani so imeli možnost odgovoriti na vprašanja na 3-točkovni skali. Anketa, ki je potekala v obdo- bju 2019-2020, je temeljila na vzorcu 92 kmetov, kar je predsta- vljalo 54,9 % kmetov preučevane statistične regije. Anketiranci so bili izbrani naključno znotraj območja, kjer se pri gojenju zelenjave uporablja tudi namakanje. Rezultati so pokazali smi- selnost uporabe faktorske analize. Z uporabo metode glavnih komponent in metode Varimax je bilo šest dejavnikov, z zače- tno lastno vrednostjo večjo od ena, razvrščenih glede na delež variabilnosti, ki jo pojasnjujejo. Ti dejavniki so: razvoj agroteh- nike, zaposlitev v kmetijstvu, možnost prodaje, naravne dano- sti, cene, proizvodni stroški, pojasnjujejo pa13,21 %, 12,65 %, 12,55 %, 11,12 %, 10,94 %, in 9,85 % celotne variabilnosti oz. vsi skupaj pojasnjuejo 70,33 % celotne variabilnosti. Ključne besede: faktorska analiza; metoda glavnih kom- ponent; dejavniki; Varimax; namakanje zelenjave Acta agriculturae Slovenica, 117/4 – 20212 M. AL-ABDALA et al. 1 INTRODUCTION Agriculture is one of the important sectors in the Syrian economy, through its contribution to employing the labor force and covering the increasing food needs, especially in the current economic crisis and economic blockade. This study applies to the Swaida Governorate, where agriculture is the main production base and its ability to absorb the workforce in all steps of production until marketing, and thus contribute to solving the un- employment problem in the governor’s countryside in particular. In spite of the fluctuation in its contribution to the local product, and the difficult economic conditions amid insecurity and the lack of available resources and weak implementation of rural development programs, the minimum level of self-sufficiency in agricultural products is sought. Factor analysis is one of the important statistical methods that enabled researchers to classify scientific phenomena in multiple fields. It is used to find out the different correlations between data and to summarize it by identifying common characteristics. Therefore, fac- tor analysis is one of the applications of the inductive approach, whereby a set of relationships can be traced back to common factors that describe and explain these relationships (Zeina, 2015). There are many studies that dealt with the exploratory factor analysis methodology in various fields such as agriculture, marketing, psycho- logical sciences, and others, as it depends on the ordinal variables and listed according to a certain scale. Pavel and Moldovan (2019) in the study exploring the role of exter- nal factors determining local economic development in rural areas in Romania, based on data collected for the 398 communes from the North-West development re- gion of Romania between 2007 and 2014, using explora- tory factor analysis of principal components, where the results showed the location from urbanization and the presence of direct contact with European roads affect the level of local economic development and there is no impact of non-refundable investment programs in infra- structure in accelerating economic development. Discovering the different dimensions of food secu- rity in relation with urban agriculture based on a sample of 360 families. The results of exploratory factor analy- sis identified three latent factors from 31 statements to which respondents indicated their level of agreement on a 5-point scale: the food availability and accessibility, adequate nutritional intake and reduction in fresh food expenditure. Contribution of each factor to the total vari- ance were the following: 25.12 %, 24.29 %, and 20.2 %) respectively, as together interpreted 69.61 % (Rezai et al., 2016). In order to exploratory factor analysis of barriers and problems affecting the development of nanotechnol- ogy in agriculture, four factors with eigenvalues greater than 1, were extracted after orthogonal rotation using the varimax technique. These factors explained 74.40 % of the total variance. According to extracted results, the financial support factor with the variance of 24.18 had the highest importance in the explanatory variables. Af- ter that, the Communication – Management, the cogni- tive learning and operating infrastructure factors were following it respectively (Ahmadi et al., 2013). To assess the performance of Agri Clinic entrepre- neurs promoted under the scheme on Agri Clinic and Agri Business Centers in India. Thus, an attempt has been made to evolve a set of factors influencing the en- trepreneurial behavior through a data reduction process of factor analysis using Varimax. The factors include planning orientation, work orientation, personal ef- ficacy, market orientation, location, business acumen, dynamism, service orientation, in-depth knowledge, achievement motivation, social networks, interest, in- ternal control, marketing strategy and innovativeness. These factors collectively explained 86.91 % of the total variance (Chandrashekar et al., 2012). There are many problems and constraints facing irrigated vegetable ag- riculture that pose great risks. These constraints lead to unstable yields. The most important of them are: the prevailing and fluctuating weather conditions, the spread of diseases and insects and the epidemically, the ineffec- tiveness of pesticides and fertilizers and their high prices, in addition to the problems related to marketing such as the lack of sales outlets or oversupply, and the monopoly of brokers. Therefore, it is necessary to identify the most important factors responsible for explaining the largest proportion of the total variation in the production of irri- gated vegetable projects in Swaida Governorate by using the exploratory factor analysis methodology. The main goal is to classify the determining factors of the prob- lems of irrigated vegetable cultivation into identifying the most important factors responsible for explaining the largest proportion of the total variation in the production of irrigated vegetable projects. 2 MATERIALS AND METHODS 2.1 STUDY AREA The study was conducted in Swaida governorate, southern Syria during the 2020 agricultural season, in the places where irrigated crops spread. Knowing that the irrigated cropping patterns in Swaida governorate are divided between summer and winter crops and fruit trees. Where the area average of the irrigated pattern, Acta agriculturae Slovenica, 117/4 – 2021 3 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria excluding the fruit trees during 2016-2018 period was about 11008.33 Dunums (area unit = 1000 m2). The sum- mer cropping pattern accounted 69.86 % by about 7690 Dunums, including tomatoes, watermelons, melons, cu- cumbers, eggplant, pepper, etc., and the winter cropping pattern represented 30.14 % by about 3318.33 Dunums, including wheat, peas, cauliflower, cabbage, onions, gar- lic and others (Ministry of Agriculture and Agrarian Re- form Statistics, 2016-2018). 2.2 DATABASE The study based on preliminary data through field visits the irrigated vegetable farmers who own wells. For the interviews, a structured questionnaire was de- signed with some of the social and economic character- istics of farmers and the concerning problems related to irrigated agriculture with multiple-choice questions using a three-point scale ranging from (1 = there is no problem), (2 = medium problem), (3 = strong prob- lem) during 2019-2020 season in Swaida Governorate. Where the sample size is consisted of 92 observations represented 54.9  % of the studied statistical commu- nity, based on a formula (Glenn, 1992) (Yamane, 1967): Where: N_ The studied community (168 wells) worked for at least three consecutive years in the irriga- tion of vegetable crops (Agricultural Extension Depart- ment, 2020), e- Precision Level ± 7% Where Confidence Level is 95 %. were randomly distributed in the study area. Data was processed using IBM Spss Statistics 26. 2.3 STATISTICAL METHODS The study relied on descriptive analysis methods to describe the study variables such as means, percentage, charts, and exploratory factor analysis. 2.3.1 Related concepts and terms Factor Analysis (FA): Is an interdependence tech- nique whose primary purpose is to define the underlying structure among the variables in the analysis. Recently was developed originally for the analysis of scores on mental tests; however, the methods are useful in a much wider range of situations, for example, analyzing sets of tests of attitudes, sets of physical measurements, and sets of economic quantities (Anderson, 2003). Principal Components Method (PCA): Is one of the most important methods of factor analysis. It can be used to analyze interrelationships among a large num- ber of variables, and explain them in terms of their com- mon underlying dimensions to find a way of condens- ing the information contained in a number of original variables into a smaller set of factors with a minimal loss of information by providing an empirical estimate of the structure of the variables considered (Hair et al., 2009). As long as PCA is used descriptively as convenient ways to summarize the relationships in a large set of observed variables, assumptions regarding the distributions of var- iables are not in Force (Tabachink & Fidell, 2013). Exploratory Factor Analysis (EPA): Is to discover the underlying structure of observed variables and iden- tifies latent factors that explain the covariation among a set of variables. Ideally, the derived factors should con- sist of relatively homogenous variables, where each item loads strongly onto one factor and minimally on the other factor(s). It is assumed that each common factor affects every observed variable and that the common fac- tors are either all correlated or uncorrelated (McDonald, 1985). 2.3.2 Mathematical Models of EPA In the EPA model, (p) is the number of variables (X1, X2, … , Xp) and (m) denotes the number of underly- ing factors (F1, F2, … , Fm). Xj is the variable represented in Eigenvalue (latent) factors. Hence, this model assumes that there are (m) underlying factors whereby each ob- served variable is a linear function of these factors to- gether. This model intends to reproduce the maximum correlations: Where: j = 1, 2, ... , p The factor loadings are aj1, aj2,…, ajm which denotes that aj1 is the factor loading of (jth) variable on the (1st) factor. The specific or unique factor is denoted by ej. The factor loadings give us an idea about how much the vari- able has contributed to the factor; the larger the factor loading the more the variable has contributed to that fac- tor (Harman, 1976). 2.3.3 Procedures in exploratory factor analysis Measure of sampling adequacy (MSA): Calculates both for the entire correlation matrix and for each indi- vidual variable evaluation the appropriateness of apply- Acta agriculturae Slovenica, 117/4 – 20214 M. AL-ABDALA et al. ing factor analysis. Values above 0.5 for either the entire matrix or an individual variable indicate appropriateness (Hair et al., 2009). Correlation matrix: When the data are appropriate, it is possible to create a correlation matrix by calculating the correlations between each pair of variables. As stated important information for the analysis in the correlation matrix are (Field, 2009): the variables have to be intercor- related, but they should not correlate too highly (extreme multicollinearity and singularity). The coefficients less than 0.90 and suggested removing one of a pair of items with bivariate correlation scores greater than 0.8), the level of significance, the determinant (which should be greater than zero), and KMO and Bartlett’s tests. Tests of Bartlett sphericity and Kaiser-Meyer-Olkin (KMO): were used to determine the level of confidence that can be expected when using EFA on data (Hair et al., 2009). The Bartlett test of sphericity is based on the statistical distribution of Chi-square and tests the null hypothesis for the overall significance of all correlation within a correlation matrix, (i.e., no correlation between the variables). Levels of significance greater than 0.1 in- dicate that the data are not suitable for the treatment with the method in question; in this case, the null hypothesis can not be rejected. The KMO test presents normalized values (between 0 and 1) and shows the proportion of common variance of the variables, or what percentage of the variables is accounted for by common factors. To interpret the results, values close to 1 indicate that the factor analysis method is perfectly suited for data pro- cessing. On the other hand, values below 0.5 indicate the inadequacy of the method (Pituch & Stevens, 2016). Orthogonal Rotation: Is ordinarily used after extrac- tion to maximize high correlations between factors and variables and minimize low ones. Numerous methods of rotation are available, but the most commonly used is va- rimax. Varimax is a variance-maximizing procedure. The goal of varimax rotation is to maximize the variance of factor loadings by making high loadings higher and low- er ones lower for each factor. (Tabachink & Fidell, 2013). 2.3.4 Criteria for the number of factors to extract Eigenvalue (Latent root) Criterion: The most com- monly used technique is the Eigenvalue criterion. Is sim- ply to apply, the rationale the latent criterion is that any individual factor should account for the variance of at least as single variable if it is to be retained for interpreta- tion. With PCA each variable contributes a value 1 root considered significant, all factors with roots less than 1 are insignificant and are disagreed (Thomapson, 2004). Communalities: are the measure of the proportion of variance explained by the extracted factors, represent- ing the amount of variance accounted by the factor so- lution for each variable, to assess whether the variables meet acceptable levels of explanation. The communali- ties should be more than 0.50 for each variable and more than 0.60 in average (Hair et al., 2009). Scree Test: As (Cattell, 1966) proposed a graphi- cal test for determining the number of factors. A scree plot graphs eigenvalue magnitudes on the vertical access, with eigenvalue numbers constituting the horizontal axis. Showing how to simplify the scree plot through dynamic graphic procedures when successive factor analyses are performed (Ledesma et al., 2015). There are four steps for applying EPA which are in- volved briefly by Tighza (2012): - Preparation of a correlation matrix between the measured variables. - Extracting the initial factors and exploration of possible data reduction. - Rotation to a terminal solution (find the interpret- able factors). - Naming the identifying factors. 3 RESULTS AND DISCUSSION 3.1 CHARACTERISTICS OF THE STUDY SAMPLE POPULATION 3.1.1 Natural properties Geographical: The sample covered into three agri- cultural settlement zones according to the amount an- nual rainfall, the largest percentage was in the second zone with about 86.96 %, and covered three administra- tive regions, Swaida region was the largest - about 60.78 % Table (1). Rainfall ratio: It ranged annually between a mini- mum of 125 mm and a maximum of 380 mm, and an average amounted about 287.2 mm. Altitude: It ranged from a minimum of 650 m to a maximum of 1470 m, and an average about 1004.5 m. 3.1.2 Economic characteristics Main job: An irrigated vegetable farming is 47.7 % of the farmers. It is followed by 28.3 % who are self-em- ployed, 15.2 % are employees in the public sector and 8.7 % have their own business. Contribution to income: The cultivation of irrigated vegetables contributes about 53.21 % of the family in- come, while other kinds of agriculture contribute about Acta agriculturae Slovenica, 117/4 – 2021 5 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria 20.33 %, and only 26.47 % income came from non-agri- culture. 3.1.3 Social Characteristics Age of the farmers: It ranged between 26 and 70 years as a min and max respectively, and about 46.4 years in average. Experience: There was a variation in the farmers’ experience regarding producing of irrigated vegetables, which ranged from 1 year to 40 years, and reached an average of about 13.55 Years. Educational levels: It  ranged between the highest percentages of those with primary education (about 64 %), then about 20 % of them with university education, and 14 % with secondary education and only 2 % were il- literate. Figure (1). 3.2 THE PROBLEMS FACING FARMERS The results of the responses about relative frequency were used to analyze the main problems that effect on cultivation of irrigated vegetable where it was found that 98.9 % of farmers emphasized the problem of high costs inputs, 83.7 % State support for production requirements and 69.6 % Infection with diseases and insects respec- tively are strong problems, Table (2). 3.3 DETERMINE THE FACTORS RESPONSIBLE FOR THE LARGEST PROPORTION OF THE TOTAL VARIANCE IN THE PRODUCTION OF IRRIGATED VEGETABLES PROJECTS, THE RESULTS OF (EPA) 3.3.1 The first Step: Analysis of the correlation matrix between the measured variables According to Table (1) in the Appendix, the value of (MSA) appears in the diagonal cells of the variable (X8) equal to 0.341, which is less than 0.5, therefore it is deleted and re-analyzed.  The  Re-analysis excluding the variable (x8) and showed the following results: Correlation coefficients: they should not exceed 0.9, as it is impossible to estimate the percentage of variance that the variables contribute to forming the extracted fac- tors. Table (3) shows the inter-correlations matrix in the upper half, and the statistical significance in the lower half, which fulfills the condition. The determinant of the matrix: which requires that the absolute value of the determinant must be greater than 0.0000, and it appears at the bottom of Table (3), Determinant = .028, meaning that the second condition is also fulfilled, and therefore the matrix does not involve the problem of exaggerated correlation between the vari- ables. Bartlett's Test of Sphericity: Table (4) shows the significance of the Bartlett test (Sig < 0.00) and the ac- ceptance of the alternative hypothesis and thus the matrix is not neutral (Identity Matrix), and there are First Settlement Zones Total %First Second Third Administrative Regions Salkad 6 22 2 30 32.61 Swaida 4 52 0 56 60.87 Shahba 0 6 0 6 6.52 Total 10 80 2 92 100 % 10.87 86.96 2.17 100 Table 1: Distribution of farmers according to the agricultural settlement zones and administrative regions in Swaida governorate during the 2020 agricultural season Source: Survey results Figure 1: Percentage distribution of farmers according to educational level Acta agriculturae Slovenica, 117/4 – 20216 M. AL-ABDALA et al. some relationships between the variables that can be ana- lyzed. Kaiser-Meyer-Olkin (KMO(: Table (4) shows that the KMO value is equal to 0.586 and is greater than 0.5, thus the reliability of the factors that will be obtained from the analysis, and that the sample size is sufficient. Measure of Sampling Adequacy (MSA): The MSA values in the diagonal cells show that all correlation coef- ficients equal or exceed the value 0.5, indicating that the level of correlation between each variable with other var- iables in the correlation matrix is sufficient for analyzing. Variables Problems Ranking (%) There Is No (1) Medium (2) Strong (3) X1 Availability Of Manpower 17.4 35.9 46.7 X2 Labor Efficiency 12.0 53.3 34.8 X3 Infection With Diseases And Insects 7.6 22.8 69.6 X4 Natural Disasters 55.4 20.7 23.9 X5 Availability Of Inputs 48.9 17.4 33.7 X6 High Costs Of Inputs 1.1 0 98.9 X7 Effectiveness Of Inputs 8.7 29.3 62.0 X8 State Support For Production Requirements 2.2 14.1 83.7 X9 Vegetable Price Fluctuations 6.5 30.4 63.0 X10 Brokers’ Control 10.9 27.2 62.0 X11 Low Selling Price 7.6 13.0 79.3 X12 Disposing Of The Product 52.2 21.7 26.1 X13 Availability Of Internal And External Markets 50.0 22.8 27.2 X14 Providing Farmers With The Necessary Expertise 57.6 18.5 23.9 X15 Availability Of Information About New Technologies 63.0 16.3 20.7 Table 2: Relative frequency of the responses to evaluate the characteristics related to the problems of irrigated vegetable produc- tion Source: Survey results x1 x2 x3 x4 x5 x6 x7 x9 x10 x11 x12 x13 x14 x15 C orrelation x1 1.00 x2 0.63 1.00 x3 0.15 0.19 1.00 x4 0.04 0.13 0.31 1.00 x5 0.16 0.00 0.03 0.15 1.00 x6 0.04 0.20 0.10 -0.04 -0.13 1.00 x7 0.15 0.20 0.10 0.09 0.08 0.09 1.00 x9 0.02 0.11 -0.01 0.14 0.28 0.27 0.17 1.00 x10 0.22 0.13 0.02 0.09 0.18 0.08 0.07 0.30 1.00 x11 0.24 0.22 0.24 0.19 -0.12 0.30 0.05 0.05 0.14 1.00 x12 0.14 0.23 0.33 0.21 0.19 0.09 0.29 0.03 -0.03 0.20 1.00 x13 0.21 0.31 0.23 0.22 0.15 0.10 0.16 0.12 0.13 0.19 0.58 1.00 x14 -0.12 -0.06 0.09 0.17 0.16 -0.17 0.17 0.03 0.09 -0.04 0.23 0.14 1.00 x15 -0.08 -0.02 0.20 0.12 0.12 -0.18 0.16 0.00 0.21 -0.09 0.24 0.27 0.75 1.00 Table 3: Correlation Matrix between the measured variables (The Problems of Irrigated Vegetable in Swaida governorate during the 2020 agricultural season) Acta agriculturae Slovenica, 117/4 – 2021 7 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria Continued Sig. (1-tailed) x1 x2 0.00 x3 0.08 0.04 x4 0.34 0.10 0.00 x5 0.06 0.49 0.38 0.07 x6 0.35 0.03 0.16 0.35 0.10 x7 0.08 0.03 0.18 0.20 0.22 0.21 x9 0.43 0.14 0.48 0.10 0.00 0.00 0.05 x10 0.02 0.11 0.42 0.19 0.04 0.23 0.25 0.00 x11 0.01 0.02 0.01 0.03 0.12 0.00 0.31 0.32 0.09 x12 0.09 0.01 0.00 0.02 0.03 0.19 0.00 0.38 0.38 0.03 x13 0.02 0.00 0.01 0.02 0.07 0.18 0.06 0.12 0.12 0.03 0.00 x14 0.13 0.29 0.20 0.05 0.06 0.06 0.05 0.38 0.19 0.36 0.01 0.10 x15 0.22 0.41 0.03 0.12 0.13 0.04 0.06 0.50 0.02 0.20 0.01 0.00 0.00 a. Determinant = .028 Source: IBM Spss Statistics 26 Output /survey results Kaiser-Meyer-Olkin Measure of Sampling Adequacy .586 Bartlett’s Test of Sphericity Approx. Chi-Square 305.846 Df 91 Sig. .000 Table 4: KMO and Bartlett’s Test Source: IBM Spss Statistics 26 Output /survey results Anti-image Matrices x1 x2 x3 x4 x5 x6 x7 x9 x10 x11 x12 x13 x14 x15 A nti-im age C ovariance x1 0.50 -0.31 -0.04 0.07 -0.15 0.08 -0.05 0.09 -0.12 -0.10 0.02 -0.01 0.03 0.03 x2 -0.31 0.51 -0.02 -0.06 0.11 -0.09 -0.05 -0.06 0.03 0.03 -0.02 -0.08 0.00 0.00 x3 -0.04 -0.02 0.75 -0.21 0.00 -0.07 0.02 0.03 0.05 -0.10 -0.13 0.05 0.09 -0.12 x4 0.07 -0.06 -0.21 0.79 -0.07 0.12 -0.01 -0.07 -0.03 -0.11 0.01 -0.07 -0.08 0.05 x5 -0.15 0.11 0.00 -0.07 0.74 0.10 0.03 -0.21 -0.07 0.15 -0.11 -0.02 -0.06 0.03 x6 0.08 -0.09 -0.07 0.12 0.10 0.74 -0.03 -0.20 -0.04 -0.17 -0.04 -0.01 0.02 0.06 x7 -0.05 -0.05 0.02 -0.01 0.03 -0.03 0.85 -0.12 0.00 0.03 -0.15 0.05 -0.03 -0.03 x9 0.09 -0.06 0.03 -0.07 -0.21 -0.20 -0.12 0.73 -0.18 0.02 0.06 -0.05 -0.02 0.04 x10 -0.12 0.03 0.05 -0.03 -0.07 -0.04 0.00 -0.18 0.75 -0.11 0.10 -0.02 0.06 -0.14 x11 -0.10 0.03 -0.10 -0.11 0.15 -0.17 0.03 0.02 -0.11 0.74 -0.06 -0.05 -0.06 0.08 x12 0.02 -0.02 -0.13 0.01 -0.11 -0.04 -0.15 0.06 0.10 -0.06 0.53 -0.27 -0.06 0.02 x13 -0.01 -0.08 0.05 -0.07 -0.02 -0.01 0.05 -0.05 -0.02 -0.05 -0.27 0.56 0.09 -0.11 x14 0.03 0.00 0.09 -0.08 -0.06 0.02 -0.03 -0.02 0.06 -0.06 -0.06 0.09 0.39 -0.27 x15 0.03 0.00 -0.12 0.05 0.03 0.06 -0.03 0.04 -0.14 0.08 0.02 -0.11 -0.27 0.34 Table 5: Measure of sampling adequacy (MSA) for the measured variables (The problems of irrigated vegetable) Acta agriculturae Slovenica, 117/4 – 20218 M. AL-ABDALA et al. 3.3.2 The second step: Extraction analysis Method: One of the statistical approaches in extract- ing factors is principal components analysis; the factors with eigenvalues (own values) > 1.0 and factorial loads > 0.4 were used as consideration criteria. The results were also combined with the orthogonal methods of rotation Varimax. Detraining the extracted factors: Table (6) shows the number of each extracted factor with Eigenvalues, and percentage of variance and cumulative variance of each of the factors. Six factors with eigenvalues greater than 1 were extracted. Factors that influence the irrigated vegetable farming, according to factors loadings after orthogonal rotation using the Varimax were classified. These factors explained 70.33 % of the total variance, and only less than 29.67 % of variance were due to factors that were not identified through factor analysis. It is noticed that the rotation distributes the variance ratios among the factors in a relatively balanced manner and does not make it concentrated in the first factor or second factor, and this is evident by comparing column (9) in Table (6) where the ratios of interpretation of variances were reached from the total variance of each factor. For exam- ple; the first factor has the highest Eigenvalue equals 1.85 and the total explained variance equals 13.21 %. The total explained variance for the first fac- tor = (Eigenvalue / the number of eigenvalues) * 100 = = (1.85/14) * 100 = 13.21 %. Commonalities: Table (7) shows the values of the communalities which is greater than 0.05 for each varia- ble, and the average for all variables is 0.703, greater than 0.60. Thus, we have obtained the values of the explained variance for each variable, for example; as the extracted value of the variable x1 (availability of manpower) equals 0.848 of the variances in the variable values are explained by the common factors. The Scree test: Is a heuristic graphic method that consists of: a) Plotting the eigenvalues (y-axis) against the com- ponents (x-axis), and b) Inspecting the shape of the resulting curve in or- der to detect the point at which the curve changes drasti- cally. The eigenvalues are plotted as a bold point within the graph, and successive values are connected by a line. Factor extraction should be stopped at the point where there is an “elbow”, or leveling of the plot (Thomapson, 2004). This plot suggests that six factors should be ex- tracted (Figure 2). 3.3.3 The third step, rotation Table (8) shows the loading of the variables on the six factors before rotation and after orthogonal rotation by the Varimax method. It is noticed that most of the items in the component matrix before rotation loaded on most of the factors. It is also showing a common loading in most of the items on A nti-im age C orrelation Continued x1 .536a -0.61 -0.07 0.12 -0.25 0.14 -0.08 0.15 -0.19 -0.17 0.04 -0.02 0.06 0.07 x2 -0.61 .617a -0.03 -0.10 0.18 -0.15 -0.08 -0.10 0.04 0.04 -0.04 -0.15 -0.01 0.01 x3 -0.07 -0.03 .646a -0.27 0.00 -0.10 0.03 0.04 0.07 -0.13 -0.20 0.07 0.16 -0.24 x4 0.12 -0.10 -0.27 .637a -0.10 0.15 -0.01 -0.09 -0.04 -0.15 0.01 -0.10 -0.14 0.10 x5 -0.25 0.18 0.00 -0.10 .499a 0.14 0.04 -0.29 -0.10 0.20 -0.17 -0.03 -0.11 0.07 x6 0.14 -0.15 -0.10 0.15 0.14 .578a -0.04 -0.27 -0.05 -0.23 -0.06 -0.02 0.03 0.11 x7 -0.08 -0.08 0.03 -0.01 0.04 -0.04 .743a -0.15 0.00 0.04 -0.23 0.08 -0.06 -0.05 x9 0.15 -0.10 0.04 -0.09 -0.29 -0.27 -0.15 .509a -0.25 0.03 0.10 -0.08 -0.04 0.08 x10 -0.19 0.04 0.07 -0.04 -0.10 -0.05 0.00 -0.25 .541a -0.15 0.15 -0.03 0.12 -0.27 x11 -0.17 0.04 -0.13 -0.15 0.20 -0.23 0.04 0.03 -0.15 .632a -0.10 -0.07 -0.11 0.17 x12 0.04 -0.04 -0.20 0.01 -0.17 -0.06 -0.23 0.10 0.15 -0.10 .655a -0.50 -0.13 0.04 x13 -0.02 -0.15 0.07 -0.10 -0.03 -0.02 0.08 -0.08 -0.03 -0.07 -0.50 .660a 0.18 -0.26 x14 0.06 -0.01 0.16 -0.14 -0.11 0.03 -0.06 -0.04 0.12 -0.11 -0.13 0.18 .536a -0.73 x15 0.07 0.01 -0.24 0.10 0.07 0.11 -0.05 0.08 -0.27 0.17 0.04 -0.26 -0.73 .522a a. Measures of sampling adequacy (MSA) Source: IBM Spss Statistics 26 Output /survey results Acta agriculturae Slovenica, 117/4 – 2021 9 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria Total Variance Explained C om ponent Initial Eigenvalues Extraction Sums of Squared Loadings Rotation Sums of Squared Loadings Total % of Variance Cumulative % Total % of Variance Cumulative % Total % of Variance Cumulative % 1 2.95 21.07 21.07 2.95 21.07 21.07 1.85 13.21 13.21 2 2.10 14.98 36.05 2.10 14.98 36.05 1.77 12.65 25.87 3 1.44 10.29 46.34 1.44 10.29 46.34 1.76 12.55 38.41 4 1.23 8.81 55.14 1.23 8.81 55.14 1.56 11.12 49.53 5 1.06 7.60 62.74 1.06 7.60 62.74 1.53 10.94 60.48 6 1.06 7.59 70.33 1.06 7.59 70.33 1.38 9.85 70.33 7 0.86 6.12 76.45 8 0.70 5.01 81.45 9 0.67 4.79 86.25 10 0.63 4.48 90.73 11 0.48 3.40 94.13 12 0.35 2.50 96.64 13 0.28 2.01 98.65 14 0.19 1.36 100.00 Extraction Method: Principal Component Analysis Table 6: Total explained variance of each extracted factors with eigenvalues Source: IBM Spss Statistics 26 Output /survey result Initial Extraction x1 1.000 .848 x2 1.000 .730 x3 1.000 .548 x4 1.000 .645 x5 1.000 .750 x6 1.000 .734 x7 1.000 .507 x9 1.000 .762 x10 1.000 .721 x11 1.000 .614 x12 1.000 .754 x13 1.000 .562 x14 1.000 .798 x15 1.000 .872 Extraction Method: Principal Component Analysis Table 7: Communalities (extracted value of variance) for the measured variables (The problems of irrigated vegetable) Source: IBM Spss Statistics 26 Output /survey results the six factors, as most of the variables were of a high load on the first factor, meaning there is a clear absence in the balance of the loading on the extracted factors, which shows the difficulty in interpretation. Whereas, after the rotation the variance explained by each factor was redistributed. As a change in the pat- tern of loading, up and down is observed on each factor, and a change in the percentage of explained variance, and it is noticed that the load values that are smaller than 0.6 are hidden and the variables are arranged according to the load, which makes the interpretation easier. The factor matrix after rotation, which includes six factors. Where all the variables were loaded after rotation on the six factors, except the variable x11. Thus, accord- ing to extracted results, the six extracted factors will be named. Table (8). Figure (3) shows a schematic representation of the rotation of the axes, an orthogonal rotation, meaning that the factors were rotated while maintaining them inde- pendent. Before rotation, all the factors were independ- ent (not completely related), and the orthogonal rotation ensures that all the factors remain unrelated. Acta agriculturae Slovenica, 117/4 – 202110 M. AL-ABDALA et al. Figure 2: Scree plot Test suggests that six factors should be extracted Component Matrixa Rotated Component Matrixa Component Component 1 2 3 4 5 6 1 2 3 4 5 6 x1 0.462 0.479 .597- 0.907 x2 0.556 0.487 0.789 x3 0.506 0.669 x4 0.459 .610- 0.772 x5 0.503 .405- .676- x6 0.512 0.586 0.701 x7 0.438 0.452 0.605 x9 0.679 0.854 x10 0.6 0.49 0.601 x11 0.431 x12 0.681 .415- 0.791 x13 0.691 0.624 x14 .745- 0.858 x15 0.434 .734- 0.909 - Extraction Method: Principal Component Analysis. - a. 6 components extracted. - Extraction Method: Principal Component Analysis. - Rotation Method: Varimax with Kaiser Normalization. - a. Rotation converged in 9 iterations. Table 8: Components matrix and loadings before rotation and after orthogonal rotation by the Varimax method, for the measured variables (The problems of irrigated vegetable) Source: IBM Spss Statistics 26 Output /survey result Acta agriculturae Slovenica, 117/4 – 2021 11 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria 3.3.4 The fourth step, naming the factors According to the results extracted from Table (8) above, the six identifying factors were named depend- ing on the percentage of each load factor. All six factors included two determinants except for the third factor (the problem of sale outlets) which included three de- terminants. Table (9). The exploratory factor analysis technique was used to correspond to the assumptions in the concerning problems with the irrigated vegetable problems in Swaida Governorate, Syria during 2019- 2020 Season, which directly effect on the production. The use of the exploratory factor analysis technique enabled the understanding of how variables are interre- lated. It allowed the adjustment of the assessment instru- ment after the removal of variables with low indicators, Figure 3: Component plot in rotated space Factor loadings %Determinants included in Factor AnalysisName assigned to factorFactor NO. 0.858 (X14): Providing farmers with the necessary expertiseThe problem of agricultural technological progress.The first 0.909 (15): Availability of information about new technologies 0.907(X1) Availability of manpowerThe problem of agricultural employment.The second 0.789(X2) Labor efficiency 0.605(X7) Effectiveness of Inputs The problem of sale outletsThe third 0.791(X12) disposing of the product 0.624 (X13) Availability of internal and external markets 0.669(X3) Infection with diseases and insectsThe problem of natural conditionsThe fourth 0.772(X4) natural disasters 0.854(X9) Vegetable price fluctuations The problem of pricesThe fifth 0.601(X10) Brokers’ control 0.676(X5) Availability of InputsThe problem of production requirementsThe sixth 0.701(X6) High costs of Inputs Table 9: The factors responsible for the largest proportion of the total variance in the production of irrigated vegetables projects Source: Depending on Table (8), survey results Acta agriculturae Slovenica, 117/4 – 202112 M. AL-ABDALA et al. namely: [State support for production requirements X8] and [Low selling price X11]. Therefore, according to the results extracted from the exploratory factor analysis technique, using the Principal components methodology and Varimax rotation the six identifying factors with an initial Eigenvalues greater than one for each and depend- ing on the percentage of each load factor were named: agricultural technological progress, agricultural employ- ment, sale outlets, natural conditions, prices, production requirements. These factors explained (13.21%, 12.65%, 12.55%, 11.12%, 1.94%, and 9.85%) of the total variance respectively, and together explained 70.33%. 4 CONCLUSIONS This research is unique because it showed the ef- fectiveness of using the exploratory factor analysis methodology in identifying the most important factors responsible for explaining the largest percentage of the total variation in the production of irrigated vegetable projects in As-Swaida Governorate. The study recom- mends policy makers to addressing all the obstacles fac- ing irrigated vegetable farming in Swaida Governorate in order to reduce their negative effects on the production process, like: marketing management through: preparing to purchase quantities of production directly from farm- ers, especially the surplus, establishing formal marketing offices to limit the control of brokers, concluding export deals with friendly neighboring countries, and setting minimum prices. Direct and indirect supervision of the production process through: supporting agricultural ex- tension, intensifying agricultural courses related to mod- ern agricultural technologies and marketing methods. Managing strict control, especially the quality of pro- duction requirements (seeds, fertilizers, irrigation water, pesticides....). 5 REFERENCES Ahmadi, H., Rezaei, R., & Kheiri, S. (2013). Factor analysis of barriers and problems affecting the development of nano- technology in agriculture. Annals of Biological Research, 4(1), 131-134. Anderson, T. W. (2003). An Introduction to Multivariate Statisti- cal Analysis. Third Edition. John Wiley & Suns, Inc. Hubo- ken, New Jersey. p.747. Cattell, R. B. (1966). The scree test for the number of factors. Multivariate Behavioral Research, 1(2), 245-276. https://doi. org/10.1207/s15327906mbr0102_10 Chandrashekar, S., Bahal, R., & V.P.H.D. (2012). Factors influ- encing the entrepreneurial behavior of agricultural gradu- ates. SAARC Journal of Agriculture. 10(2), 31‐39. https:// doi.org/10.3329/sja.v10i2.18321 Field, A. (2009). Discovering Statistics using Spss. 3th edition. London: SAGE publications Ltd. Singapore. p. 854. Glenn, D. I. (1992). Determining Sample Size. Florida coop- erative extension service. University of Florida. Fact sheet peod-6. Harman, H. H. (1976). Modern Factor Analysis. Third edition. Revised. Chicago, University of Chicago Press. P. 508. Hair, J. F. Jr., Black, W. C., Babin, B. J., & Anderson, R. E. (2009). Multivariate Data Analysis. Seventh edition. Published by Pearson. Mcdonald, R. P. (1985). Factor Analysis and Related Methods. Hillside, NJ: Lawrence Erlbaum associates, inc. Ledesma, D. R., Mora, P. M. V., & Macbeth, G. (2015). The scree test and the number of factors: a dynamic graphics ap- proach. Spanish Journal of Psychology, 18(11), 1–10. https:// doi.org/10.1017/sjp.2015.13 Pavel, A. & Octavian, M. (2019). Determining local econom- ic development in the rural areas of Romania. Exploring the role of exogenous factor. Sustainability, 11, 282; DOI: 10.3390 / su11010282. https://doi.org/10.3390/su11010282 Pituch, K. A. & Stevens, J. P. (2016). Applied Multivariate Sta- tistics For The Social Sciences Analyses With SAS and SPSS. Sixth edition. Routledge (Taylor & Francis group). New York and London. Rezai, G., Shamsudin, M. N., & Mohamed, Z. (2016). Urban agriculture: a way forward to food and nutrition security in Malaysia, Procedia - Social and Behavioral Sciences, 216, 39 – 45. https://doi.org/10.1016/j.sbspro.2015.12.006 Ministry of Agriculture and Agrarian Reform. Directorate of planning and international cooperation. Annual agricultur- al statistical abstracts (2016-2018). Damascus, Syria. Ministry of Agriculture and Agrarian Reform. Directorate of agricultural extension unpublished data 2020. Swaida, Syr- ia. Tabachnik, B. G. & Fidell, L. S. (2013). Using Multivariate Statis- tics. Sixth edition, Pearson education, Inc. New Jersey. USA. P.1018. Tighza, M. B. (2012). Exploratory Factor Analysis and Confirm- atory, Concepts and Methods Using Spss and LIREl. First edi- tion. Dar Almasera. Aman, Jordan. P. 399. Thompson, B. (2004). Exploratory and Confirmatory Fac- tor Analysis Understanding Concepts and Applications. American Psychological Association. P.185. https://doi. org/10.1037/10694-000 Yamane, T. (1967). An Introductory Analysis. Second edition. New York: Harper and Row. Zeina, M. (2015). The important of exploratory factor analysis to achieve the factorial structure of psychological tests. Geel Journal of Social and Human Sciences, 14, 40-31. Acta agriculturae Slovenica, 117/4 – 2021 13 Classification of determinant factors of irrigated vegetable problems using exploratory factor analysis in Swaida governorate, Syria Anti-image Matrices x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 x12 x13 x14 x15 A nti-im age C orrelation x1 .532a -0.61 -0.09 0.10 -0.24 0.15 -0.10 0.12 0.15 -0.14 -0.18 0.01 0.03 0.09 0.03 x2 -0.61 .566a 0.03 -0.05 0.17 -0.18 -0.01 -0.30 -0.08 -0.05 0.08 0.03 -0.26 -0.09 0.10 x3 -0.09 0.03 .734a -0.24 0.00 -0.11 0.06 -0.18 0.05 0.01 -0.11 -0.16 -0.01 0.10 -0.17 x4 0.10 -0.05 -0.24 .638a -0.10 0.13 0.01 -0.13 -0.09 -0.08 -0.13 0.04 -0.15 -0.17 0.14 x5 -0.24 0.17 0.00 -0.10 .515a 0.14 0.03 0.02 -0.29 -0.09 0.19 -0.17 -0.02 -0.10 0.06 x6 0.15 -0.18 -0.11 0.13 0.14 .548a -0.06 0.12 -0.27 -0.01 -0.24 -0.08 0.03 0.06 0.07 x7 -0.10 -0.01 0.06 0.01 0.03 -0.06 .738a -0.19 -0.14 -0.06 0.06 -0.18 -0.01 -0.10 0.01 x8 0.12 -0.30 -0.18 -0.13 0.02 0.12 -0.19 .341a -0.04 0.30 -0.13 -0.21 0.41 0.25 -0.32 x9 0.15 -0.08 0.05 -0.09 -0.29 -0.27 -0.14 -0.04 .511a -0.25 0.03 0.11 -0.09 -0.05 0.09 x10 -0.14 -0.05 0.01 -0.08 -0.09 -0.01 -0.06 0.30 -0.25 .469a -0.18 0.08 0.10 0.18 -0.34 x11 -0.18 0.08 -0.11 -0.13 0.19 -0.24 0.06 -0.13 0.03 -0.18 .589a -0.07 -0.12 -0.14 0.20 x12 0.01 0.03 -0.16 0.04 -0.17 -0.08 -0.18 -0.21 0.11 0.08 -0.07 .641a -0.54 -0.18 0.11 x13 0.03 -0.26 -0.01 -0.15 -0.02 0.03 -0.01 0.41 -0.09 0.10 -0.12 -0.54 .512a 0.27 -0.36 x14 0.09 -0.09 0.10 -0.17 -0.10 0.06 -0.10 0.25 -0.05 0.18 -0.14 -0.18 0.27 .475a -0.75 x15 0.03 0.10 -0.17 0.14 0.06 0.07 0.01 -0.32 0.09 -0.34 0.20 0.11 -0.36 -0.75 .465a a. Measures of Sampling Adequacy (MSA) Appendix 1 Source: IBM Spss Statistics 26 Output /survey result Acta agriculturae Slovenica, 117/4, 1–13, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2176 Original research article / izvirni znanstveni članek Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes Leyla NAZARI 1, 2, Ebrahim DEHGHANIAN 3, Afshar ESTAKHR 1, Azim KHAZAEI 4, Behzad SORKH- ILALEHLOO 4, Mohammad Reza ABBASI 5 Received April 15, 2021; accepted December 09, 2021. Delo je prispelo 15. aprila 2021, sprejeto 9. decembra 2021 1 Crop and Horticultural Science Research Department, Fars Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Shiraz, Iran 2 Corresponding author, e-mail: l.nazari@areeo.ac.ir 3 Agricultural Engineering Research Department, Fars Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Shiraz, Iran 4 Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 5 Crop and Horticultural Science Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes Abstract: Sorghum (Sorghum bicolor (L.) Moench) is the fifth important cereal considered a drought-tolerant crop. However, its reduction of grain yield considerably occurs in a shortage of water. In the current study, 10 sorghum genotypes were assessed for their grain yield under normal irrigation and water deficit irrigation. As well, the efficacy of several drought indices was evaluated for the selection of high-yield and drought-tolerant genotypes. The experiment was conducted as a split-plot considering three irrigation levels as main-plot and 10 genotypes as sub-plot. Correlation among the indices, clustering of the genotypes along with principal component analysis was employed. Yield production was significantly and positively correlated with indices MP (mean productivity), STI (stress tolerance index), GMP (geometric productivity), HM (harmonic mean), and YI (yield index) in all the irrigation lev- els. Therefore, these indices are more effective in the selection of high-yielding genotypes under different water conditions. Rank means of stress indices for each genotype revealed that genotype TN-04-79 in mild deficit irrigation and genotypes KGS23 and TN-04-79 in severe deficit irrigation were the most tolerant. Key words: sorgum; drought stress; grain yield; water productivity; drought response indices Uvajanje najboljših kriterijev za ovrednotenje tolerance na sušo pri genotipih sirka Izvleček: Navadni sirek (Sorghum bicolor (L.) Moench) je peto najpomembnejše na sušo odporno žito, a se kljub temu njegov pridelek zrnja znantno zmanjša ob pomanjkanju vode. V tej raziskavi je bilo ocenjenih 10 genotipov navadnega sirka glede na pridelek zrnja ob normalnem namakanju in v razme- rah vodnega deficita. Ocenjeni so bili tudi različni indeksi to- lerance na sušo pri izboru genotipov z velikimi pridelki zrnja in dobre tolerance na sušo. Poskus je bil izveden kot puskus z deljenkami, kjer so bila obravnavanja z namakanjem na glavnih ploskvah in 10 genotipov na podploskvah. Uporabljene so bile korelacije med indeksi in združevanje genotipov glede na glav- no komponento. Velikost pridelka je bila značilno pozitivno povezana z indeksi MP (poprečna produktivnost), STI (indeks tolerance na stres), GMP (geometrična produktivnost), HM (harmonično poprečje) in YI (indeks pridelka) pri vseh načinih namakanja. Ti indeksi so torej bolj učinkoviti pri izboru visoko donosnih genotipov v razmerah različne preskrbe z vodo. Po- prečje rangov stresnih indeksov za vsak genotip je odkrilo, da je genotip TN-04-79 najučinkoviteši ob blagem pomanjkanju vode, genotipa KGS23 in TN-04-79 pa sta bila najbolj odporna na sušo. Ključne besede: navadni sirek; sušni stres; pridelek zrnja; učinkovitost izrabe vode; indeksi odziva na sušni stres Acta agriculturae Slovenica, 117/4 – 20212 L. NAZARI et al. 1 INTRODUCTION Sorghum (Sorghum bicolor  (L.) Moench) is a C4 and drought-tolerant crop used for food, feed, and fiber (Ludlow et al., 1990). Its tolerance to drought can be at- tributed to morphological characteristics (e.g. deep root system and thick leaf wax), physiological responses (e.g. stay green and osmotic adjustment), and adaptive mech- anisms allowing tolerance under extreme drought con- ditions (reviewed in Tari et al., 2013). In the dry region of Asia and the Middle East, drought is one of the most important abiotic stresses, leading to the limitation of plant growth and yield productivity (Zhang et al., 2018). Therefore, improving yield production per unit of water (water productivity) is an efficient strategy in dry regions (Ali and Talukder, 2008). Blum (2005) suggested that the selection of geno- types should mainly focus on high yield under non-stress conditions and secondly under water stress conditions. The selection of genotypes that have tolerant genes is difficult as drought tolerance is a quantitative trait with intricate heritability. Therefore, despite the lack of infor- mation on drought tolerance mechanisms, researchers have proposed the utility of different selection indices to screen drought-tolerant genotypes (Anwaar et al., 2019). Hence, we have employed the following selection criteria for screening drought-tolerant genotypes and introduc- ing the best indices. Several indices based on the yield under control (Yp) and stress (Ys) have been introduced for the selection of drought-tolerant genotypes. Among these, the indices employed in various stress conditions are stress tolerance (TOL) and mean productivity (MP) introduced by Ros- ielle and Hamblin (1981), Stress susceptibility index (SSI) by (Fischer and Maurer, 1978), stress tolerance index (STI) and geometric mean productivity (GMP) by Fer- nandez (1992), Harmonic mean of yield (HM) by Jafari et al. (2009), yield index (YI) by Gavuzzi et al. (1997), yield stability index (YSI) by Bouslama and Schapaugh (1984), yield reduction ratio (YRR) by Golestani-Araghi and Assad (1998). Selection of high-yield genotypes in both normal and deficit irrigation using a combination of these indices is preferred. Therefore, different statistical analyses including analysis of variance (ANOVA), corre- lation, principal component analysis (PCA), and cluster analysis were performed. The study aimed to investigate the efficiency of the mentioned indices for screening tol- erant genotypes of sorghum to drought stress. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE The experiment was conducted at the Research Farm of Fars Agricultural and Natural Resources Research and Education Center, Shiraz, Iran (52°42’ E, 29°46’ N, 1.604 m elevation) with a semi-arid environment (Fig. 1a). It is characterized by mean annual precipitation of 345 mm copper ppm magnesium ppm zinc ppmclay %silt %sand % potassium ppm phosphorus ppmO.C %EC*103pH soil depths cm 0.907.600.8033.446.220.443411.20.950.978.00-30 0.968.500.9636.842.820.43104.20.812.157.930-60 Table 1: The chemical properties of the soil in the experimental area EC: electrical conductivity; O.C: organic carbon; extractable phosphorus was measured according to Olson method Fig. 1: The spatial position of the experimental site captured on 07/11/2021 (a); minimum and maximum of temperature (b) and ET0 (c) during the growth season (2018) conducted at Zargan, Iran (52°42’E, 29°46’N) Acta agriculturae Slovenica, 117/4 – 2021 3 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes and an annual temperature of 15.8  °C. Minimum and maximum temperature and ET0 during growth season are presented in Fig. 1b,c. The soil is characterized by fine, carbonatic, active, thermic Typic Calcixerepts (soil taxonomy, 2014) and Cambic Calcisol (Lomic, Ochric) (WRB, 2015). The fertilizers were distributed based on soil test results (Table 1). 2.2 EXPERIMENTAL DESIGN The plants (10 sorghum genotypes, supplemen- tary Table 1) were cultivated manually as split-plot in a randomized complete block design with three replicates on 6 June 2018. Water deficit treatment was considered as the main factor and genotype as the sub-factor. Sub- plots were 12 m2 including 4 rows of 5 m long with a row distance of 0.6 m. Tinning was performed 4 weeks after sowing with a target of 10 plants per linear meter. Weed control was performed manually during the season. Irrigation treatments were applied to the main plots at three levels of normal irrigation, mild and severe water-deficit irrigation defined as irrigation when the evaporation rates from pan class A exceeded 60, 120, and 180 mm, respectively. Water stress was started from the 5 leaves stage and continued during the season. Irrigation was applied using a tape drip and the irri- gation volume was recorded by using a volumetric coun- ter. FAO-CROPWAT 8.0 as a decision support system (DSS) was used to calculate the reference crop evapo- transpiration (ETo) (Clarke, 2001) and schedule differ- ent levels of irrigation. The accuracy of this method was demonstrated by comparing it to original crop water re- quirements (Surendran et al., 2019). Meteorological data were taken daily from the Zargan Meteorological Station near the experimental field. The irrigation requirement was calculated according to Doorenbos and Pruitt (1977) (Table 2). 2.3 MEASUREMENTS AND DROUGHT INDICES Agronomic characteristics including plant height (PH), panicle length (PL), stem diameter (SD), and the number of leaves per plant (NoL) were recorded for 10 plants per plot from the middle two-row of each plot. As well, 1000 seed mass (1000 SM), dry matter yield (DMY), and harvest index (HI) were recorded. Water productiv- ity (WP) was calculated as Ali and Talukder (2008) (Ta- ble 2). Drought tolerance indices were calculated accord- ing to the equations in Table 2. Ranking of the genotypes In de x de sc rip tio n eq ua tio n Re fe re nc e C ro p W at er R eq ui re m en t (m m /d ay ) K c : w at er re qu ire m en t c oe ffi ci en t c ha ng in g w ith th e gr ow th st ag es o f so rg hu m ; E T o : t he re fe re nc e ev ap ot ra ns pi ra tio n of p la nt u nd er sp ec ifi ed co nd iti on s m ea su re d by p an e va po ra tio n. ET c = K c × E T o D oo re nb os a nd P ru itt , 1 97 7; D oo re nb os a nd K as sa m , 1 98 6 W at er p ro du ct iv ity (W P) cr op p ro du ct io n pe r u ni t v ol um e of w at er ; h ig h va lu es a re m or e de sir ab le A li an d Ta lu kd er , 2 00 8 To le ra nc e in de x (T O L) Lo w v al ue s i nd ic at e m or e st ab ili ty u nd er d efi ci t i rr ig at io n Ro sie lle a nd H am bl in (1 98 1) M ea n pr od uc tiv ity (M P) H ig h va lu es a re m or e de sir ab le Ro sie lle a nd H am bl in (1 98 1) St re ss su sc ep tib ili ty in de x (S SI ) Va lu es < 1 a re m or e to le ra nt Fi sc he r a nd M au re r ( 19 78 ) St re ss to le ra nc e in de x (S TI ) H ig h va lu es in di ca te m or e to le ra nt Fe rn an de z ( 19 92 ) G eo m et ric p ro du ct iv ity (G M P) H ig h va lu es a re m or e de sir ab le K ris tin e t a l. (1 99 7) H ar m on ic m ea n of y ie ld (H M ) H ig h va lu es a re m or e de sir ab le Ja fa ri et a l. (2 00 9) Yi el d in de x (Y I) H ig h va lu es in di ca te m or e to le ra nt G av uz zi e t a l. (1 99 7) Yi el ds st ab ili ty in de x (Y SI ) H ig h va lu es in di ca te m or e st ab ili ty u nd er n or m al a nd d efi ci t i rr ig at io n Bo us la m a an d Sc ha pa ug h (1 98 4) Yi el d re du ct io n ra tio (Y RR ) Lo w v al ue s i nd ic at e m or e su ita bl e fo r d efi ci t i rr ig at io n G ol es ta ni -A ra gh i a nd A ss ad (1 99 8) Ta bl e 2: D es cr ip tio n, e qu at io n an d re fe re nc e of cr op w at er re qu ire m en t, w at er p ro du ct iv ity , a nd d ro ug ht to le ra nc e in di ce s Acta agriculturae Slovenica, 117/4 – 20214 L. NAZARI et al. based on the indices was performed according to the method of Mickky et al. (2019). The means of grain yield and the indices were ranked considering that indices with higher values are more de- sirable except TOL, SSI, and YRR. Afterward, rank mean (R’) and standard deviation of rank (SDR) were calcu- lated. Rank mean is defined as the average of ranking val- ues across all drought tolerance indices of each genotype. Rank sum (RS) of each genotype was then determined by the addition of rank mean (R’) and standard deviation of rank (SDR). 2.4 STATISTICAL ANALYSIS Analysis of variance (ANOVA) and mean compari- son were performed using SAS release 9.2 (SAS Institute, Cary, NC, USA). Before doing ANOVA, normality tests were conducted. Provided that F-values were significant, a mean comparison was done (Duncan’s test, p ≤ 0.05). Drought stress indices, principal component analysis (PCA) and Pearson’s correlation between the indices were performed using iPASTIC that is an online tool kit for the estimation of plant abiotic stress indices (Khalili et al., 2016). Genotypes were clustered using Ward’s hier- archical clustering. Treatments PH cm PL cm SD mm NoL 1000 SM g Yield kg ha-1 DMY kg m-3 HI % WP kg m-3 Irrigation level normal irrigation 175.2a 29.8a 26.4a 16.9a 29.4a 5847.2a 27874.1a 23.2a 0.65a mild deficit irrigation 152.0b 27.6ab 24.5b 14.0a 28.5ab 4026.4b 22260.9b 21.3b 0.58b severe deficit irrigation 138.7c 25.3b 23.6c 14.6a 26.5b 2759.2c 21480.3b 16.5c 0.43c Genotype MGS2 105.3g 30.7c 25.9c 12.8bc 2.9f 2910.1f 16188.9e 17.7e 0.37e KGS23 94.0h 18.9c 24.0d 10.9bc 4.7bc 4683bc 16466.6e 29.2b 0.63b TN-04-78 120.0f 24.0d 30.1b 16.7b 3.5e 3491.1e 24276.2c 14.4e 0.45d TN-04-79 221.1a 10.0g 24.9cd 15.2bc 6.5a 6517.6a 30906.7b 21.3c 0.88a TN-04-129 91.9h 22.3d 33.4a 13.4bc 4.3c 4304.2cd 20498.8d 20.8d 0.57c TN-04-134 201.6b 14.6f 21.5e 22.7a 6.2b 4974.3b 28825.6b 16.7e 0.64b TN-04-142 227.0a 7.7g 21.7e 28.4a 1.6g 1558.7g 48770.0a 3.2f 0.19f TN-04-59 159.3d 53.4a 24.5cd 10.4bc 5.0b 5047.8b 18799.6de 26.8b 0.67b TN-04-86 151.2e 40.9b 21.2e 9.8c 4.7bc 4685.4bc 15641.6e 30.3a 0.63b TN-04-90 180.6c 53.2a 21.1e 11.4bc 3.9d 3937.2d 18343.6de 22.6c 0.538c Table 3: The main effects of irrigation level and genotype on morphological traits, yield, and water productivity of 10 sorghum genotypes PH: plant height; PL: panicle length; SD: stem diameter; NoL: number of leaves; 1000 SM: 1000 seed mass; DMY: dry matter yield; HI: harvest index; WP: water productivity. Means followed by the same letter in a column do not differ by Duncan’s test at 5 % probability 3 RESULTS AND DISCUSSION Here, we evaluated 10 sorghum genotypes for drought tolerance collected from different parts of Iran and kept at The National Plant Gene-Bank of Iran, SPII. Natural genetic diversity may play an important role in food security through pre-breeding programs or the introduction of important traits or genes into existing cultivars (Priyanka et al., 2021). As well, the efficacy of drought stress indices for screening of these genotypes was scrutinized. Using the yield values of Yp and Ys, vari- ous indices were calculated (Table 4) and the genotypes were ranked for each index (Table 5). 3.1 MORPHOLOGICAL TRAITS Significant differences were observed between ir- rigation regimes for all traits (p < 0.01 for PH, SD, and DMY; p < 0.05 for PL and 1000 SM) except NoL. There were significant differences between genotypes for all traits (p < 0.01) indicating significant variation among the genotypes. The interaction effect of deficit irrigation × genotype was significant (p < 0.01) except for NoL and 1000 SM. Acta agriculturae Slovenica, 117/4 – 2021 5 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes The average PH reduced 13.2 % and 20.8 % under mild and severe deficit irrigation, respectively (Table 3). The reduction ratios for PL were 7  % and 15  % under mild and severe deficit irrigation, respectively (Table 3). Ashraf and Foolad (2007) indicated a reduction in tur- gidity and cell growth and development under water shortage observed as a reduction in PH or panicle size. A significant effect of drought stress on the PH of for- age sorghum has also been demonstrated (Mutava et al., 2011). Stem diameter (SD) decreased 7.2  % and 10.6  % under mild and severe stress, respectively (Table 3). Our results were in line with Almodares et al. (2013) dem- onstrating that the stem diameter of sorghum decreased proportionally to water deficit intensity. The reduction of SD in sugarcane under water deficit has been proven (Silva et al., 2008). Controversial results were reported by other studies pointing out no reduction in SD under drought stress (Almodares et al., 2013; Fracasso et al., 2016; Ottman et al., 2001). The 1000 SM reduction was 9.8 % from normal irrigation to severe deficit irrigation (Table 3). Deficit irrigation resulted in a notable fall in DMY equal to 79.9 % and 77.1 % under mild and severe stress, respectively (Table 3). Genotypes TN-04-79 and TN-04-142 exhibited the highest PH value and genotypes KGS23 and TN-04-129 were the lowest in PH (Table 3). Generally, taller sor- ghum genotypes are favored for small-scale farms that mechanical harvests are not employed (Devnarain et al., 2016). Genotypes TN-04-90 and TN-04-59 had the high- est PL and genotypes TN-04-79 and TN-04-142 ranked the last (Table 3). Different values of SD were obtained with the highest value for the genotype TN-04-129 (Ta- ble 3). The highest NoL belonged to TN-04-134 and TN- 04-142. Genotypes TN-04-79 and TN-04-90 ranked the highest and the lowest 1000 SM, respectively (Table 3). The genotype TN-04-142 produced the highest DMY. There were no significant differences between MGS2, KGS23, TN-04-59, TN-04-86, and TN-04-90 in DMY as the lowest rank (Table 3). 3.2 GRAIN YIELD, HARVEST INDEX, AND WA- TER PRODUCTIVITY (WP) The effects of deficit irrigation, genotype, and their interaction on yield, HI, and WP were significant (p < 0.01). Grain yield and HI decreased significantly in re- sponse to water deficit, resulting in lower values equal to 52.8 % and 28.9 %, respectively (Table 3). The mean of WP under severe deficit irrigation was reduced by 31.7 % compared to normal irrigation (Table 3). Chimonyo et al. (2016) reported no significant reduction in sorghum yield under deficit irrigation in comparison to full irriga- tion (3160 kg ha-1 vs. 3240 kg ha-1), indicating sorghum as drought tolerant, which is suitable for marginal lands. However, our results noted that sub-optimal irrigation resulted in sub-optimal WP. Hence, an important point to farmers is the benefit of irrigating sorghum consider- ing the water supply. The highest grain yield and WP under normal irri- gation belonged to genotype TN-04-79 followed by TN- 04-134 (Fig. 2). Under mild deficit irrigation, genotype TN-04-79 had the highest grain yield and WP, because WP of this genotype under mild deficit irrigation was slightly higher (not statistically significant) than that val- ue under normal irrigation. The highest values of grain yield and WP, when severe deficit irrigation was im- posed, were related to genotype KGS23 followed by TN- 04-79 and TN-04-86. Moreover, the highest WP obtained for genotype TN-04-79 under mild deficit irrigation and the lowest value of WP belonged to genotype TN-04-142 under severe stress (Fig. 2). It has been reported that sorghum WP was in a range of 1.24-1.34 kg m-3 in Nebraska under normal ir- rigation (Maman et al. 2003). Grain WP in the trial of Hadebe et al. (2020) was relatively lower in a range of 0.75-1.1 kg m-3 for three different genotypes. Moreover, they attributed high WP under irrigation to high yield proportional to water applied in the field. The effect of genotype, duration, and extent of water stress may ac- count for the variation of results in this study with those of other studies. 3.3 VALUES AND RANKS OF DROUGHT INDICES Mean comparison of ranking values (R), ranking mean values (R’) and rank sum (RS) under mild deficit irrigation showed that genotype TN-04-79 performed superiorly except for TOL (ranked 3) (Table 5). The su- perior genotype based on the TOL index was genotype TN-04-129. On the other hand, genotype TN-04-142 performed inferiorly based on Yp, Ys, and all drought tol- erance indices except for TOL ranked 8 (Table 5). A dif- ferent trend in the response of the genotypes to severe deficit irrigation was observed. While genotype TN-04- 79 performed superiorly based on Yp, MP, STI, GMP, and HM, genotype KGS23 was superior when considering Ys, TOL, SSI, YSI, YRR, and YI indices (Table 5). It could be concluded that different drought-tol- erance indices presented herein introduced different genotypes as drought tolerant. Similar results have been reported for the screening of drought-tolerant genotypes based on various indices (Nikneshan et al., 2019; Abd El- Mohsen et al. 2015). Therefore, the selection of tolerant Acta agriculturae Slovenica, 117/4 – 20216 L. NAZARI et al. genotypes was adopted by the ranking method based on ranking mean values (R’), standard deviation of ranks (SDR), and rank sum (RS). According to values of R’ and RS calculated based on the yield under normal irrigation and mild deficit irrigation, genotype TN-04-79 exhibited the first mean rank value and sum rank value followed by genotype TN- 04-129 indicating that these genotypes can be primarily categorized as the most tolerant to mild deficit irrigation. Whilst, genotype TN-04-142, MGS2, and TN-04-78 ex- hibited the worst mean rank and rank sum, respectively, that can be considered as the most susceptible to mild water deficit irrigation (Table 5). On the other hand, R’ and RS calculated based on yield in normal irrigation and severe deficit irrigation presented different results. The first mean rank and sum rank value belonged to gen- Figure 2: Grain yield and water productivity (WP) of 10 grain sorghum genotypes under normal irrigation, mild and severe defi- cit irrigation. Means followed by the same letter are not significantly different in each level of irrigation treatment (Duncan’s test, p < 0.05) otype KGS23, while genotype TN-04-142 was inferior in R’ and RS (Table 5). 3.4 CORRELATIONS AMONG DROUGHT INDI- CES Pearson’s correlation coefficients (r) between Yp, Ys, and the indices were determined to select the best indices for the screening of drought-tolerant genotypes (Fig. 3). A positive significant correlation between Yp and Ys un- der mild and severe deficit irrigation was recorded (Fig. 3). This may imply that high yielding potential under normal irrigation is necessarily accompanied by reasona- ble yield under mild and severe deficit irrigation. Similar Acta agriculturae Slovenica, 117/4 – 2021 7 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes ge no ty pe C al cu la te d ba se d on y ie ld u nd er n or m al a nd m ild d efi ci t i rr ig at io n Y p Y S TO L M P SS I ST I G M P H M YS I YR R YI M G S2 4. 39 ±0 .3 6 2. 67 ±0 .2 8 1. 71 ±0 .3 2 3. 53 ±0 .2 8 0. 74 ±0 .1 0 0. 34 ±0 .0 6 3. 42 ±0 .2 8 3. 32 ±0 .2 9 0. 61 ±0 .0 6 0. 39 ±0 .0 6 0. 97 ±0 .1 0 KG S2 3 5. 58 ±0 .2 7 4. 28 ±0 .5 4 1. 31 ±0 .2 8 4. 93 ±0 .4 0 0. 45 ±0 .1 2 0. 70 ±0 .1 2 4. 88 ±0 .4 3 4. 84 ±0 .4 5 0. 76 ±0 .0 6 0. 24 ±0 .0 6 1. 55 ±0 .2 0 TN -0 4- 78 6. 07 ±0 .6 9 2. 70 ±0 .5 3. 38 ±0 .7 3 4. 38 ±0 .4 8 1. 04 ±0 .1 5 0. 48 ±0 .1 2 4. 03 ±0 .4 9 3. 72 ±0 .5 3 0. 45 ±0 .0 8 0. 55 ±0 .0 8 0. 98 ±0 .1 8 TN -0 4- 79 8. 18 ±0 .5 7 7. 30 ±0 .3 2 0. 88 ±0 .3 9 7. 74 ±0 .4 2 0. 20 ±0 .0 8 1. 75 ±0 .1 8 7. 73 ±0 .4 1 7. 71 ±0 .4 1 0. 89 ±0 .0 4 0. 11 ±0 .0 4 2. 65 ±0 .1 2 TN -0 4- 12 9 5. 98 ±0 .4 5 5. 23 ±0 .3 6 0. 75 ±0 .7 8 5. 60 ±0 .1 2 0. 23 ±0 .2 3 0. 91 ±0 .0 3 5. 58 ±0 .1 1 5. 56 ±0 .1 0 0. 88 ±0 .1 2 0. 12 ±0 .1 2 1. 89 ±0 .1 3 TN -0 4- 13 4 7. 91 ±0 .2 3 4. 06 ±0 .6 2 3. 85 ±0 .4 7 5. 99 ±0 .4 0 0. 92 ±0 .1 3 0. 94 ±0 .1 7 5. 66 ±0 .4 9 5. 35 ±0 .5 8 0. 51 ±0 .0 7 0. 49 ±0 .0 7 1. 47 ±0 .2 2 TN -0 4- 14 2 3. 47 ±0 .6 0 0. 72 ±0 .1 1 2. 76 ±0 .5 0 2. 10 ±0 .3 5 1. 50 ±0 .0 3 0. 07 ±0 .0 2 1. 58 ±0 .2 5 1. 19 ±0 .1 9 0. 21 ±0 .0 1 0. 79 ±0 .0 1 0. 26 ±0 .0 4 TN -0 4- 59 6. 68 ±0 .2 9 4. 91 ±0 .1 9 1. 78 ±0 .4 2 5. 79 ±0 .1 2 0. 50 ±0 .1 0 0. 96 ±0 .0 4 5. 72 ±0 .1 2 5. 65 ±0 .1 2 0. 74 ±0 .0 5 0. 26 ±0 .0 5 1. 78 ±0 .0 7 TN -0 4- 86 5. 45 ±0 .6 3 4. 69 ±0 .4 3 0. 76 ±0 .2 3 5. 07 ±0 .5 2 0. 26 ±0 .0 5 0. 75 ±0 .1 6 5. 06 ±0 .5 2 5. 04 ±0 .5 1 0. 86 ±0 .0 3 0. 14 ±0 .0 3 1. 70 ±0 .1 6 TN -0 4- 90 4. 75 ±0 .1 1 3. 71 ±0 .3 7 1. 03 ±0 .2 8 4. 23 ±0 .2 3 0. 41 ±0 .1 2 0. 52 ±0 .0 6 4. 20 ±0 .2 5 4. 16 ±0 .2 6 0. 78 ±0 .0 6 0. 22 ±0 .0 6 1. 35 ±0 .1 3 ge no ty pe C al cu la te d ba se d on y ie ld u nd er n or m al a nd se ve re d efi ci t i rr ig at io n Y p Y s TO L M P SS I ST I G M P H M YS I YR R YI M G S2 4. 39 ±0 .3 6 1. 67 ±0 .1 9 2. 72 ±0 .5 5 3. 03 ±0 .0 9 1. 16 ±0 .1 5 0. 21 ±0 .0 1 2. 70 ±0 .0 5 2. 41 ±0 .1 4 0. 39 ±0 .0 8 0. 61 ±0 .0 8 0. 61 ±0 .0 7 KG S2 3 5. 58 ±0 .2 7 4. 19 ±0 .3 7 1. 39 ±0 .5 2 4. 89 ±0 .1 9 0. 47 ±0 .1 6 0. 68 ±0 .0 6 4. 83 ±0 .2 1 4. 78 ±0 .2 4 0. 75 ±0 .0 8 0. 25 ±0 .0 8 1. 52 ±0 .1 3 TN -0 4- 78 6. 07 ±0 .6 9 1. 70 ±0 .2 8 4. 37 ±0 .7 2 3. 89 ±0 .3 9 1. 35 ±0 .1 0 0. 30 ±0 .0 7 3. 21 ±0 .3 4 2. 65 ±0 .3 6 0. 28 ±0 .0 5 0. 72 ±0 .0 5 0. 62 ±0 .1 0 TN -0 4- 79 8. 18 ±0 .5 7 4. 07 ±0 .4 7 4. 11 ±0 .6 2 6. 13 ±0 .4 2 0. 95 ±0 .1 1 0. 97 ±0 .1 5 5. 76 ±0 .4 4 5. 42 ±0 .4 8 0. 50 ±0 .0 6 0. 50 ±0 .0 6 1. 47 ±0 .1 7 TN -0 4- 12 9 5. 98 ±0 .4 5 1. 71 ±0 .1 8 4. 27 ±0 .6 1 3. 84 ±0 .1 6 1. 34 ±0 .0 9 0. 30 ±0 .0 2 3. 19 ±0 .0 9 2. 65 ±0 .1 8 0. 29 ±0 .0 5 0. 71 ±0 .0 5 0. 62 ±0 .0 6 TN -0 4- 13 4 7. 91 ±0 .2 3 2. 95 ±0 .1 0 4. 96 ±0 .2 3 5. 43 ±0 .1 3 1. 18 ±0 .0 3 0. 68 ±0 .0 3 4. 83 ±0 .1 2 4. 30 ±0 .1 2 0. 37 ±0 .0 1 0. 63 ±0 .0 1 1. 07 ±0 .0 4 TN -0 4- 14 2 3. 47 ±0 .6 0 0. 48 ±0 .0 2 2. 99 ±0 .5 9 1. 98 ±0 .3 1 1. 62 ±0 .0 4 0. 05 ±0 .0 1 1. 29 ±0 .1 3 0. 85 ±0 .0 4 0. 14 ±0 .0 2 0. 86 ±0 .0 2 0. 17 ±0 .0 1 TN -0 4- 59 6. 68 ±0 .2 9 3. 55 ±0 .2 8 3. 13 ±0 .5 0 5. 12 ±0 .1 3 0. 88 ±0 .1 1 0. 69 ±0 .0 5 4. 87 ±0 .1 6 4. 63 ±0 .2 1 0. 53 ±0 .0 6 0. 47 ±0 .0 6 1. 29 ±0 .1 0 TN -0 4- 86 5. 45 ±0 .6 3 3. 91 ±0 .3 1 1. 54 ±0 .6 6 4. 68 ±0 .3 7 0. 52 ±0 .1 8 0. 62 ±0 .0 9 4. 61 ±0 .3 4 4. 54 ±0 .3 2 0. 72 ±0 .1 0 0. 28 ±0 .1 0 1. 42 ±0 .1 1 TN -0 4- 90 4. 75 ±0 .1 1 3. 35 ±0 .2 2 1. 39 ±0 .3 0 4. 05 ±0 .0 9 0. 55 ±0 .1 1 0. 46 ±0 .0 3 3. 99 ±0 .1 1 3. 93 ±0 .1 3 0. 71 ±0 .0 6 0. 29 ±0 .0 6 1. 21 ±0 .0 8 Ta bl e 4: M ea n va lu es ± st an da rd d ev ia tio n of g ra in y ie ld (t on /h a) a nd d ro ug ht to le ra nc e in di ce s o f t en so rg hu m g en ot yp es u nd er n or m al ir rig at io n, m ild a nd se ve re w at er d efi ci t irr ig at io n Yp = g ra in y ie ld u nd er n or m al ir rig at io n, Y s = g ra in y ie ld u nd er d efi ci t i rr ig at io n, T O L = to le ra nc e i nd ex , M P = m ea n pr od uc tiv ity , S SI = st re ss su sc ep tib ili ty in de x, S TI = st re ss to le ra nc e i nd ex , G M P = ge om et ric p ro du ct iv ity , H M = h ar m on ic m ea n of y ie ld , Y SI = y ie ld st ab ili ty in de x, Y RR = y ie ld re du ct io n ra tio , Y I = y ie ld in de x Acta agriculturae Slovenica, 117/4 – 20218 L. NAZARI et al. M ild d efi ci t i rr ig at io n R R’ SD R RS G en ot yp e Y p Y s TO L M P SS I ST I G M P H M YS I YR R YI M G S2 9 9 6 9 7 9 9 9 7 7 9 8. 18 1. 17 9. 35 KG S2 3 6 5 5 6 5 6 6 6 5 5 5 5. 45 0. 52 5. 98 TN -0 4- 78 4 8 9 7 9 8 8 8 9 9 8 7. 91 1. 45 9. 36 TN -0 4- 79 1 1 3 1 1 1 1 1 1 1 1 1. 27 0. 65 1. 92 TN -0 4- 12 9 5 2 1 4 2 4 4 3 2 2 2 2. 55 1. 29 3. 84 TN -0 4- 13 4 2 6 10 2 8 3 3 4 8 8 6 5. 55 2. 73 8. 28 TN -0 4- 14 2 10 10 8 10 10 10 10 10 10 10 10 9. 82 0. 60 10 .4 2 TN -0 4- 59 3 3 7 3 6 2 2 2 6 6 3 4. 00 1. 84 5. 84 TN -0 4- 86 7 4 2 5 3 5 5 5 3 3 4 4. 18 1. 40 5. 58 TN -0 4- 90 8 7 4 8 4 7 7 7 4 4 7 6. 09 1. 70 7. 79 Se ve re d efi ci t i rr ig at io n R R’ SD R RS G en ot yp e Y p Y s TO L M P SS I ST I G M P H M YS I YR R YI M G S2 9 9 4 9 6 9 9 9 6 6 9 7. 73 1. 85 9. 58 KG S2 3 6 1 1 4 1 3 4 2 1 1 1 2. 27 1. 74 4. 01 TN -0 4- 78 4 8 9 7 9 7 7 7 9 9 8 7. 64 1. 50 9. 14 TN -0 4- 79 1 2 7 1 5 1 1 1 5 5 2 2. 82 2. 23 5. 05 TN -0 4- 12 9 5 7 8 8 8 8 8 8 8 8 7 7. 55 0. 93 8. 48 TN -0 4- 13 4 2 6 10 2 7 4 3 5 7 7 6 5. 36 2. 46 7. 82 TN -0 4- 14 2 10 10 5 10 10 10 10 10 10 10 10 9. 55 1. 51 11 .0 5 TN -0 4- 59 3 4 6 3 4 2 2 3 4 4 4 3. 55 1. 13 4. 67 TN -0 4- 86 7 3 3 5 2 5 5 4 2 2 3 3. 73 1. 62 5. 35 TN -0 4- 90 8 5 2 6 3 6 6 6 3 3 5 4. 82 1. 83 6. 65 Ta bl e 5: R an ki ng v al ue s ( R) , r an ki ng m ea n va lu es (R ’), st an da rd d ev ia tio n of ra nk s ( SD R) a nd ra nk su m (R S) o f g ra in y ie ld o f t en so rg hu m g en ot yp es u nd er n or m al ir rig at io n an d w at er d efi ci t i rr ig at io n aft er 1 20 m m e va po ra tio n fr om P an cl as s A Yp = g ra in y ie ld u nd er n or m al ir rig at io n, Y s = g ra in y ie ld u nd er d efi ci t i rr ig at io n, T O L = to le ra nc e in de x, M P = m ea n pr od uc tiv ity , S SI = st re ss su sc ep tib ili ty in de x, S TI = st re ss to le ra nc e in de x, G M P = ge om et ric p ro du ct iv ity , H M = h ar m on ic m ea n of y ie ld , Y SI = y ie ld st ab ili ty in de x, Y RR = y ie ld re du ct io n ra tio , Y I = y ie ld in de x Acta agriculturae Slovenica, 117/4 – 2021 9 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes results of the wheat response to drought were previously recorded by Abebe et al. (2020). While there was a significant correlation between Ys (mild and severe deficit irrigation) and all the indi- ces, there was no correlation between Yp and SSI, YSI, and YRR. There was no correlation between Yp and TOL under mild deficit irrigation. On the other hand, a posi- tive significant correlation was obtained between Yp and TOL calculated based on yield in severe deficit irrigation suggesting that selection based on the low score of TOL may lead to enhanced yield under severe deficit irriga- tion but reduced yield under normal irrigation (Fig. 3). Also, yield in all irrigation treatments was significantly and positively correlated with MP, STI, GMP, HM, and YI (Fig. 3). Thus, it can be concluded that these indices were more efficient in the selection of genotypes with high yield potential under different water conditions. Indices being significantly correlated with grain yield under both normal irrigation and water deficit ir- rigation are suitable for the screening of genotypes (Mi- tra, 2001). Therefore, indices MP, STI, GMP, HM, and YI which were positively correlated with both Yp and Ys at p ≤ 0.01 (Fig. 3) may be considered as better predictors of yield in different irrigation. As well, sorghum genotypes with high values of MP, STI, GMP, HM, and YI can be thus regarded as drought tolerant. Our results are some- what in agreement with those findings of Nouri et al. (2011) and Golabadi et al. (2006) who found a correla- tion between either Ys or Yp and MP, GMP, and STI. A perfect positive correlation (r = 1) was noted be- tween Ys and YI and between SSI and YRR. On the other hand, a perfect negative correlation (r = -1) was noted between SSI and YSI, YSI, and YRR in both mild and se- vere water deficit irrigation (Fig. 3). A similar finding was recorded by Mickky et al. (2019) who evaluated 10 wheat cultivars based on drought tolerance indices under nor- mal irrigation (Yp) and deficit irrigation (Ys). 3.5 CLUSTER ANALYSIS Classification of genotypes according to Yp, Ys, and various indices under normal irrigation and mild defi- cit irrigation categorized 10 sorghum into three groups; group 1 including MGS2, TN-04-90, TN-04-78 and TN- 04-14; group 2 including KGS23, TN-04-86, TN-04-12, TN-04-99 and TN-04-13; and group 3 including geno- type TN-04-79 (Fig. 4a). Clustering based on yield and drought tolerance indices under normal irrigation and mild deficit irrigation grouped the genotypes into toler- ant, semi-tolerant/susceptible, and susceptible. The first group with the lowest value of R’ and RS (TN-04-79) can be distinguished as tolerant to mild deficit irrigation. The second group had mean values of R’ (2.75-5.67) and RS (3.97-8.44) considered as semi-sensitive/tolerant and the third group with higher R’ and RS was the most suscepti- ble genotypes to mild deficit irrigation (Fig. 4 a). Fig. 3: Heat map based on the actual values of indices (Pearson’s correlation analysis) across 10 sorghum genotypes produced us- ing iPASTIC online tool kit. Yp, yield under normal irrigation; Ys, yield under mild deficit irrigation for (a) and under severe deficit irrigation for (b); TOL, tolerance index; MP, mean productivity, GMP, geometric mean probability; HM, Harmonic mean; SSI, stress susceptibility index; STI, stress tolerance index; YI, yield index; YSI, yield stability index; RSI, relative stress index Acta agriculturae Slovenica, 117/4 – 202110 L. NAZARI et al. Fig. 4: Dendrograms of the cluster analysis and similarity coefficients among 10 sorghum genotypes based on Yp, Ys, and the drought tolerance indices under normal irrigation and mild deficit irrigation (a) and under normal irrigation and severe deficit irrigation (b) On the other hand, three different clusters were observed based on Yp, Ys, and drought tolerance indices under normal irrigation and severe deficit irrigation (Fig. 4b). Genotypes MGS2, TN-04-14, TN-04-78, and TN- 04-12 were classified into group 1; KGS23, TN-04-86, and TN-04-90 into group 2; and TN-04-79, TN-04-13, and TN-04-59 into group 3. The first and second groups included the genotypes with the lowest to medium values of R’ and RS and thus were considered to be tolerant or semi-tolerant. The genotypes of the third group had the highest values of R’ and RS indicating the most suscepti- ble to severe deficit irrigation. Cluster analysis has been extensively employed for the determination of genetic diversity and classification of genotypes under various abiotic stresses (Golabadi et al. 2006; Mohammadi et al. 2011). 3.6 PRINCIPAL COMPONENT ANALYSIS (PCA) AND BIPLOT The PCA results revealed that the first two prin- cipal components accounted for 98.51 % (PC1:81.03 %, PC2:4.63 %) of the total variation in yield performance and nine yield-based indices calculated under normal ir- rigation and mild deficit irrigation. Biplot showed that the PC1 was positively correlated with yield (Yp and Ys under mild stress) and all indices except TOL and SSI, whereas PC2 was positively correlated with yield (Yp and Ys under mild deficit irrigation) and all indices excluding RSI and YSI (Fig. 5a). On the other hand, the PCA biplot for yield (Yp and Ys under severe deficit irrigation) and drought tolerance indices of sorghum genotypes was reflecting 99.28  % (PC1:74.6 %, PC2:24.63 %) of the total variability in data (Fig. 5b). The biplot categorized the indices into three groups (Fig. 5). The first group was those with high PC1 and PC2 (Yp, MP, STI, GMP, HM, YI, and YS in Fig. 5a and Yp, MP, STI, GMP, and HM in Fig 5b). The second group was indices with low PC1 but high PC2 (SSI and TOL) (Fig. 5a,b) and the third group were those with high PC1 but low PC2 including RSI (Fig. 5a,b) and Ys and YI (Fig. 5b). The cosine of the angle between the vectors of any two indices in a biplot is an indicator of the correlation coefficient. Therefore, we can note that those indices whose vector has been placed between the vectors of Yp and Ys are appropriate for the selection of drought-toler- ant genotypes. It can be implied that MP, GMP, STI, HM, and YI allocating between Yp and Ys are the best indices to distinguish tolerant from susceptible genotypes. Herein, the results obtained from PCA (Fig. 5) confirmed those obtained from correlation coefficients (Fig. 3). The results of our study showed that TN-04-79 and TN-04-59 are tolerant genotypes with the highest values for the MP, GMP, STI, and HM indices, while genotypes KGS23, TN-04-129, TN-04-86, and TN-04-90 under mild stress and genotypes KGS23, TN-04-86, TN-04-90 with the highest values for YSI and RSI were the most Acta agriculturae Slovenica, 117/4 – 2021 11 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes stable genotypes (Table 4). Introduction of these tolerant genotypes into the sorghum breeding programs may be suggested to policymakers to release new cultivars toler- ant to drought stress. It has been noted that increasing harvest index can improve yield stability (Kashiwagi et al., 2015). The reduction of grain yield under deficit irri- gation could lead to a lower harvest index. We also found that indices including MP, GMP, HM, STI, YI, and YSI are strongly correlated with sorghum yield. Thus, these drought-tolerant indices should benefit the breeders in breeding programs. 4 CONCLUSIONS In the current study, eight sorghum genotypes col- lected from different parts of Iran along with two prom- ising lines reported as drought-tolerant were compared in terms of response to deficit irrigation. The grain yield and water productivity of the genotypes were signifi- cantly influenced by water deficit irrigation. The relative efficacy of selection indices could be an advantage using two or more traits simultaneously than using single traits independently. Thus, indices including MP, STI, GMP, HM, and YI, highly correlated with Yp and Ys, may be more suitable for the selection of drought-tolerant geno- types. Screening of tolerant genotypes to water deficit ir- rigation using the ranking method and cluster analysis discriminated genotypes as the most tolerant, semi-tol- erant/sensitive, and susceptible. Therefore they are rec- ommended to be used in breeding programs as parents for improvement of drought tolerance in commercial cultivars. Further evaluation of these genotypes based on drought indices across multiple locations and years is still demanded to validate their stability for developing sorghum cultivars. 5 ACKNOWLEDGMENTS The present work was supported by Agricultural Organization of Fars [Project number 24-50-03-332- 961709]. 6 ABBREVIATIONS ANOVA: Analysis of variance; PCA: principal com- ponent analysis; PH: plant height; PL: panicle length; SD: stem diameter; NoL: number of leaves per plant; 1000 SM: 1000 seed mass; DMY: dry matter yield; HI: harvest index; WP: Water productivity; Yp: grain yield under normal irrigation; Ys: grain yield under deficit irrigation; MP: mean productivity; TOL: tolerance index; SSI: stress susceptibility index; STI: stress tolerance index; GMP: geometric productivity; HM: harmonic mean of yield; Fig. 5: Principal components (PC) analysis based on the correlation matrix of yield under normal irrigation (Yp) and yield under mild deficit irrigation (a) and severe deficit irrigation (b) (Ys) and nine tolerance and susceptibility indices calculated using iPASTIC online tool kit. Yp, yield under normal irrigation; Ys, yield under mild deficit irrigation for (a) and under severe deficit ir- rigation for (b); TOL, tolerance index; MP, mean productivity, GMP, geometric mean probability; HM, Harmonic mean; SSI, stress susceptibility index; STI, stress tolerance index; YI, yield index; YSI, yield stability index; RSI, relative stress index. Acta agriculturae Slovenica, 117/4 – 202112 L. NAZARI et al. YSI: yield stability index; YRR: yield reduction ratio; YI: yield index. 7 REFERENCES Abd El-Mohsen, A. A., Abd El-Shafi, M. A., Gheith, E. M. S., Suleiman, H. S. (2015). Using different statistical proce- dures for evaluating drought tolerance indices of bread wheat genotypes. Advance in Agriculture and Biology, 4, 19- 30. https://doi.org/10.15192/PSCP.AAB.2015.4.1.1930 Abebe, T., Belay, G., Tadesse, T., Keneni, G. (2020). Selec- tion efficiency of yield based drought tolerance indices to identify superior sorghum [Sorghum bicolor (L.) Moench] genotypes under two-contrasting environments. African Journal of Agricultural Research, 15(3), 379-392. https://doi. org/10.5897/AJAR2020.14699 Ali, M. H., Talukder, M. S. U. (2008). Increasing water produc- tivity in crop production: a synthesis. Agricultural Water Management, 95, 1201-1213. https://doi.org/10.1016/j.ag- wat.2008.06.008 Almodares, A., Hotjatabady, R. H., Mirniam, E. (2013). Effects of drought stress on biomass and carbohydrate contents of two sweet sorghum cultivars. Journal of Environmental Bi- ology, 34, 585-589. Anwar, H. A., Perveen, R., Mansha, M., Abid, M., Sarwar, Z. M., Aatif, H. M., Umar, U., Sajid, M., Aslam, H. M. U., Alam, M. M., Rizwan, M., Ikram, R. M., Alghanem, S. M. S., Rashid, A., Khan, K. A. (2020). Assessment of grain yield indices in response to drought stress in wheat (Triticum aestivum L.). Saudi Journal of Biological Sciences, 27(7), 1818-1823. https://doi.org/10.1016/j.sjbs.2019.12.009 Ashraf, M., Foolad, M. R. (2007). Roles of glycine, betaine and proline in improving plant abiotic stress resistance. Envi- ronmental and Experimental Botany, 59, 206-216. https:// doi.org/10.1016/j.envexpbot.2005.12.006 Blum, A. 1988. Plant breeding for stress environments. CRC Press, Boca Raton, Florida, USA. Rosielle A. A., Hamblin J. (1981). Theoretical aspects of selection for yield in stress and non-stress environments. Crop Science, 21, 943-946. https://doi.org/10.2135/cropsci1981.0011183X002100060 033x Bouslama, M., Schapaugh, W. T. (1984). Stress tolerance in soybean. Part 1: evaluation of three screening techniques for heat and drought tolerance. Crop Science, 24, 933-937. https://doi.org/10.2135/cropsci1984.0011183X002400050 026x Chimonyo, V. G. P., Modi, A. T., Mabhaudhi, T. (2016). Water use and productivity of a sorghum-cowpea-bottle gourd intercrop system. Agricultural Water Management, 165, 82- 96. https://doi.org/10.1016/j.agwat.2015.11.014 Clarke, D., Smith, M., El-Askari, K. (2001). CropWat for Win- dows: User Guide; University of Southampton: Southamp- ton, UK. Clarke, J. M., DePauw, R. M., Townley Smith, T. F. (1992). Eval- uation of methods for quantification of drought tolerance in wheat. Crop Science, 32, 423-428. https://doi.org/10.2135/cr opsci1992.0011183X003200030029x Devnarain, N., Crampton, B. G., Chikwamba, R., Becker, J. V. W., & O’Kennedy, M. M. (2016). Physiological responses of selected African sorghum landraces to progressive water stress and re-watering. South African Journal of Botany, 103, 61-9. https://doi.org/10.1016/j.sajb.2015.09.008 Doorenbos, J., Kassam, A. H. (1986). Yield Response to Water, Irrigation and Drainage Paper 33. Food and Agricultural Organization of the United Nations: Rome. Doorenbos, J., Pruitt, W. O. (1977). Crop water requirements. Revised 1977. FAO Irrig Drain. Paper 24. FAO of the Unit- ed Nations, Rome, pp 144. Fernandez, G. C. J. (1992). Effective selection criteria for as- sessing plant stress tolerance. In: Proceedings of the Inter- national Symposium on Adaptation of Vegetables and other Food Crops in Temperature and Water Stress, Taiwan 13-16 August 1992, 257-270 p. Fischer, R. A., Maurer, R. (1978). Drought resistance in spring wheat cultivars. I. Grain yield responses. Australian Jour- nal of Agricultural Research, 29, 897-912. https://doi. org/10.1071/AR9780897 Fracasso, A., Trindade, L., Amaducci, S. (2016). Drought toler- ance strategies highlighted by two Sorghum bicolor races in a dry-down experiment. Journal of Plant Physiology, 190, 1-14. https://doi.org/10.1016/j.jplph.2015.10.009 Gavuzzi, P., Rizza, F., Palumbo, M., Campaline, R. G., Ricciardi, G. L., Borghi, B. (1997). Evaluation of field and laboratory predictors of drought and heat tolerance in winter cereals. Canadian Journal of Plant Science, 77, 523-531. https://doi. org/10.4141/P96-130 Golabadi, M. A., Arzani, S. A., Maibody, M. (2006). Assessment of drought tolerance in segregating populations in durum wheat. African Journal of Agricultural Research, 1(5), 62- 171. Golestani Araghi, S., Assad, M. T. (1998). Evaluation of four Screening Techniques for Drought Resistance and Their Relationship to Yield Reduction Ratio in Wheat. Euphytica. 103, 293-299. https://doi.org/10.1023/A:1018307111569 Hadebe, S. T., Mabhaudhi, T., Modi, A. T. (2020). Water pro- ductivity of selected Sorghum genotypes under rainfed con- ditions. International Journal of Plant Production, 14, 259- 272. https://doi.org/10.1007/s42106-019-00082-4 Jafari, A., Paknejad, F., Jami Al-Ahmadi, M. (2009). Evaluation of selection indices for drought tolerance of corn (Zea mays L.) hybrids. International Journal of Plant Production, 3(4), 33-38. https://doi.org/10.22069/IJPP.2012.661 Kashiwagi, J., Krishnamurthy, L., Purushothaman, R., Upad- hyaya, H. D., Gaur, P. M., Gowda, C. L. L., et al. (2015). Scope for improvement of yield under drought through the root traits in chickpea (Cicer arietinum L.). Field Crop Re- search, 170, 47-54. https://doi.org/10.1016/j.fcr.2014.10.003 Khalili, M., Pour-Aboughadareh, A., Naghavi, M. R., Moham- madAmini, E. (2014). Evaluation of drought tolerance in safflower genotypes based on drought tolerance indices. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 42, 214- 218. https://doi.org/10.15835/nbha4219331 Ludlow, M. M., Muchow, R. C. A Critical Evaluation of Traits for Improving Crop Yields in Water-Limited Environ- ments1. In: Brady N. C., editor. Advances in Agronomy. Vol. 43. Academic Press; Cambridge, MA, USA: 1990. pp. Acta agriculturae Slovenica, 117/4 – 2021 13 Introduction of the best criterion for evaluation of tolerance to drought stress in sorghum’s genotypes 107-153. https://doi.org/10.1016/S0065-2113(08)60477-0 Maman, N., Lyon, D. J., Mason, S. C., Galusha, T. D., Higgins, R. (2003). Pearl millet and grain sorghum yield response to water supply in Nebraska. Agronomy Journal, 95, 1618- 1624. https://doi.org/10.2134/agronj2003.1618 Mickky, B., Aldesuquy, H., Elnajar, M. (2019). Uni- and multi- variate assessment of drought response yield indices in 10 wheat cultivars. Journal of Crop Science and Biotechnology, 22, 21-29. https://doi.org/10.1007/s12892-018-0221-0 Mitra, J. (2001). Genetics and genetic improvement of drought resistance in crop plants. Current Science, 80, 758-762. Mohammadi, M., Karimizadeh, F., Abdipour, M. (2011). Evalu- ation of drought tolerance in bread wheat genotypes under dryland and supplemental irrigation conditions. Australian Journal of Crop Science, 5, 487-493. Mutava, R. N., Prasad, P. V. V., Tuinstra, M. R., Kofoid, M. D., Yu, j. (2011). Characterization of sorghum genotypes for traits related to drought tolerance. Field Crops Research, 123, 10-18. https://doi.org/10.1016/j.fcr.2011.04.006 Nikneshan, P., Tadayyon, A., Javanmard, M. (2019). Evaluating drought tolerance of castor ecotypes in the center of Iran. Heliyon, 5, e01403. https://doi.org/10.1016/j.heliyon.2019. e01403 Nouri, A., Etminan, A., Jaime, A., Silva, T. D., Mohammadi, R. (2011). Assessment of yield, yield related traits and drought tolerance of durum wheat genotypes (Triticum turjidum var. durum Desf.). Australian Journal of Crop Science, 5, 8-16. Ottman, M. J., Kimball, B. A., Pinter, P. J. Jr., Wall, G. W., Van- derlip, R. L., Leavitt, S. W., LaMorte, R. L., Matthias, A. D., Brooks, T. J. (2001). Elevated CO2 increases sorghum bio- mass under drought conditions. New Phytologist, 150, 261- 273. https://doi.org/10.1046/j.1469-8137.2001.00110.x Priyanka, V., Kumar, R., Dhaliwal, I., Kaushik, P. (2021). Germ- plasm conservation: Instrumental in agricultural biodi- versity-A review.  Sustainability, 13(12), 6743. https://doi. org/10.3390/su13126743 Silva, M. D. A., Silva, J. A. G. D., Enciso, J., Sharma, V., Jifon, J. (2008). Yield components as indicators of drought toler- ance of sugarcane. Scientia Agricola, 65, 620-627. https:// doi.org/10.1590/S0103-90162008000600008 Surendran, U., Raja, B. A. P., Kumar, V., Rajan, K., Jayakumar, M. (2019). Analysis of drought from humid, semi-arid and arid regions of India using DrinC model with different drought indices. Water Resources Management, 33, 1521- 1540. https://doi.org/10.1007/s11269-019-2188-5 Tari, I., Laskay, G., Takács, Z., Poór, P. (2013). Response of sor- ghum to abiotic stresses: A review. Journal of Agronomy and Crop Science, 199, 264-274. https://doi.org/10.1111/ jac.12017 Zhang, X., Lei, L., Lai, J., Zhao, H., Song, W., (2018). Effects of drought stress and water recovery on physiological re- sponses and gene expression in maize seedlings. BMC Plant Biology, 18, 68. https://doi.org/10.1186/s12870-018-1281-x Acta agriculturae Slovenica, 117/4, 1–7, Ljubljana 2021 doi:10.14720/aas.2021.117.4.1601 Original research article / izvirni znanstveni članek Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard Mersija DELIĆ 1, Fikreta BEHMEN 2, Saša MATIJAŠEVIĆ 3, Šaćira MANDAL 4, Saud HAMIDOVIĆ 2, Senad MURTIĆ 5, 6 Received April 08, 2020; accepted October 30, 2021. Delo je prispelo 8. aprila 2020, sprejeto 30. oktobra 2021 1 University of Sarajevo, Faculty of Agriculture and Food Sciences, Department of Viticulture, Sarajevo, Bosnia and Herzegovina 2 University of Sarajevo, Faculty of Agriculture and Food Sciences, Department of Horticulture, Sarajevo, Bosnia and Herzegovina 3 University of Belgrade, Faculty of Agriculture, Department of Viticulture, Belgrade, Serbia 4 University of Sarajevo, Faculty of Pharmacy, Department of Natural Sciences in Pharmacy, Sarajevo, Bosnia and Herzegovina 5 University of Sarajevo, Faculty of Agriculture and Food Sciences, Department of Plant Physiology, Sarajevo, Bosnia and Herzegovina 6 Corresponding author, e-mail: murticsenad@hotmail.com Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard Abstract: The aim of this study was to study the impact of crop load on the yield and grape quality of ‘Merlot’ and ‘Vra- nac’ (Vitis vinifera L.) in Trebinje vineyard. The crop load levels studied in this trial were 9 buds (V1) and 12 buds (V2) per vine at each variety trained on Lenz-Moser bilateral cordon system. The impact was determined by measurements of yield per vine and grape quality characterized by the contents of total soluble solids, titratable acidity, total phenolics, total flavonoids and by total antioxidant capacity. The measured parameters of grape quality of ‘Merlot’ and ‘Vranac’ were not influenced significant- ly by crop load levels. V2, compared to the V1, showed the po- tential for increasing grape yield only for Merlot variety under experimental conditions. The results of this study also showed a positive correlation between total phenolics/flavonoids and total antioxidant capacity of grape berries in both varieties, re- gardless of crop loads applied. Key words: flavonoids; total phenolics; pruning; yield Vpliv obtežitve na pridelek in kakovost grozdja sort Merlot in Vranac (Vitis vinifera L.) v vinogradu v Trebinju Izvleček: Namen raziskave je bil preučiti vpliv obtežitve trt na pridelek in kakovost sort Merlot in Vranac (Vitis vini- fera L.) v vinogradih Trebinja. Velikost obremenitve trt v tem poskusu so bile trte z 9 (V1) in 12 brsti (V2) na vsako sorto, go- jeno na Lenz-Moser bilateralnem kordonu. Vpliv je bil določen z meritvami pridelka na trto, kakovost grozdja je bila določena z vsebnostjo celokupne suhe snovi, titrabilne kislosti, vsebnosti celokupnih fenolov in flavonoidov in celokupne antioksidacij- ske sposobnosti. Izmerjeni parametri kakovosti grozdja sort Merlot in Vranac niso bili znalično vplivani z obremenitvijo trt. V2 je v primerjavi z V1 pokazala zmožnost povečanja pridelka grozdja samo pri sorti Merlot v razmerah te raziskave. Rezultati te raziskave so pokazali še pozitivno korelacijo med vsebnostjo celokupnih fenolov/flavonoidov in celokupno antioksidacijsko kapaciteto grozdnih jagod pri obeh sortah ne glede na obreme- nitev trt. Ključne besede: flavonoidi; celokupni fenoli; rez; pride- lek Acta agriculturae Slovenica, 117/4 – 20212 M. DELIĆ et al. 1 INTRODUCTION The grape yield and quality are influenced by many factors such as climatic conditions, soil chemi- cal and physical properties variety, viticulture manage- ment practices, crop load, leaf removal, irrigation, and rootstock-scion relationship (Pachnowska and Ochmian, 2018). Among the factors affecting vine growth and de- velopment, winter pruning has the greatest impact on the grape yield and quality of the growing season due to its impact on bud fertility and nutrient reserves in grapevine (Qiu et al., 2019). Winter pruning refers to the removal of non-beneficial plant parts during the late dormant season, and retaining only selected buds for the next season’s fruiting. In some wine regions, such as France, the exact number of buds for each variety is outlined by the France’s Appellation d’Origine Contrôlée regulations (Gangjee, 2012). However, if to many buds are left at win- ter pruning, the vine will produce many shoots leading to a dense canopy and opposite, if a few buds left, the shoots may grow too vigorously leading to low yield (Collins et al., 2020). In order to achieve the right balance between vegetative growth and grape production it is essential to determine an optimal crop load for each grapevine va- riety (Pellegrino et al., 2014). Optimal crop load varies from one variety to another, depending mainly on vari- ety itself, vineyard location, soil type, climatic conditions, etc. (Raj Kumar et al., 2017). Crop loads also impacts chemical grape composition and thus the wine quality. From an economic point of view, grape growers must find the correct balance between quantity, fruit quality and long-term vine health when determining appropri- ate crop levels on their vines (Čuš, 2004; Schamel and Schubert, 2016). Accordingly, permanent necessity for achieving optimal crop loads is always present, especially in regions where the connections among pruning prac- tices and grape production are poorly understood. Lenz-Moser bilateral cordon is the most commonly used vine training system for ‘Merlot’ and ‘Vranac’ (Vitis vinifera L.) in Trebinje region in Bosnia and Herzego- vina. Crop load levels commonly used within this train- ing system in Trebinje region are 9 and 12 buds per vine. The effect of crop load treatments on yield of ‘Merlot’ and ‘Vranac’ has been extensively investigated. Bogičević et al. (2015) examined the effect of early leaf removal and cluster thinning treatments on berry growth and grape composition of Vranac variety. Results showed that early leaf removal followed by cluster thinning resulted in a lower berry mass and number of berries per cluster, and thus a lower yield. Peppi et al. (2017) noted that an ad- justment in mechanical pruning with regard to cutting height is a feasible alternative to obtain regular and sus- tainable yields in ‘Merlot’ with considerably lower labour inputs in the vineyard. On the other hand, the effect of crop load treatments on the content of bioactive com- pounds such as total phenolics and flavonoids in berries of Merlot and Vranac varieties has been less studied. Therefore, this study is primarily intended to evalu- ate the impact of crop load on total phenolics, total flavo- noids and total antioxidant capacity of grapevine varie- ties Merlot and Vranac in Trebinje vineyard. The study also included the measurements of the following grape quality parameters: total soluble solids, titratable acid- ity as well as yield. The hypotheses tested were: (1) lower crop load can effectively increase total phenolic and fla- vonoid contents and total antioxidant capacity in berries of Merlot and Vranac varieties as compared to higher crop load; (2) total soluble solids, titratable acidity as well as yield in both varieties Merlot and Vranac will differ de- pending on the crop loads. Understanding the relation- ship between the crop loads and the grape production in ‘Merlot’ and ‘Vranac’ can provide valuable information for management decisions that need to be made in the vineyard. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE AND PLANT MATE- RIAL This study was conducted during 2018 at a com- mercial vineyard planted with ‘Merlot’ and ‘Vranac’ vines grafted onto Richter 110 rootstock. The vineyard was located in a village Zagradinje (42°38′N 18°14′E), 10 kilometers away from Trebinje (Herzegovina region), in a zone of altered Mediterranean climate. In contrast to the narrow coastal area, Trebinje region is characterized by warmer and drier summers, while winters are more humid. According to Köppen and Geiger, climate in Tre- binje region is classified as Csa (hot-summer Mediterra- nean climate). The average annual temperature in stud- ied area is 14.2 °C, and the precipitations average is 1,338 mm. The rain in Trebinje falls mostly in the winter, with relatively little rain in the summer. Vineyard was planted in 2008 on a sandy-loam soil, with planting distance of 2.5 m between rows and 1.0 m between vines in the row. Training system at both Merlot and Vranac was a Lenz-Moser bilateral cordon system. The crop load treatments applied at the study were as fol- lows: (V1) - 9 buds/vine (spurs with 2-3 buds; 3 spurs/ cordon) and (V2) - 12 buds/vine (spurs with 2-3 buds; 5 spurs/cordon). Each crop load treatment consisted of 4 plots located in two different rows (12 vines each; 48 vines per treatment) with one guard row between treat- ments. Crop loads used in this study are the most preva- Acta agriculturae Slovenica, 117/4 – 2021 3 Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard lent for Merlot and Vranac in Trebinje vineyards and are therefore selected for this study. Vranac is an autochthonous grapevine variety of Montenegro. The clusters are medium in size (cluster mass is in the range of 180 to 220 g) and are well filled with large, thin-skinned berries. The wine of this variety has a pleasant taste, velvety sweetness and intensive dark red colour (Šuković et al., 2020). Merlot is a red grape- vine variety from Bordeaux, France. It produces medium to large clusters (cluster mass is in the range of 220 to 300 g) with berries medium large in size and round with blue colour. The Merlot wine is velvety-red, fruity and pleas- ant taste and very refreshing (Renouf et al., 2010). 2.2 GRAPE YIELD AND QUALITY ANALYSIS Grape yield was weighted at harvest and expressed as kg per vine. Total soluble solids (TSS) were meas- ured using an Atago PAL-1 digital refractometer and expressed in degrees Brix (ISO, 2003). Titratable acid- ity (TA) was measured by titrating 10 ml of grape juice with 0.1 mol l-1 NaOH, using phenolphthalein as indica- tor (AOAC, 2000) and expressed as grams of tartaric acid per litre of grape juice (g l-1). 2.2.1 Extraction of phenolics from grape berries The extraction of phenolic compounds from the ful- ly ripened and matured grape berries was performed us- ing a 30 % aqueous ethanol solution (Canals et al., 2005). Before extraction, a fresh grape berries were oven dried at 50 °C until constant mass, ground and then sieved to pass a 2-mm sieve. The average moisture content was 77.8 % for Vranac and 79.0 % for Merlot. Detailed extraction procedures were as follows: 1 g of air-dried grape sam- ple was placed into 100 ml Erlenmeyer flask and mixed with 40 ml 30 % aqueous ethanol solution. The flask was heated in a water bath at 35-37 °C for 1 h. After heating, the flask was cool down to room temperature, and then the mixture was filtered through filter paper into 50 ml flask and diluted to the mark with 30 % aqueous etha- nol solution. The extract thus obtained was used for the analysis of total phenolic content, total flavonoid content and total antioxidant capacity. 2.2.2 Total phenolic content The total phenolic content (TPC) of the extract was measured by the Folin-Ciocalteu assay (Ough and Amerine, 1988) with slight modifications. The test sam- ple (0.25 ml of extract) was mixed into 25 ml flask with 15 ml of distilled water and 1.25 ml of Folin Ciocalteu reagent (diluted by distilled water in the ratio 1:2). After 5 min, 3.75 ml of saturated sodium carbonate solution (8 % w/v in water) was added. The flask was filled to the mark with a 30 % aqueous ethanol solution and heated in a water bath at 50 °C for 30 min. After heating, the flask was cool down to room temperature, and the ab- sorbance of blue colour was measured using a UV Spec- trophotometer (Amersham, Ultrospec 2100 pro) at 765 nm. TPC was calculated from a standard curve of gallic acid (5 - 500 mg l-1) and the results were expressed as mg of gallic acid equivalent per 100 g fresh mass (mg GAE 100 g-1 FM). 2.2.3 Total flavonoid content The total flavonoid content (TFC) of the extract was measured by the aluminium chloride colorimetric assay (Zhishen et al., 1999). The test sample (1 ml of extract) was mixed into 10 ml flask with 4 ml of distilled water and 0.3 ml 5 % NaNO2. After 5 min, 0.3 ml 10 % AlCl3 was added. The flask was incubated at room temperature for 6 min, and thereafter 2 ml of 1 mol l-1 NaOH was added. The flask was filled to the mark with distilled wa- ter and after 15 min the absorbance of red colour was measured at 510 nm. TFC was calculated from a stand- ard curve of catechin (0-100 mg l-1) and the results were expressed as mg of catechin equivalent per 100 g of fresh mass (mg CE 100 g-1 FM). 2.2.4 Total antioxidant capacity The total antioxidant capacity (TAC) of the extract was determined by ferric reducing antioxidant power (FRAP) assay (Benzie and Strain, 1996). The test sam- ple (80 μl of extract), 240 μl of distilled water, and 2080 μl of FRAP reagent (reagent was obtained by mixing 0.3 mol l-1 acetate buffer (pH = 3.6), 10 mmol l-1 TPTZ (2,4,6-tripyridyl-s-triazine) and 20 mmol l-1 FeCl3 x 6 H2O in ratio 10 : 1 : 1) were added into 10 ml Erlenmeyer flask and heated in a water bath at 37 °C for 5 min. After heating, the flask was cool down to room temperature, and the absorbance of blue colour was measured at 595 nm. TAC was calculated from a standard curve of FeSO4 x 7H2O (0 - 2 mmol l -1) and the results were expressed as mmol Fe2+ per 100 g fresh mass (mmol Fe2+ 100 g-1 FM). Acta agriculturae Slovenica, 117/4 – 20214 M. DELIĆ et al. 2.3 STATISTICAL ANALYSIS All the chemical measurements of TPC, TFC and TAC were conducted in triplicates and the results were expressed as the mean ± standard deviation. Experimen- tal data were subjected to analysis of variance (ANOVA) using Microsoft Excel 2013 statistical program. In order to interpret the relationships between total TPC / TFC and TAC, Pearson’s correlation coefficient analysis were conducted using the Microsoft Excel 2013 software. 3 RESULTS AND DISCUSSION The results of the analysis of yield and grape quality parameters of ‘Merlot’ and ‘Vranac’, depending on crop load treatments are presented in Table 1 and Table 2, re- spectively. The results of this study showed that the yield of the Vranac variety in the crop load treatment with nine buds/ vine was significantly lower compared with the higher crop load treatment (twelve buds/vine). This result is expected, since the lower crop load reduces the number of fruitful buds and consequently the number of grape clusters per vine, and thus the yield (Rubio and Yuste, 2002). Interestingly, this hypothesis for the ‘Merlot’ has not been confirmed in this study. The probably reason for that is less sensitivity of the ‘Merlot’ to differences in the number of buds in relation to the ‘Vranac’. Contra- rily, a higher crop load levels allows the vine to produce many grape clusters, which often results in a higher yield (Aipperspach et al., 2020). However, if to many buds left, then vine may produce many shoots that are outwardly observable as a large, leafy canopy leading to poor fruit quality and a weakening of the vine (Keller, 2010). Ac- cordingly, achieving optimal crop load for each variety is essential for vine growth and development and thus for grape production. In this study, there was no observed change in the quality of the grapes in the both varieties: Merlot and Vranac, regarding to the crop loads. These results are inconsistent with most other studies (Petri and Clingel- effer, 2006; Reynolds et al., 2007; Brighenti et al., 2017; Drenjančević et al., 2017). Khamis et al. (2017) noted the grape quality parameters have inverse correlation with the number of buds per vine, that is, the lower crop loads increase the total soluble solids and phenolic con- tent. Positive correlation between lower crop load levels and total soluble solids or phenolic contents in grape has been reported in many other studies (Peña-Neira et al., 2007; Gil-Muñoz et al., 2009; Bubola et al., 2011). However, some of the studies failed to find an associa- tion between crop removal treatment and some of the above-mentioned grape quality parameters (Karoglan et al., 2014; Mawdsley et al., 2018). In our study, there is no evidence that lower crop load level increases total soluble solids, total phenolics and total flavonoids in grape ber- ries. Unfortunately, the drawback of the present study is that only two crop load levels were used in the experi- ment and it is very difficult to draw conclusions from. Our hypothesis was that lower crop load level (9 buds/ vine) would significantly increase total soluble solids, to- tal phenolics and total flavonoids in grape berries of both cultivars as compared to higher crop load level (12 buds/ vine), however, the study results did not confirm it. The results of the present study have also shown Crop load treatments Yield (kg per vine) TSS (˚Brix) TA (mg l-1) TPC (mg GAE 100 g-1 FM) TFC (mg CE 100 g-1 FM) TAC (mmol Fe2+ 100 g-1 FM) 9 buds/vine 3.32 ± 0.51 20.83 ± 0.8 7.14 ± 0.06 147.4 ± 9.1 80.7 ± 7.3 2.10 ± 0.21 12 buds/vine 3.57 ± 0.76 21.30 ± 1.2 7.16 ± 0.04 154.2 ± 9.7 84.3 ± 5.1 2.27 ± 0.37 Table 1: Yield, total soluble solids (TSS), titratable acidity (TA), total phenolic content (TPC), total flavonoid content (TFC) and total antioxidant capacity (TAC) of ‘Merlot’ Values expressed as an average ± standard deviation Crop load treatments Yield (kg per vine) TSS (˚Brix) TA (mg l-1) TPC (mg GAE 100 g-1 FM) TFC (mg CE 100 g-1 FM) TAC (mmol Fe2+ 100 g-1 FM) 9 buds/vine 3.32 ± 0.44b 22.13 ± 1.1 7.07 ± 0.11 166.8 ± 14.1 75.3 ± 6.3 2.26 ± 0.33 12 buds/vine 4.18 ± 0.53a 21.80 ± 0.9 7.16 ± 0.14 154.1 ± 19.6 74.3 ±8.1 2.08 ± 0.29 Table 2: Yield, total soluble solids (TSS), titratable acidity (TA), total phenolic content (TPC), total flavonoid content and total an- tioxidant capacity (TAC) of ‘Vranac’ Values expressed as an average ± standard deviation. Different letters in each column represent significant difference among variants Acta agriculturae Slovenica, 117/4 – 2021 5 Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard crop load treatments should be done to confirm the con- clusions of this study. 5 LITERATURE Aipperspach, A., Hammond, J., Hatterman-Valenti, H. (2020). Utilizing pruning and leaf removal to optimize ripening of Vitis riparia-based ‘Frontenac Gris’ and ‘Marquette’ wine grapes in the Northern great plains. Horticulturae, 6(1), 18. https://dx.doi.org/10.3390/horticulturae6010018 AOAC, (2000). Acidity (Titratable) of fruit products (Method No. 942.15). Retrieved from http://www.eoma.aoac.org/ methods/ Balea, Ş. S., Pârvu, A. E., Pop, N., Marín, F. Z., Pârvu, M. (2018). Polyphenolic compounds, antioxidant, and cardioprotective effects of pomace extracts from Fetească Neagră cultivar. Oxidative Medicine and Cellular Longevity, 2018, 8194721. http://dx.doi.org/10.1016/j.pathophys.2018.07.026 Benzie, I. F., Strain J. J. (1996). Ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP as- say. Analytical Biochemistry, 239(1), 70-76. http://dx.doi. org/10.1006/abio.1996.0292 Bogičević, M., Maras, V., Mugoša, M., Kodžulović, V., Raičević, J., Šućur, S., Failla, O. (2015). The effects of early leaf re- moval and cluster thinning treatments on berry growth and grape composition in cultivars Vranac and Cabernet Sauvi- gnon. Chemical and Biological Technologies in Agriculture, 2, 13. http://dx.doi.org/10.1186/s40538-015-0037-1 Borges, G., Degeneve, A., Mullen, W., Crozier, A. (2010). Iden- tification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cran- berries. Journal of Agricultural and Food Chemistry, 58(7), 3901-3909. http://dx.doi.org/10.1021/jf902263n Brighenti, A. F., Cipriani, R., Malinovski, L. I., Vanderlinde, G., Allebrandt, R., Feldberg, N. P., Silva, A. L. (2017). Ecophysiology of three Italian cultivars subjected to two pruning methods in Santa Catarina, Brazil. Acta Horticul- turae, 1157, 381-388. http://dx.doi.org/10.17660/ActaHor- tic.2017.1157.53 Bubola, M., Peršurić, Đ., Kovačević Ganić, K. (2011). Impact of cluster thinning on productive characteristics and wine phenolic composition of ‘Merlot’. Journal of Food Agricul- ture and Environment, 9(1), 36-39. Canals, R., Llaudy, M. C. Valls, J., Canals, J. M., Zamora F. (2005). Influence of ethanol concentration on the extrac- tion of color and phenolic compounds from the skin and seeds of ‘Tempranillo’ grapes at different stages of ripening. that the total soluble solids, titratable acidity and total phenolics of grape berries fell within the range expected for ‘Vranac’ and ‘Merlot’. For example, in the study con- ducted by Mitić et al. (2012) total phenolics in grape of ‘Vranac’ and ‘Merlot’ were 158.6 ± 1.9 and 169.2 ± 2.7 mg GAE 100 g-1 FM, respectively. However, numerous studies reported much higher contents of total pheno- lics in berries of ‘Vranac’ and ‘Merlot’ than those de- termined in the present research (Pajović et al., 2014; Franco-Bañuelos et al., 2017). Scientists generally agree that total phenolic contents as well as other grape qual- ity parameters depends on many factors such as cultivar, viticulture practices and environmental conditions in the vine-growing regions. Toscano et al. (2019) reported that sub-optimal environmental conditions can influence the biosynthesis and accumulation of many secondary me- tabolites in plants, including phenolic compounds. The results of this study also showed a positive cor- relation between total phenolics / flavonoids and total antioxidant capacity of grape berries in both varieties, regardless of crop loads applied (Table 3). Similar cor- relations were also determined in other published studies (Garrido et al., 2016; Cosme et al., 2018). These results suggest that the phenolic compounds are carriers of antioxidant activity in the plant, and this hypothesis has, in fact, been confirmed by many scien- tists (Borges et al., 2010; Stanković et al., 2012; Balea et al., 2018). 4 CONCLUSIONS Higher crop load level (12 buds/vine) as compared to lower crop load level (9 buds/vine) within the Lenz- Moser bilateral cordon system showed potential for in- creasing grape yield only for ‘Merlot’ under experimen- tal conditions. However, the quality grape parameters of ‘Merlot’ and ‘Vranac’ were not influenced significantly by crop loads. Unfortunately, this study has some limita- tions, which have to be pointed out. The main limitation of the present study is (1) one year was included in the study and (2) small differences among crop load treat- ments. Therefore, further investigations involving more Table 3: Pearson’s correlation coefficient between total phenolics (TPC)/total flavonoids (TFC) contents and total antioxidant capacity (TAC) Merlot Vranac 9 buds/vine 12 buds/vine 9 buds/vine 12 buds/vine TPC vs. TAC 0.935 0.922 0.911 0.915 TFC vs. TAC 0.944 0.951 0.932 0.926 Acta agriculturae Slovenica, 117/4 – 20216 M. DELIĆ et al. Journal of Agricultural and Food Chemistry, 53(10), 4019- 4025. https://dx.doi.org/10.1021/jf047872v Collins, C., Wang, X., Lesefko, S., De Bei, R., Fuentes, S. (2020). Effects of canopy management practices on grapevine bud fruitfulness. OENO One, 54(2), 313-325. https://dx.doi. org/10.20870/oeno-one.2020.54.2.3016 Cosme, F., Pinto, T., Vilela, A. (2018). Phenolic compounds and antioxidant activity in grape juices: A chemical and sensory view. Beverages, 4, 22. https://dx.doi.org/10.3390/bever- ages4010022 Čuš, F. (2004). Influence of crop load on yield and grape quality of `Chardonnay`. Acta Agriculturae Slovenica, 83(1), 73-83. Drenjančević, M., Jukić, V., Zmaić, K., Kujundžić, T., Rastija, V. (2017). Effects of early leaf removal on grape yield, chemi- cal characteristics, and antioxidant activity of grape variety Cabernet Sauvignon and wine from eastern Croatia. Acta Agriculturae Scandinavica, Section B - Soil & Plant Science, 67(8), 705-711. http://dx.doi.org/10.1080/09064710.2017.1 332238 Franco-Bañuelos, A., Contreras-Martínez, C., S., Carranza- Téllez, J., Carranza-Concha, J. (2017). Total phenolic con- tent and antioxidant capacity of non-native wine grapes grown in Zacatecas, Mexico. Agrociencia, 51(6), 661-671. Gangjee, D. (2012). The Appellation of Origin in France. In Re- locating the Law of Geographical Indications, (pp. 77-126). Cambridge: Cambridge University Press. http://dx.doi. org/10.1017/CBO9781139030939.005 Garrido, I., Uriarte, D., Hernández, M., Llerena, J. L., Valdés, M. E., Espinosa, F. (2016). The evolution of total phenolic com- pounds and antioxidant activities during ripening of grapes (Vitis vinifera ‘Tempranillo’) grown in semiarid region: Effects of cluster thinning and water deficit. International Journal of Molecular Sciences, 17(11), 1923. https://dx.doi. org/10.3390/ijms17111923 Gil-Muñoz, R. R., Vila-Lopez, J. I., Fernandez, J. I., Martinez- Cutillas, A. (2009). Effects of cluster thinning on antho- cyanin extractability and chromatic parameters of ‘Syrah’ and ‘Tempranillo’ grapes and wines. OENO One, 43, 45-53. http://dx.doi.org/10.20870/oeno-one.2009.43.1.786 International Organization for Standardization. (2003). Fruit and vegetable products: Determination of soluble solids, Re- fractometric method (ISO Standard No. 2173). Retrieved from https://www.iso.org/standard/35851.html Karoglan, M., Osrečak, M., Maslov Bandić, L., Kozina, B. Effect of cluster and berry thinning on ‘Merlot’ and ‘Cabernet Sau- vignon’ wines composition. Czech Journal of Food Sciences, 32(5), 470-476. http://dx.doi.org/10.17221/598/2013-CJFS Keller, M. (2010). The Science of Grapevines. London: Elsevier, Academic Press. Khamis, M. A., Atawia, A. A. R., El-Badawy, H. E. M., Abd El- Samea, A. A. M. (2017). Effect of buds load on growth, yield and fruit quality of superior grapevines. Middle East Jour- nal of Agriculture Research, 6(1), 152-160. Mawdsley, P. F. W., Dodson Peterson, J. C., Casassa, L. F. (2018). Agronomical and chemical effects of the timing of cluster thinning on ‘Pinot Noir’ (Clone 115) grapes and wines. Fermentation, 4(3), 60. https://dx.doi.org/10.3390/fermen- tation4030060 Mitić, M., Souquet, J-M., Obradović, M., Mitić, S. (2012). Phy- tochemical profiles and antioxidant activities of Serbian table and wine grapes. Food Science and Biotechnology, 21, 1619-1626. http://dx.doi.org/10.1007/s10068-012-0215-x Ough C. S., Amerine M. A. (1988). Methods for analysis of must and wines. New York, NY: John Wiley & Sons. Pachnowska, K.., Ochmian, I. (2018). Cane pruning intensity of vine as a substantial factor influencing physico-chemi- cal attributes of berries cultivar ‘Regent’. Folia Pomeranae Universitatis Technologiae Stetinensis Agricultura, Alimen- taria, Piscaria et Zootechnica, 343, 43-54. http://dx.doi. org/10.21005/AAPZ2018.47.3.04 Pajović, R., Raičević, D., Popović, T., Sivilotti, P., Lisjak, K., Vanzo, A. (2014). Polyphenolic characterisation of ‘Vranac’, ‘Kratosija’ and ‘Cabernet Sauvignon’ (Vitis vinifera L.) grapes and wines from different vineyard locations in Mon- tenegro. South African Journal for Enology and Viticulture, 35(1), 139-148. http://dx.doi.org/10.21548/35-1-994 Pellegrino, A., Clingeleffer, P., Cooley, N. E., Walker, R. (2014). Management practices impact vine carbohydrate status to a greater extent than vine productivity. Frontiers in Plant Sci- ence 5, 283. http://dx.doi.org/10.3389/fpls.2014.00283 Peña-Neira, A., Cáceres, A., Pastenes, C. (2007). Low molecu- lar weight phenolic and anthocyanin composition of grape skins from ‘Syrah’ (Vitis vinifera L.) in the Maipo valley (Chile): Effect of clusters thinning and vineyard yield. Food Science and Technology International, 13, 153-158. http:// dx.doi.org/10.1177/1082013207077920 Peppi, M. C., Kania, E., Talep, R., Castro, P., Reginato, G. (2017). Effect of different cutting heights of mechanically pruned grapevines ‘Merlot’ over three consecutive seasons. South African Journal of Enology & Viticulture, 38(2), 221-227. http://dx.doi.org/10.21548/38-2-1609 Petri, P. R., Clingeleffer P. R. (2006). Crop thinning (hand versus mechanical), grape maturity and anthocyanin concentration: Outcomes from irrigated ‘Cabernet Sauvi- gnon’ (Vitis vinifera L.) in a warm climate. Australian Jour- nal of Grape and Wine Research, 12, 21-29. http://dx.doi. org/10.1111/j.1755-0238.2006.tb00040.x Qiu, Z., Chen, G., Qiu, D. (2019). Pruning and dormancy break- ing make two sustainable grape-cropping productions in a protected environment possible without overlap in a single year. PeerJ. 7, e7412. http://dx.doi.org/10.7717/peerj.7412 Raj Kumar, A., Parthiban, S., Subbiah, A., Sangeetha, V. (2017). Effect of severity of pruning on yield and quality characters of grapes (Vitis vinifera L.): A review. International Journal of Current Microbiology and Applied Sciences, 6(4), 818-835. https://dx.doi.org/10.20546/ijcmas.2017.604.103 Renouf, V., Trégoat, O., Roby, J-P., van Leeuwen, C. (2010). Soils, rootstocks and grapevine varieties in prestigious Bordeaux vineyards and their impact on yield and quality. OENO One, 44(3), 127-134. http://dx.doi.org/10.20870/ oeno-one.2010.44.3.1471 Reynolds, A. G., Schlosser, J., Sorokowsky, D., Roberts, R., Will- werth, J., de Savigny, C. (2007). Magnitude of viticultural and enological effects. II. Relative impacts of cluster thin- ning and yeast strain on composition and sensory attributes of ‘Chardonnay Musqué’. American Journal of Enology and Viticulture, 58, 25-41. Rubio, J. A., Yuste J. (2002). Effects of cluster-thinning and the Acta agriculturae Slovenica, 117/4 – 2021 7 Influence of crop load on the yield and grape quality of Merlot and Vranac (Vitis vinifera L.) varieties in Trebinje vineyard water regime in the yield, development, must and physi- ological behavior of ‘Tempranillo’ driven in a trellis. Viti- cultura y Enología Profesional, 83, 82-92. Schamel, G. H., Schubert, G. H. (2016). An optimal con- trol model of crop thinning in viticulture. BIO Web of Conferences 7, 03022. https://dx.doi.org/10.1051/bio- conf/20160703022 Sharma, A., Shahzad, B., Rehman, A., Bhardwaj, R., Landi, M., Zheng, B. (2019). Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules, 24(13), 2452. http://dx.doi.org/10.3390/mol- ecules24132452 Stanković, M. S., Niciforović, N., Mihailović, V., Topuzović, M., Solujić, S. (2012). Antioxidant activity, total phenolic con- tent and flavonoid concentrations of different plant parts of Teucrium polium L. subsp. polium. Acta Societatis Botani- corum Poloniae, 81(2), 117-122. http://dx.doi.org/10.5586/ asbp.2012.010 Šuković, D., Knežević, B., Gašić, U., Sredojević, M., Ćirić, I., Todić, S., Mutić, J., Tešić, Ž. (2020). Phenolic profiles of leaves, grapes and wine of grapevine variety Vranac (Vitis vinifera L.) from Montenegro. Foods, 9(2), 138. https://doi. org/10.3390/foods9020138 Toscano, S., Trivellini, A., Cocetta, G., Bulgari, R., Francini, A., Romano, D., Ferrante, A. (2019). Effect of preharvest abi- otic stresses on the accumulation of bioactive compounds in horticultural produce. Frontiers in plant science, 10, 1212. https://dx.doi.org/10.3389/fpls.2019.01212 Zhishen, J., Mengcheng, T., Jianming, W. (1999). The determi- nation of flavonoid contents in mulberry and their scaveng- ing effects on superoxide radicals. Food Chemistry, 64, 555- 559. http://dx.doi.org/10.1016/S0308-8146(98)00102-2 Acta agriculturae Slovenica, 117/4, 1–10, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2119 Original research article / izvirni znanstveni članek Inheritance of plant height, straw yield and flag leaf area in MBB x Gavi- ota durum wheat (Triticum durum Desf.) cross Manel SALMI 1, 2, Zine El Abidine FELLAHI 3, Abdelkader BENBELKACEM 4, Amar BENMAHAMMED 5, Hamenna BOUZERZOUR 5 Received March 02, 2021; accepted November 02, 2021. Delo je prispelo 2. marca 2021, sprejeto 2. novembra 2021 1 University of Mostefa Ben Boulaid, Life and Natural Sciences Faculty, Ecology and Environment Department, Batna, Algeria 2 Corresponding author, e-mail: salmi.mnl@gmail.com 3 University of Mohamed El Bachir El Ibrahimi, Natural, Life and Earth Sciences and the Universe Faculty, Agronomy Department, Bordj Bou Arréridj, Algeria 4 Algerian National Institute of Agronomic Research (INRAA), Research Unit of Constantine, Algeria 5 University of Ferhat Abbas, Life and Natural Sciences Faculty, Ecology and Plant Biology Department, Setif, Algeria Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross Abstract: Plant height, straw mass and flag leaf area are recognized by physiologists as morphological markers of drought stress tolerance. Developing varieties intended for arid and semi-arid zones need to select for these traits. Understand- ing the genetic control of a given trait helps breeder to handle the segregating populations under study in a more efficient and consistent manner by choosing the best breeding method avail- able to realize significant genetic advance. For this purpose, six generations: parents, F1, F2, BC1, BC2, derived from MBB x ‘Gaviota’ durum wheat (Triticum durum Desf.) cross were grown to investigate the nature of gene action involved in the inheritance pattern of the three traits. The results indicated that the six-parameter model fitted the best the data related to the variability present in the generation means of the studied traits. Generation mean analysis indicated that non-allelic interac- tions were important factors controlling the expression of these characters with complementary type of gene action governing FLA and STW inheritance. High heritability estimates, moder- ate to high expected responses to selection, significant genetic correlations with grain yield and greater role of non-additive effects in controlling the inheritance of the three studied traits suggested that breeding methods exploiting both fixable and non-fixable components be applied to break unfavorable link- age and to accumulate useful genes in the base population, fol- lowed by mono-trait or index based selection in late advanced generations. Key words: gene effects; non-allelic interaction; durum wheat; plant height; straw yield; flag leaf area; heritability Višina rastlin, masa slame in površina lista zastavičarja so od fiziologov prepoznane morfološke lastnosti, ki nakazujejo to- leranco na sušni stres Izvleček: Višina rastlin, masa slame in površina lista za- stavičarja so od fiziologov prepoznane morfološke lastnosti, ki nakazujejo toleranco na sušni stres. Sorte, vzgojene za sušna območja morajo biti izbrane glede na lastnosti, ki omogočajo prenašanje suše. Razumevanje genetske kontrole za določeno lastnost pomaga žlahtniteljem uravnavati različne populacije v raziskavi na bolj učinkovit in verodostojen način pri izbiri najboljše metode žlahtnenja za dosego pomembne genetske prednosti. V ta namen je bilo gojeno šest generacij rastlin iz križanja starševske generacije, F1, F2, BC1, BC2 z MBB x ‘Ga- viota’ trde pšenice (Triticum durum Desf.) kot osnova za pre- učevanje delovanja genov, ki so vključeni v vzorec dedovanja treh lastnosti. Rezulatati so pokazali, da se je model šestih para- metrov najbolje prilegal podatkom, povezanih z variabilnostjo preučevanih lastnosti kot povprečje v generaciji. Analiza gene- racijskih povprečij je pokazala, da so bile nealelske interakcije pomemben dejavnik za nadzor izražanja tistih lastnosti, ki so komplementarne delovanju genov in ki vodijo FLA in STW dedovanje. Velike vrednosti dednosti, zmerne glede na priča- kovan odziv selekcije, značilna genetska korelacija s pridelkom zrnja in večja vloga neaditivnih učinkov pri kontroli dednosti treh preučevanih lastnosti nakazujejo, da bi žlahtniteljske me- tode, ki uporabljajo vezane in nevezane komponente lahko bile uporabljene za prekinitev nezaželjenih povezav genov in pospe- šitev uporabnih genov v osnovnih populacijah, ki bi jim sledil izbor posameznih ali indeksiranih lastnosti v kasnejših izbolj- šanih generacijah. Ključne besede: učinki genov; nealelske interakcije; trda pšenica; višina rastlin; pridelek slame; površina lista zastavičar- ja; dednost Acta agriculturae Slovenica, 117/4 – 20212 M. SALMI et al. 1 INTRODUCTION In the arid and semi-arid regions, rainfed grown du- rum wheat (Triticum durum Desf.) crop suffers from the combined effects of drought and heat stresses, undergo- ing substantial grain yield losses (Royo et al., 2014; Lui et al., 2015). To minimize yield decline, drought toler- ance improvement is then seen as a key breeding com- ponent for the development of cultivars devoted to these environments, where reducing wind erosion and meet- ing livestock feeding requirement have heightened the importance of increasing straw production along with grain yield. In fact, crop residues are usually not burned but either maintained to protect soil from wind erosion or grazed and straw is balled, stored and fed to livestock during the winter months (Annichiarico et al., 2005; Chenaffi et al., 2011). In this context plant height, a straw yield correlated trait, is seen as an important character- istic influencing cultivars adoption under such growing conditions (Rabti et al., 2020; Jatayev et al., 2020; Had- dad et al., 2021). Because of their straw yield advantage, tall varieties, derived from land-races, are still cultivated and not replaced by newly released reduced height cul- tivars (Rabti et al., 2020). For a full expression of their potential, dwarf wheat varieties need to be grown in fa- vorable, well-watered conditions which permit applica- tion of relatively high levels of nitrogen fertilizer which is usually not the case in arid and semi-arid environments. This type of plant material becomes, under drought stress conditions, too short, yielding less than tall varieties, showing reduced flag leaf area, kernel size, kernel Zn, Fe, Mg and Mn concentration, coleoptiles, and roots length (Aziz et al., 2017; Velu et al., 2017; Jatayev et al., 2020; Rabti et al., 2020). Positive relationship between plant height and grain yield, under drought stress growing conditions, implied that there is a minimum height be- low which grain yield limitation becomes evident (Slafer et al., 2005; Royo et al., 2014). in this context, Yani and Rashidi (2012) reported that straw yield and plant height had positive and significant role in the expression of grain yield under drought conditions. Asadi et al. (2019) noted that straw yield explained 65 % of grain yield variation and exerted substantial direct effect on grain yield under water deficit conditions. Belagrouz et al. (2018) reported significant correlation between grain yield water use effi- ciency, harvest index and plant height, suggesting that se- lection for plant height and harvest index could improve both water use efficiency and grain yield under drought prone environments. Flag leaf is the main photosynthetic organ providing the major assimilate required for spike growth. It senses environmental signals and consequent- ly adapts to surrounding environment by minimizing area reduction and delaying senescence, caused by termi- nal drought stress (Farook et al., 2009; Belkherchouche et al., 2015). Joshi et al. (1984) found that flag leaf size was positively correlated with grain yield, suggesting that optimal flag leaf dimensions could be an important breeding target under drought prone environments. In- formation on straw yield, plant height and flag leaf area inheritance can assist developing adapted cultivars for areas practicing cereal-livestock farming systems and conservation agriculture (Chenaffi et al., 2011; Jatayev et al., 2020). Genetic variation for quantitative characters in segregating population is of prime concern to breeders. It determines selection efficiency which depends upon the nature and magnitude of genetic variability available. Genetic models devoted to the estimation of different genetic effects have been developed. Among these mod- els, generation means analysis provides information on the relative importance of gene effects due to additive, dominance deviations and non-allelic interactions, in determining generation means (Mather and Jinks, 1982; Shayan et al., 2018; Salmi et al., 2019). Plant height was found to be controlled mainly by over dominance while flag leaf area was reported to be mostly under additive control combined with partial dominance and epistatic gene actions (Saleem et al., 2005; Shabbir et al., 2012; Yang et al., 2016). Salmi et al. (2019) found that domi- nance acted in the direction of increased plant height. Joshi and Sharma (1984) reported that genes affecting smaller leaves are partially dominant over genes affecting larger leaves. Evidence for non-allelic interactions was reported and linkage among loci appeared to be an im- portant component of flag leaf dimensions heredity (De Pace et al., 2001). The present study aimed to investigate the inheritance pattern of straw yield, plant height and flag leaf area in a durum wheat cross involving a tall and a semi-dwarf varieties. 2 MATERIALS AND METHODS 2.1 SITE, PLANT MATERIAL, AND EXPERIMEN- TAL DESIGN Two durum wheat (Triticum durum Desf.) varieties, namely Gaviota (GTA), a semi-dwarf cultivar derived from ‘Crane’ /4/ ‘Polonicum PI 185309’//Triticum gluti- nosum enano/2 * ‘Tehuacan 60’/3/’Grulla’ cross (http:// wgb.cimmyt.org/gringlobal/AccessionDetail.aspx?id= 1783 ), and a tall cultivar, Mohammed Ben Bachir (MBB), a head-row selection derived from a land race adapted to Setif ’s high plateaus region (Algeria), were hybrid- ized during the 2015-2016 cropping season at the Field Crop Institute, Agricultural Experimental Station of Setif (ITGC, AES, 36°12’N 05°24’E, 1080 m above sea level, Acta agriculturae Slovenica, 117/4 – 2021 3 Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross Setif, Algeria). During the 2016-2017 cropping season, F1 was selfed to produce F2, and crossed to the parents to obtain back cross generations (BC1 and BC2). The parents were crossed again to get the first filial generation (F1). The following cropping season (2017-2018), the six ba- sic generations were grown in a randomized completed block design, with five replications. Parents, F1, and BC’s generations were sown in one row, 2 m long, 20 cm inter- row spacing and 10 cm plant-plant spacing in the row. F2 generation was sown in thirty rows 2 m long. Recom- mended cultural practices for the area were followed as reported in Salmi et al. (2019). 2.2 DATA COLLECTION AND ANALYSIS Data were collected from 5, 5, 10, and 30 plants per replication for the parents, F1, BC’s, and F2 generations, respectively. Prior to harvest, length of the main stem of each plant was measured from the ground level to the base of the spike and recorded as plant height estimate in cm (PHT, cm). Straw mass (STM, g plant-1) was deter- mined as the difference between above ground plant bio- mass (BIO, g plant-1) and plant grain yield (GY, g plant-1). Flag leaf area (FLA, cm²) was estimated by the product of leaf length x leaf width x 0.749 (Spagnoletti-Zeuli and Qualset, 1990). Collected data were subjected to an analysis of variance using Cropstat software (2007) to test generation effect. Whenever this effect, tested against the residual mean square, was significant, genetic analysis for the specific trait was undertaken. To test the presence of additive vs. dominance genes effects, contrast method (Steel and Torrie, 1982) was applied to check the signifi- cance of the following comparisons: F1 vs. mid-parent, P1 vs. P2, F2 vs. average BC’s, and BC1 vs. BC2. The notations adopted for gene effects were [m], [d], [h], [i], [j], and [l] representing main, additive and dominance gene effects, and additive × additive, additive × dominance, and dom- inance × dominance epistatic interactions, respectively. The appropriate genetic model (three vs. six parameters) was also determined using both ABCD and joint scal- ing tests. These tests provide information regarding the absence or the presence of gene interactions (Mather and Jinks, 1982). Significance of any one or both scaling tests implies inadequacy of the additive–dominance model. The C and D scaling tests provide check for dominance x dominance (l) and additive x additive (i) types of epis- tasis, respectively. The genetic parameters [m], [d], [h], [i], [j], and [l] were estimated by weighted least square method. The purpose of using weights was to account for differential precision with which means of different generations were estimated based on varying sample size. Gene effects were tested for significance using the t-test (Kearsey and Pooni, 1996). Three vs six-parameter models testing ware performed using GENMEANS sub- routine implemented in Tnaustat software (Manivannan, 2014). Genotypic and environmental variance compo- nents, of the measured traits, were estimated by equating the observed values of the different generations, accord- ing to Mather and Jinks, (1982) as follows: σ²E = ¼ (σ²P1 + σ²P2 + 2σ²F1), σ²D = (2σ²F2 – σ²BC1 – σ²BC2) and σ²H = 4 (σ²F2 -½σ²D - σ²E). The significance of the mean value of a particular parameter was tested against its correspond- ing standard error, via a Student’s t-test, as suggested by Mather and Jinks (1982). Broad-sense heritability (H² bs) was calculated according to Kearsey and Pooni, (1996), as follow: H²bs = (σ2D + σ 2 H) / (σ 2 D + σ 2 H + σ 2 E) = (σ 2 G) / (σ2P), where σ 2 D, σ 2 H, σ 2 E, σ 2 G, and σ 2 P stand for the ad- ditive, dominance, environmental variance components, genetic, and phenotypic variances, respectively. Narrow sense heritability (h²ns) was estimated according to Hal- lauer and Miranda Filho (1989) as follow: h²ns = σ 2 D / (σ 2 D + σ2H +σ 2 E) = (σ 2 D) / (σ 2 P), Standard errors (SE) of these estimates were calculated as: SE (h2bs) = [SE (σ 2 G)] / (σ 2 P) and SE (h2ns) = [SE (σ 2 D)] / (σ 2 P). Significance of these pa- rameters, h²bs and h²ns was tested using a t-test equals to the ratio of heritability over its standard error (Halloran et al., 1979). The expected response to selection (ERS) was derived according to Sing and Chaudhary (1999) as follows: ERS = 2.06 * h² bs * √σ²F2, and expressed as per- cent of the over mean (Xbar) of the given trait: ERS (%) = (100 * ERS) / Xbar. Relationship between studied traits and grain yield was inspected through genotypic corre- lation coefficient (rg), which was derived as the ratio of covariance to the square root of the product of the cor- responding variance of the two traits considered. Geno- typic covariance was determined using the property of the analysis of variance of the sum of two variables as suggested by Kwon and Torrie (1964) and described in Mansouri et al. (2018), using Past software (Hammer et al., 2001). The standard error of rg was derived using the formulae of Reeves (1955), reported by Koots and Gibson (1996), as follows: SErg = [(1- rg²) / √2] * [(√SEh²i * SEh²j) / (√h²i*√h²j)], where h²i and h²j are the traits heritability’s. Student’s t-test was used to determinate the significance of the correlation coefficient. 3 RESULTS AND DISCUSSION 3.1 VARIATION AND MEAN PERFORMANCES Significant generation effect was revealed for flag leaf area (FLA), plant height (PHT) and straw mass (STM) by the analysis of variance, indicating the pres- ence of substantial genetic variability and allowing to Acta agriculturae Slovenica, 117/4 – 20214 M. SALMI et al. carry out in deep bio-metrical analysis (Table 1). A sig- nificant generation effect is a prerequisite to perform the inheritance study of the targeted traits applying genera- tion means analysis model. Among generations traits mean estimates, varied from 19.67 to 29.70 cm² for FLA, from 82.00 to 136.25 cm for PHT, and from 12.32 to 31.98 g plant-1 for STM (Figures 1, 2 and 3). Contrast analysis indicated that deviations between parental mean values for the studied traits were significant (Table 1). ‘Gavi- ota’ had significantly greater FLA (29.70 cm²) than MBB (26.08 cm²), while MBB showed significantly greater PHT (136.25 cm) and STM (31.55 g plant-1) than ‘Gavi- ota’ whose mean values for both traits were 86.87 cm and 15.24 g/plant, respectively (Figure 1, 2 and 3). These results suggested that the crossed parents carry different allelic combinations involved in the ge- netic control of the studied traits. On average, F1 ex- hibited equal FLA (29.35 cm²) to the best parent GTA (29.70 cm²), equal PHT (82.00 cm) to the semi-dwarf parent GTA (86.87 cm) and equal STM (31.98 g plant-1) to the tall parent MBB (31.55 g plant-1), within the lim- its of their standard errors (Figures 1, 2 and 3). Further- more, F1 means differed significantly from mid-parent average for PHT and STM but not for FLA, suggesting that dominance was predominantly involved in the ge- netic control of PHT and STM; while additive genetic control was predominantly expressed for FLA (Table 1, Figures 1, 2 and 3). Dominance acted in the direction of Sources of variation DF FLA (cm²) PHT (cm) STM (g/plant) Generations 5 89.56** 2091.65** 398.08** Replications 4 9.56 8.81 25.00 Homogeneous (Homo) 2 21.56** 4504.12** 455.19** P1 vs P2 1 8.10** 1190.50** 18.10** F1 vs ½(P1+P2) 1 2.55ns 568.92** 6.69* Heterogeneous (Het) 2 20.38ns 579.97** 164.99ns F2 vs ½(BC1+BC2) 1 4.95* 177.96** 8.88** BC1 vs BC2 1 16.57** 9.17** 1.11ns Homo vs Het 1 89.86** 56.58** 20.43** Residual 20 4.05 5.12 36.72 Table 1: Mean square deviations of the analysis of variance for flag leaf area, plant height and straw mass in Gaviota x MBB durum wheat cross Ns, *, **: non-significant and significant effects at 5 % and 1 % probability level, respectively. FLA: Flag leaf area, PHT: Plant height; STM : Straw mass Figure 1: Mean of flag leaf area of the basic generations Acta agriculturae Slovenica, 117/4 – 2021 5 Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross reduced PHT and increased STM, suggesting that GTA carries more dominant genes controlling PHT, while MBB carries more dominant genes controlling STM. Based F1 data analysis, these results agreed with find- ings of several authors who reported that non-additive genetic effects appeared as an important component of the genetic architecture of PHT and STM, while additive gene effects were prevalent for FLA (Saleem et al., 2005; Shabbir et al., 2012; Yang et al., 2016). Mean values of the F2 generation deviated significantly from the average of BCs generations for the three studied traits, being sig- nificantly higher and laying within the parental range for PHT and STM and outside of this range for FLA (Table 1, Figures 1, 2 and 3). 3.2 GENE EFFECTS ABCD and joint scaling tests, applied to appraise presence of epistasis, were found significant, invaliding the additive–dominance model adequacy for explain- ing the inheritance pattern of PHT, STM and FLA, and suggesting the adoption of higher than three-parameter model (Table 2). These results indicate that higher order Figure 2: Mean of straw yield of basic generations Figure 3: Mean of plant height of the basic generations Acta agriculturae Slovenica, 117/4 – 20216 M. SALMI et al. inter-allelic interactions played an important role in the expression of the measured traits, and additive–domi- nance model alone will not be sufficient to deal with the inheritance pattern of such traits. Estimates of the gene effects derived from this six-parameter model are given in Table 2. Gene main effect [m] was significant for all three analyzed traits, indicating that these traits are con- trolled by minor genes and quantitatively inherited. For PHT, additive [d] and dominance [h] gene affects, and additive * additive [i] and additive * dominance [j] non- allelic interactions were significantly involved in the in- heritance of this trait. Dominance [h] gene effects and additive * additive [i] non-allelic interactions came out as the salient features of the genetic control of this char- acter as this is indicated by the high absolute values of the genetic parameters. The negative sign of the additive * dominance [j] component indicated that genes involved in the control of this trait were dispersed the parents. These results agreed with Novoselović et al. (2004), Ojaghi and Akhundova (2010), Mohamed et al. (2013), Dorri et al. (2014) and Fellahi et al. (2016) who re- ported that non-additive gene effects played an impor- tant role in the inheritance of PHT. Salmi et al. (2019) found that dominance acted in the direction of in- creased plant height But Akhtar and Chowdhry (2006) as well as Hannachi et al. (2013) reported that additive gene effects were predominant in the genetic control of this character. For STM, the additive [d] gene effects were not significant while dominance [h] gene effects, additive * additive [i], additive * dominance [j] and dominance * dominance [l] allelic interactions were sig- nificant. The gene effects [h], additive * additive [i], and dominance * dominance [l] allelic interactions exhibited the largest effects. Being significant and of the same sign, dominance [h] gene effects and dominance * dominance [l] non-allelic interactions suggested the implication of complementary type of epistasis in the genetic control of this trait. A greater magnitude of dominance [h] com- pared to additive [d] gene effects, as this is the case in the present study, for this trait, arises, according to Ke- arsey and Pooni (1996), when genes are dispersed in the parents. For FLA, the additive * dominance [j] epistatic component was not significant while the additive [d] and dominance [h] gene effects, the additive * additive [i], and dominance * dominance [l] allelic interactions were significant. The dominance * dominance [l] com- ponent exhibited the largest effect. The implication of complementary type of epistasis in the genetic control Traits PHT STM FLA Scaling test A 9.65 ± 4.96ns 37.14 ±1.11** 17.72 ± 1.71**   B 31.25 ± 2.56** 20.44 ± 2.18** 15.86 ± 0.66**   C 84.03 ± 4.41** 26.38 ±2.65** 17.04 ± 1.35**   D 21.55 ± 3.54** 41.99 ± 1.69** 8.12 ± 1.11** Joint scaling test X² 860.27** 569.90** 745.72** Genetic parameters m 117.79 ± 1.09** 34.25 ± 0.62** 24.51 ± 0.32**   [d] 13.87 ± 2.78** 0.19 ± 1.14ns 2.89 ± 0.90**   [h] - 72.67 ± 7.09** 75.39 ± 3.42** 14.49 ± 2.23**   [i] - 43.11 ± 7.09** 83.98 ± 3.39** 16.24 ± 2.22**   [j] - 10.84 ± 2.79* 8.35 ± 1.21** 1.08 ± 0.91ns   [l] 2.18 ± 11.99ns 141 ± 5.27** 49.54 ± 3.85** Type of gene actions ----- Complementary Complementary H²bs±SE 0,98 ± 0.40* 0,99 ± 0.41* 0,98 ± 0.40* h²ns ±SE 0,57 ± 0.23* 0.76 ± 0.31* 0,39 ± 0.16* rg GY/ -0.57 ± 0.15* 1.21 ± 0.17* 0.91 ± 0.07* ERS 8.25 22.72 7.03 ERS% 6.99 99.44 29.69 Table 2: Scaling tests, gene action types, heritability (H²bs, h²ns), expected response to selection (ERS) and genotypic correlation (rgGY/…) estimates for plant height, straw yield and flag leaf area in MBB x GTA durum wheat cross PHT = Plant height, STM = straw mass, FLA = Flag leaf area. m = mean main effect, [d] = assistive effect, [h] = dominance effect, [i] = additive x additive effect, [j] = additive x dominance effect, [1] = dominance x dominance effect. Ns, * and ** = non-significant and significant effects at 5 % and 1 % probability levels, respectively Acta agriculturae Slovenica, 117/4 – 2021 7 Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross of this trait is suggested by the dominance gene effects [h] and the dominance * dominance [l] allelic interaction which were significant and of the same sign. Dominance [h] component was greater than additive [d] gene effects suggesting that genes controlling FLA are dispersed in the parents. In this context Saleem et al. (2005); Inamul- lah et al. (2006); Munir et al. (2007); Ijaz et al. (2013) and Yang et al. (2016) found that FLA was mostly under addi- tive genetic control combined to partial dominance and epistasis type of gene actions. Joshi and Sharma (1984) mentioned that dominance acted in the direction of re- duced FLA. Shayan et al. (2019) reported that the additive-dom- inance model fitted best the variation present among generation means for FLA, while for PHT and STM, the six-parameter model was adequate implying the pres- ence of non-allelic interactions in the inheritance of these two traits. Divergence in the result among various studies seems to indicate that genetic model adequacy as well as the preponderance of significant gene effects and non-allelic interactions are dependent upon the cross combination genetic background and the experimen- tal growth conditions experienced. The complementary epistasis type implicated in the inheritance of STM and FLA suggested the possibility of heterosis expression for these two traits. In fact, Punia et al. (2011), referring to Jinks and Jones (1958), mentioned that heterosis is likely to be expressed with greater magnitude in crosses where complementary type of interaction is expressed. The fact that generation means variation fitted a digenic epistatic model suggested that improvement of PHT, STM and FLA would be fairly difficulty compared to the situation where the additive-dominance model was the most ad- equate. Furthermore, Sirohi and Gupta (1993) suggested that traits showing high magnitude of dominance [h] than additive [d] gene effects, as this is the case for PHT, STM and FLA, in the present study, can be improved through conventional breeding approaches. But selec- tion need to be delayed until later generations when the dominance effects would have diminished and desirable segregants become available. 3.3 HERITABILITY, EXPECTED RESPONSE TO SE- LECTION, AND GENOTYPIC CORRELATIONS Being significant, broad sense heritability estimates for FLA, PHT and STM were appreciably high, taking values of 98.00, 98.00 and 99.00  %, respectively. Their corresponding narrow sense counterparts were also significant, but of lower magnitude, being still high for STM (76.00  %), moderate for PHT (57.00  %) and low for FLA (39.00  %). High h²ns values indicate that the environment influences less the expression of the given character. In fact, estimates of heritability are useful for a breeder to weigh the proportion of variation which is inheritable from that which is non-inheritable. Heritabil- ity values observed in the present study were in the range of those reported in similar studies. Fellahi et al. (2020) reported h²bs values of 86.50 % for PHT and 77.40 % for FLA. Novoselovic et al. (2004) reported h²bs values rang- ing from 0.54 to 0.81 for PHT of several crosses. These high H²bs values suggested that these traits are less im- pacted by environmental variation, and then are easily amenable to improvement. In this context, Johnson et al. (1966) mentioned that since plant height and straw mass heritability were sufficiently high then selection in the F2 for these traits could be effective. So, based on the herita- bility estimates observed in the present study, STM, FLA and PHT appeared amenable to significant improvement applying early selection. Genotypic correlation coefficients, relating grain yield (GY) to FLA, PHT and STM, found in this study, were significant, taking values of 0.91, -0.57 and 1.21, re- spectively (Table 2). while PHT was posirivelly correlated with STM (r = 0.315**), but negatively correlated with FLA (r = -0.153*), and STM was positivelly correlated with FLA (r = 0.269**). These correlation coefficients in- dicated that selection of high values for FLA and STM will be accompanied by increased GY, but selection to increase PHT had a negative impact on GY and on FLA. The negative correlation relating PHT to GY, observed in the present study, contradicted Ataei et al. (2017) results which showed that GY was positively and highly corre- lated with PHT and peduncle under drought stress con- ditions, emphasizing the importance of PHT as selection criterion to improve drought tolerance. In this context and according to Davidson et al. (1992) and Belkher- chouche et al. (2015) wheat peduncle is a transient source of water-soluble carbohydrates, playing a crucial role in minimizing grain yield decline under drought stress conditions. Similarly, Mohsin et al. (2009) found that grain yield correlated positively with FLA, PHT, biomass (BIO), under drought stress. Under rainfed growing conditions, Mansouri et al. (2018) reported that above ground plant biomass exhibited significant and positive correlation coefficients with GY, STM and PHT. The ex- pected response to selection estimates were low, being less than 10 % for PHT (8.24 cm or 6.99 %, XbarF2 = 117.87 cm) and high, above 20 %, for STM (22.74 g plant-1 or 99.4 % XbarF2 = 22.85 g plant -1), FLA (7.03 cm2 or 26.6 %, XbarF2 = 23.67 cm 2) (Table 2). These results indicated that, based on the magnitude of the variability expressed by each trait, moderate to appreciable genetic gain could be made via mono trait selection. Durum wheat production is often impacted by Acta agriculturae Slovenica, 117/4 – 20218 M. SALMI et al. drought stress, particularly in the arid and semi-arid re- gions. To overcome this situation, it is necessary to devel- op improved varieties devoted to these specific environ- ments. Morphological characters like plant height, flag leaf area, and straw yield, had been identified and pro- posed as morphological markers for drought tolerance. Breeding procedure for drought tolerance depends upon the pattern of inheritance and the nature of actions of the genes involved in the genetic control of the drought related traits. A better understanding of the complexi- ties of the genetic control of these traits will be useful for cultivar improvement. Globally, from the results of this study, it can be summarized that the additive–dominance model was inadequate, suggesting the adoption of a six- parameter genetic model. Additive [d] and dominance [h] gene effects, and non-allelic interactions ([i], [j] and [l]) were involved in the inheritance of PHT, with the predominance of [h] and [i] components. Complemen- tary type of epistasis was implicated in the inheritance of STM and FLA. These results were in lines with findings of some studies (Novoselovic et al., 2004; Ojaghi and Ak- hundova, 2010; Mohamed et al., 2013; Dorri et al., 2014; Fellahi et al., 2016) and diverged from those of others studies (Inamullah et al., 2006, Munir et al., 2007; Ijaz et al., 2013; Yang et al., 2016; Shayan et al., 2019), suggesting that genetic model adequacy as well as the preponderance of significant gene effects and non-allelic interactions, governing the inheritance of a given trait, are dependent upon the cross combination genetic background and the experimental growth conditions experienced. Due to the presence of non-allelic interactions PHT, STM and FLA improvement would be fairly difficulty, requiring the implementation of conventional breeding approaches such as the inclusion of F2’s showing high performances in multiple crosses for further improvement of the stud- ied traits in order to synthesize a dynamic population accumulating most of the favorable genes. This mating procedure seems to be a good technique to disrupt link- age, to generate usefull recombination and to accumulate favorable genes in the base population. Selection need to be delayed until later generations when the dominance effects would have diminished and desirable segregants become available. This strategy is supported by the high heritability estimates, the moderate to high expected ge- netic gains and the significant genotypic correlation coef- fcicients relating the studied traits to grain yield. 4 CONCLUSION The results of the present study indicated that non- allelic interactions, in addition to additive and dominant gene effects are important factors controlling the expres- sion of PHT, STM and FLA. Application of conventional selection procedure may not be rewarding for the im- provement of these characters. But inter mating among the selected segregants followed by few generations of selfing could be useful to break the undesirable linkage and allow accumulation of favorable alleles for improve- ment of these traits. 5 REFERENCES Akhtar, N., & Chowdhry, M. A. (2006). Genetic analysis of yield and some other quantitative traits in bread wheat. Interna- tional Journal of Agriculture and Biology, 4, 523–527. Annicchiarico, P., Abdellaoui, Z., Kelkouli, M., Zerargui. H. (2005). Grain yield, straw yield and economic value of tall and semi-dwarf durum wheat cultivars in Algeria. Journal of Agricultural Science, 143, 57–64. https://doi.org/10.1017/ S0021859605004855 Asadi, A., Valizadeh, M., Mohammadi, S. A., Khodarahmi. M. (2015). Genetic analysis of some physiological traits in wheat by generation means analysis under normal and wa- ter defict conditions. Biological forum, 7, 722-733. Ataei R., Gholamhoseini, M., Kamalizadeh, M. (2017). Genetic analysis for quantitative traits in bread wheat exposed to ir- rigated and drought stress conditions. Phyton, 86, 228-235. https://doi.org/10.32604/phyton.2017.86.228 Aziz, T., Mahmood, Z., Mahmood, K., Shazadi, A., Kazi, M., Rasheed, A. (2018). Genotypic variation and genotype x environment interaction for yield-related traits in syn- thetic hexaploid wheats under a range of optimal and heat- stressed environments. Crop Science, 58, 295-303. https:// doi.org/10.2135/cropsci2017.01.0035 Belagrouz A., Chennafi, H., Bouzerzour, H., Hakimi, M., Ra- zem, R., Hadj Sahraoui, A. (2018). Relationships among water use efficiency and the physio-agronomic traits in durum wheat (Triticum durum Desf.) cultivars assessed under rainfed conditions of the eastern high plateaus of Al- geria. The Journal Agriculture and Forestry, 64(3), 159-172. https://doi.org/10.17707/AgricultForest.64.3.14 Belkherchouche, H., Benbelkacem, A., Bouzerzour, H., Benma- hammed. A. (2015). Flag leaf and awns ablation and spike shading effects on spike yield and kernel weight of durum wheat (Triticum turgidum L. var. durum) under rainfed conditions. Advances in Environmental Biology, 9(8), 184- 191. Chennafi, H., Hannachi, A., Touahria, O., Fellahi, Z.E.A., Makhlouf, M., Bouzerzour, H. (2011). Tillage and residue management effect on durum wheat [Triticum turgidum (L.) Thell. ssp. turgidum conv. durum (Desf.) Mackey] growth and yield under semi-arid climate. Advances in En- vironmental Biology, 3231–3241. Cropstat. (2007). CropStat for Windows 7.2. Dapo, Manila IRRI - International Rice Research Institute. Davidson, D. J. and Chevalier, P. M. (1992). Storage and remo- bilization of water-soluble carbohydrates in stems of spring wheat. Crop Science, 32, 186-190. https://doi.org/10.2135/cr opsci1992.0011183X003200010038x Acta agriculturae Slovenica, 117/4 – 2021 9 Inheritance of plant height, straw yield and flag leaf area in MBB x Gaviota durum wheat (Triticum durum Desf.) cross De Pace, C., Snidaro, D., Ciaffi, M., Vittori, D., Ciofo, A., Cenci, A. (2001). Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphyt- ica, 117, 67–75. https://doi.org/10.1023/A:1004095705460 Dorri, P., Khorasani, S. K., Shahrokhi, M. (2014). Generation means analysis. A case study of variance components in KSC 500 generations of maize (Zea mays L.). International Research Journal of Applied and Basic Sciences, 8(2), 194- 200. Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S. M. A. (2009). Plant drought stress: effects, mechanisms and man- agement. Agronomy for sustainable development, Spring- er Verlag/EDP Sciences/INRA, 29, 85-212. https://doi. org/10.1007/978-90-481-2666-8_12 Fellahi, Z. E. A., Hannachi, A., Bouzerzour. H. (2020). Expected genetic gains from mono trait and index-based selection in advanced bread wheat (Triticum aestivum L.) populations. Revista Facultad Nacional de Agronomía Medellín, 73, 9131- 9141. https://doi.org/10.15446/rfnam.v73n2.77806 Fellahi, Z. E. A., Hannachi, A., Bouzerzour, H., Dreisigacker, S., Yahyaoui, A., Sehgal, D. (2017). Genetic analysis of morpho-physiological traits and yield components in F2 partial diallel crosses of bread wheat (Triticum aestivum L.). Revista Facultad Nacional de Agronomía Medellín, 70, 8237–8250. https://doi.org/10.15446/rfna.v70n3.61927 Haddad, L., Bachir, A., Yakhlef, N., Benmahammed, A., Bou- zerzour, H. (2021). Durum wheat [Triticum turgidum (L.) Thell ssp turgidum conv. durum (Desf.) Mackey] during the past 70-year in Algeria: Performance assessment of a set of historical varieties under rainfed conditions of the eastern high plateaus. Jordan Journal of Biological Sciences, 14. (in press). Hallauer, A. R., & Mirinda Filho, J. B. (1989). Quantitative ge- netics in maize breeding. Second edition, Ames, IOWA State University Press. 468 p. Halloran, G. M., Knight, R., McWhirter, K. S., Sparrow, D. H. B. (1979). Plant breeding. Knight, R. (Ed.). Poly-Graphics Pty. Ltd. Brisbane, Australia. pp. 61 - 62. Hammer, O., Harper, D. A. T., Ryan, P. D. (2001). Paleontologi- cal statistics software package for education and data analy- sis. Palaeontologia Electronica, 4(9). Hannachi, A., Fellahi, Z. E. A. Bouzerzour, H., Boutakrabt. A. (2013). Diallel-cross analysis of grain yield and stress tolerance-related traits under semi-arid conditions in Durum wheat (Triticum durum Desf.). Electron- ic Journal of Plant Breeding, 4, 1027-1033. https://doi. org/10.1155/2013/201851 Ijaz U., Miullah, S., Kashif. M. (2013). Generation means analy- sis for five physiological traits of bread wheat under rain- fed condition. Universal Journal of Plant Science, 1, 21-26. https://doi.org/10.13189/ujps.2013.010103 Inamullah, A., Hussain, M., Hassan, G. F., Gul, R. (2006). Dial- lel analysis of the inheritance pattern of agronomic traits of bread wheat. Pakistan Journal of Boutany, 38, 1169-1175. Jatayev, S., Sukhikh, I., Vavilova, V., Smolenskaya, S. E., Gon- charov, N. P., Kurishbayev, A., Zotova L. (2020). Green revolution ‘stumbles’ in a dry environment: Dwarf wheat with Rht genes fails to produce higher grain yield than tall- er plants under drought. Plant, Cell and Environment, 43, 2355-2364. https://doi.org/10.1111/pce.13819 Johnson, V. A., Biever, K. J., Haunold, A., Schmidt, J. W. (1966). Inheritance of plant height, yield of grain, and other plant and seed characteristics in a cross of hard red winter wheat, Triticum aestivum L.. Crop Science, 6, 307-312. https://doi. org/10.2135/cropsci1966.0011183X000600040011x Joshi, S. K., Sharma, S. N., Singhania, D. L., Sain. R. S. (2004). Combining ability in the F1 and F2 generations of diallel cross in hexaploid wheat (Triticum aestivum L. em. Thell). Hereditas, 141, 115-121. https://doi.org/10.1111/j.1601- 5223.2004.01730.x Kearsey, M. J. and Pooni, H. S. (1996). The genetic analysis of quantitative traits. Chapman and Hall, London. 396 p. https://doi.org/10.1007/978-1-4899-4441-2 Koots, K. R., Gibson. J. P. (1996). Realized sampling variances of estimates of genetic parameters and the difference be- tween genetic and phenotypic correlations. Genetics, 143, 1409-1416. https://doi.org/10.1093/genetics/143.3.1409 Kwon, S. H. and Torrie, J. H. (1964). Heritability and inter-re- lationship among traits of two soybean populations. Crop Science, 4, 196-198. https://doi.org/10.2135/cropsci1964.00 11183X000400020023x Liu, H., Searle, I. R., Mather, D. E., Able, A. J., Able. J. A. (2015). Morphological, physiological and yield responses of durum wheat to pre-anthesis water-deficit stress are genotype-de- pendent. Crop and Pasture Science, 66, 1024–1038. https:// doi.org/10.1071/CP15013 Manivannan, N. (2014). TNAUSTAT- Statistical package: htt- ps://sites.google.com/site/tnaustat. Mansouri, A., Oudjehih, B., Benbelkacem, A. Fellahi, Z. E. A., Bouzerzour, H. (2018). Variation and relationships among agronomic traits in durum wheat [Triticum turgidum (L.) Thell. ssp. turgidum conv. durum (Desf.) Mackey] under south Mediterranean growth conditions: Stepwise and path analyses. International Journal of Agronomy, 1-11. https:// doi.org/10.1155/2018/8191749 Mather, K., & Jinks, J. L. (1982). Biometrical Genetics. The Study of Continuous Variation. Third edition. Chapman and Hall, London – New York, pp. 279. https://doi.org/10.1007/978- 1-4899-3406-2 Mohsin, T., Khan, N., Naqvi, F. N. (2009). Heritability, pheno- typic correlation and path coefficient studies for some agro- nomic characters in synthetic elite lines of wheat, Journal of Food, Agriculture and Environment, 7, 278–282. Munir, M., Chowdhry, M. A., Ahsan, M. (2007). Generation mean studies in bread wheat. Internaional Journal of Ag- riculure and Biology, 9, 282-286. Novoselović, D., Barić, M., Drenzer, G., Gunjača, J., Lalić, A. (2004). Quantitative inheritance of some wheat plant traits. Genetics and Molecular Biology, 27, 92-98. https://doi. org/10.1590/S1415-47572004000100015 Ojaghi, J., & Akhundova, E. (2010). Genetic analysis for yield and its components in doubled haploid wheat. African Jour- nal of Agricultural Research, 5, 306-315. Punia, S. S., Baldev, R. Koli, N. R. Ranwah, B. R. Rokadia, P., Maloo, S. R. (2011). Genetic architecture of quantitative traits in field pea. Journal of Food Legumes, 24, 299–303. Rabti, A., Mekaoussi, R., Fellahi, Z. E. A., Hannachi, A., Ben- Acta agriculturae Slovenica, 117/4 – 202110 M. SALMI et al. belkacem, A., Benmahammed, A., Bouzerzour, H. 2020. Characterization of old and recent durum wheat [Triticum turgidum (L.) Tell. convar. durum (Desf.) Mackey] varieties assessed under south Mediterranean conditions. Egyptian Journal of Agronomy, 42, 307-320. https://doi.org/10.21608/ agro.2020.43329.1230 Rad, M. R. N., Kadir, M. A., Yusop, M. R., Jaafar, H. Z., Danaee, M. (2013). Gene action for physiological parameters and use of relative water content (RWC) for selection of tolerant and high yield genotypes in F2 population of wheat. Austo- ralian Journal of Crop Science, 7, 407-413. Royo, C., Nazco, R., Villegas, D. (2014). The climate of the zone of origin of Mediterranean durum wheat (Triticum durum Desf.) landraces affects their agronomic performance. Ge- netic Resources and Crop Evolution, 61, 1345–1358. https:// doi.org/10.1007/s10722-014-0116-3 Saleem, M., Aslam, M., Muhammad, C., Kashif, K., Khaliq. M. (2005). Inheritance pattern of plant height, grain yield and some leaf characteristics of spring wheat. International Journal of Agriculture and Biology, 7, 1019-1025 Salmi, M., Benmahammed, A., Benderradji, L., Fellahi, Z. E. A., Bouzerzour, H., Oulmi, A., Benbelkacem. A. 2019. Genera- tion means analysis of physiological and agronomical tar- geted traits in durum wheat (Triticum durum Desf.) cross. Revista Facultad Nacional de Agronomía Medellín, 72, 8971- 8981. https://doi.org/10.15446/rfnam.v72n3.77410 Shabbir, G., Kiran, T., Akram, Z., Ijaz, M., Shah. K. N. (2012). Genetics of some biometric traits in bread wheat (Triticum aestivum L.). Journal of Agricultural Research, 50, 457-468. Shayan, S., Moghaddam Vahed, M., Norouzi, M., Mohammadi, A., Tourchi, M., Molaei. B. (2018). Inheritance of agronom- ical and physiological traits in the progeny of Moghan3 and Arg bread wheat varieties cross. Plant Genetic Research, 4, 43-60. https://doi.org/10.29252/pgr.4.2.43 Singh, R. P., Huerta-Espino, J., Rajaram, S., Crossa, J. (2001). Grain yield and other traits of tall and dwarf isolines of modern bread and durum wheats. Euphytica, 119, 241–244. https://doi.org/10.1023/A:1017541805454 Sirohi, A., & Gupta, V. P. (1993). Additive, dominance and epi- static components of variation for seed protein content in pea (Pisum sativum L.). Indian Journal of Genetics, 53, 252- 256. Slafer, G. A., Araus, J. L. Royo, C., Del Moral, L. F. G. (2005). Promising eco-physiological traits for genetic improvement of cereal yields in Mediterranean environments. Annals of Applied Biology, 146, 61–70. https://doi.org/10.1111/j.1744- 7348.2005.04048.x Spagnoletti-Zeuli, P. L., & Qualset, C. O. (1990). Flag leaf variation and the analysis of diversity in du- rum wheat. Plant Breeding, 105, 189–202. https://doi. org/10.1111/j.1439-0523.1990.tb01196.x Steel, R. G. D., & Torrie, J. H. (1960). Principles and procedures of statistics, McGraw-Hill Books, New York. 481 p. Velu, G., Singh, R. P. Huerta, J., Guzman C. (2017). Genetic impact of Rht dwarfing genes on grain micronutrients concentration in wheat. Field Crops Research, 21, 373–377. https://doi.org/10.1016/j.fcr.2017.09.030 Yang, D., Liu, Y., Cheng, H., Chang, L., Chen, J., Chai, S., Li, M. (2016). Genetic dissection of flag leaf morphology in wheat (Triticum aestivum L.) under diverse water regimes. BMC Genetics, 17, 1-15. https://doi.org/10.1186/s12863- 016-0399-9 Yani, S. C., & Rashidi, V. (2012). Selection indices in the im- provement of wheat grain yield on drought stress condi- tions, the success of selection in the cross populations depend on the knowledge of inheritance pattern of the desired traits. African Journal of Agricultural Research, 7, 1177-1183. https://doi.org/10.5897/AJAR11.1616 Acta agriculturae Slovenica, 117/4, 1–12, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2241 Review article / pregledni znanstveni članek Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline Primož ŽIGON 1, 2, Franci Aco CELAR 3 Received June 12, 2021; accepted October 12, 2021. Delo je prispelo 12. junija 2021, sprejeto 12. oktobra 2021 1 Kmetijski inštitut Slovenije, Oddelek za varstvo rastlin, Ljubljana 2 Korespondenčni avtor, e-naslov: primoz.zigon@kis.si 3 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo, Ljubljana Endophytic fungi as biological control agents and their indi- rect effects on plants Abstract: The use of entomopathogenic fungi represents one of the most important non-chemical alternatives for pest control in crop production. In addition to their pathogenicity to arthropods, they have many other important effects that fa- vor their use in biological control. They live in plants as endo- phytes and have an inhibitory effect on plant pathogens. They inhabit the rhizosphere of many plants in natural and agricul- tural ecosystems and have a stimulatory effect on their growth and development. These recently acquired ecological functions are not yet fully understood, but point to the broader potential of using entomopathogenic endophytic fungi in crop produc- tion, not only as biopesticides but also as mycofungicides and growth stimulants (biostimulants). To achieve the full poten- tial of entomopathogenic endophytic fungi in daily agricultural practice, practical application should be considered in the de- velopment of commercial products and the application tech- niques of entomopathogenic endophytic fungi that allow suc- cessful colonization of plants should be considered. Key words: entomopathogenic fungi; endophyte; benefi- cial organisms; bioinsecticides; Beauveria; Metarhizium Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline Izvleček: Uporaba entomopatogenih gliv predstavlja enega temeljnih ukrepov nekemičnega varstva rastlin pred škodljivci. Poleg patogenosti za členonožce imajo te glive tudi druge lastnosti, zaradi katerih so širše uporabne v biotičnem varstvu rastlin. V rastlinah živijo kot endofiti in lahko deluje- jo zaviralno tudi na različne povzročitelje rastlinskih bolezni. Naseljujejo rizosfero številnih rastlin v naravnih in kmetijskih ekosistemih ter delujejo spodbujevalno na njihovo rast in ra- zvoj. Te v zadnjem času dognane ekološke funkcije še niso podrobneje raziskane, vendar kljub temu nakazujejo na širši potencial uporabe entomopatogenih endofitnih gliv pri pride- lavi rastlin, ne le kot sredstev za biotično zatiranje škodljivcev, pač pa tudi kot mikofungicidov in sredstev za krepitev rasti in razvoja (biostimulantov). Za umestitev uporabe entomopato- genih endofitnih gliv v vsakdanjo kmetijsko prakso je potreb- no pri razvoju komercialnih pripravkov upoštevati praktičnost uporabe in preučiti tehnike nanosa entomopatogenih endofi- tnih gliv, ki omogočajo uspešno kolonizacijo rastlin. Ključne besede: entomopatogene glive; endofiti, koristni organizmi, bioinsekticidi; Beauveria, Metarhizium Acta agriculturae Slovenica, 117/4 – 20212 P. ŽIGON in F. A. CELAR 1 UVOD 1.1 ENDOFITI Endofiti so različne vrste gliv in bakterij, ki živijo v notranjosti rastlinskega tkiva ter so z rastlinami v mutu- alističnem odnosu (Wilson, 1995). Dokaze za obstoj en- dofitizma so odkrili v fosiliziranih ostankih rastlin, kar kaže na vzpostavitev tovrstnih odnosov že vse od poja- va prvih kopenskih rastlin (Field in sod., 2015a; Field in sod., 2015b). Endofiti z gostiteljskimi rastlinami tvorijo kompleksne večplastne interakcije, ki imajo pozitivne učinke na rastline. Z namenom prilagoditve in preživetja znotraj rastlin mikroorganizmi vzpostavijo mutualistični odnos v okviru katerega imata oba organizma medseboj- no korist (Kogel in sod., 2006; Hardoim in sod., 2015). Kolonizacija rastlinskega tkiva brez povzročanja vidnih posledic vzpostavitve tega odnosa, ki bi se odražal v ob- liki poškodb rastlinskega tkiva, poteka vsaj del življenj- skega kroga endofitnega organizma. Gre torej za okužbo rastlin, ki se ne odraža v pojavu vidnih bolezenskih zna- menj (Petrini, 1991). Endofiti so lahko prisotni v rast- linskih tkivih po celotni rastlini ali posameznih organih kot so korenine, steblo, listi, cvetovi, semena itd. Njihov življenjski prostor znotraj rastlin obsega notranjost celic (intercelularno) in/ali se nahajajo le v medceličnem pros- toru (intracelularno) (Hardoim in sod., 2015). Endofitni mikroorganizmi pripomorejo k odzivu in prilagoditvi rastlin na biotske in abiotske strese ter z vzbujanjem raz- ličnih mehanizmov inducirane odpornosti omogočajo le tem premostitev stresnih situacij (Rodriguez in Red- man, 2008; Rho in sod., 2018). Endofiti namreč stimu- lirajo tvorbo signalnih molekul kot so etilen, jasmonska kislina in salicilna kislina, s čimer vplivajo na aktivacijo mehanizmov inducirane odpornosti (Robert-Seilaniantz in sod., 2011). Eden izmed pomembnih odzivov rastlin na stres je tudi tvorba sekundarnih metabolitov. Endo- fiti posredno vplivajo na povečano tvorbo rastlinskih sekundarnih metabolitov z aktivacijo genov za njihovo sintezo, določeni endofiti pa sekundarne metabolite, ki so udeleženi v obrambnih odzivih rastlin, tvorijo tudi sami (Van Wees in sod., 2008; Zamioudis in Pieterse, 2012). Endofiti lahko vplivajo na večjo dostopnost v tleh vezanih hranil in mineralov ter sproščanje le teh v talno raztopino, s čimer rastlinam omogočajo njihov privzem in tako vplivajo na boljše fiziološko stanje ter prehran- jenost gostiteljskih rastlin. Druge prilagoditve rastlin na abiotski stres, ki jih omogočajo endofiti, so povezane s tvorbo rastlinskih hormonov, stresnih proteinov, antiok- sidantov in encimov, ki povečujejo toleranco rastlin na stresne razmere (Rho in sod., 2018). Po drugi strani je vzpostavitev mutualističnega od- nosa z rastlinami ključna za preživetje in obstoj nekate- rih endofitov, obligatnih heterotrofov, ki so odvisni od privzema hranil iz rastlinskega tkiva (Bamisile in sod., 2018). Rastline endofitnim organizmom nudijo ustrezno okolje za rast, zagotavljajo vir ogljika in drugih hranil ter v nekaterih primerih preko prenosa s semenom go- stiteljskih rastlin omogoča njihov obstoj in ohranjanje (Hardoim in sod., 2015). Rastline s koreninskimi izločki v območje korenin privabljajo množico mikroorganiz- mov. Mikroorganizmi v tleh, patogeni ali nepatogeni, imajo enak potencial za okužbo rastlin. Ta je odvisna od razmer v rizosferi, vrste in odziva gostitelja ter sposob- nosti mikroorganizma, da zaobide njegov imunski odziv (Philippot in sod., 2013). Endofiti iz rizosfere vstopajo v rastlinsko tkivo preko koreninskih laskov in naravnih odprtin. Vstop endifitov in kolonizacijo rastlinskega tki- va pogojuje tvorba specifičnih encimov, signalnih mo- lekul (na primer flavonoidov) in drugih metabolitov, ki omogočajo premostitev obrambnih mehanizmov rastlin in razlikovanje koristnih endofitnih organizmov od dru- gih potencialno patogenih mikroorganizmov v rizsoferi (Kogel in sod., 2006; Hardoim in sod., 2015). 1.1.1 Endofitne glive Izraz endofiti je prvič uporabil Heinrich Anton de Bary in z njim označil glive, katerih hife preraščajo celice in tkiva avtotrofnih organizmov (Bary, 1866). Prisotnost endofitnih gliv (EG) v rastlinskem tkivu je prvič doka- zal E. M. Freeman (Freeman in Ward, 1904), ki je izoliral glivo iz rodu Epichloë iz semen omotne ljuljke (Lolium temulentum L.). Do danes so EG našli v številnih rast- linskih vrstah, ki rastejo praktično na vseh območjih in vrstah rastišč od severnega do južnega pola zemeljske Slika 1: Vpliv endofitov na rast in odziv rastlin na biotske in abiotske stresne dejavnike (Sharma in sod., 2020) Acta agriculturae Slovenica, 117/4 – 2021 3 Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline oble (Saikkonen in sod., 2004; Arnold, 2007; Rodrigu- ez in sod., 2009). EG tvorijo različne vrste sekundarnih metabolitov kot so alkaloidi, cikloheksani, flavonoidi, kinoni in terpeni, ki imajo protimikrobne, antioksida- tivne, antikancerogene in citotoksične učinke (Rana in sod., 2019; Mantzoukas in Eliopoulos, 2020). Imajo tudi pomembno vlogo pri odzivu rastlin na okužbo/napad rastlinskih patogenov in herbivorov, omogočajo boljšo prehranjenost rastlin, saj povečujejo dostopnost makro in mikro hranil ter omogočajo vezavo atmosferskega du- šika (Bacon, 1993; Behie in sod., 2013). Za vzpostavitev simbiotskega odnosa EG tvorijo hidrolitične encime (ce- lulaze, lipaze, proteaze, oksidaze), ki omogočajo premos- titev obrambnih mehanizmov rastline in s tem uspešno kolonizacijo rastlinskih tkiv. Znotraj rastlin hife gliv rastejo skozi parenhim tudi vse do ksilema. Sistemična kolonizacija rastlin je značilna za akropetalno (od spodaj navzgor) rastoče hife gliv, torej predvsem v primeru, da gliva izvira iz okuženega semena ali pa vstopa v rastlino preko korenin (Mantzoukas in Eliopoulos, 2020). Uspeš- nost kolonizacije rastlin je odvisna od razmer v rizosferi (kompeticija mikroorganizmov), okoljskih razmer (vla- ga, temperatura), rastlinske vrste in njenega fiziološkega stanja (Carroll, 1988). Številne vrste EG imajo pomemb- no vlogo pri odzivu rastlin na okužbo/napad rastlinskih patogenov in škodljivcev. Za EG velja, da imajo načeloma pozitiven vpliv na rastline, saj se okužba, ki jo povzroča- jo, ne odrazi v bolezenskih znamenjih. Kljub temu za ne- katere vrste EG velja, da lahko latentna okužba pod dolo- čenimi pogoji, ki so lahko posledica okoljskih sprememb, stresa ali obdobja senescence rastlin, povzroči tudi pojav bolezni (Saikkonen in sod., 2004; Bamisile in sod., 2018). Na podlagi do sedaj opravljenih raziskav ugotavljajo veliko vrstno pestrost EG, ki izhajajo iz debel zaprtotro- snic (Ascomycota) in prostotrosnic (Basidiomycota) ter poddebla Mucoromycotina. Asimptomatske okužbe so med drugim vzrok za slabo poznavanje in manjšo raz- iskanost interakcij med glivami in rastlinami, zato je bilo izoliranih in taksonomsko opisanih relativno malo vrst. Endofiti so lahko specializirani na ravni družine, ali pa okužujejo širši krog gostiteljev na različnih rasti- ščih. Taksonomsko lahko EG razvrščamo v dve skupini glede na družino, ki ji posamezne vrste pripadajo: vrste iz družine Clavicipitaceae (Ascomycota, Hypocreales) kolonizirajo relativno ozek krog gostiteljskih rastlin iz družine trav (Poaceae) kot so Festuca spp., Lolium spp. idr. V drugo skupino sodijo druge glive, ki ne izhajajo iz družine Clavicipitaceae in kolonizirajo širši krog gosti- teljskih rastlin (Carroll, 1988; Rodriguez in sod., 2009; Hardoim in sod., 2015). Za enostavnejše razumevanje poznanih kompleksnih interakcij med EG in njihovimi gostiteljskimi rastlinami jih lahko razvrščamo glede na način prenosa med gostiteljskimi rastlinami (horizontal- no ali vertikalno), glede na način kolonizacije in širje- nja v po rastlini (sistemično ali omejeno na posamezne organe oziroma rastlinska tkiva) ali glede na način pre- hranjevanja (nekrotrofi in biotrofi) (Rodriguez in sod., 2009). Poleg morfološke identifikacije so za določevanje gliv vsekakor pomembne novejše molekulske tehnike na osnovi DNK, ki omogočajo natančnejšo klasifikacijo in filogenetsko opredelitev EG ( Vega in sod., 2009; Bamisi- le in sod., 2018). 1.1.2 Entomopatogene glive Glive so prevladujoči patogeni členonožcev, med nj- imi žuželk in pajkovcev, med katerimi so tudi pomemb- ni rastlinski škodljivci. Entomopatogene glive (EPG) so namreč tekom evolucije razvile prefinjene mehanizme izkoriščanja žuželk za zadovoljevanje svojih prehranskih potreb. EPG so paraziti teh organizmov in pri njih po- vzročajo bolezenska stanja. Med EPG uvrščamo preko 700 različnih vrst, od katerih večina okužuje širok krog gostiteljev, njihovi posamezni sevi pa so bolj patogeni za določene vrste žuželk in pršic (Vega in sod., 2009; Sandhu in sod., 2012). Redke vrste EPG so bolj specia- lizirane in okužujejo ožji krog gostiteljev, kot na primer Aschersonia aleyrodes Webber 1897, ki okužuje le ka- parje (Coccoidea) in ščitkarje (Aleyrodidae) (Humber, 2008). Insekticidno učinkovanje EPG je specifično glede na gostiteljske organizme kot so npr. gosenice metuljev (Lepidoptera), uši (Aphidae), resarji (Thrips spp.) in dru- ge kozmopolitske vrste, ki so znani škodljivci kmetijskih rastlin. Do okužbe pride po naključju, ko spore EPG z vetrom ali vodo pridejo v stik z gostiteljem in kalijo na površju njegovega telesa. Vanj prodrejo skozi naravne odprtine ali neposredno prek zunanje kutikule. Uspeš- nost in hitrost kalitve spor je odvisna od okoljskih dejav- nikov kot sta vlaga in temperatura, dostopnosti hranil, kisika, pH ter vsebnosti protimikrobnih snovi na kuti- kuli gostitelja (Sandhu in sod., 2012). Prodor EPG skozi zunanjo epikutikulo poteka mehansko, v večini prime- rov na podlagi tvorbe specifičnih kaveljčkom podobnih struktur ali apresorijev. Sestava notranjega dela kutikule (prokutikula) je kompleksnejša, sestavljajo jo predvsem hitinske fibrile, beljakovine in lipidi (Hackman, 1953). Prodor glive je zato poleg mehanskega učinka odvisen tudi od biokemičnega vpliva in tvorbe hidrolitičnih enci- mov, ki razgrajujejo celične strukture v kutikuli (Pedrini in sod., 2007). Okužba je odvisna tudi od učinkovitos- ti različnih strategij, na podlagi katerih EPG zaobidejo imunski odziv gostitelja. Pri številnih vrstah pride do spremembe načina rasti in tvorbe blastospor, preko kate- rih pride do uspešne kolonizacije hemocela in privzema hranil iz hemolimfe. Posledica je postopno izčrpavanje, Acta agriculturae Slovenica, 117/4 – 20214 P. ŽIGON in F. A. CELAR podhranjenost in s tem onemogočanje vitalnih funkcij gostitelja (Sandhu in sod., 2012). Poleg tega pri številnih EPG pride do tvorbe toksičnih metabolitov, ki pospešijo pogin gostitelja, olajšajo prodor hif in omogočajo širjenje blastospor v hemolimfi (Vidal in Jaber, 2015). Hife nato prepredejo notranjost telesa in prehajajo na površje ka- davra, kjer tvorijo nove spore, ki širijo okužbo naprej. O insekticidnih lastnosti EPG in njihovem poten- cialu za zatiranje škodljivcev so govorili že v 19. sto- letju, potem ko je italijanski entomolog Agostino Bassi dokazal, da je okužba z glivo Beuveria bassiana (Bals.- Criv.)  Vuill.  (1912) povzročila bolezensko stanje svilo- prejke (Vega in sod., 2009). Z začetkom proizvodnje in množične uporabe kemičnih insekticidov sredi prejšnje- ga stoletja, prave potrebe po uporabi biotičnih agensov za varstvo rastlin ni bilo, zato do nadaljnjega razvoja tega področja ni prišlo. Danes je pri iskanju okoljsko sprejeml- jivejših načinov zatiranja rastlinskih škodljivcev uporaba EPG ena ključnih alternativ uporabi kemičnih insektici- dov. Prednosti uporabe EPG v primerjavi s sintetičnimi insekticidi so predvsem v zmanjšanju neželenih učinkov na neciljne organizme in s tem ohranjanje biotske pes- trosti agroekosistema ter zmanjšanju ostankov kemijskih spojin v okolju ter s tem manjši vpliv na zdravje ljudi in živali (Vega in sod., 2009; Lacey in sod., 2015). Upora- ba entomopatogenih gliv v biotičnem varstvu večinoma temelji na t. i. pristopu preplavnega biotičnega varstva, pri katerem gre za ciljni vnos z namenom čimprejšnjega zatrtja škodljivca pri čemer nadaljnje razmnoževanje in ohranjanje glive v prostoru ni pomembno. EPG se upo- rabljajo v obliki tehničnih mikoinsekticidnih formulacij pri katerih je bistvena hitrost učinkovanja, da je doseže- na čim prejšnja smrtnost tarčnega organizma (Faria in Wraight, 2007). Za namene masovne proizvodnje pri- pravkov mora biti produkcija EPG enostavna in cenovno sprejemljiva, pripravki pa morajo dosegati konstantno učinkovitost v poljskih razmerah. Kljub temu, da je več kot 700 vrst EPG iz približno 90 rodov, se v praksi v glav- nem uporabljajo le glive iz rodov Beauveria, Metarhizium, Lecanicillium in Isaria, ki ustrezajo prej omenjenim kri- terijem (Vega in sod., 2009). Več kot 60 % komercialnih mikoinsekticidov temelji na vrstah Beauveria bassiana in Metarhizium anisopliae (Metchnikoff)  Sorokin  (1883), ki okužujeta številne rastlinske škodljivce in sta najbolje preučeni vrsti EPG, saj sta se med prvimi uporabljali za namene biotičnega varstva (Sandhu in sod., 2012). Ko- mercialne biopesticidne formulacije pretežno vsebujejo EPG v obliki blastokonidijev, ki se jih nanaša na površje rastlin. Učinkovitost EPG je v tem primeru v naravnih razmerah omejena, saj so spore občutljive na UV svetlo- bo in pomanjkanje vlage (Vega in sod., 2009). 1.1.3 Entomopatogene endofitne glive Vloga EPG pa ni omejena zgolj na zatiranje škodl- jivcev, pač pa je njihova ekosistemska funkcija, ki po- sredno vpliva na rast rastlin, mnogo širša. V številnih nedavnih študijah namreč ugotavljajo, da nekatere vrste EPG z rastlinami tvorijo različne interakcije, kar naka- zuje na njihovo endofitno naravo v smislu kolonizacije rastlinskih tkiv (Mantzoukas in Eliopoulos, 2020). Poleg sposobnosti okuževanja rastlinskih škodljivcev imajo entomopatogeni endofiti (EPGE) lahko tudi antagonis- tične lastnosti proti povzročiteljem rastlinskih bolezni in z naselitvijo v rizosferi posredno vplivajo na izboljšan- je razmer za rast rastlin (Jaber in Enkerli, 2017; Jaber in Ownley, 2018). Ta njihova večstranska vloga in multipli- kativni učinki na rastline kažejo na večji potencial EPGE v biotičnem varstvu rastlin, ne le kot sredstev za zatiranje rastlinskih škodljivcev, pač pa tudi v varstvu pred po- vzročitelji bolezni in posrednimi vplivi na spodbujanje rasti gostiteljskih rastlin (Vega, 2018). Iz rastlinskih tkiv so bile v preteklih raziskavah izolirane številne glive, ki so znani patogeni rastlinskih škodljivcev in na ta način dokazali njihovo naselitev v rastlinskem tkivu. Za zna- ne pomembnejše vrste EPG kot so Beauveria bassiana, Beauveria brongniartii (Sacc.) Petch, Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams, Cordyceps farinosa (Holmsk.) Kepler, B. Shrestha & Spatafora, Ne- otyphodium spp., Cladosporium spp., Acremonium spp. in Akanthomyces lecanii (Zimm.) Spatafora, Kepler & B. Shrestha je bila na podlagi postopkov izolacije iz rastlin- skih tkiv dokazana njihova v naravi prisotna endofitna vloga (Preglednica 1). Izmed naštetih je najbolj preučena B. bassiana, ki je splošno razširjena talna gliva in fakul- tativni endofit, ki okužuje preko 700 vrst žuželk in pr- šic. B. bassiana se v tleh širi kot saprofit, ki pa ima slabo tekmovalno sposobnost za odmrlo organsko snov, ki ji predstavlja osnovni vir hranil za nadaljnjo rast (Hajek, 1997). Vzpostavitev mutualistične interakcije z rastlina- mi je zato velikega pomena za obstoj in širjenje glive. Ci- tološke študije kažejo, da nekatere EPGE pretežno rastejo na površju korenin in kolonizirajo le celice epiderma in koreninske skorje ter tako le v manjši meri vstopajo ne- posredno v globje tkivo gostitelja. Z različnimi postopki inokulacije je bila sposobnost kolonizacije rastlinske- ga tkiva uspešno dokazana tudi za nekatere druge vrste gliv, kot so Metarhizium anisopliae (Metschn.) Sorokī, Fusarium oxysporum Schltdl., Trichoderma lixii (Pat.) P. Chaverri, Fusarium fujikuroi Nirenberg in Trichoderma asperellum Samuels, Lieckf. & Nirenberg, ki so v različnih raziskavah izkazovale insekticidno delovanje (Akello in Sikora, 2012; Batta, 2013; Muvea in sod., 2014). Acta agriculturae Slovenica, 117/4 – 2021 5 Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline 2 ENTOMOPATOGENE ENDOFITNE GLIVE KOT BIOTIČNI AGENSI ZA VAR- STVO PRED RASTLINSKIMI ŠKODLJIVCI EPGE po napadu rastlinskih škodljivcev na različne načine vplivajo na njihovo prehranjevanje in fiziološko stanje. Med pogostejšimi negativnimi posledicami pre- hranjevanja z rastlinami, ki so okužene z EPGE se ka- žejo v upočasnitvi rasti in razvoja, motnjah hranjenja in zmanjšani sposobnosti razmnoževanja in manjšem šte- vilu odloženih jajčec (Vidal in Jaber, 2015). Insekticidne učinke EPGE na škodljive žuželke in pršice so dokazali ob prisotnosti v različnih gostiteljskih vrstah (Pregledni- ca 1). Opravljene so bile številne raziskave, ki pa nima- jo enotnih zaključkov o vzrokih za negativne učinke na škodljivce. Hife gliv preraščajo rastlinska tkiva, vendar ni konkretnejših dokazov, da bi znotraj tkiv prihajalo tudi do sporulacije, zato ob prehranjevanju rastlinskih škodljivcev s koloniziranim tkivom, ki ga preraščajo hife EPGE ne pride do neposredne okužbe škodljivca in na- stanka mikoz (Vega, 2008). EPG v vlogi endofitov na ško- dljivce posredno vplivajo preko drugih mehanizmov kot so antibioza, antiksenoza ali krepitev inducirane odpor- nosti rastlin. S povečano tvorbo signalnih molekul, zlasti jasmonske kisline, se sproži sinteza inhibitorjev in dru- gih snovi, na primer polifenol oksidaze, ki na škodljivce delujejo toksično. EPGE po napadu škodljivcev lahko vplivajo na hitrejšo aktivacijo rastlinskih obrambnih me- hanizmov in močnejši obrambni odziv rastlin (Ownley in sod., 2010; Dara, 2019). V številnih raziskavah ome- njajo tudi kopičenje mikotoksinov ali drugih sekundar- nih metabolitov, ki jih EPGE izločajo v rastlinskem tkivu in povzročajo različne posledice na škodljivcih (Carroll, 1988; Vega, 2008; Gurulingappa in sod., 2011). Eden od obrambnih mehanizmov, ki ga lahko sprožajo EPGE je tudi sprememba v sestavi ali zmanjšanje tvorbe hlapnih komponent – kairomonov, ki so za škodljivce predstavlja- jo pomemben orientir pri iskanju gostitelja (Vega, 2018). 2.1 UPORABA ENTOMOPATOGENIH ENDOFIT- NIH GLIV V BIOTIČNEM VARSTVU RASTLIN PRED ŠKODLJIVCI Sposobnost kolonizacije rastlin je bila dokazana za večino vrst EPG, zaradi česar je njihov potencial za upo- rabo v biotičnem varstvu še posebej izrazit (Vega, 2018). Življenje znotraj rastlinskega tkiva omogoča manjšo od- visnost od okoljskih razmer in daljše obdobje varstva pred škodljivci. Uspešnost okužbe in kolonizacije rastlinskega tkiva je odvisna od biotskih in abiotskih dejavnikov, kot sta vlaga in temperatura (Vega, 2008). Na okužbo vpliva- jo tudi interakcije z drugimi mikroorganizmi, lastnosti rastnega medija-substrata, vrsta in starost gostiteljskih rastlin ter velikost glivnega inokluluma (vcepka). Za na- mene načrtnega vnosa EPGE v rastline se uporabljajo različne metode inokulacije rastlin kot so foliarni nanos, zalivanje s suspenzijo EPGE, namakanje koreninskega sistema pred sajenjem, pomakanje semen v suspenzijo glive ali njeno neposredno injiciranje v rastlinsko tkivo (Bamisile in sod., 2018; Vega, 2018). Foliarni nanos su- spenzije konidijev je enostavna in največkrat uporabljena metoda aplikacije v raziskavah in pri uporabi komerci- alnih bioinsekticidov. Pri takem načinu vnosa EPGE ve- činoma pride do lokalne kolonizacije tkiva, na katerega je bila suspenzija nanešena in se ne izrazi v sistemični okužbi rastline. Pri tem načinu je vstop EPGE skozi listno povrhnjico lahko omejen zaradi manjše zastopanosti na- ravnih odprtin, kot so na primer listne reže in drugih morfoloških lastnosti listov, ki onemogočajo prodor v rastlinsko tkivo (Tefera in Vidal, 2009). Inokulacija ra- stlin v začetnih fazah razvoja, bodisi z uporabo semen, okuženih z EPGE ali njenim nanosom na površje semen (seed coating), omogoča manjšo izpostavljenost neugo- dnim vremenskim razmeram in vzpostavitev ustreznih okoljskih razmer za okužbo v tleh (Vidal in Jaber, 2015). Pred sajenjem je inokulacijo sadik z EPGE možno izvesti tudi s pomakanjem sadik v glivno suspenzijo. Inokulacija rastlin ob začetku rasti, ima poleg potencialno dolgotraj- nejšega varstva rastlin v občutljivejših fazah razvoja, tudi pozitiven vpliv na rast in hitrejši mladostni razvoj rastlin (Bamisile in sod., 2018). Okužba z EPGE pred sajenjem ali med rastjo z zalivanjem omogoča varstvo semena in podzemnih delov rastline pred talnimi škodljivci, proti katerim je s foliarnim nanosom EPGE skorajda nemo- goče učinkovito ukrepati. Po drugi strani so EPGE, ki jih vnašamo v tla z zalivanjem ali neposredno inokulacijo podzemnih delov rastlin, podvržene tekmovanju z dru- gimi mikroorganizmi v rizosferi in izpostavljene poten- cialnim antagonistom. Prav tako ni nujno, da v primeru uspešne kolonizacije korenin pride kasneje tudi do siste- mične okužbe nadzemnega dela rastline (Parsa in sod., 2013). 3 ENTOMOPATOGENI ENDOFITI KOT BIOTIČNI AGENSI ZA VARSTVO PRED RASTLINSKIMI PATOGENI Za nekatere EPGE je bilo ugotovljeno, da poleg vpli- va na škodljive žuželke in pršice, rastline varujejo tudi pred patogenimi organizmi. V ospredju nedavno opra- vljenih raziskav je bilo zlasti preučevanje vplivov vrst B. bassiana in Lecanicillium spp. za katere so v različnih raziskavah dokazali antagonistično delovanje predvsem proti različnim povzročiteljem glivičnih bolezni (Ownley Acta agriculturae Slovenica, 117/4 – 20216 P. ŽIGON in F. A. CELAR En to m op at og en a gl iv a G os tit el jsk a ra st lin a Šk od lji ve c N ač in in ok ul ac ije Sa ro cla di um st ric tu m (W . G am s) S um m er b. pa ra di žn ik (S ol an um ly co pe rs icu m L .) ju žn a pl od ov rt ka (H eli co ve rp a ar m ig er a [H üb ne r, 18 08 ]) Z (J al lo w in so d. , 2 00 8) A sp er gi llu s fl av us L in k cv et ač a (B ra ss ica ol er ac ea v ar . b ot ry tis ) Sp od op te ra li tu ra F ab ric iu s, 17 75 F (K au r i n so d. , 2 01 5) A sp er gi llu s b ra sil ie ns is Va rg a, F ris va d & S am so n cv et ač a (B ra ss ica ol er ac ea L . v ar . b ot ry tis ) Sp od op te ra li tu ra F (K au r i n so d. , 2 01 5) Be au ve ria b as sia na bo b (V ici a fa ba L .) gr ah ov a uš (A cy rt ho sip ho n pi su m H ar ris , 1 77 6 ) ST (A ke llo in S ik or a, 2 01 2) čr na fi žo lo va u š ( Ap hi s f ab ae S co po li, 1 76 3) ST (A ke llo in S ik or a, 2 01 2) ju žn a pl od ov rt ka (H eli co ve rp a ar m ig er a) F (V id al in Ja be r, 20 15 ) ja go dn ja k (F ra ga ria x an an as sa D uc he sn e) siv a br es ko va u š ( M yz us p er sic ae [S ul ze r, 17 76 ]) Z , R I (D ar a in D ar a, 2 01 3) pš en ic a (T rit icu m ae sti vu m L .) st ru ne (L im on iu s c al ifo rn icu s [ M an ne rh ei m , 18 43 ], H yp on oi du s b ico lo r [ Es ch sc ho ltz ]) Z, S C (R ed dy , Z ha o, in so d. , 2 01 4) bo m ba ž ( G os sy pi um h irs ut um L .) H eli co ve rp a ze a Bo dd ie , 1 85 0 ST , F (L op ez in S w or d, 2 01 5) pa ra di žn ik (S ol an um ly co pe rs icu m ) ju žn a pl od ov rt ka (H eli co ve rp a ar m ig er a) F, I, RI (Q ay yu m in so d. , 2 01 5) pa ra di žn ik ov m ol j ( Tu ta a bs ol ut a [M ey ric k, 1 91 7] ) F (K lie be r i n Re in ek e, 20 16 ) to ba ko v šč itk ar (B em isi a ta ba ci [G en na di us , 1 88 9] ) F, I (E l-D ee b in so d. , 2 01 2) pa pr ik a (C ap sic um a nn um L .) siv a br es ko va u š ( M yz us p er sic ae ) Z (J ab er in A ra j, 20 18 ) cv et ač a (B ra ss ica ol er ac ea va r. bo tr yt is) ka pu so v m ol j ( Pl ut ell a xy lo ste lla [L in na eu s, 17 58 ]) F (G au ta m in so d. , 2 01 6) ka pu so va m uh a (D eli a ra di cu m [L in na eu s, 17 58 ]) Z (R az in ge r i n so d. , 2 01 4) ko ru za (Z ea m ay s L .) ko ru zn a ve šč a (O str in ia n ub ila lis ) F, I, RI (L ew is in B in g, 1 99 1) am er išk a ko ru zn a so vk a (S po do pt er a fr ug ip er da Sm ith & A bb ot , 1 79 7) F, SC (R am ire z- Ro dr ig ue z i n Sá nc he z- Pe ña , 2 01 6) m el on a (C uc um is m elo L .) to ba ko v šč itk ar (B em isi a ta ba ci ) F (G ar rid o- Ju ra do in so d. , 2 01 7) vi ns ka tr ta (V iti s v in ife ra L .) sm ok vi n vo ln at i k ap ar (P la no co cc us fi cu s [ Si gn or et , 1 87 5] ) F (R on do t i n Re in ek e, 20 18 ) ze le ni šk rž at ek (E m po as ca v iti s [ G öt he , 1 87 5] ) F (R on do t i n Re in ek e, 20 18 ) ol jn a og rš či ca (B ra ss ica na pu s L . v ar . n ap us ) ju žn a pl od ov rt ka (H eli co ve rp a ar m ig er a) F (V id al in Ja be r, 20 15 ) pr av i d at lje ve c ( Ph oe ni x da ct yl ife ra L .) pa lm ov ri lč ka r ( Rh yn ch op ho ru s f er ru gi ne us [A .G .O liv ie r, 17 91 ]) I (G óm ez -V id al in so d. , 2 00 6) Be uv er ia b ro ng ia rt ii fiž ol (P ha se ul us v ul ga ri s L .) / F (J ab er in E nk er li, 2 01 7) Pr eg le dn ic a 1: E nt om op at og en e en do fit ne g liv e, za k at er e je b ila v p re te kl ih ra zi sk av ah n a po dl ag i r az lič ni h na či no v in ok ul ac ije u go to vl je na sp os ob no st k ol on iz ac ije ra zl ič ni h vr st g os tit el jsk ih ra st lin in u či nk ov ito st p ro ti nj ih ov im šk od lji vc em Acta agriculturae Slovenica, 117/4 – 2021 7 Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline N ad al je va nj e Cl on os ta ch ys ro se a če bu la (A lli um ce pa L .) to ba ko v re sa r ( Th rip s t ab ac i [ Li nd em an , 1 88 9] ) ST , R I (M uv ea in so d. , 2 01 4) Fu sa riu m o xy sp or um fiž ol (P ha se ul us v ul ga ri s) Li rio m yz a hu id ob re ns is (B la nc ha rd , 1 92 6) ST (A ku ts e in so d. , 2 01 3) Fu sa riu m fu jik ur oi bo b (V ici a fa ba ) Li rio m yz a hu id ob re ns is ST (A ku ts e in so d. , 2 01 3) Tr ich od er m a lix ii bo b (V ici a fa ba ) Li rio m yz a hu id ob re ns is ST (A ku ts e in so d. , 2 01 3) Co rd yc ep s f um os or os ea pa pr ik a (C ap sic um a nn um ) siv a br es ko va u š ( M yz us p er sic ae ) Z (M an tz ou ka s i n La go gi an ni s, 20 19 ) Ak an th om yc es le ca ni i na va dn a bu ča (C ur cu rb ita m ax im a L. ) bo m ba že vč ev a uš (A ph is go ss yp ii [G lo ve r, 18 77 ]) F (G ur ul in ga pp a in so d. , 2 01 1) Le ca ni cil liu m lo ng isp or um (P et ch ) Z ar e & W . G am s ku m ar a (C uc um is sa tiv us L .) bo m ba že vč ev a uš (A ph is go ss yp ii) F (K im in so d. , 2 00 8) M et ar hi zi um a ni so pl ia e fiž ol (P ha se ul us v ul ga ri s) gr ah ov a uš (A cy rt ho sip ho n pi su m ) ST (A ke llo in S ik or a, 2 01 2) čr na fi žo lo va u š ( Ap hi s f ab ae ) ST (A ke llo in S ik or a, 2 01 2) O ph io m yi a ph as eo li (T ry on , 1 89 5) ST (M ut un e in so d. , 2 01 6) cv et ač a (B ra ss ica ol er ac ea v ar . b ot ry tis ) ka pu so va m uh a (D eli a ra di cu m ) Z (R az in ge r i n so d. , 2 01 4) ol jn a og rš či ca (B ra ss ica na pu s L . v ar . n ap us ) ka pu so v m ol j ( Pl ut ell a xy lo ste lla ) F (B at ta , 2 01 3) M et ar hi zi um b ru nn eu m P et ch ko ru za (Z ea m ay s) st ru ne (A gr io te s o bs cu ru s) ST (K ab al uk in E ric ss on , 2 00 7) pa pr ik a (C ap sic um a nn um ) siv a br es ko va u š ( M yz us p er sic ae ) Z (J ab er in A ra j, 20 18 ) cv et ač a (B ra ss ica o ler ac ea va r. bo tr yt is) ka pu so va m uh a (D eli a ra di cu m ) Z (R az in ge r i n so d. , 2 01 4) m el on a (C uc um is m elo L .) to ba ko v šč itk ar (B em isi a ta ba ci ) F (G ar rid o- Ju ra do in so d. , 2 01 7) pš en ic a (T rit ic um a es tiv um ) st ru ne (L im on iu s c al ifo rn icu s, H yp on oi du s b ico lo r) Z, S C (R ed dy , T an gt ra ku lw an ic h, in so d. , 2 01 4) M et ar hi zi um ro be rt sii J. F. Bi sc h. , S. A . R eh ne r & H um be r pš en ic a (T rit ic um a es tiv um ) st ru ne (L im on iu s c al ifo rn icu s, H yp on oi du s b ico lo r) Z, S C (R ed dy , T an gt ra ku lw an ic h, in so d. , 2 01 4) sir ek (S or gh um b ico lo r L .) Se sa m ia n on ag rio id es L ef eb vr e, 18 27 F (M an tz ou ka s i n so d. , 2 01 5) ko ru za (Z ea m ay s) / SC Pu rp ur eo cil liu m li la cin um (Th om Lu an gs a- ar d, H ou br ak en , H yw el -Jo ne s & S am so n bo m ba ž ( G os sy pi um hi rs ut um ) H eli co ve rp a ze a ST (L op ez in S w or d, 2 01 5) Tr ich od er m a ha rz ia nu m R ifa i če bu la (A lli um ce pa ) to ba ko v re sa r ( Th rip s t ab ac i) ST , R I (M uv ea in so d. , 2 01 4) F = fo lia rn i n an os , Z = za liv an je , S C = o pl aš če nj e se m en a, S T = na m ak an je se m en a, I = in jic ira nj e, RI = p om ak an je k or en in , / = n i b ilo p os eb ej p re uč en o za za tir an je ci ljn eg a or ga ni zm a Acta agriculturae Slovenica, 117/4 – 20218 P. ŽIGON in F. A. CELAR in sod., 2010; Jaber in Ownley, 2018). Kako EPGE vpli- vajo na zmanjšanje okužb in poškodb zaradi glivičnih in drugih povzročiteljev bolezni (še) ni povsem znano, naj- verjetneje pa gre za sočasen odziv na več ravneh. Načini delovanja antagonističnih gliv temeljijo na več mehaniz- mih, ki posamično ali sinergistično delujejo na povzroči- telje rastlinskih bolezni preko tekmovanja (kompeticije) za življenjski prostor in hrano, mikoparazitizma, antibi- oze in vzpodbujanja inducirane sistemične odpornosti rastlin (Ownley in sod., 2010). Za EPGE Trichoderma spp. in Lecanicillium spp. je značilno, da nekatere patogene glive ob neposrednem stiku parazitirajo (mikoparazitizem) ter jih na ta način slabijo in preprečujejo njihovo rast. Na zmanjšanje okužb z glivnimi patogeni vpliva že sama uspešnost kolonizacije rastlinskih tkiv z EPGE, ki s patogenimi glivami tekmu- jejo za prostor in hranila (kompeticija). Torej, če je EPGE predčasno prisoten v rastlini, je za patogeno glivo znotraj rastlinskega tkiva na voljo manj prostora in hranil, zato je možnost okužbe in širjenja manjša. Poleg tega predho- dna kolonizacija v rastlinah sproži sintezo lignina in dru- gih komponent v celičnih stenah, ki krepijo mehansko odpornost in onemogočajo penetracijo glivnih hif (Jaber in Ownley, 2018). Antibioza temelji na tvorbi toksičnih hlapnih in nehlapnih organskih molekul in tvorbi enci- mov, ki so vključeni v razgradnjo celičnih struktur dru- gih mikroorganizmov. Poleg tega so EPGE tudi bogat vir sekundarnih metabolitov, ki delujejo protimikrobno in citotoksično proti povzročiteljem bolezni. B. bassiana na primer tvori beauvericin, ki ima široko učinkovanje pro- ti številnim mikroorganizmov. Tvorijo ga tudi nekatere druge EPG (Ownley in sod., 2010). Sistemična inducira- na odpornost označuje obrambni odziv rastlin na stresne razmere zaradi biotskih ali abiotskih dejavnikov, ki ga spodbudijo nepatogeni organizmi, tudi EPGE. Dokaza- no je, da predčasna kolonizacija rastline z EPGE poleg vpliva na zmanjšanje posledic napada škodljivcev, lahko vpliva tudi na zmanjšanje okužb in bolezenskih znamenj s strani povzročiteljev bolezni (Dara, 2019). Kolonizacija rastlin z EPGE namreč v rastlinah lahko poveča vsebnost salicilne kisline in s tem vpliva na izražanje genov za sin- tezo protimikrobnih encimov kot so hitinaze in glukozi- daze (Jaber in Ownley, 2018). Na ta način so obrambni mehanizmi rastline predčasno v stanju pripravljenosti, kar omogoča rastlinam hitrejši in odločnejši odziv proti povzročiteljem bolezni. Antagonistične učinke Beauveria spp. so ugotovili ob prisotnosti v različnih rastlinah proti različnim pov- zročiteljem glivičnih bolezni kot so Botrytis cinerea Pers., Fusarium oxysporum, Gaeumannomyces graminis (Sacc.) Arx & D.L. Olivier, Phytium sp., Rhizoctonia solani J.G. Kühn in Septoria sp. (Renwick in sod., 1991; Bark in sod., 1996; Sang Myeong in sod., 1999). Pri paradižniku in bombažu so ob tretiranju semen z B. bassiana prepreči- li okužbe kalečih rastlin z talnima patogenima glivama Pythium myriotylum Drechsler in Rhizoctonia solani. Uporaba istega seva B. bassiana je pri bombažu vplivala tudi na zmanjšanje virulentnosti bakterije Xanthomonas axonopodis pv. malvacearum (Smith 1901) Vauterin et al., 1995 (Ownley in sod., 2008). Podobno je B. bassia- na omogočila varstvo čebulic čebule pred okužbami s talno glivo Fussarium oxysporum, povzročiteljico fuza- rijske gnilobe (Flori in Roberti, 1993). Foliarni nanos suspenzije konidijev komercialnega seva ATCC 74040 glive B. bassiana je imel značilen vpliv na zmanjšanje pojavnosti simptomov okužb z virusom rumenega mo- zaika na bučkah (ZYMV) in peronospore na vinski trti (Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni) (Jaber in Salem, 2014; Jaber, 2015). V eni izmed študij so preučevali tudi vpliv glive Lecanicillium leccanii na zmanjšanje okužb z mokro gnilobo Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish. in pepelovko Podosphaera fuliginea (Schltdl.) U. Braun & S. Takam. na bučah (Benhamou in Brodeur, 2001). 4 ENTOPOMATOGENI ENDOFITI KOT SPODBUJEVALCI RASTI Vse večje število raziskav potrjuje, da lahko EPGE pomembno vplivajo na hitrejšo rast in razvoj rastlin. Okužba z EPGE namreč lahko stimulira rast koreninske- ga sistema, s čimer se izboljša privzem hranil in vode v rastline. Kolonizacija glavnatega zelja z B. bassiana je v lončnem poskusu vplivala na boljšo rast rastlin, ki se je kazala v večji biomasi rastlin in večjim pridelkom, zaradi boljšega privzema hranil (Dara in sod., 2017). Inokulacija krompirja z glivo M. brunneum je povzročila povečanje vsebnosti dušika in fosforja v rastlinah, kar se je odrazilo v povečanju biomase, površine listov in pridelka (Krell in sod., 2018). Aplikacija gliv B. bassiana, M. brunneum in C. fumosorosea z zalivanjem je vplivala na boljšo rast ze- lenih delov rastline ter značilno povečanje pridelka jagod (Dara, 2016). Z izboljšanjem mineralne prehrane EPGE vplivajo na boljšo vitalnost rastlin ter s tem pripomorejo k blaženju ali preprečevanju posledic abiotskih in biot- skih stresnih dejavnikov, med drugim tudi povzročiteljev bolezni. Tako je kolonizacija bučk z glivo B. bassiana za- radi vpliva na izboljšanje rasti zmanjšala posledice okužb z virusom rumenega mozaika bučke (ZYMV) (Jaber in Salem, 2014). Podobno je na zmanjšanje simptomov ko- reninske bolezni, ki jo povzroča gliva Fusarium phaseoli (Burkh.) T. Aoki & O‘Donnell, vplivala tudi inokulacija fižola z glivo Metarhizium robertsii (Sasan in Bidochka, 2013). Posledica kolonizacije rastlin z EPGE je poveča- nje tvorbe beljakovin vključenih v fotosintetske reakcije Acta agriculturae Slovenica, 117/4 – 2021 9 Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline in presnovo (Raad in sod., 2019). Okrepljena rast rastlin je lahko tudi posledica povečanja tvorbe fitohormonov v rastlinah in sideroforov (molekul bogatih z Fe3+ ioni), ki jih tvorijo EPGE in tako vplivajo na boljšo oskrbo rastlin z železom (Rana in sod., 2019). Zanimivi so izsledki razi- skav, ki kažejo, da B. bassiana in nekatere vrste Metarhizi- um spp. lahko oskrbujejo rastline z dušikom iz kadavrov parazitiranih žuželk in pršic (Behie in sod., 2012; Behie in Bidochka, 2014). Po okužbi in poginu gostiteljskih žu- želk EPGE vzpostavijo endofitno interakcijo, na podlagi katere pride do privzema dušika v rastline. Na ta način lahko EPGE izdatno vplivajo na oskrbo rastlin s tem hra- nilom in kroženje dušika v ekosistemu. Domnevajo, da gre pri tej interakciji med EPGE in rastlinami za mutu- alističen odnos pri katerem rastline v zameno za dušik EPGE oskrbujejo z ogljikom. Vpliv EPGE na povečanje rasti je odvisen tudi od načina inokulacije. Vpliv B. bassi- ana, B. brongniartii in M. brunneum na boljšo rast rastlin je bil ugotovljen le pri inokulaciji semen, medtem ko pri foliarni aplikaciji EPGE ta odziv ni bil dosežen (Jaber in Enkerli, 2017). 5 ZAKLJUČEK Uporaba EPG za zatiranje škodljivcev je bila preu- čena v številnih raziskavah. Med najpogosteje uporablje- nimi EPG v biotičnem varstvu rastlin so vrste Beauveria bassiana, Metarhizium spp. in nekatere druge, ki so v viš- jih rastlinah prisotne tudi kot endofiti. Endofitizem EPG omogoča kolonizacijo rastlinskih tkiv in s tem sistemično varstvo rastlin pred škodljivci skozi daljše časovno obdo- bje. Poleg tega, da vplivajo na zmanjšanje poškodb zaradi napada škodljivcev, lahko EPGE v gostiteljskih rastlinah omogočijo tudi boljšo oskrbo le teh s hranili in s tem nji- hovo boljšo rast, lažjo premostitev stresnih razmer zaradi abiotskih dejavnikov ter zmanjšanje okužb s patogenimi organizmi. Med drugim se okužba z EPGE v rastlinah lahko odraža tudi v povečani tvorbi sekundarnih meta- bolitov, ki delujejo toksično na druge organizme, tudi na ljudi. Pri preučevanju načinov inokulacije in posrednih vplivov na gostiteljske rastline ostaja še veliko neznank, prav tako je za širšo uporabo EPGE ključno razumevanje abiotskih in biotskih dejavnikov, ki vplivajo na uspešnost kolonizacije rastlinskega tkiva. Pomemben kriterij pri ra- zvoju in uporabi komercialnih biopesticidov na podlagi EPGE je nepredvidljiva učinkovitost in nekonsistentnost, ki je posledica vpliva številnih dejavnikov na vzpostavi- tev interakcije z gostiteljem. Do sedaj je bila večina razi- skav v zvezi s preučevanjem EPGE kot biotičnih agensov opravljena v laboratorijskih razmerah, zato je za razvoj celovite strategije zatiranja škodljivcev potrebno več po- zornosti nameniti tudi preučevanju ustreznih okoljskih razmer in ustreznih metod inokulacije, ki omogočajo razvoj endofitizma in dolgotrajnejše pozitivne učinke na nivoju celotne rastline. 6 LITERATURA Akello, J., Sikora, R. (2012). Systemic acropedal influence of en- dophyte seed treatment on Acyrthosiphon pisum and Aphis fabae offspring development and reproductive fitness. Bio- logical Control, 61(3), 215–221. https://doi.org/10.1016/j. biocontrol.2012.02.007 Akutse, K. S., Maniania, N. K., Fiaboe, K. K. M., Van den Berg, J., Ekesi, S. (2013). Endophytic colonization of Vicia faba and Phaseolus vulgaris (Fabaceae) by fungal pathogens and their effects on the life-history parameters of Liriomyza huidobrensis (Diptera: Agromyzidae). Fungal Ecology, 6(4), 293–301. https://doi.org/10.1016/j.funeco.2013.01.003 Arnold, A. E. (2007). Understanding the diversity of foliar en- dophytic fungi: progress, challenges, and frontiers. In Fun- gal Biology Reviews (Vol. 21, Issues 2–3, pp. 51–66). Else- vier. https://doi.org/10.1016/j.fbr.2007.05.003 Bacon, C. W. (1993). Abiotic stress tolerances (moisture, nutri- ents) and photosynthesis in endophyte-infected tall fescue. Agriculture, Ecosystems and Environment, 44(1–4), 123– 141. https://doi.org/10.1016/0167-8809(93)90042-N Bamisile, Bamisope S., Dash, C. K., Akutse, K. S., Keppanan, R., Wang, L. (2018). Fungal endophytes: Beyond herbivore management. In Frontiers in Microbiology (Vol. 9, Issue MAR, p. 544). Frontiers Media S.A. https://doi.org/10.3389/ fmicb.2018.00544 Bamisile, Bamisope Steve, Dash, C. K., Akutse, K. S., Keppanan, R., Afolabi, O. G., Hussain, M., Qasim, M., Wang, L. (2018). Prospects of endophytic fungal entomopathogens as bio- control and plant growth promoting agents: An insight on how artificial inoculation methods affect endophytic colonization of host plants. Microbiological Research, 217, 34–50. https://doi.org/10.1016/j.micres.2018.08.016 Bark, Y. G., Lee, D. G., Kim, Y. H., Kang, S. C. (1996). Antibi- otic properties of an entomopathogenic fungus, Beauveria bassiana, on Fusarium oxysporum and Botrytis cinerea. The Plant Pathology Journal, 12(2), 245–250. Bary, A. de (Anton). (1866). Morphologie und Physiologie der Pilze, Flechten und Myxomyceten . W. Engelmann,. https:// www.biodiversitylibrary.org/item/211464. https://doi. org/10.5962/bhl.title.120970 Batta, Y. A. (2013). Efficacy of endophytic and applied Metarhi- zium anisopliae (Metch.) Sorokin (Ascomycota: Hypocre- ales) against larvae of Plutella xylostella L. (Yponomeuti- dae: Lepidoptera) infesting Brassica napus plants. Crop Protection, 44, 128–134. https://doi.org/10.1016/j.cro- pro.2012.11.001 Behie, S. W., Zelisko, P. M., Bidochka, M. J. (2012). Endophytic insect-parasitic fungi translocate nitrogen directly from in- sects to plants. Science, 336(6088), 1576–1577. https://doi. org/10.1126/science.1222289 Behie, Scott W., Bidochka, M. J. (2014). Ubiquity of insect- derived nitrogen transfer to plants by endophytic insect- Acta agriculturae Slovenica, 117/4 – 202110 P. ŽIGON in F. A. CELAR pathogenic fungi: An additional branch of the soil nitro- gen cycle. Applied and Environmental Microbiology, 80(5), 1553–1560. https://doi.org/10.1128/AEM.03338-13 Behie, Scott W, Padilla-Guerrero, I. E., Bidochka, M. J. (2013). Nutrient transfer to plants by phylogenetically diverse fungi suggests convergent evolutionary strategies in rhizos- pheric symbionts. Communicative Integrative Biology, 6(1), e22321–e22321. https://doi.org/10.4161/cib.22321 Benhamou, N., Brodeur, J. (2001). Pre-inoculation of Ri T- DNA transformed cucumber roots with the mycoparasite, Verticillium lecanii, induces host defense reactions against Pythium ultimum infection. Physiological and Molecular Plant Pathology, 58(3), 133–146. https://doi.org/10.1006/ pmpp.2001.0322 Carroll, G. (1988). Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology, 69(1), 2–9. https://doi.org/10.2307/1943154 Dara, S. K. (2016). Impact of entomopathogenic fungi and beneficial microbes on strawberry growth , health , and yield. 1–4. https://ucanr.edu/blogs/blogcore/postdetail. cfm?postnum=22709 Dara, S. K. (2019). Non-entomopathogenic roles of en- tomopathogenic fungi in promoting plant health and growth. In Insects (Vol. 10, Issue 9). MDPI AG. https://doi. org/10.3390/insects10090277 Dara, S. K., Dara, S. S. (2013). Endophytic colonization and pest management potential of Beauveria bassiana in strawber- ries. Journal of Berry Research, 3(4), 203–211. https://doi. org/10.3233/JBR-130058 Dara, S. K., Dara, S. S. R., Dara, S. S. (2017). Impact of en- tomopathogenic fungi on the growth, development, and health of cabbage growing under water stress. Ameri- can Journal of Plant Sciences, 8, 1224–1233. https://doi. org/10.4236/ajps.2017.86081 El-Deeb, H. M., Lashin, S. M., Arab, Y. A. S. (2012). Reaction of some tomato cultivars to tomato leaf curl virus and evalu- ation of the endophytic colonisation with Beauveria bassi- ana on the disease incidence and its vector, Bemisia tabaci. Archives of Phytopathology and Plant Protection, 45(13), 1538–1545. https://doi.org/10.1080/03235408.2012.681246 Faria, M. R. d., Wraight, S. P. (2007). Mycoinsecticides and My- coacaricides: A comprehensive list with worldwide cover- age and international classification of formulation types. Bi- ological Control, 43(3), 237–256. https://doi.org/10.1016/j. biocontrol.2007.08.001 Field, K. J., Pressel, S., Duckett, J. G., Rimington, W. R., Bidar- tondo, M. I. (2015a). Symbiotic options for the conquest of land. Trends in Ecology Evolution, 30(8), 477–486. https:// doi.org/10.1016/J.TREE.2015.05.007 Field, K. J., Rimington, W. R., Bidartondo, M. I., Allinson, K. E., Beerling, D. J., Cameron, D. D., Duckett, J. G., Leake, J. R., Pressel, S. (2015b). Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromyco- tina fungi under a simulated Palaeozoic CO2 decline. The ISME Journal, 10(6), 1514–1526. https://doi.org/10.1038/ ismej.2015.204 Flori, P., Roberti, R. (1993). Treatment of onion bulbs with antagonistic fungi for the control of Fusarium oxysporum f.sp. cepae. Difesa Delle Piante, 16(4), 5–12. https://eurekamag.com/research/002/724/002724654.php Freeman, E. M., Ward, H. M. (1904). The seed-fungus of Lo- lium temulentum L., the darnel. Philosophical Transactions of the Royal Society of London. Series B, Containing Papers of a Biological Character, 196(214–224), 1–27. https://doi. org/10.1098/rstb.1904.0001 Garrido-Jurado, I., Resquín-Romero, G., Amarilla, S. P., Ríos- Moreno, A., Carrasco, L., Quesada-Moraga, E. (2017). Transient endophytic colonization of melon plants by entomopathogenic fungi after foliar application for the control of Bemisia tabaci Gennadius (Hemiptera: Aleyro- didae). Journal of Pest Science, 90(1), 319–330. https://doi. org/10.1007/s10340-016-0767-2 Gautam, S., Mohankumar, S., Kennedy, J. S. (2016). Induced host plant resistance in cauliflower by Beauveria bassiana. Journal of Entomology and Zoology Studies, 4(2), 476–482. Gómez-Vidal, S., Lopez-Llorca, L. V., Jansson, H. B., Salinas, J. (2006). Endophytic colonization of date palm (Phoenix dac- tylifera L.) leaves by entomopathogenic fungi. Micron, 37(7), 624–632. https://doi.org/10.1016/j.micron.2006.02.003 Gurulingappa, P., McGee, P. A., Sword, G. (2011). Endophytic Lecanicillium lecanii and Beauveria bassiana reduce the sur- vival and fecundity of Aphis gossypii following contact with conidia and secondary metabolites. Crop Protection, 30(3), 349–353. https://doi.org/10.1016/j.cropro.2010.11.017 Hackman, R. H. (1953). Chemistry of insect cuticle. I. The wa- ter-soluble proteins. The Biochemical Journal, 54(3), 362– 367. https://doi.org/10.1042/bj0540362 Hajek, A. (1997). Ecology of terrestrial fungal entomopatho- gens. Advances in Microbial Ecology, 15, 193–249. https:// doi.org/10.1007/978-1-4757-9074-0_5 Hardoim, P. R., van Overbeek, L. S., Berg, G., Pirttilä, A. M., Compant, S., Campisano, A., Döring, M., Sessitsch, A. (2015). The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews : MMBR, 79(3), 293–320. https://doi.org/10.1128/ MMBR.00050-14 Humber, R. A. (2008). Evolution of entomopathogenicity in fungi. Journal of Invertebrate Pathology, 98(3), 262–266. https://doi.org/10.1016/j.jip.2008.02.017 Jaber, L. R. (2015). Grapevine leaf tissue colonization by the fungal entomopathogen Beauveria bassiana s.l. and its ef- fect against downy mildew. BioControl, 60(1), 103–112. https://doi.org/10.1007/s10526-014-9618-3 Jaber, L. R., Araj, S. E. (2018). Interactions among endophyt- ic fungal entomopathogens (Ascomycota: Hypocreales), the green peach aphid Myzus persicae Sulzer (Homop- tera: Aphididae), and the aphid endoparasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae). Biologi- cal Control, 116, 53–61. https://doi.org/10.1016/j.biocon- trol.2017.04.005 Jaber, L. R., Enkerli, J. (2017). Fungal entomopathogens as en- dophytes: can they promote plant growth? Biocontrol Sci- ence and Technology, 27(1), 28–41. https://doi.org/10.1080/ 09583157.2016.1243227 Jaber, L. R., Ownley, B. H. (2018). Can we use entomopathogen- ic fungi as endophytes for dual biological control of insect pests and plant pathogens? In Biological Control (Vol. 116, pp. 36–45). Academic Press Inc. https://doi.org/10.1016/j. Acta agriculturae Slovenica, 117/4 – 2021 11 Endofitne glive v biotičnem varstvu rastlin pred škodljivimi organizmi in njihov posreden vpliv na rastline biocontrol.2017.01.018 Jaber, L. R., Salem, N. M. (2014). Endophytic colonisation of squash by the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) for managing Zucchini yellow mosaic virus in cucurbits. Biocontrol Science and Technol- ogy, 24(10), 1096–1109. https://doi.org/10.1080/09583157. 2014.923379 Jallow, M. F. A., Dugassa-Gobena, D., Vidal, S. (2008). Influence of an endophytic fungus on host plant selection by a poly- phagous moth via volatile spectrum changes. Arthropod- Plant Interactions, 2(1), 53–62. https://doi.org/10.1007/ s11829-008-9033-8 Kabaluk, J. T., Ericsson, J. D. (2007). Metarhizium anisopliae seed treatment increases yield of field corn when applied for wireworm control. Agronomy Journal, 99(5), 1377– 1381. https://doi.org/10.2134/agronj2007.0017N Kaur, T., Singh, B., Kaur, A., Kaur, S. (2015). Endophyte-me- diated interactions between cauliflower, the herbivore Spodoptera litura, and the ectoparasitoid Bracon hebe- tor. Oecologia, 179(2), 487–494. https://doi.org/10.1007/ s00442-015-3358-7 Kim, J. J., Goettel, M. S., Gillespie, D. R. (2008). Evaluation of Lecanicillium longisporum, Vertalec® for simultaneous sup- pression of cotton aphid, Aphis gossypii, and cucumber powdery mildew, Sphaerotheca fuliginea, on potted cu- cumbers. Biological Control, 45(3), 404–409. https://doi. org/10.1016/j.biocontrol.2008.02.003 Klieber, J., Reineke, A. (2016). The entomopathogen Beauveria bassiana has epiphytic and endophytic activity against the tomato leaf miner Tuta absoluta. Journal of Applied Ento- mology, 140(8), 580–589. https://doi.org/10.1111/jen.12287 Kogel, K. H., Franken, P., Hückelhoven, R. (2006). Endophyte or parasite - what decides? In Current Opinion in Plant Biol- ogy (Vol. 9, Issue 4, pp. 358–363). Elsevier Current Trends. https://doi.org/10.1016/j.pbi.2006.05.001 Krell, V., Unger, S., Jakobs-Schoenwandt, D., Patel, A. V. (2018). Endophytic Metarhizium brunneum mitigates nutrient deficits in potato and improves plant productivity and vi- tality. Fungal Ecology, 34, 43–49. https://doi.org/10.1016/j. funeco.2018.04.002 Lacey, L. A., Grzywacz, D., Shapiro-Ilan, D. I., Frutos, R., Brownbridge, M., Goettel, M. S. (2015). Insect pathogens as biological control agents: Back to the future. Journal of Invertebrate Pathology, 132, 1–41. https://doi.org/10.1016/j. jip.2015.07.009 Lewis, L. C., Bing, L. A. (1991). Bacillus thuringiensis Berliner and Beauveria bassiana (Balsamo) Vuillimen for european corn borer control: Program for immediate and season- long suppression 1. The Canadian Entomologist, 123(2), 387–393. https://doi.org/10.4039/Ent123387-2 Lopez, D. C., Sword, G. A. (2015). The endophytic fungal en- tomopathogens Beauveria bassiana and Purpureocillium lilacinum enhance the growth of cultivated cotton (Gossyp- ium hirsutum) and negatively affect survival of the cotton bollworm (Helicoverpa zea). Biological Control, 89, 53–60. https://doi.org/10.1016/j.biocontrol.2015.03.010 Mantzoukas, S., Chondrogiannis, C., Grammatikopoulos, G. (2015). Effects of three endophytic entomopathogens on sweet sorghum and on the larvae of the stalk borer Sesa- mia nonagrioides. Entomologia Experimentalis et Applicata, 154(1), 78–87. https://doi.org/10.1111/eea.12262 Mantzoukas, S., Eliopoulos, P. A. (2020). Endophytic en- tomopathogenic fungi: A valuable biological control tool against plant pests. In Applied Sciences (Switzerland) (Vol. 10, Issue 1, p. 360). MDPI AG. https://doi.org/10.3390/ app10010360 Mantzoukas, S., Lagogiannis, I. (2019). Endophytic coloniza- tion of pepper (Capsicum annum) controls aphids (My- zus persicae Sulzer). Applied Sciences (Switzerland), 9(11), 2239. https://doi.org/10.3390/app9112239 Mutune, B., Ekesi, S., Niassy, S., Matiru, V., Bii, C., Maniania, N. K. (2016). Fungal endophytes as promising tools for the management of bean stem maggot Ophiomyia phaseoli on beans Phaseolus vulgaris. Journal of Pest Science, 89(4), 993–1001. https://doi.org/10.1007/s10340-015-0725-4 Muvea, A. M., Meyhöfer, R., Subramanian, S., Poehling, H. M., Ekesi, S., Maniania, N. K. (2014). Colonization of onions by endophytic fungi and their impacts on the biology of Thrips tabaci. PLoS ONE, 9(9), 108242. https://doi.org/10.1371/ journal.pone.0108242 Ownley, B. H., Griffin, M. R., Klingeman, W. E., Gwinn, K. D., Moulton, J. K., Pereira, R. M. (2008). Beauveria bassiana: Endophytic colonization and plant disease control. Jour- nal of Invertebrate Pathology, 98(3), 267–270. https://doi. org/10.1016/j.jip.2008.01.010 Ownley, B. H., Gwinn, K. D., Vega, F. E. (2010). Endophytic fungal entomopathogens with activity against plant patho- gens: Ecology and evolution. BioControl, 55(1), 113–128. https://doi.org/10.1007/s10526-009-9241-x Parsa, S., Ortiz, V., Vega, F. E. (2013). Establishing fungal en- tomopathogens as endophytes: towards endophytic biolog- ical control. Journal of Visualized Experiments : JoVE, 74, 50360. https://doi.org/10.3791/50360 Pedrini, N., Crespo, R., Juárez, M. P. (2007). Biochemistry of insect epicuticle degradation by entomopathogenic fungi. In Comparative Biochemistry and Physiology - C Toxicol- ogy and Pharmacology (Vol. 146, Issues 1-2 SPEC. ISS., pp. 124–137). Elsevier Inc. https://doi.org/10.1016/j. cbpc.2006.08.003 Petrini, O. (1991). Microbial ecology of leaves. Spring-Verlag, New York, 179–197. https://doi.org/10.1007/978-1-4612- 3168-4_9 Philippot, L., Raaijmakers, J. M., Lemanceau, P., van der Put- ten, W. H. (2013). Going back to the roots: the microbial ecology of the rhizosphere. Nature Reviews. Microbiology, 11(11), 789–799. https://doi.org/10.1038/nrmicro3109 Qayyum, M. A., Wakil, W., Arif, M. J., Sahi, S. T., Dunlap, C. A. (2015). Infection of Helicoverpa armigera by endophy- tic Beauveria bassiana colonizing tomato plants. Biological Control, 90, 200–207. https://doi.org/10.1016/j.biocon- trol.2015.04.005 Raad, M., Glare, T. R., Brochero, H. L., Müller, C., Rostás, M. (2019). Transcriptional reprogramming of Arabidopsis thaliana defence pathways by the entomopathogen Be- auveria bassiana correlates with resistance against a fungal pathogen but not against insects. Frontiers in Microbiology, 10(MAR), 615. https://doi.org/10.3389/fmicb.2019.00615 Ramirez-Rodriguez, D., Sánchez-Peña, S. R. (2016). Endo- Acta agriculturae Slovenica, 117/4 – 202112 P. ŽIGON in F. A. CELAR phytic Beauveria bassiana in Zea mays: Pathogenicity against larvae of fall armyworm, Spodoptera frugiperda. Southwestern Entomologist, 41(3), 875–878. https://doi. org/10.3958/059.041.0330 Rana, K. L., Kour, D., Sheikh, I., Dhiman, A., Yadav, N., Yadav, A. N., Rastegari, A. A., Singh, K., Saxena, A. K. (2019). Endophytic Fungi: Biodiversity, Ecological Significance, and Potential Industrial Applications (pp. 1–62). Springer In- ternational Publishing. https://doi.org/10.1007/978-3-030- 10480-1_1 Razinger, J., Lutz, M., Schroers, H. J., Palmisano, M., Wohler, C., Urek, G., Grunder, J. (2014). Direct plantlet inoculation with soil or insect-associated fungi may control cabbage root fly maggots. Journal of Invertebrate Pathology, 120, 59–66. https://doi.org/10.1016/j.jip.2014.05.006 Reddy, G. V. P., Tangtrakulwanich, K., Wu, S., Miller, J. H., Ophus, V. L., Prewett, J., Jaronski, S. T. (2014). Evaluation of the effectiveness of entomopathogens for the management of wireworms (Coleoptera: Elateridae) on spring wheat. Journal of Invertebrate Pathology, 120, 43–49. https://doi. org/10.1016/j.jip.2014.05.005 Reddy, G. V. P., Zhao, Z., Humber, R. A. (2014). Laboratory and field efficacy of entomopathogenic fungi for the manage- ment of the sweetpotato weevil, Cylas formicarius (Cole- optera: Brentidae). Journal of Invertebrate Pathology, 122, 10–15. https://doi.org/10.1016/j.jip.2014.07.009 Renwick., A., Campbell, R., Coe, S. (1991). Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathology, 40(4), 524– 532. https://doi.org/10.1111/j.1365-3059.1991.tb02415.x Rho, H., Hsieh, M., Kandel, S. L., Cantillo, J., Doty, S. L., Kim, S.-H. (2018). Do endophytes promote growth of host plants under stress? A meta-analysis on plant stress mitigation by endophytes. Microbial Ecology, 75(2), 407–418. https://doi. org/10.1007/s00248-017-1054-3 Robert-Seilaniantz, A., Grant, M., Jones, J. D. G. (2011). Hor- mone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annual Review of Phy- topathology, 49, 317–343. https://doi.org/10.1146/annu- rev-phyto-073009-114447 Rodriguez, R. J., White, J. F., Arnold, A. E., Redman, R. S. (2009). Fungal endophytes: Diversity and functional roles: Tansley review. In New Phytologist (Vol. 182, Issue 2, pp. 314–330). John Wiley Sons, Ltd. https://doi.org/10.1111/ j.1469-8137.2009.02773.x Rodriguez, R., Redman, R. (2008). More than 400 million ye- ars of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis. Journal of Experimental Botany, 59(5), 1109–1114. https://doi. org/10.1093/jxb/erm342 Rondot, Y., Reineke, A. (2018). Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.) reduces infestation with pier- cing-sucking insects. Biological Control, 116, 82–89. https://doi.org/10.1016/j.biocontrol.2016.10.006 Saikkonen, K., Wäli, P., Helander, M., Faeth, S. H. (2004). Evolu- tion of endophyteplant symbioses. Trends in Plant Science, 9(6), 275–280. https://doi.org/10.1016/j.tplants.2004.04.005 Sandhu, S. S., Sharma, A. K., Beniwal, V., Goel, G., Batra, P., Kumar, A., Jaglan, S., Sharma, A. K., Malhotra, S. (2012). Myco-biocontrol of insect pests: Factors involved, mecha- nism, and regulation. Journal of Pathogens, 2012, 1–10. htt- ps://doi.org/10.1155/2012/126819 Sang Myeong, L., Woon Hong, Y., Hyeong Jin, J., Sang Chul, S., Yil Seong, M. (1999). Effect of entomopathogenic fungi on growth of cucumber and Rhizoctonia solani. FRI Journal of Forest Science Seoul, 62, 118–125. https://eurekamag.com/ research/003/417/003417068.php Sasan, R. K., Bidochka, M. J. (2013). Antagonism of the endo- phytic insect pathogenic fungus Metarhizium robertsii aga- inst the bean plant pathogen Fusarium solani f. sp. phaseoli. Canadian Journal of Plant Pathology, 35(3), 288–293. htt- ps://doi.org/10.1080/07060661.2013.823114 Tefera, T., Vidal, S. (2009). Effect of inoculation method and plant growth medium on endophytic colonization of sor- ghum by the entomopathogenic fungus Beauveria bas- siana. BioControl, 54(5), 663–669. https://doi.org/10.1007/ s10526-009-9216-y Van Wees, S. C., Van der Ent, S., Pieterse, C. M. (2008). Plant immune responses triggered by beneficial microbes. In Current Opinion in Plant Biology (Vol. 11, Issue 4, pp. 443– 448). Elsevier Current Trends. https://doi.org/10.1016/j. pbi.2008.05.005 Vega, F. E. (2008). Insect pathology and fungal endophytes. Journal of Invertebrate Pathology, 98(3), 277–279. https:// doi.org/10.1016/j.jip.2008.01.008 Vega, F. E. (2018). The use of fungal entomopathogens as en- dophytes in biological control: a review. In Mycologia (Vol. 110, Issue 1, pp. 4–30). NLM (Medline). https://doi.org/10. 1080/00275514.2017.1418578 Vega, F. E., Goettel, M. S., Blackwell, M., Chandler, D., Jackson, M. A., Keller, S., Koike, M., Maniania, N. K., Monzón, A., Ownley, B. H., Pell, J. K., Rangel, D. E. N., Roy, H. E. (2009). Fungal entomopathogens: new insights on their ecology. In Fungal Ecology (Vol. 2, Issue 4, pp. 149–159). Elsevier. htt- ps://doi.org/10.1016/j.funeco.2009.05.001 Vidal, S., Jaber, L. R. (2015). Entomopathogenic fungi as en- dophytes: plantendophyteherbivore interactions and pro- spects for use in biological control. Current Science, 109(1), 46–54. http://www.jstor.org/stable/24905690 Wilson, D. (1995). Endophyte: The evolution of a term, and cla- rification of its use and definition. Oikos, 73(2), 274–276. https://doi.org/10.2307/3545919 Zamioudis, C., Pieterse, C. M. J. (2012). Modulation of host im- munity by beneficial microbes. Molecular Plant-Microbe In- teractions : MPMI, 25(2), 139–150. https://doi.org/10.1094/ MPMI-06-11-0179 Acta agriculturae Slovenica, 117/4, 1–4, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2066 Short communication / kratek prispevek Development of efficient integrated management package against sweet potato weevil (Cylas formicarius [Fabricius, 1798]) Mohammad Monirul Hasan TIPU 1, 2, Raunak JAHAN 3, Jubaidur RAHMAN 4, Md Mizanur RAHMAN 5, Md Ariful ISLAM 6, Md Mosiur Rahman Bhuyin APU 7 Received January 26, 2021; accepted September 10, 2021. Delo je prispelo 26. januarja 2021, sprejeto 10. septembra 2021 1 Plant Pathology Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh 2 Corresponding author, email: tipubari2013@yahoo.com 3 Department of Aquaculture, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh 4 Agronomy Division, Regional Agricultural Research Station, Bangladesh Agricultural Research Institute (BARI), Jamalpur, Bangladesh 5 Tuber Crops Research Centre, Regional Agricultural Research Station, Bangladesh Agricultural Research Institute (BARI), Jamalpur, Bangladesh 6 Oilseed Research Centre, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh 7 Horticulture Research Centre, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh Development of efficient integrated management package against sweet potato weevil (Cylas formicarius [Fabricius, 1798]) Abstract: The sweet potato weevil (Cylas formicarius, 1798) is one of the most damaging sweet potato pests. To pre- vent an economic crop loss, it is very important to develop a suitable and efficient integrated pest management strategy. A field experiment was set up with three replications at Ja- malpur to select the best integrated management package from eight different treatments against sweet potato weevil. The results showed that the lowest percentage of infestation by number (2.94  %) and mass (3.22  %) was found when the crop was planted on November 01; earthing-up for two times, Carbofuran 5G was sprayed @ 15 kg ha-1 at 60 days after planting with irrigation and tuber was harvested at 130 days after planting. The marketable yield (23.75 kg) and percent in- crease of yield than control (50.86  %) performed the highest in the same combination. These findings suggested an effec- tive integration of different management strategies to reduce sweet potato weevil infestation in Bangladesh successfully. Key words: sweet potato; sweet potato weevil; integrated management; marketable tuber yield Razvoj učinkovitega integriranega načina zatiranja hrošča Cylas formicarius (Fabricius, 1798) na sladkem krompirju Izvleček: Hrošč Cylas formicarius (Fabricius, 1798) je najpomembnejši škodljivec sladkega krompirja. Za prepreči- tev izpada pridelka je potrebno razviti ustrezen in učinkovit način integriranega zatiranja škodljivca. V ta namen je bil v Jamalpurju izveden poljski poskus s tremi ponovitvami za izbor najustreznejšega načina integriranega zatiranja ško- dljivca med osmimi obravnavanji. Rezultati so pokazali, da je bil najmanjši odstotek napada, tako v številčnosti škodljivca (2,94  %) kot v masi pridelka (3,22  %) ugotovljen v obravna- vanju, ko je bil sladki krompir posajen prvega novembra in dvakrat osipan, poškropljen s karbofuranom 5G 15 kg ha-1 60 dni po saditvi, z namakanjem in spravilom gomoljev 130 dni po saditvi. Tržni pridelek (23,75 kg) in odstotek povečanja pridelka v primerjavi s kontrolo (50,86  %) sta bila največja v istem obravnavanju. Te ugotovitve nakazujejo učinkovito vključevanje različnih načinov zatiranja za učinkovito zmanj- šanje napada sladkega krompirja od hrošča Cylas formicarius v Bangladešu. Ključne besede: sladki krompir; škodljivec sladkega krompirja; integrirano varstvo; tržni pridelek gomoljev 2 Acta agriculturae Slovenica, 117/4 – 2021 M. M. H. TIPU et al. 1 INTRODUCTION The sweet potato (Ipomoea batatas (L.) Lam.) has an important role in transforming the nutrition and food security for developing countries significantly in recent years (Korada et al., 2010). The scientific in- formation developed in the sweet potato research has enabled the growers to boost productivity and quality. There are some fundamental needs facing farmers in all major sweet potato producing countries, but there are other significant needs specific to certain regions. The sweet potato weevil (SPW) (Cylas formicarius [Fab- ricius, 1798]) has become widely dispersed, mainly in tropical and subtropical areas of the world (Hue and Low, 2015), and it recently has been found in higher latitude areas as well. It is the most severe pest of sweet potato in Bangladesh. It causes damage both in the field and in storage. The larvae mine the sweet potato tu- ber and damage the inside tissue. The tuber becomes spongy in appearance, riddled with cavities, and dark in color (Uritaini et al., 1975; Kyereko et at., 2019). The sweet potato larvae make a tunnel inside the root tis- sue, which is the primary cause of inviting several soil- borne pathogens. Once these pathogens enter the tuber, they become responsible for causing further damage like secondary infection by different pathogenic bacte- ria and fungi (Onwueme and Charles, 1994). Besides, the sweet potato weevil larvae have an ability to cause damage to the vascular system of the plant. As a result, the number and size of tuber roots those are stored for the future become drastically reduced (Hue and Low, 2015). Sweet potato weevil is causing about 50 to 100 % yield loss in the field (Sorensen, 2009). It is challenging to deal with sweet potato weevils when they are already in the crop. Cultural practices have proven to be effective control against the sweet potato weevil, but insecticide applications remain the primary basis of control (Muruvanda et al., 1986; Sutherland, 1986). Management of this pest through the shifting of the planting dates could be one of the best ways. The weevil population reaches a peak at the beginning of the dry season because of the high tem- perature and rainfall (Ladanyi and Hufnagel, 2006; Gomi et al., 2007). So, if it may be possible to harvest two weeks earlier, it may reduce the yield loss. Another way of reducing the sweet potato weevil infestation is to hail-up of soil by re-ridging around the plant-base to fill soil cracks (Beyene, 2015). Pheromone traps are usually used as monitoring, training, and management tools. Many effective traps have been designed by farm- ers using locally available materials. Different traps are so delicate that they fail to catch weevils make mislead- ing information that the pest is not present (Beyene, 2015). Many insecticides control sweet potato weevil as a foliar spray or basal granular applications. The only chemical method cannot solve the weevil infestation, but good husbandry can control them by prevent- ing spreading. So, management of this pest by using a suitable integrated management strategy is important to save the environment. Yet, no effective integrated management practice against sweet potato weevil has so far been developed or recommended. Therefore, we designed the present study to select the best integrated management package against sweet potato weevil for higher yield. 2 MATERIALS AND METHODS The study was conducted during the winter sea- son of 2015 at Regional Agricultural Research Station, BARI, Jamalpur, as it was reported to be the hot spot area. Jamalpur is located between 24°55′10″ North and 89°56′53″ East, and the soil is neutral in pH and silty loam in texture. The experiment comprising eight treat- ments were replicated thrice following RCBD. Eight treatments namely, T1 (Earthing-up one time + Plant- ing 01 Nov. + Pheromone trap + harvest 130 DAP), T2 (Earthing-up one time + Planting 15 Nov. + Pheromone trap + harvest 120 DAP), T3 (Earthing-up two times + Planting 01 Nov. + Carbofuran 5G @ 15kg ha-1 at 60 days after sowing with irrigation + harvest 130 DAP), T4 (Earthing-up two times + Planting 15 Nov. + Carbo- furan 5G @ 15kg ha-1 at 60 days after sowing with irri- gation + harvest 120 DAP), T5 (Earthing-up three times + Planting 01 Nov. + harvest 130 DAP), T6 (Earthing- up three times + Planting 15 Nov. + harvest 120 DAP), T7 (Farmer’s practice) and T8 (Control) were evaluated. BARI SP-8 sweet potato variety was used for this ex- periment. The spacing between plants was 30 cm and rows 60 cm. The plot size for each treatment was 3 m x 3 m. All plantings were from vine cuttings, and stand- ard horticultural procedures were followed. The roots in each plot were counted and weighed, and evaluated for severity of weevil damage. A sampling of adult wee- vils using a sweeping-net was carried out six times at 30 days intervals starting from 30 days after planting. The stem and roots were taken as samples from different plants of respective plots and then they were dissected to count the number of adult weevils, pupae, and larvae. The data on the extent of damage on root tubers and stem (vines) was recorded according to the rating scale described by Rangi et al. (1994). The data on the infes- tation percentage on the stem (vine) and tuber were calculated. Data were statistically analyzed in the MStat program, and means were separated by DMRT. 3Acta agriculturae Slovenica, 117/4 – 2021 Development of efficient integrated management package against sweet potato weevil (Cylas formicarius [Fabricius, 1798]) 3 RESULTS AND DISCUSSION The different integrated treatments were tested for evaluating the efficacy in controlling sweet potato weevil. The effects of all treatment combination on root infestation by sweet potato weevil were presented in Table 1. Significant variation in controlling sweet po- tato weevil was observed in the combination of various management packages. In the case of percent infestation of the root by number, the lowest percentage of infestation was found in T3 (2.94%), which was statistically identical with T6 (3.56  %) and followed by T1 (5.53  %), T2 (4.38  %), T4 (6.71 %) and T5 (4.42 %), respectively. Correspondingly, the lowest percentage of infestation of the root by mass was also found in T3 (3.22  %), which was followed by T1 (8.20 %), T2 (7.52 %), T4 (9.81 %), T5 (5.78 %), and T6 (5.32  %), respectively. Among all the treatments, marketable yield per plot was ensured significantly the highest in T3 (23.75 kg), which was followed by T2 (18.36 kg), T4 (20.26 kg), T5 (20.33 kg), and T6 (21.51 kg), respectively. The lowest yield per plot was found in T8 (11.67 kg), which was statistically identical with T7 (13.17 kg) and followed by T1 (15.04 kg) and T2 (18.36 kg), respectively. Among all the treatments, the percent increase of yield over control was found the highest in T3 (50.86 %) and the lowest in T7 (11.39 %). Our present study suggested that early planning on November 01, earthing up for two times, applying Carbofuran 5G @ 15kg ha-1 at 60 DAP with irrigation and harvesting after 130 days of planting worked the best to manage sweet potato weevil successfully. Bohinc et al. (2019) found combination of calcium cyanamide (1000 kg ha-1), propolis (5 ml 1-1 H2O) and limestone dust (345 kg ha-1) was effective against different potato pests in summer. Hue and Low (2015) described earth- ing up as an excellent approach that prevented the entry of weevils into tuber and oviposition by female weevils. Palaniswami and Mohandas (1994) also observed that the weevil infestation was significantly reduced by this method. Timely harvesting also reduces weevil infes- tation at a significant level. Ebregt et al. (2005) found that harvesting 14 days earlier decrease the yield loss of sweet potato by weevil attack. The findings of the pre- sent study were strongly supported by Taye and Tadesse (2013), where they reported that carbofuran could ef- ficiently manage sweet potato weevil infestation when this chemical was used with other pesticides. Table 1: Effect of different integrated treatments against sweet potato weevil infestation at Jamalpur In a column, treatment means having a common letter(s) are statistically identical by LSD at 5 % level of significance. Figure in the parenthesis indicates square root transformation Treatments Infestation by number (%) Infestation by mass (%) Marketable yield/plot (kg) Increase/decrease yield over control (%) T1 (Earthing-up (One time) + Planting 01 Nov. + Pheromone trap + harvest 130 DAP) 5.53 bc (2.32) 8.20 bc (2.78) 15.04 bc 22.41 T2 (Earthing-up (One time) + Planting 15 Nov. + Pheromone trap + harvest 120 DAP) 4.38 bc (2.09) 7.52 bc (2.71) 18.36 abc 36.44 T3 (Earthing-up (Two times) + Planting 01 Nov. + Carbofuran 5G @ 15kg/ha at 60 DAP with irrigation + harvest 130 DAP) 2.94 c (1.65) 3.22 c (1.75) 23.75 a 50.86 T4 (Earthing-up (Two times) + Planting 15 Nov. + Carbofuran 5G @ 15kg/ha at 60 DAP with irrigation + harvest 120 DAP) 6.71 bc (2.46) 9.81 bc (3.01) 20.26 ab 42.40 T5 (Earthing-up (Three times) + Planting 01 Nov. + harvest 130 DAP) 4.42 bc (2.09) 5.78 bc (2.40) 20.33 ab 42.60 T6 (Earthing-up (Three times) + Planting 15 Nov. + harvest 120 DAP) 3.56 c (1.80) 5.32 bc (2.20) 21.51 ab 45.75 T7 (Farmer’s practice) 9.13 b (2.98) 12.39 b (3.47) 13.17 c 11.39 T8 (Control) 15.12 a (3.88) 19.82 a (4.42) 11.67 c 4 Acta agriculturae Slovenica, 117/4 – 2021 M. M. H. TIPU et al. 4 CONCLUSION Understanding the insights of sweet potato wee- vil and their infestation is crucial so that a precise pre- ventive method could be designed. Integrating several cultural practices and chemicals like early planting, earthing up at the proper time, timely harvesting, and appropriate chemical insecticide can manage sweet po- tato weevil infestation in the crop field. The combina- tion of various IPM strategies that we explained in the study could be an efficient package to prevent the wee- vil infestation for achieving the nation’s fundamental demand of ensuring food and nutrition security. 5 ACKNOWLEDGMENTS This experiment was funded by Bangladesh Agri- cultural Research Institute, Gazipur, Bangladesh from the GoB fund. 6 REFERENCES Beyene, K. (2015). Destitution, biology, yield loss and manage- ment of sweet potato weevils (Cylas formicaries (Fabrcius) Insecta: Coleoptera) in Ethiopia. Journal of Biology, Agri- culture and Healthcare, 5(22), 65-72. Bohinc, T., Vučajnk, F., & Trdan, S. (2019). The efficacy of en- vironmentally acceptable products for the control of major potato pests and diseases. Zemdirbyste, 106(2), 135-142. https://doi.org/10.13080/z-a.2019.106.018 Ebregt, E., Struik, P.C., Odongo, B., & Abidin, P.E. (2005). Pest damage in sweet potato, groundnut and maize in north-eastern Uganda with special reference to damage by millipedes (Diplopoda). NJAS - Wageningen Journal of Life Sciences, 53(1), 49-69. https://doi.org/10.1016/S1573- 5214(05)80010-7 Gomi, T., Nagasaka, M., Fukuda, T., & Higahara, H. (2007). Shifting of the life cycle and life-history traits of the fall webworm in relation to climate change. Entomologia Ex- perimentalis et Applicata, 125(2), 179-184. https://doi. org/10.1111/j.1570-7458.2007.00616.x pHue, S.M., & Low, M.Y. (2015). An insight into sweet potato weevils management: A review. Psyche, Article ID 849560. https://doi.org/10.1155/2015/849560 Korada, R.R., Naskar, S.K., Palaniswami, M.S., & Ray, R.C. (2010). Management of sweet potato weevil [Cylas formi- carius (Fab.)]: An overview. Journal Root Crops, 36, 14-26. Kyereko, W.T., Hongbo, Z., Amoanimaa-Dede, H., Meiwei, G., & Yeboah, A. (2019). The major sweet potato weevils; management and control: A review. Entomology, Ornithol- ogy & Herpetology: Current Research, 8, 218. https://doi. org/10.35248/2171-0983.8.218 Ladanyi, M., & Hufnagel, L. (2006). The effect of climate change on the population of sycamore lace bug (Corythuca ciliata, Say, Tingidae Heteroptera) based on a simulation model with phenological response. Applied Ecology and Environ- mental Research, 4(2), 85-112. https://doi.org/10.15666/ aeer/0402_085112 Muruvanda, D.A., Beardsley, J.W., & Mitchell, W.C. (1986). In- secticidal control of sweet potato weevil in Hawaii. Tropi- cal Agriculture, 63(2), 155-157. Onwueme, I.C., & Charles, W.B. (1994). “Tropical root and tu- ber crops: production, perspectives and future prospects,” FAO Plant Production and Protection Paper. 126, 129, Food and Agriculture Organization of the United Nations, Rome, Italy. Palaniswami, M.S., & Mohandas, N. (1994). Reridging as a cultural method for the management of sweet potato weevil Cylas formicarius F. Journal Root Crops, 20, 101-105. Rangi, D.K., Allard, G.B., Smit, N.J.M., & Gethi, M. (1994). Sampling methods for sweet potato in Africa. In G. B. Al- lord, L. J. Skoglund, P. Neuenschwander and R. J. Murphy (ed.), Root and tuber pest management in East and Southern Africa. Proceeding of regional workshop held in Mombassa (pp. 108-109). Kenya. Sorensen, K. (2009). Sweetpotato Insects: Identification, Biol- ogy and Management. In G. Loebenstein & G. Thottappilly (eds), The Sweetpotato (pp. 161-188). Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-9475-0_10 Sutherland, J.A. (1986). A review of the biology and con- trol of sweetpotato weevil Cylas formicarius (Fab.). Tropical Pest Management, 32, 304-315. https://doi. org/10.1080/09670878609371084 Taye, A., & Tadesse, E. (2013). Screening chemical pesticides for the management of sweet potato weevil, Cylas puncticol- lis (Bohemann). International Journal of Advance Agricul- tural Research, 1, 48-57. Uritani, I., Saito, T., Honda, H., & Kim, W.K. (1975). Induc- tion of furano-terpenoids in sweet potato roots by the lar- val components of the sweet potato weevils. Agricultural and Biological Chemistry, 37(7), 1862-1875. https://doi. org/10.1271/bbb1961.39.1857