AActa agriculturae Slovenica • eISSN 1854-1941 • 118 – 3 • Ljubljana, oktober 2022 118•3 2022ACTA AGRICULTURAE SLOVENICA Acta agriculturae Slovenica Letnik / Volume 118 · Številka / Number 3 · 2022 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), Iryna BANDURA (Melitopol, Ukraine), Michael BLANKE (Bonn, Germany), 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), Salim LEBBAL (Khenchela, Algeria), Mario LEŠNIK (Maribor, Slove- nia), 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), Ola- lekan Suleiman 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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Trenutno revija ne zaračunava stroškov za predložitev in obdelavo vključenih prispevkov. / The journal does not charge APCs or submission charges. 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: Arbuskularna mikoriza v koreninah koruze (Zea mays L.). Temno modro obarvane zgostitve so arbuskuli, ki so pomembni za izmenjavo hranil med arbuskularnimi mikoriznimi (AM) glivami in rastlinami (Foto: Irena Maček, 1–13) Cover: Arbuscular mycorrhiza in roots of maize (Zea mays L.). In dark blue are arbuscules, which are important for nutrient exchange between arbuscular mycorrhizal (AM) fungi and plants (Photo: Irena Maček, 1–13) Acta agriculturae Slovenica Volume / Letnik 118 · Number / Številka 3 · 2022 Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers Genetska karakterizacija lokalnih sort koruze (Zea mays L.) gojenih na Kosovu ovrednotena z MITE-Hbr označevalci Barbara PIPAN, Sali ALIU, Dukagjin ZEKA, Vladimir MEGLIČ 1–7 Efficacy of Bacillus subtilis (Ehrenberg1835) Cohn1872, in suppressing Fusarium oxysporum Schlecht. emend. Snyder & Hansen, the causal agent of root rot of date palm offshoots (Phoenix dactylifera L.) in Iraq Učinkovitost bakterije Bacillus subtilis Ehrenberg 1835) Cohn 1872 pri zatiranju glive Fusarium oxysporum Schlecht. emend. Snyder & Hansen, kot povzročiteljice koreninske gnilobe pri dateljevi palmi (Phoenix dactylifera L.) v Iraku Naji Salim JASSIM, Muntaha Abd. ATI 1–10 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Klonsko razmnoževanje vrste Tetragonolobus palaestinus Bioss: jordanske zdravilne rastline Mawadda MHERAT, Mohammad SHATNAWI, Rida SHIBLI, Tamara QUDAH, Saida ABU MALLOH, Tamadour AL-QUDAH 1–9 Isolation of salt-tolerant Pseudomonas strains with potential for alleviation of salt stress in peanut plant (Arachis hypogaea L.) Izolacija na sol tolerantnih sevov bakterij iz rodu Pseudomonas s potencialom zmanševanja solnega stresa pri arašidu (Arachis hypogaea L.) Quang Trung DO, The Anh LUU, Minh Truong DAO, Quoc Nam HOANG, Trong Tri NGUYEN 1–9 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Količinsko ovrednotenje apokarotenoidov v komercialnih vzorcih indijskega (kašmirskega) žafrana z analizo UV-Vis spektroskopije in HPLC Tahir ul Gani MIR, Jaskaran SINGH, Saurabh SHUKLA 1–9 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits Predtretiranje semen s cinkovimi nano delci je zmanjšalo izražanje genov tolerance na slanost pri soji (Glycine max L.) in izboljšalo lastnosti pridelka Reda Mohamed GAAFAR, Mohamed Lotfi HALAWA, Adel Ramadan EL-SHANSHORY, Abdelhamid Abdelrahim EL-SHAER, Rana Hosny DIAB, Marwa Mahmoud HAMOUDA 1–14 Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley Identifikacija genov na ravni celotnega genoma za kinaze AGC in njihovo izražanje kot odziv na vročinski in hladni stres pri ječmenu Zohreh HAJIBARAT, Abbas SAIDI 1–11 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress Medsetev vzpodbuja fiziološko in morfološko prilagodljivost oljne ogrščice in ječmena v sušnem stresu Noushin SADEGHZADEH, Roghieh HAJIBOLAND, Charlotte POSCHENRIEDER 1–17 Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test Viabilnost semen dveh sort kavovca (Coffea arabica L.) z uporabo različnih predobravnavanj pred tetrazolijevim testom José Duván BUENDÍA CONTRERAS, Laura Yolima MORENO ROZO, Seir Antonio SALAZAR MERCADO 1–6 Evaluation of forage maize yield and soil organic matter content under green manure cultivation Ovrednotenje pridelka silažne koruze in vsebnosti organske snovi v tleh v razmerah zelenega podora Elias ARAZMJOO, Alireza MOGHRI FERIZ, Mohammad Ali BEHDANI, Sohrab MAHMOODI 1–8 Fruit collapse incidence and quality of pineapple as affected by biopesticides based on Pseudomonas fluorescens and Trichoderma harzianum Vpliv uporabe biopesticidov na osnovi bakterije Pseudomonas fluorescens in glive Trichoderma harzianum na propad in kakovost plodov ananasa Diego Mauricio CANO-REINOSO, Loekas SOESANTO, Kharisun KHARISUN, Condro WIBOWO 1–13 Enhance the phytoremediation efficiency of Echinochloa colona for Pb-contaminated soil by phosphorus solubilizing bacteria Povečanje fitoremediacijske učinkovitosti vrste Echinochloa colona z bakterijami, ki sproščajo fosfor v tleh, onesnaženih s svincem Quang Trung DO 1–9 Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. Vpliv nanodelcev srebra (Ag) na fiziološke in biokemične lastnosti kalusa dveh vrst materine dušice (Thymus sp.) in vrste Zataria multiflora Boiss. Nima MOSAVAT, Maryam YOUSEFIFARD, Pooran GOLKAR, Rabia JAVED 1–8 Field resistance phenotyping of durum wheat to fusarium head blight in Algeria Ugotavljanje odpornosti trde pšenice na fuzariozo klasov na prostem v Alžiriji Salah HADJOUT, Zouaoui BOUZNAD, Leila MEKLICHE, Mohamed ZOUIDI 1–9 Flight activity of Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae) infesting two Algerian olive varieties in north-west Algeria Let oljčne muhe (Bactrocera oleae (Rossi, 1790), Diptera: Tephritidae) na dveh alžirskih sortah oljke v severozahodni Alžiriji Zineb BOURAKNA, Kada RIGHI, Fatiha ASSIA, Abdelkader ELOUISSI 1–8 Phytochemical analysis, antioxidant and photoprotective activities of aqueous extract of Euphorbia retusa Forssk. different parts from Algeria Kemična analiza, antioksidacijska in fotoprotektivna aktivnost vodnih izvlečkov iz različnih delov vrste mlečka Euphorbia retusa Forssk. iz Alžirije Salwa LAHMADI, Mohamed Seif Allah KECHEBAR, Samira KAROUNE, Chawki BENSOUICI, Lynda GALI, Latifa KHATTABI, Hasna BOURAL, Amina CHOUH, Somia SAAD 1–10 Pollen quality and sensory attributes of Algerian jujube (Ziziphus lotus (L.) Lam.) honeys Kakovost peloda in senzorične lastnosti medu iz alžirske vrste čičimaka (Ziziphus lotus (L.) Lam.) Scherazad MEKIOUS, Carine MASSEAUX, Wahida LOUCIF-AYAD, Monica VERCELLI 1–7 Review Article / Pregledni znanstveni članek Development of research methods to characterise arbuscular mycorrhizal fungal communities and potential effects of fungal endophyte biodiversity on vegetation Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv biodiverzitete glivnih endofitov na vegetacijo Irena MAČEK 1–13 Short Scientific Article / Kratki znanstveni prispevek Ultraviolet disinfection of water in recirculating aquaculture system: a case study at sturgeon caviar fish farm Razkuževanje vode z ultravijolično svetlobo v recirkulacijskem akvakulturnem sistemu reje: primer ribogojnice jesetrov za prirejo iker, namenjenih za proizvodnjo kaviarja Anatolii SEMENOV, Kateryna SEMENOVA 1–4 Acta agriculturae Slovenica, 118/3, 1–7, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2344 Original research article / izvirni znanstveni članek Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers Barbara PIPAN 1, Sali ALIU 2, 3, Dukagjin ZEKA 2, Vladimir MEGLIČ 1 Received September 15, 2021; accepted June 29, 2022. Delo je prispelo 15. septembra 2021, sprejeto 29. junija 2022 1 Crop Science Department, Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia 2 Department of Crop Science, Faculty of Agriculture and Veterinary, Prishtina, University of Prishtina, Kosovo 3 Corresponding author,e-mail: sali.aliu@uni-pr.edu Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers Abstract: The aim of this study was to examine and de- scribe genetic structure on autochthonous maize germplasm (flint types) from different localities in Kosovo using Hbr mark- ers. The genetic characterization of 6-8 individual seedlings per each of 20 landraces was conducted by Hbr display calculated per selective base, the most efficient genetic diversity estimator to distinguish between landraces was primer combination Hbr- Int5-F/MseI+T. The strongest genetic relatedness (r = 55.57) had landrace ACC4 having orange colored seeds, showing the highest genetic uniformity when compared to other accessions. Clustering analysis using the Bayesian approach generated two genetic clusters for observed landraces. As a measure of population structure influenced by genetic drift and migration, Fst values for each genetic cluster were obtained. Higher Fst (0.4027) was calculated within the first genetic group compar- ing to the second one (0.2001), reflecting a higher levels of out-crossing and conservation between landraces from the first genetic cluster. A similar distribution of genetic linkages was observed from dendrogram, constructed using Dice coefficient and neighbour-joining (NJ) algorithm with minor deviations for landraces ACC6 and ACC28. Genotypes of ACCmk land- race reveal the highest genetic distinction compared to other genotypes, reflecting the highest number of bands (241) and the highest number of private bands (10) as the number of bands unique to a single population, respectively. Key words: genetic variability; heartbreaker family mark- ers; maize Genetska karakterizacija lokalnih sort koruze (Zea mays L.) gojenih na Kosovu ovrednotena z MITE-Hbr označevalci Izvleček: Namen raziskave je bil preučiti in opisati genet- sko strukturo avtohtone dednine trdinke iz različnih lokalitet Kosova s Hbr označevalci. Genetska karakterizacija 6-8 sejank od vsake lokalne sorte je bila izvedena s prikazom nabora Hbr profilov, izračunanega na osnovi selektivne baze. Najučinko- vitejši določitelj genetske raznovrstnosti za razločevanje med lokalnimi sortami je bila kombinacija primerjev Hbr-Int5-F/ MseI+T. Najmočnejšo genetsko povezanost med genotipi (r = 55.57) je imela lokalna sorta ACC4 z oranžnimi zrni, ki je iz- kazovala največjo genetsko izenačenost v primerjavi z drugimi akcesijami. Klasterska analiza z uporabo modela aposteriorne verjetnosti (Bayesian approach) je za vključene lokalne sorte oblikovala dve genetski skupini. Kot merilo za analizo popu- lacijske structure, na katero vplivata genetski zdrs in migracija, so bile izračunane vrednosti Fst za obe genetski skupini. Večja vrednost Fst (0,4027) je bila izračunana znotraj prve genetske skupine v primerjavi z drugo (0,2001), kar kaže na večji delež navskrižnega križanja in ohranjanja raznolikosti med lokalnimi sortami prve genetske skupine grozda. Podobna porazdelitev genetskih povezav je bila določena na dendrogramu, izdelanem z uporabo Dice-ovega koeficienta podobnosti in algoritma raz- vrščanja po metodi združevanja najbližjih sosedov (NJ) z manj- šim odstopanjem za akcesiji ACC6 in ACC28. Genotipi akcesije ACCmk so se genetsko najbolj razlikovali od drugih na osnovi največjega števila prisotnih namnožkov (241) in največjega šte- vila prisotnih “privatnih namnožkov” (10) in v številu namnož- kov, ki so bili omejeni samo na eno populacijo. Ključne besede: genetska raznolikost; družina “Heart- breaker” označevalcev; koruza Acta agriculturae Slovenica, 118/3 – 20222 B. PIPAN et al. 1 INTRODUCTION Maize (Zea mays L.) is one of the most genetical- ly diverse and widespread crops in South-East Europe (Đalović et al., 2015; Ignjatović-Micić et al., 2015). Maize landraces have been largely replaced by commercial maize hybrids in Kosovo. The share of hybrids was 4 % in the 1960’s and already at 90 % in the early 2000’s due to their high yield potential. Studies of specific combining abilities of inbred lines and physiological traits of some hybrids were already performed in agroecological condi- tions of Kosovo (Aliu et al., 2008, 2010). Maize became widely investigated plant species regarding different ap- plications; its water use efficiency (Wang et al., 2013), morphological, physiological and biochemical response to SiO2 nanoparticles (Sharifi-Rad et al., 2016), different irrigation regimes and planting methods (Singh Brar et al., 2016). There are also different marker systems, ap- plied to assess genetic characterization of maize germ- plasm, including RAPD (Random Amplified Polymor- phic DNA) (Srdić et al., 2007), SSR (Simple Sequence Repeats) (Ignjatović-Micić et al., 2015) and MITE-Hbr (Miniature Inverted Repeat Transposable Element from the family Heartbreaker) (Casa et al. (2000; 2002) and Kavar et al. (2007)). MITE-Hbr markers have been firstly exploited and developed as a new marker system (modification of the AFLP-Amplified Fragment Length Polymorphism procedure) for evaluation of the maize genome (Casa et al. (2000; 2002)). Kavar et al. (2007) used Hbr markers to evaluate the genetic diversity of Slo- venian maize germplasm, originating from Western Bal- kan (former Yugoslavia). Related to those three reports, MITE-Hbr markers were proven to be stable, highly polymorphic, cost-effective, easily mapped and evenly distributed throughout the maize genome. SSRs are highly applicable markers in our genetic studies of different plant species, among other grape- vines (Rusjan etal., 2012, 2015) sweet potato (Pipan et al., 2016), brassicas (Pipan et al., 2011, 2013), and beans (Maras et al., 2015). In a study by Ignjatović-Micić et al. (2015), using SSR markers, they reported that higher genetic variation was observed among flint genotypes, comparing to dent ones. They also suggest that landraces from Western Balkans are highly adapted to specific en- vironmental conditions and uses and therefore could be a valuable source of genetic variability. The adaptation to diverse agro-ecological conditions is a result of natural and selection by farmers. The aim of this study was to examine and describe genetic structure of autochthonous maize germplasm from Kosovo using Hbr marker system and to employ Hbr display to show genetic differences and associations between and within observed flint landraces of maize collected in different localities of Kosovo, with a possibil- ity to detect conservation of gene flow into the maize ge- nome. The knowledge of genetic characteristics and lan- drace-specific background of maize germplasm would be of a benefit for future breeding and germplasm improve- ment programmes in Kosovo. 2 MATERIALS AND METHODS Twenty maize landraces, collected from differ- ent locations in Kosovo (Table 1), were screened using MITE-Hbr markers. DNA from 6 - 8 individuals of each landrace was extracted from each individual seedling us- ing BioSprint 15 DNA Plant Kit (Qiagen) and MagMax Express Magnetic Particle Processor (Life Technologies, Grand Island, NY) following manufacturer’s instruc- tions. Hbr display with some modifications was per- formed as described by Casa et al. (2000, 2002) and Ka- var et al. (2007). 600 ng of genomic DNA was digested for 3 h at 65 °C in 20 µl of 10x Tango buffer containing MseI (Fermentas). Adaptors (5’gacgatgagtcctgag and 5’tactcag- gactcat) to the digested DNAs and aliquots of the restric- tion/ligation reactions were visualized on 0.9 % agarose gels to check the quality of DNA digestion. Pre-selective amplification was performed using primers Hbr-Int5-E (5’gattctccccacagccagattc) and MseI+0 (5’gacgatgagtcct- gagtaa). Selective amplification was performed with each of the three selective primer combinations (MseI+C, MseI+G and MseI+T) with a fluorescently labeled Hbr internal primer (5’-6FAM-agccagattttcagaaaagctg). Frag- ment analysis was performed on the 3130XL Genetic Analyzer (Applied Biosystems), and sizing of fluorescent fragments/bands was determined by comparison with size standard GeneScan-500 ROX (Applied Biosystems) using GeneMapper 4.0 (Applied Biosystems). A binary matrix was constructed by scoring fragments as either present (1) or absent (0) in each DNA sample. Principal Coordinate Analysis (PCoA), Analysis of Molecular Variance (AMOVA), number of different alleles (Na), number of effective alleles (Ne) and Shan- non’s information index (I) across landraces for each selective base was calculated in GenAlEx v.6.4 (Peakal and Smousse, 2006). Genetic similarities were calculated on the basis of a binary matrix using the Dice similar- ity index (Dice, 1945). These coefficients were used to construct the clustering using the neighbur-joining (NJ) algorithm by 100 bootstraps in FreeTree (Pavliček et al., 1999) and visualized using TreeView (Page, 1996) soft- ware. Genetic diversity parameters between and within landraces including AMOVA, band patterns (num- ber of bands, number of private bands, number of lo- cally common bands alleles occurring in 50 % or fewer Acta agriculturae Slovenica, 118/3 – 2022 3 Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers landraces, expected heterozygosity) and mean within landrace pairwise values (r) were conducted using Ge- nAlEx v.6.4 (Peakal and Smousse, 2006). Structure 2.3.3 software (Pritchard et al., 2009) was employed for infer- ring landrace structure using a Bayesian approach. Ten independent runs for each K (from1 to 7) in the case of admixture model were performed and burning period of 10,000 followed by 100,000 Markov Chain Monte Carlo repeats was used. The ideal K-value was selected based on the increases in likelihood ratios between runs us- ing Evanno’s delta K statistic (Evanno et al., 2005) im- plemented in a Structure Harvester (Earl and von Holdt, 2011). The estimation of flowering time was made in the same year at different locations, which are presented in Table 1. 3 RESULTS Kosovo landraces included in our study are all of a flint type with white kernel (fruit) color, except for a lan- drace ACC4, which kernels are orange (Table 1). Genetic characterization of 6-8 individual seedlings per each landrace was conducted using Hbr display. A total of 498 markers, ranging in size from 60-500 bp, were generated using three primer combinations: Hbr- Int5-F/MseI+T, Hbr-Int5-F/MseI+C, and Hbr-Int5-F/ MseI+G. Regarding genetic diversity estimators (PCoA, AMOVA, Na, Ne and I) calculated per selective base (T, C, G), the most efficient primer combination to distin- guish between landraces from Kosovo, was Hbr-Int5-F/ MseI+T, and Hbr-Int5-F/MseI+C respectively (Table 2). First three axes in PCoA (via covariance distance ma- trix) cumulatively explained 70 % of genetic variability for Hbr-Int5-F/MseI+T; 67 % for Hbr-Int5-F/MseI+C and 59 % for Hbr-Int5-F/MseI+G (Table 2). Percent of molecular variability among landraces in Hbr screen- ing varied from 10 (Hbr-Int5-F/MseI+C) to 18 (Hbr- Int5-F/MseI+T), depending on selective primer applied (Table 2). Additionally, the highest values of Na (7.400), Ne (1.067) and I (0.074) were calculated for Hbr-Int5-F/ MseI+T (Table 2). Landrace-specific genetic diversity was estimated by applying different algorithms to compare a genetic composition among and within autochthonous landrac- es from Kosovo. The summary of mean within landrace pairwise values, based on genetic distance, is presented in Figure 1. The lowest mean r value was calculated Landrace label Locality Latitude [° ‘ ‘’] Longitude [° ‘ ‘’] Altitude [m] Vernacular name Landscape Kernel type Kernel Color Kernel shape Flowering time [days] ACC2 Ferizaj 42.25.21 21.09.06 555 Bardhosh Flat Flint White Oval 69 ACC4 Shtime 42.26.40 21.42.96 642 Kolomboq Mountain Flint Orange Oval 72 ACC6 Skenderaj 42.44.39 20.48.04 603 Miser Valley Flint White Oval 71 ACC8 Skenderaj 42.44.39 20.47.39 597 Miser Valley Flint White Oval 75 ACC12 Skenderaj 42.45.00 20.48.23 623 Miser Flat Flint White Oval 76 ACC14 Drenas 42.39.30 20.42.46 565 Kolomboq Flat Flint White Oval 76 ACC16 Drenas 42.39.21 20.42.32 586 Kolomboq Flat Flint White Oval 75 ACC26 Vushtrri 42.48.38 20.58.30 518 Kolomboq Flat Flint White Oval 72 ACC28 Suharekë 42.21.45 20.49.02 388 Miser Valley Flint White Oval 77 ACC30 Vushtrri 42.50.46 20.59.26 557 Kolomboq Flat Flint White Oval 71 ACC32 Drenas 42.34.50 20.54.06 585 Kolomboq Flat Flint White Oval 71 ACC34 Podujevë 42.53.39 21.12.12 598 Kolomboq Mountain Flint White Oval 75 ACCmk Lipjan 42.31.45 21.07.20 551 Miser Flat Flint White Oval 69 ACC38 Kamenicë 42.33.56 21.31.32 812 Kolomboq Mountain Flint White Longi 67 ACC40 Kamenicë 42.34.16 21.31.32 766 Kolomboq Mountain Flint White Oval 72 ACC42 Prishtinë 42.35.35 21.20.40 824 Kolomboq Mountain Flint White Oval 65 ACC44 Drenas 42.41.21 20.45.31 691 Kolomboq Flat Flint White Oval 71 ACC46 Malisheve 42.27.56 20.43.22 576 Miser Mountain Flint White Oval 72 ACC48 Malisheve 42.28.01 20.44.04 562 Miser Mountain Flint White Oval 70 ACC50 Drenas 42.41.50 20.44.43 567 Kolomboq Flat Flint White Oval 76 Table 1: Characteristics of maize landraces from Kosovo Acta agriculturae Slovenica, 118/3 – 20224 B. PIPAN et al. across ACC50 (39.03) where r was also outside U and L limits (Figure 1) reflecting the weakest genetic related- ness of genotypes within ACC50 landrace. The strongest genetic relatedness (r = 55.57) was reached within orange colored seeds of ACC4 landrace (Figure 1) showing the highest genetic uniformity of included genotypes within ACC4 compared to other landraces. The genetic structure of observed landraces, described by Bayesian clustering approach in Figure 2 shows higher uniformity of ACC4, with 98.1 % probability that landrace ACC4 belongs to the first (red) genetic cluster and lower genetic uniform- ity within ACC50 with 79.9 % probability that genotypes Cumulative % in PCoA (first 3 axes) AMOVA (% among landraces) Na Ne I Hbr-Int5-F/MseI+T 70 18 7.400 1.067 0.074 Hbr-Int5-F/MseI+C 67 10 6.850 1.066 0.072 Hbr-Int5-F/MseI+G 59 16 7.100 1.065 0.069 Table 2: Analysis of genetic diversity among landraces by selective bases from ACC50 belong to the second genetic cluster (green) which is also confirmed by r value (Figure 1). In general, clustering analysis using the Bayesian method generated two genetic clusters (ideal K, con- ducted using Structure Harvester) for observed landraces (Figure 2). Similary colorored segments represents the estimat- ed membership to the genetic cluster. The first genetic cluster (red) posses 0.0778 of expected heterozygosity between genotypes and 0.0925 was calculated for the second one (green), respectively. Regarding their genetic structure, landraces ACC6, ACC12, ACC14, ACC34, Notes: Na-The number of alleles; Ne- The number of detected effective alleles; I-Shannon’s information index Figure 1: Mean within landrace pairwise values (r) according to genetic distance. Upper (U) and lower (L) confidence limits bound the 95 % confidence interval about the null hypothesis of ‘No difference ‘ across the landraces as determined by permuta- tion (99) Figure 2: Structure plot of maize landraces from Kosovo Acta agriculturae Slovenica, 118/3 – 2022 5 Genetic characterization of maize (Zea mays L.) landraces grown in Kosovo assessed by MITE-Hbr markers ACC28, ACC4, ACC46, ACC38, ACC16, ACC44, ACC2, and ACC8 belong to the first cluster of landraces (red); meanwhile landraces ACC30, ACC40, ACC42, ACC32, ACC48, ACCmk, ACC50, and ACC26 comprise the sec- ond genetic cluster (green) (Figure 2). A similar distribu- tion of genetic relations between landraces was observed in a dendrogram, constructed using Dice coefficient (Dice, 1945) and the NJ algorithm with minor deviations for landraces ACC6 and ACC28 (Figure 3). Figure 3: Genetic linkages of observed landraces from Kosovo using Dice coefficient and neighbour-joining (NJ) algorithm. (Green and red colours assign cluster colours from structure plot on Figure 2) Figure 4: Band patterns across maize landraces from Kosovo We have calculated landrace-specific parameters of genetic diversity (Figure 4) to compare genetic character- istics between all maize landraces collected, applying Hbr display. Genotypes of landrace ACCmk reveal the high- est genetic distinction compared to genotypes within other landraces, reflecting the highest number of bands (241) and the highest number of private bands (10) (Fig- ure 4), respectively. A number of common bands with a frequency of > 5 %, which are found in the 50 % assessed landraces, reached the highest values for ACC40 (115), ACC32 (114) and ACCmk (111), respectively (Figure 4). Evaluation of genetic differentiation within and be- tween landraces, applying Hbr markers, provided use- ful information about genetic the structure, relatedness and genetic diversity of autochthonous maize germplasm from Kosovo. Genetic uniformity of genotypes within landraces is high, regardless to low values of expected heterozygosity (max He = 0.067, data not shown) as a measure of genetic diversity within landraces. Genotypes within the second (green) cluster reveal lower genetic di- versity (Fst = 0.2001) compared to the first (red) cluster (Fst = 0.4027). 4 DISCUSSION Results presented in Table 2 indicate that Hbr- Int5-F/MseI+T is the most informative selective primer provided by Hbr display to distinguish twenty maize landraces collected from different locations in Kosovo. In the study by Kavar et al. (2007) evaluating Slovenian maize landraces, the most informative primer combi- nation was Hbr-Int5-F/MseI+G, revealing the highest number of loci (103) scored using Hbr display. On the other hand, calculated genetic diversity values for maize landraces from Kosovo are similar using Hbr display. This was as well the case evaluating Slovenian accessions by Kavar et al. (2007), where similar values of scored loci (73-103) were obtained applying different selec- tive primers. Cluster analysis using Bayesian approach revealed no genetic relatedness (regarding their genetic Acta agriculturae Slovenica, 118/3 – 20226 B. PIPAN et al. structure) between landraces from the same geographic origin (locality and landscape), a name of landrace and kernel shape (no difference for ACC38 which is oval), respectively. Structure plot also shows that individual plants from landraces ACC14 (87.3 % red, 12.7 % green), ACC50 (20.1 % red; 79.9 % green), ACC26 (28.0 % red, 72.0 % green), and ACC28 (75.0 % red, 25.0 % green) are sharing on average higher proportion of germplasm belonging to both genetic clusters (Figure 2). According to the data in Table 1, four landraces named Kolomboq (vernacular name or local name), originating from flat landscape have the longest flowering period of more than 72 days. There is one exception in this cluster, landrace ACC28 Miser (local name), originating from the valley (Suharekë) with the longest flowering period (77 days). A difference in landrace distributions for genetic cluster- ing, when comparing the structure plot (Figure 2) and dendrogram (Figure 3) could be a logical consequence of different algorithms/approaches applied for specific pur- pose, required for genetic diversity assessment. According to the landrace-specific parameters of genetic diversity (Figure 4), landrace ACCmk represents a potentially interesting source for further genetic studies and germplasm improvement. A high number of private bands actually represents unique copies of Hbr transpo- sons that ACCmk landrace harbors compared to private alleles observed in other studies using SSR markers. Pri- vate alleles in those cases could reflect accumulation and conservation of introduced genes via out-crossing along generations to the plant genome (Pipan et al., 2013). Cal- culated number of common bands with a frequency > 5 % (Figure 4) for landraces ACC40, ACC32 and even ACCmk, are sharing the highest number of scored bands with other landraces, even though that the three landrac- es belong to the second (green) genetic cluster/group (Figures 2 and 3). Evaluation of maize landraces from Kosovo was successfully assessed by a rare type of marker system, miniature inverted repeat transposable element - Hbr marker, using three selective primer combinations. To distinguish genotypes between and within different landraces, application of only one selective marker could be sufficient, as reported Kavar et al. (2007). Applica- tion of SSR markers as a codominant marker system is also used in genetic diversity studies of maize landraces (Ignjatović-Micić et al., 2015). According to the results presented, there are strong genetic relations between dif- ferent landraces, comprising two genetic groups, which could be assigned to the two general micro centers of diversity in Kosovo. As a measure of a population struc- ture influenced by genetic drift and migration, Fst values for each genetic cluster generated using Bayesian cluster analysis, were obtained. Higher Fst (0.4027) was calcu- lated within the first (red) genetic group compared to the second (green) one (0.2001), reflecting higher levels of out-crossing and conservation between landraces from the first genetic cluster. This result could be also a con- sequence of simultaneous cultivation and uncontrolled gene flow between observed maize landraces listed in Table 1 within the same production area of Kosovo in the past. 5 CONCLUSIONS Related to mean r value, there is 29.7 % of a variable genetic part, which is dispersed along included landraces from Kosovo. It is important to point out that landraces evaluated, originated from different localities with a di- verse landscape (flat, mountain, and valley) and from various production areas. According to the results, there are strong genetic relations between different landraces, comprising from two genetic groups, which could in- dicate on two micro diversification locations of flint type in Kosovo. The results provided, we can conclude that MITE-Hbr markers are highly applicable and cost- effective tool for maize genetic diversity studies and as in this case for a genetic distinction between and within landraces collected in Kosovo. 6 ACKNOWLEDGEMENT This work was in part financially supported by grant P4-0072 (Agrobiodiversity programme) from the Slo- venian Research Agency. We are thankful to the mag. Romana Rutar providing seedlings, dr. Katarina Rudolf- Pilih for an extra hand with DNA extraction and to the dr. 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Delo je prispelo 31. marca 2022, sprejeto 3. julija 2022 1 Plant pathology, Date Palm Research Center, University of Basrah, Basrah, Iraq 2 Corresponding author, e-mail: ahmidnaji916@gmail.com Efficacy of Bacillus subtilis (Ehrenberg1835) Cohn1872, in suppressing Fusarium oxysporum Schlecht. emend. Snyder & Hansen, the causal agent of root rot of date palm offshoots (Phoenix dactylifera L.) in Iraq1 Abstract: Date palm root rot disease is one of the most important diseases of date palms and offshoots. It is caused by many soil-borne pathogenic fungi. Pathogenicity assays of the isolated fungi showed that the major causative agents of root rot disease in date palm plantlets were Fusarium oxysporum Schlecht. emend. Snyder & Hansen, F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg S1, F. proliferatum S2, Gib- berella fujikuroi (Sawada) Wollenw., and Rhizoctonia solani J.G. Kühn. The most virulent fungus was F. oxysporum with a sever- ity index of 82.16 % of root rot, while R. solani was the least harmful with a disease severity rate of 12.42 %. In laboratory tests, Bacillus subtilis reduced the radial mycelial growth of F. oxysporum on PDA medium by 86.6 %. The application of B. subtilis in combination with F. oxysporum substantially inhib- ited the severity of root rot disease relative to plantlets treated with only F. oxysporum. In addition, B. subtilis application in the presence or absence of F. oxysporum improved the plant physiology of plantlets, including total chlorophyll, total carot- enoid, antioxidant enzyme levels (catalase and peroxidase), and total proline content. Key words: B. subtilis; date palm; F. oxysporum; plant physiology Učinkovitost bakterije Bacillus subtilis Ehrenberg  1835) Cohn 1872 pri zatiranju glive Fusarium oxysporum Schlecht. emend. Snyder & Hansen, kot povzročiteljice koreninske gni- lobe pri dateljevi palmi (Phoenix dactylifera L.) v Iraku Izvleček: Koreninska gniloba je najpomembnejša bolezen dateljeve palme. Povzročajo jo številne talne patogene glive. Preiskus patogenosti z izolati gliv na sadikah dateljeve palme je pokazal, da so bili glavni povzročitelji njene koreninske gnilobe naslednje glive: Fusarium oxysporum Schlecht. emend. Snyder & Hansen, F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg S1, F. proliferatum S2, Gibberella fujikuroi (Sawada) Wollenw., and Rhizoctonia solani J.G. Kühn. Najbolj virulentna je bila gliva F. oxysporum, z indeksom virulentnosti 82,16  % med tem, ko je bila gliva R. solani najmanj škodljiva z indeksom povzročitve koreninske gnilobe 12,42  %. V laboratorijskem poskusu je bakterija B. subtilis na PDA gojišču zmanjšala radial- no rast micelija glive F. oxysporum za 86,6 %. Uporaba bakterije B. subtilis je v kombinaciji z glivo F. oxysporum znatno zavrla razvoj koreninske gnilobe na sadikah dateljeve palme v prim- erjavi s sadikami, ki so bile tretirane samo z glivo. Dodatno je uporaba bakterije B. subtilis v prisotnosti ali odsotnosti glive F. oxysporum izboljšala fiziološke parametre sadik kot so vsebnost celokupnega klorofila in karotenoidov, aktivnost antioksidaci- jskih encimov katalaze in peroksidaze ter vsebnost celokupnega prolina Ključne besede: B. subtilis; dateljeva palma; F. oxyspo- rum; fiziološki parametri rastline Acta agriculturae Slovenica, 118/3 – 20222 N. S. JASSIM and M. A. ATI 1 INTRODUCTION Date palm (Phoenix dactylifera L.), Palmaceae (Are- cacea), is a tropical and subtropical plant native to south- ern Asia and Africa. Selective breeding over thousands of years has resulted in the 3,000 variants presently farmed across the world in areas where the date palm thrives in hot, dry climates (Zaid, 2002). Dates are high in nutrients and provide a wonderful source of energy. Date fruits are composed of 70 % carbohydrates, primarily sugars, and 15 %-30 % water. Dates are also a good source of miner- als, including iron, potassium, and calcium, and are low in salt and fats (Thabet et al., 2010; Dayani et al., 2012). Date palms are infected by many soil-borne patho- genic fungi that threaten mature trees and offshoots, re- sulting in substantial tree damage and yield losses across the world (El-Morsi et al., 2009; Maitlo et al., 2013). In several locations, pathogenic fungi of root rot and wilt disease caused by Fusarium oxysporum, Fusarium solani, Fusarium moniliform, and Rhizoctonia solani have been isolated from young offshoots and adults of the date palm (Phoenix dactylefera L.) (Alwahshi et al., 2019; Arafat et al., 2012; Baraka et al., 2011). Chemical pesticides used to manage soil-borne diseases can result in pathogen resistance, negative ef- fects on people and beneficial soil organisms, and pol- lution. Several soil fumigants and fungicide compounds are expected to be phased out soon. However, in order to achieve optimal plant development and production, soil pathogens will still need to be managed (Gerhard- son, 2002). As a result, efficient beneficial microbes that can be used as an alternate method for controlling soil pathogens need to be discovered. For managing many soil-borne diseases, microbial antagonists such as bacte- ria and fungi have the potential to be a low-cost, healthy, and ecologically friendly solution (Caron et al., 2002; Gravel et al., 2004). Bacillus species proliferate quickly, are resistant to harsh environmental conditions, and have been identi- fied as beneficial microorganisms. Antibiotics; compe- tition with pathogens for space or resources; destruc- tion of pathogen hyphae; synthesis of siderophores and phytohormones that stimulate plant development; and induced systemic resistance (ISR) in the host plant are all modes of action by which Bacilus subtilis suppresses plant pathogens (Cao et al., 2012; Chen et al., 2020; Li et al., 2013). By induction of systemic resistance in plants, B. subtilis generates volatile chemicals that influence plant development and activate the plant defense mecha- nism (Hashem et al., 2019; Wang et al., 2018).  Bacillus spp. also produce endospores, which allow the bacteria to live in harsh environments, allow for germination in response to varied environmental circumstances, allow for long-period storage of biopesticide, and make the for- mulation process easier (Collins & Jacobsen, 2003). The US Food and Drug Administration (USFDA) classifies B. subtilis as “generally recognized as healthy” (GRAS) for use in the food processing industry. This work aimed to measure the efficacy of B. sub- tilis strains against F. oxysporum under greenhouse con- ditions. Second, levels and activity of some physiologi- cal and biochemical components were measured in date palm during infection by F. oxysporum in the presence and absence of B. subtilis, and compared to the control treatments.  2 MATERRIALS AND METHODS 2.1 PATHOGENICITY ASSAY OF ISOLATED FUNGI Representative fungal isolate strains used through- out this study were obtained from a previous study car- ried out in the Biology Department, College of Science, University of Basrah, Basrah by Kazaal (2019). These isolates are Fusarium oxysporum, Fusarium proliferatum S1, Fusarium proliferatum S2, and Fusarium fujikuroi. Rhizoctonia solani isolate obtained from the lab of date palm diseases/Date Palm Research Center/University of Basrah. Infection trials with the recovered isolates run in a greenhouse trial at the Date Palm Research Center. Six- month-old plantlets (grown from seeds of the Halawii cultivar) were planted into plastic pots (2 kg pots) filled with sterilized soil (1 : 1 peat moss + sand). To prepare the fungi inoculant, each isolate was cultivated on PDA for 5–15 days at 27 °C. The spore suspension of each isolate was made by flooding plates of 15-day-old cultures with sterile distilled water, scraping with a sterilized glass rod, filtering, and adjusting to a 106 spore ml-1 concentration using a Neubauer haemocytometer before adding to the potted soil. Potted soil was injected with each fungus inoculum at a concentration of 106 spores ml-1 with ir- rigation water (Al-Ani et al., 2012). Each fungus (treat- ment) has five pots (replicates) with three plantlets, and a control treatment (uninfected soil). The pots were in the greenhouse under favorable conditions. For three months, pots were kept at 90 % soil humidity. The pots were carefully watered every time at the level of the field capacity. The percentage of disease severity is calculated after 60 days from inoculation using the following scale (with little modulation) of 0–5: where 0 = healthy; 1 = 1-25 % of the plant has a few spots on the roots; 2 = 26- 50 % of the roots have spots and one leaf is wilting; 3 = 51-75 % of the roots have big black spots and all leaves are Acta agriculturae Slovenica, 118/3 – 2022 3 Efficacy of Bacillus subtilis ..., in suppressing Fusarium oxysporum ..., the causal agent of root rot of date palm offshoots ... in Iraq wilting; 4 = up to 76 % of roots are rotted and all leaves are wilting, and5 = dead plants (Abdou et al., 2003). The disease severity index (DSI) of each replicate was calculated according to the method described by Liu et al. (1995) as follows: DSI = Σd/(dmax × n) × 100, where d = the disease rating of each plantlet, dmax = the maxi- mum rate of disease, and n = the total number of plant- lets in each replication assessed. 2.2 EFFECT OF B. SUBTILIS AGAINST THE GROWTH OF F. OXYSPORUM IN VITRO The purpose of the experiment was to determine the antagonistic connection between the most virulent fungus, F. oxysporum and B. subtilis (Bacillus subtilis was isolated by serial dilution technique on nutrient agar me- dium (NAM). A 0.5 g of BioHealth biopesticide was sepa- rated and vortexed for 15 minutes in 10 ml of distilled wa- ter. From 10-1 to 10-6, the suspension was serially diluted. 1 ml of suspension was pipette out and distributed with a glass rod in an L shape onto nutrient agar plates and incubated at 37 °C for 24 hours. For subsequent research, the most conspicuous colonies were separated and kept at 4 °C. The in vitro effect of B. subtilis on colony growth of F. oxysporum was assessed by the dual culture method. A 0.7 cm dia. disc from the F. oxysporum culture was cho- sen from the colony’s edge (5 days) and was placed in the center of the PDA plate. After that, four-discs (0.7 cm dia. each) were taken from a three-day old B. subtilis colony on nutrient ager (NA) medium and placed at the periph- ery of the petri dish with equal dimension to each other and 1.5 cm from the edge of the petri dish. For the con- trol treatment, a 0.7 cm dia. disc from the same pathogen colony was added to the sterilized PDA plate (without adding B. subtilis). For both the antagonism treatment and control, there were five plates (= replicates). All the plates were incubated at 28 °C. After incubation, in the antagonism treatment, the radial growth mycelium of the fungus was measured when the radial growth mycelium in the control reached the edge of the growth plates. The percentage of fungal growth inhibition (FGI) was calcu- lated as the ratio of growth between fungal growth in the treatment opposite to the control: FGI % = [1 – (FG in antagonism/FG in control)] 100. 2.3 THE NATURE OF EXPERIMENT This experiment was conducted in the greenhouse of the Date Palm Research Center, University of Basrah, during the 2019-2020 growing season. The experiment was repeated twice. Plantlets were 12 months old (they were grown from seeds of the Halawii cultivar) and were planted in plastic pots (4 kg each) filled with sterilized soil (1 : 1 peat moss + sand). The pots (=replicates) ex- periments were arranged and conducted in a completely randomized design. The pots in the experiment were di- vided into four treatments: (1) controls (no added any bacterial or fungal inoculation); (2) plantlets inoculated with B. subtilis only; (3) plantlets inoculated with F. ox- ysporum only; and (4) plantlets inoculated first with B. subtilis at a concentration of 108 spores ml-1 (B. subtilis inoculum concentration used according to the manufac- ture recommendation, 0.5 g of BioHealth biopesticide 10 mL of water, the concentration of B. subtilis was 108 spores ml-1) and, after 48 hours, also inoculated with F. oxysporum at a concentration of 10 6 spores ml-1 (for both organisms, the inoculum was added at a rate of 150 ml/ pot with irrigation water) (Al-Ani et al., 2012). All pots were placed in the greenhouse under favorable condi- tions (28–30 °C, watering and fertilization). Treatments were applied to the plantlets, which were then held for 28 days. Each treatment was replicated five times (each replicate having one pot with three plantlets). At the end of the experiment (day 28), we measured four response parameters: (1) photosynthetic pigment content, (2) an- tioxidant enzyme levels, and (3) total soluble proline, as described in detail below: Photosynthetic pigment content was measured ac- cording to the protocol of Metzner et al. (1965). The total chlorophyll and carotenoids were deter- mined spectrophotometrically (CECL, 2021 spectropho- tometer, UK).The absorbance was calculated against a blank of pure 85 % aqueous acetone at 452 and 663 nm, represented as mg. g fresh mass (FM). Using the follow- ing equations: total chlorophyll and total carotenoids: photosynthetic pigments represented as mg. g FM-1.  For antioxidants, the activity of catalase (CAT) was determined according to Luck (1974), and the activity was expressed as a unit/mg of protein. Peroxidase (POD) activity was estimated according to Kara & Mishra (1976). The amount was evaluated by the absorbance at 420 nm, and the enzyme activity was expressed as a unit/ mg of protein. The protocol of Bates et al. (1973) was used to deter- mine the total soluble proline leaf content. The toluene reagent was aspirated from the aqueous phase, and the solution absorbance was measured at 520 nm. Proline content was determined by measuring it from a standard curve and was calculated as mg. g dry mass-1 (DM). Acta agriculturae Slovenica, 118/3 – 20224 N. S. JASSIM and M. A. ATI 2.4 DATA ANALYSIS The experimental design was completely rand- omized. The statistical analysis data was carried out by analysis of variance ANOVA using SPSS-21 software, the differences in the means were determined by the least significant difference test (LSD) (p < 0.05). 3 RESULTS 3.1 PATHOGENICITY TESTS OF ISOLATED FUNGI Fusarium oxysporum, F. proliferatum S1, F. prolif- eratum S2, F. fujikuroi, and R. solani, were responsible for root rot infections in date palm plantlets (Table1). F. oxysporum was the most pathogenic fungus, causing 82.16 % of root rot severity, with highly significant differ- ences compared with other fungi, followed by F. prolifera- tum S1, F. proliferatum S2, and F. fujikuroi, which caused 30.12 %, 24.26 %, and 18.56 % severity, respectively. R. solani was the least harmful species as it showed a disease severity 12.42 %. 3.2 EFFECT OF B. SUBTILIS AGAINST THE GROWTH OF F. OXYSPORUM IN VITRO Bacillus subtilis reduced colony spread of F. oxyspo- rum on PDA by 86.6 % (Fig.1) (1.2 cm mean radial col- ony growth with B. subtilis versus 9.0 cm mean colony growth without B. subtilis) after 6 days of incubation at 27 °C. 3.3 EFFECTS ON TOTAL CHLOROPHYLL AND TOTAL CAROTENOIDS The results showed that date palm plantlets treated with B. subtilis in the presence and absence of F. oxyspo- rum resulted in a highly significant increase in total chlo- rophyll and total carotenoid, in comparison to the patho- gen alone (F. oxysporum). Plantlets treated with B. subtilis had the highest scores, while the pathogen treatment re- sulted in low values (Fig.2). B. subtilis significantly raised Disease severity of root rot disease Fungi tested D DSI % Plant survival F. oxysporum 4 82.16 a 12.82 a F. proliferatum S1 3 30.12 b 68.64 b F. proliferatum S2 3 24.26 c 76.10 c F. fujikuroi 2 18.56 c 82.20 d R. solani 2 12.42 d 90.00 d Control(untreated) 0 _ 100 e LSD at p = 0.05 NA 2.86 4.93 Table 1: Pathogenicity of fungal isolates recovered from date palm plantlets after greenhouse inoculations, 60 days post- inoculation Average scores for 15 plantlets for each treatment, where; D: disease rating scale and, DSI: disease severity index Fig. 1: In vitro inhibition of mycelial growth of F. oxysporum by B. subtilis on PDA (A: antagonism, B: F. oxysporum) Acta agriculturae Slovenica, 118/3 – 2022 5 Efficacy of Bacillus subtilis ..., in suppressing Fusarium oxysporum ..., the causal agent of root rot of date palm offshoots ... in Iraq the total chlorophyll and total carotenoids in comparison with the F. oxysporum treatment. When plantlets infect- ed with F. oxysporum were treated with the bacterium B. subtilis, chlorophyll levels were restored to roughly 90 % of control levels, and carotenoid levels were restored to control levels. 3.4 LEVELS OF CATALASE (CAT) AND PEROXI- DASE (POD) ENZYMES The obtained data showed that B. subtilis augment- ed the levels of the antioxidant enzymes CAT and POD significantly in F. oxysporum-treated plantlets, about 31.2 % and 5.2 %, respectively, compared to inoculated plantlets with F. oxysporum alone (Fig.3). Plantlets in- oculated with F. oxysporum had lower levels of catalase (CAT) and peroxidase (POD) enzyme activity than those in the control. The data analyses showed highly signifi- cant enhancement of the antioxidant enzyme activities (CAT and POD) as a result of treating the plantlets with the B. subtilis strain in the presence and absence of the pathogenic fungus F. oxysporum. 3.5 EFFECT ON TOTAL PROLINE CONTENT Data from our study revealed that total proline was reduced in response to inoculation with F. oxyspo- rum compared with un-inoculated plantlets (control). Infection by F. oxysporum reduced proline levels, but co-application of B. subtilis restored proline to normal levels seen in the control (Fig.4). Data from the ANO- VA table showed that the value of total proline content was increased in plantlets inoculated with B. subtilis in the presence of F. oxysporum by about 47.6 % compared with plantlets inoculated with F. oxysporum alone. F. ox- ysporum reduced total proline content by approximately 48.2 % (compared to the control (healthy). 4 DISCUSSION 4.1 PATHOGENICITY TESTS The results of the pathogenicity test showed that Fusarium isolates were highly pathogenic to date palm plantlets. Species of Fusarium are known to produce tox- ins such as fumosisin, fusaric acid, fusaproliferin, fusa- rin, zearalenone, and others, which aid in the attack and parasitism of plant hosts (Hernandez et al., 2010). Ac- cording to El Modafar & El Bostani (2000), F. oxysporum releases cell wall hydrolytic enzymes that hydrolyze host ingredients, allowing the pathogen to move into root tis- sues, and these enzymes are linked with disease progres- sion. Our results agree with those of other researchers, showing that date palm trees and offshoots are attacked by many soil-borne pathogenic fungi capable of causing severe losses and degradation (Arafat et al., 2012; Baraka et al., 2011), including F. oxysporum, F. solani, F. monili- forme, F. smitectum, R. solani, and Thielaviopsis paradoxa (De Seynes) Höhn.(Ahmed, 2018; El-Morsi et al., 2012; Maitlo et al., 2013). Fig. 2: Effectiveness of B. subtilis in presence and absence of F. oxysporum on: A: total chlorophyll content and, B: Total carotenoid content.(Each value is the mean of five replicates, means in the columns followed by the different letters are significantly different at p < 0.05 test) Acta agriculturae Slovenica, 118/3 – 20226 N. S. JASSIM and M. A. ATI 4.2 EFFECT OF B. SUBTILIS AGAINST THE GROWTH OF F. OXYSPORUM IN VITRO The results of a double culture in Petri dishes con- taining PDA medium revealed that Bacillus subtilis has the ability to suppress radial mycelial growth to a large extent. Bacillus subtilis suppresses pathogen growth di- rectly by the synthesis of many secondary metabolites, such as hormones, cell wall degrading enzymes, and an- tioxidants. Our results were in agreement with studies by Cao et al. (2012), who observed that B. subtilis pro- duces many antibiotic compounds, including fengycin, iturin, and bacillomycin, and these compounds inhibit mycelial growth and spore germination of the fungal pathogen F. oxysporum. Jassim (2015) showed that B. subtilis completely inhibited the mycelial growth of the pathogen F. moniliforme in the PDA medium. Siala et al. (2016) found that an endophytic strain of B. subtilis slowed the growth of Fusarium species on PDA and that this strain produced proteases, contributing to the deg- radation of the cell walls of fungal pathogens. Isolate B. subtilus 174 has been shown to have strong biocontrol activity and to cause significant suppression of disease severity in Fusarium wilt disease in tomato plants caused by F. oxysporum, likely due to induced resistance (Akarm and Anjum, 2011). Several important plant pathogens, including Fusarium sp. (Zhao et al., 2013), Rhizoctonia solani (Kumar et al., 2012), and Verticillium dahliae Kleb (Li et al., 2013), can be suppressed by B. subtilis. Bhusal & Mmbaga (2020) examined three Bacillus spp. isolates as biological control agents against the pathogen Phy- tophthora capsici Leonian. These isolates suppressed the mycelial growth of P. capsici in vitro and reduced the in- cidence of disease in plants grown in soil infested with Phytophthora inoculum under greenhouse conditions. 4.3 EFFECTS ON TOTAL CHLOROPHYLL AND TOTAL CAROTENOIDS Our results show that plantlets inoculated with F. oxysporum reduced photosynthetic activity (mostly pho- tosynthesis), perhaps due to reduced levels of key pro- teins in the thylakoid membranes and/or the reduction of RuBPC-specific leaf soluble proteins (Weintraub & Jones, Fig. 3: Effectiveness of Bacillus subtilis in presence and absence of Fusarium oxysporum on catalase enzyme, and peroxidase en- zyme (Each value is mean of five replicates, means in the columns followed by the different letters are significantly different at p < 0.05 test) Fig. 4: Effectiveness of Bacillus subtilis in the presence or and absence of Fusarium oxysporum on proline content (Each value is mean of five replicates: means in the columns followed by the different letters are significantly different at p < 0.05 test) Acta agriculturae Slovenica, 118/3 – 2022 7 Efficacy of Bacillus subtilis ..., in suppressing Fusarium oxysporum ..., the causal agent of root rot of date palm offshoots ... in Iraq 2010). Huang et al. (2012) found that stress and infec- tion by fungi led to a decline in leaf chlorophyll that was due to increased chlorophyllase activity, increased active oxygen products, and destabilization of ionic equilibri- um. Reduction in chlorophyll may be due to the toxic ac- tion of compounds released by pathogenic fungus; such compounds lead to necrosis and chlorosis due to their toxic effects on chloroplasts in the host cells (Bashan et al., 1995). The reduction in the absorption of minerals (e.g., magnesium) required for chlorophyll synthesis can also indirectly reduce the chlorophyll content in plants infected by pathogens (Murkute et al., 2006; Sheng et al., 2008). Nevertheless, in response to F. oxysporum infec- tion, B. subtilis greatly increased the production of anti- oxidant enzymes, several secondary metabolites, growth regulator hormones, and enzymes to degrade cell walls (Hashem et al., 2019). According to Cazorla et al. (2007), because B. subtilis can emit antibiotics and hydrolytic en- zymes, it may change its environment for the better and develop resistant endospores to survive in harsh environ- ments. In mung beans, B. subtilis alleviated symptoms of infection by the fungal pathogen Macrophomina phaseo- lina (Tassi) Goid., reducing charcoal rot disease and en- hancing total chlorophyll (Hashem et al., 2017). Shi et al. (2010) showed that B. subtilis elevated the photosynthetic capacity and total content of chlorophyll of sugar beet, resulting in a consequently enhanced synthesis of car- bohydrates. In date palms, B. subtilis increased the total chlorophyll and total carotenoid in plants under abiotic stress and prevented the harmful effects of stress (Jassim et al., 2020). F. oxysporum infection in date palm plantlets generates reactive oxygen species (ROS), such as radicals of superoxide(O-2), hydroxyl (OH-), and molecules of hydrogen peroxide (H2O2). The accumulation of ROS in infected plant cells causes major and important injuries in all plant functions (Manhas & Kaur, 2016). 4.4 LEVELS OF CATALASE (CAT) AND PEROXI- DASE (POD) ENZYMES Antioxidant enzymes mitigate the damage level from reactive oxygen species (ROS) in plants, which is a source of oxidative stress from pathogen infection (As- ada, 1999). Catalase (CAT) is one of the most prevalent detoxifying enzymes in plants, and it plays an important role in regulating ROS generation and buildup. In con- trast, the catalase enzyme converts H2O2 into H2O and O2, so any increase in CAT activity is more likely to re- sult in a decrease in H2O2 generation, as shown at low concentration in the plantlet treatments inoculated with both B. subtilis in the presence or absence of F. oxyspo- rum. At high concentrations, plantlets inoculated with F. oxysporum alone showed the opposite effect, with an increase in H2O2 production accompanying a decrease in CAT activity. The increase in antioxidant enzyme activ- ity levels enhances disease resistance in the host plants (Shi et al., 2010). Selvaraj & Chellappan (2006) explain that POD enzymes play an important role in producing ethylene, resisting disease, promoting wound healing, and lignin formation, as well as in building cell walls by changing polymerizing hydroxyl and methoxy cinnamic alcohols into lignin. Xie et al. (2021) found that B.subtilis strain LZ88 induced plant resistance with an enhanced expression in tobacco leaves of the antioxidant enzymes peroxi- dase (POD) and polyphenol oxidase (PPO). B. subtilis induced systemic resistance and alleviated the harmful effects of pathogens by increasing the activity of antioxi- dant enzymes on plants (Hashem et al., 2019).In a recent study on date palms, Jassim et al. (2020) showed that B. subtilis protected date palms from the harmful effects of salt stress and increased the activity of antioxidant en- zymes (CAT and POD). In our study we found that F. oxysporum, in the absence of B. subtilis, increased the lev- els of CAT and POD activity, which confirms that oxida- tive damage is associated with ROS scavenging, while the B. subtilis bacteria inhibited the activity of the pathogen fungus F. oxysporum and significantly reduced the nega- tive effects on the vital processes in the plant, which re- flected its effect on the plant, and that led to mitigation in the antioxidant enzymes compared to the pathogen treatment alone. 4.5 EFFECT ON TOTAL PROLINE CONTENT There were significant differences in the total pro- line content among plants treated with B. subtilis and F. oxysporum compared with F. oxysporum. This can be at- tributed to the ability of B. subtilis to limit the activity of the fungus pathogen through the ability to excrete anti- biotics such as subtilin, bacteriocins, iturins, and bacilo- mycin, which act to inhibit the growth of fungus (Meena & Kanwar, 2015; Wang et al., 2015). Proline is essential in plants and accumulates during pathogen attacks in a variety of species (Rehman et al., 2014). According to the study by Wang et al. (2012), the inoculation of cucumber plants (Cucumis sativa L.) with a mixture of three plant growth promoting rhizobacteria (PGPR) strains (B. ce- reus AR156, B. subtilis SM21, and Serratia sp. XY21) el- evated leaf proline content by 3–4 fold compared to un- inoculated plants. Proline catabolism plays an essential role in controlling the cellular ROS balance and can also control various other regulatory pathways. It has also Acta agriculturae Slovenica, 118/3 – 20228 N. S. JASSIM and M. A. ATI been demonstrated that proline accumulation activates the pathways of alternative detoxification by the mainte- nance and duration of ROS-eliminating enzymes (Hayat et al., 2012). Plants inoculated with B. subtilis showed less damage from the harmful effects of M. phaseoline and an increase in the accumulation of sugars, proline, and free amino acids, which are considered to be the key osmolytes for sustaining the content of cellular water to protect the structures and functions of cell organelles (Hashem et al., 2017). 5 CONCLUSION Pathogenicity studies showed that Fusarium ox- ysporum is the most causative agent of root rot disease in date palm plantlets, with a severity index of 82.16 % for root rot. The Bacillus subtilis strain reduced the mycelial growth of F. oxysporum in vitro as well as in vivo. Our results show that B. subtilis is a beneficial microorganism for controlling the root rot disease of date palm plantlets caused by F. oxysporum. B. subtilis inhibited oxidative damage caused by the pathogenic fungus and signifi- cantly improved all measured physiological characteris- tics that were adversely affected by the fungus pathogen. 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BioControl, 58, 283–292. htt- ps://doi.org/10.1007/s10526-012-9496-5 Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.1208 Original research article / izvirni znanstveni članek Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Mawadda MEHERAT 1, Mohammad SHATNAWI 1, Rida SHIBLI 2, Tamara QUDAH 3, 4, Saida ABU MALLOH 3, Tamadour AL-QUDAH 5 Received July 03, 2019; accepted July 05, 2022. Delo je prispelo 20. julija 2019, sprejeto 5. julija 2022 1 Faculty of Agricultural technology, Al Balqa Applied University, Salt, Jordan 2 Al Ahliyya Amman University (and The University of Jordan), Faculty of Agricultural technology, Amman, Jordan 3 Hamdi Mango Centre for Scientific Research, University of Jordan, Amman 4 Corresponding author,e-mail: t.alqudah@ju.edu.jo 5 Department of Nutrition and Food Technology, Faculty of Agriculture, Mutah University, Karak, Jordan Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Abstract: Tetragonolobus palaestinus Bioss (Aljalaton) is one of the Jordanian medicinal plants that can be used to treat stomach pain and some infections. This study was done in order to establish optimal in vitro propagation method for T. palaestinus. Factors of in vitro shooting, rooting, and ac- climatization of the in vitro Tetragonolobus palaestinus seed- lings were studied using different growth regulators. For in vitro shooting, different cytokinins including benzylamino purine (BAP), kinetin, TDZ, and zeatin were used in increas- ing concentrations (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, and 2.0 mg l-1). Using benzylamino purine (BAP produced a maximum of 2.0 shoots/explants on Murashige and Skoog (MS) medium supplemented with 0.3 mg l-1. Moreover, the effect of different concentrations of IBA (indole-3-butyric acid), IAA (indole- 3-acetic acid), andnaphthalene acetic acid (NAA) was evalu- ated for in vitro rooting. The highest number of roots (4.06 roots/explant) was obtained on MS medium supplemented with 0.3 mg l-1 IBA. All of the plants (100 %) were grown nor- mally after the acclimatization process. Based on these results simple protocol of T. palaestinus in vitro culture was optimized for the first time which can be utilized to do more studies on cell culture and production of active secondary metabolites. Key words: acclimatization;, in vitro; shoot multiplica- tion;, rooting Klonsko razmnoževanje vrste Tetragonolobus palaestinus Bioss: jordanske zdravilne rastline Izvleček: Vrsta Tetragonolobus palaestinus Bioss (Alja- laton) je jordanska zdravilna rastlina, ki se lahko uporablja za blaženje bolečin v želodcu in zdravljenje nekaterih okužb. Namen raziskave je bil vzpostaviti optimalen način in vitro razmnoževanja te rastline. Preučevani so bili dejavniki in vi- tro gojenja (vkoreninjenja, tvorbe poganjkov) in aklimatizacije sadik te rastline z uporabo različni rastnih regulatorjev. Za in vitro tvorbo poganjkov so bili uporabljeni različni citokinini in sicer benzilamino purin (BAP), kinetin (TDZ) in zeatin v naraščajoči koncentraciji (0,0; 0,3; 0,6; 0,9; 1,2; 1,5 in 2,0 mg.l- 1). Uporaba benzilamino purina (0,3 mg l-1) je dala pri gojenju na Murashige in Skoog (MS) gojišču največ poganjkov, dva na izseček. Učinek različnih koncentracij rastnih regulator- jev (IBA-indol-3-maslene kisline, IAA -indol-3-ocetne kisline in naftalen ocetne kisline NAA) je bil ovrednoten pri in vitro vkoreninjenju. Največje število korenin (4,06 korenin/izseček) je bilo dobljeno na MS gojišču, z dodatkom 0,3 mg l-1 IBA. Vse rastline (100 %) so po obdobju aklimatizacije rastle normalno. Na osnovi teh rezultatov je bil prvič optimiziran enostaven pro- tokol za in vitro gojenje te vrste, ki bi se lahko uporabil v na- daljnih raziskavah na celičnih kulturah in produkciji aktivnih sekundarnih metabolitov. Ključne besede: aklimatizacija; in vitro; namnoževanje poganjkov; vkoreninjanje 2 Acta agriculturae Slovenica, 118/3 – 2022 M. MEHERAT et al. 1 INTRODUCTION Tetragonolobus palaestinus is a wild plant from the Fabaceae family. Its natural habitat can be found in the northern parts of Jordan on the rocks and meadow ar- eas (Afifi & Abu-Irmaileh, 2000). T. palaestinus is a her- baceous plant, it starts to grow after seasonal rainfall in the winter, and has a red flower and a small fruit (pod) which can be served as fresh or boiled to eat, it is well-known among the people as Aljlatoun (Al-Ka- raki, 2000). Most Jordanian wild plants are becoming endangered due to the expansion of urban and rural settlements, uncontrolled deforestation, illegal collec- tion, industrial pollution, and low level of environmen- tal awareness (Al-Bakri et al., 2011). Therefore, to solve such problem, alternative methods for massive plant propagation like plant tissue culture techniques and other biotechnological approaches are used for pro- ducing medicinal plants, isolating medicinal secondary products, conserving and rapid propagating valuable, rare, and endangered plant species (Arafeh et al., 2006; Al-Mahmood et al., 2012; Qrunfleh et al., 2013; Shatna- wi , 2013). Propagation methods of T. palaestinus using seeds are not preferred due to the low germination per- centage (Al-Karaki, 2000). In vitro culture of T. palaestinus can solve propaga- tion problems as it guarantees mass production of plant material without compromising the natural resources and it also improves and conserves this plant (Alenizi et al., 2020; Ebrahim et al., 2007; Shatnawi 2006, Shib- li et al., 2018). The use of in vitro culture technique is the best solution to overcome T. palaestinus propaga- tion problems and can also enhance mass production without threatening the natural resources (Shibli et al., 2003; Makhadmeh & Shatnawi, 2008; Shatnawi et al., 2011, Al Qudah et al., 2011). Also, it is an important tool in both basic and applied studies and for commercial applications (Arafeh et al., 2006; Ahmad et al., 2010). Using micropropagation; the plant developed from this technique is true to type or genetically uniform with the mother plant (Shibli et al., 2003). Production of a large number of genetically uniform disease-free plants is known to be a reliable technique system. The con- ventional method of propagation is done by vegeta- tive methods through root suckers or terminal cutting which is classified as very slow (George et al., 2008). In vitro propagation plays a major role in the rapid production of disease-free planting material of newly improved varieties year rounded basis (Ebrahim et al., 2007; Shatnawi 2006, Shatnawi et al., 2011). Shoot tip culture is a relatively simple in vitro technique for the rapid propagation of selected pathogen-free plant ma- terials. Therefore, many simple protocols have been de- veloped for the rapid multiplication of newly released commercially important genotypes through apical meristem cultures. Successful commercial micropro- pagation protocol depends on successful rooting and acclimatization of in vitro derived plantlets (Ebrahim et al., 2007; Shatnawi 2006, Shatnawi et al., 2011). Till now; there are no available data on the in vitro propagation of T. palaestinus. So, this study was initiated to develop an applicable and simple protocol for in vitro establish- ment, multiplication, rooting, and acclimatization of T. palaestinus. 2 MATERIAL AND METHODS 2.1 ESTABLISHMENT OF IN VITRO CULTURE Plant seeds of wild T. palaestinus were collected in mid of April in north Jordan – “Al-Sareeh, Irbid” (32.3306° N latitude and 35.8951° E Longitude). First- ly, surface sterilization of seeds was done by wash- ing seeds with tap water for 5 min. After that, seeds were immersed in (4  %) sodium hypochlorite for 15 min. The following sterilization steps were performed under sterile conditions in a laminar air flow cham- ber; the seeds were washed 3 times in sterile distilled water, then soaked in 70  % ethanol solution for 30 s and washed several times with sterile distilled water. Seeds were cultured on the surface of hormone free Murashige and Skoog (MS) medium (1962) inside Petri dishes (five seeds/ Petri dish). Murashige and Skoog (MS) medium was supplemented with vitamins and 30 g l-1of sucrose. After the final volume of the MS media was adjusted to 1 l and the pH to 5.75, 8 g of agar was added to the media mixture with constant stirring and heating until the agar was completely dissolved. After that, 100 ml of medium was poured into Erlenmeyer flasks. Then flasks were plugged and autoclaved at 121 ºC for 15 min. After that cultures of seeds were kept in a growth room in the dark and moderate temperature 24 ± 2 ºC for four weeks until full germination. The germinated seedlings were transferred to light condi- tions in the growth room under the light regime (16/8 h (light/dark) and a light intensity of 50 μmol m-2s-1. Afterward, cultures were transferred to the new medi- um and further grown. Then, cultures were transferred to MS medium provided with growth regulators, i.e. 0.3 mg l-1 benzyl amino purine (BAP) and 0.05 mg l-1 naphthalene acetic acid (NAA) with 30 g l-1 sucrose, to increase the growth of the cultures. 3 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Acta agriculturae Slovenica, 118/3 – 2022 2.2 SHOOT PROLIFERATION Microshoots of 10 mm in length, were treated with different concentrations of cytokinins for shoot prolif- eration experiments. MS media were supplemented with 0.0, 0.3, 0.6, 0.9, 1.2, 1.5 or 2.0 mg l-1 of BAP, Ki- netin, Thidiazuron (TDZ) or Zeatin. Five replications with three microshoots were used for each treatment. Data were collected after five weeks for the microshoots growth parameters as shown in Table 1. 2.3 ROOT FORMATION OF IN VITRO CULTURES Microshoots, 10 mm in length, were treated with different concentrations of auxins. For root formation MS media were supplemented with 0.0, 0.3, 0.6, 1.2, 1.5 or 2.0 mg l-1 of indole-3-butyric acid (IBA), indole ace- tic acid (IAA) or naphthalene acetic acid (NAA). Ten replications were used with one microshoot / replicate. Data were collected for the number of axillary shoots/ explant, shoot length, root length, and rooting (%) after five weeks. 2.4 ACCLIMATIZATION The fully in vitro rooted microshoots were hard- ened gradually from in vitro tubes. Firstly, the tubes plugs were removed for three days and the cultures were left in the growth room. After that microshoots were gently transferred from test tubes and washed un- til all agar residues were removed and grown in plastic pots that have a suitable mixture of (1 peat : 3 perlite). Cultures were covered with perforated plastic bags for 3 days with continuous wetting with sterile distilled wa- ter. After that, plastic bags were removed and the pots were left for extra 2 weeks under growth room condi- tions with continuous wetting. At the end of the accli- matization experiment, the survival percentage of the acclimatized plants was registered. 2.5 EXPERIMENTAL DESIGN The completely randomized design (CRD) was used with all the experiments. Data were analyzed in SPSS Software with Tukey HSD Multiple Range test at p ≤ 0.05. Means and standards error of means were cal- culated. 3 RESULTS AND DISCUSSION In this study, significant differences in the shoots growth parameters were obtained using BAP, Kinetin, TDZ, or zeatin at different concentrations. At 0.3 mg l-1 of BAP, maximum shoots numbers (2.0 shoot per ex- plants) were obtained (Table 1 and Fig. 1). Additionally, the length of shoots and the number of leaves on aver- age increase up to two- fold (30.0 mm and 12.2 leaves, respectively) in comparison with controls (18.0 mm, 7.0 leaves, respectively). At 0.9 mg l-1 BAP, a maximum fresh mass of 112.0 mg was obtained and it was 1.7-fold higher compared to controls (66.0 mg). Shoot length increased up to 30 mm and 26 mm at the lowest and the highest concentrations of BAP (0.3, 2.0 mg l-1; re- spectively), comparing with the control (18.0 mm). But; at 1.2 and 1.5 mg l-1 BAP the length of the shoots was significantly smaller compared to the length of shoots in medium with 0.3 mg l-1 BAP. Furthermore; BAP at (0.3 and 0.6 mg l-1) concentrations resulted in the highest number of leaves 12 (leaves). One of the best cytokinins that can be used to induce in vitro shoot formation is 6-benzylaminopurine (Singh et al., 2019). Thảo et al. (2013) reported the highest shoot induction in common bean (Phaseolus vulgaris L.) when BAP was used with NAA in media. Similarly, in Securidaca longipedunculata (Fresen) the combinations between BAP and IBA at (1.5 mg and 0.1 mg l-1; respectively) produced a better short number and length per explant than other growth regulator combinations (Lijalem and Feyissa, 2020). Besides that, BAP has been reported in many previous studies for shoot multiplications. For example; Trichosanthes dioica Roxb. was established from nodal explants on MS medium containing 1.0 mg l-1 BAP (Tiwari et al., 2010). BAP also; gave the best re- sults for Prosopis cineraria (L.) Druce in vitro establish- ment (Kumar and Singh, 2010). BAP gave the best out- come for shoot induction in the in vitro grain legume, Phaseolus vulgaris (Malik and Saxena, 1992 ) Similarly, kinetin at 0.3 mg l-1 had increased the shoot length up to 32.0 mm which was 1.7-fold longer than control (18.0 mm) (Table 1, Fig 1). Moreover, maxi- mum dry and fresh mass (140 and 114 mg; respectively) of in vitro Tetragonolobus palaestinus explants were ob- tained at 0.3 g l-1 of kinetin. Increasing concentrations of kinetin inhibited the growth of the shoots in length and their appearance was swelling and short. Kinetin induced expansion of growth by swelling rather than elongation, this was confirmed previously by Naeem (2004). Ahmadi et al. (2011) reported that using kinetin at 2.0 mg l-1 increased the in vitro shoot induction in 4 M. MEHERAT et al. Acta agriculturae Slovenica, 118/3 – 2022 Matthiola incana (L.) W.T.Aiton. In Moringa stenopetala (Baker f.) Cufod.; maximum number of shoots per ex- plant (3.43 ± 1.41) and 7.97 ± 4.18 leaves per explant were obtained on MS medium containing 0.5 mg l-1 ki- netin with 0.01 mg l-1 NAA. (Adugna et al., 2020). The addition of 0.3 mg l-1 TDZ to MS medium re- sulted in longer shoots (32.0 mm) compared to con- trols (18.0 mm), and the highest number of leaves per explant (14.8 leaves per explant) was obtained on MS medium supplemented with 1.2 mg l-1 TDZ. The Growth regulator TDZ had been used in previous stud- ies in order to promote in vitro propagation of differ- ent plants species of the Fabaceae family; such as, in vitro Psophocarpus tetragonolobus (L.) D.C. (Singh et al., 2014); and common bean (Veltcheva et al., 2005). The results from the present work demonstrated that TDZ at low concentration was effective compared to other cytokinins (Table 1). However, it was found to be effective at low concentration. Low concentrations of TDZ (0.01 mg l−1) were the most appropriate for shoot regeneration in Abelmoschus moschatus Medik (Sharma & Shahzad, 2008). The effect of TDZ on growth param- eters is not entirely clear, and more studies are needed to understand its role in plant tissue cultures. (Ugand- har et al., 2012). TDZ in combination with NAA pro- duced relatively shorter shoots when used with Secu- ridaca longipedunculata (Fresen) (Lijalem and Feyissa, 2020). Furthermore; TDZ had been reported to have an adverse effects with Vitex trifolia L. (Ahmed and Anis, 2012). When zeatin was used, a maximum number of shoots (1.4 shoots per explant) was obtained on MS medi- um supplemented with 0.3, 0.9, and 1.2 mg l-1 Zeatin (Ta- ble 1). While the maximum shoot length (28.0 mm) was produced on MS medium supplemented with 0.9 mg l-1 Zeatin. On the other hand, Vikram et al. ( 2012) reported that Zeatin at 1.2 mg l-1 produced a maximum number of multiple shoot formation in Lycopersicum esculentum L..  In addition, a highly efficient organogenesis protocol for in vitro regeneration of eggplant was developed using zeatin (García-Fortea et al., 2020). This may be due to that, zeatin suppress apical dominance which leads to in- crease numbers of multiple shoots and reduce the length of the shoot. 3.1 IN VITRO ROOTING The in vitro rooting of T. palaestnius was signifi- cantly induced at a concentration of 0.3 mg l-1 of IBA with (4.06 roots/microshoot). The rooting percentage was 40 % with 3.33 mm/root long at 0.3 mg l-1 of IBA. Meanwhile; control and other concentrations of IBA showed lower in vitro rooting; as we can show in (Table 2 & Fig 2). Low concentrations of IBA (1.0 mg l-1) also resulted in the highest in vitro rooting in Cicer micro- phyllum Benth. (Singh et al., 2019) and in common bean (Phaseolus vulgaris L.) (Thảo et al., 2013). For rooting with IAA growth regulator; the maxi- mum number of roots per microshoots was (2.14 roots/ explant) obtained at 1.2 mg l-1 IAA with a maximum root length of 2.94 mm. The maximum root percentage (30 %) was also recorded on media supplemented with 1.2 mg l1 IAA. Using 0.3 mg l-1 NAA resulted in 0.5 developed root length of 1.94 mm (Table 2). No callus occurred at mi- croshoots bases. The current study showed that auxin is essential for the induction of root formation of in vitro T. palaestnius cultures. This is because auxin exerts a primary role in root formation by its involvement in successive and inter- dependent phases (Mineo, 1990). The rooting of legumi- nous species is dependent on the auxin type (Dewir et al., Figure 1: In vitro shoot formation of Tetragonolobus palaestinus after five weeks. A) MS with 0.3 mg l-1 (BAP). B) MS with 0.6 mg l-1 (BAP). C) MS medium with 0.3 mg l-1 Kinetin. Bars represent 1.0 cm Acta agriculturae Slovenica, 118/3 – 2022 5 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant 2016). Using IBA or NAA at a low concentrations such as 0.3 mg l-1 increased cell division and root primordial formation and proved to enhanced rooting percentage, number of roots per rooted explants and, root length as compared to IAA or IBA. Some studies found that NAA was the best rooting auxin in Vigna mungo (L.) Hepper (Mony et al., 2010). Furthermore; different legumes of the Fabaceae family have been in vitro rooted using dif- ferent auxins types. For example; the in vitro rooting in Clitoria ternatea L. which is known as the butterfly pea plant was obtained by the addition of 1.50 mg l-1 NAA with the highest number of adventitious roots (12.86 ± 2.14) (Lee et al., 2021). While IBA had been used in the in vitro rooting of Lotononis bainesii Baker (Fabaceae) at the concentration of 0.049 μM IBA (Vidoz et al., 2012). Also, 83 % of in vitro rooting in Thermopsis turcica Kit Tan, Vural & Küçük. (Fabaceae) was attained on pulsed- IBA treated shoots (Cenkci et al., 2008). The growth reg- ulator IAA, was used at 0.005–0.01 mg l-1 to induce in vitro rooting in Psoralea corylifolia L.(Fabaceae) which is Dry mass/five explants (mg) Fresh mass/five explants (mg) Number of leaves/ explant Shoot length (mm) Number of axillary shoots/explant Concentrations mg L-1 34.0 ± 2.4 c66.0 ± 2.0 b7.0 ± 0.32 c18.0 ± 1.22 c1.0 ± 0.0 bControl 0.0 BAP 34.0 ± 2.4 c66.0 ± 2.0 b12.2 ± 0.74 a30.0 ± 3.1 a2.0 ± 0.32 a0.3 56.0 ± 5.0 b90.0 ± 2.0 ab12.0 ± 0.44 a28.0 ± 2.0 ab1.6 ± 0.20 ab0.6 106.9 ± 15.0 a112.0 ± 22.0 a11.2 ± 0.37 ab26.0 ± 2.4 ab1.6 ± 0.40 ab0.9 52.0 ± 5.0 b92.0 ± 4.0 ab10.8 ± 0.37 b22.0 ± 2.0 b1.0 ± 0.24 b1.2 20.0 ± 5.0 d52.0 ± 0.0 c11.4 ± 0.51 ab22.0 ± 2.0 b1.6 ± 0.00 ab1.5 62.0 ± 5.0 b88.0 ± 17.6 ab11.8 ± 0.37 ab26.0 ± 2.4 ab1.8 ± 0.20 ab2.0 Kinetin 114.0 ± 25.0 a140.0 ± 24.4 a12.4 ± 1.12 a32.0 ± 3.7 a1.2 ± 0.20 a0.3 64.0 ± 9.21 ab96.0 ± 14.7 ab11.8 ± 0.48 a22.0 ± 0.0 b1.2 ± 0.20 a0.6 66.0 ± 5.12 ab92.0 ± 3.7 ab11.8 ± 0.48 a24.0 ± 2.4 ab1.2 ± 0.2 a0.9 20.0 ± 3.1 c44.0 ± 4.0 d8.0 ± 0.20 c15.0 ± 0.0 d1.0 ± 0.0 b1.2 66.0 ± 4.0 ab88.0 ± 2.0 b10.2 ± 0.37 b20.0 ± 0.0 b1.4 ± 0.24 a1.5 14.0 ± 2.0 c30.0 ± 0.0 d4.0 ± 0.24 d15.0 ± 0.0 d1.0 ± 0.0 b2.0 TDZ 58.0 ± 7.3 c84.0 ± 4.4 b10.8 ± 0.37 b32.0 ± 2.0 a1.6 ± 0.24 a0.3 64.0 ± 5.1 b90.0 ± 4.4 ab12.8 ± 0.58 ab20.0 ± 0.0 ab1.8 ± 0.37 a0.6 85.0 ± 5.8 ab84.0 ± 5.1 b10.8 ± 0.37 b22.0 ± 2.0 b1.2 ± 0.20 b0.9 112.0 ± 24.1 a136.0 ± 26.1 a14.8 ± 0.24 a30.0 ± 4.4 a1.8 ± 0.37 a1.2 60.0 ± 5.4 c92.0 ± 2.0 ab10.6 ± 0.40 b24.0 ± 2.4 ab1.6 ± 0.24 a1.5 66.0 ± 6.7 b90.0 ± 6.7 ab11.8 ± 0.41 b22.0 ± 2.0 b1.6 ± 0.24 a2.0 Zeatin 106.9 ± 24.0 a66.0 ± 1.8 b15.4 ± 0.81a26.0 ± 2.45 a1.4 ± 0.25 a0.3 56.0 ± 4.8 b92.0 ± 2.0 ab10.4 ± 0.24 ab20.0 ± 0.0 ab1.2 ± 0.20 ab0.6 74.0 ± 5.1 ab112.0 ± 22.0 a15.4 ± 1.02 a28.0 ± 3.74 a1.4 ±0.40 a0.9 52.0 ± 1.9 b92.0 ± 5.0 ab10.0 ± 0.95 ab24.0 ± 2.44 ab1.4 ± 0.24 a1.2 20.0 ± 6.0 d52.0 ± 2.0 c7.6 ± 0.51 c11.0 ± 1.00 c1.0 ± 0.00 b1.5 62.0 ± 3.0 b88.0 ± 4.0 ab11.60 ± 0.88 ab20.0 ± 00 ab1.2 ± 0.20 ab2.0 Table 1: The effect of different concentrations of the cytokinins on in vitro grown Tetragonolobus palaestinus after five weeks of in- cubations. *Values represent means ± standard error. *Means within the column for each growth regulator having different letters are significantly different according to Tukey HSD at p ≤ 0.05 Acta agriculturae Slovenica, 118/3 – 20226 M. MEHERAT et al. a rare and endangered herbaceous medicinal plant (Rout et al., 2010). We can see from this, that in vitro rooting depend on plant species and the type of auxin used. 3.2 ACCLIMATIZATION The acclimatization process of Tetragonolobus pa- laestinus microshoots was proved its ability to the pro- duction of healthy acclimatized micro shoots. The mix- ture of (Peatmoss: Perlite) was suitable for roots growth and new leaves were formed after 2 weeks (Fig 3). All of the rooted plantlets were survived after acclimatiza- tion process after acclimatization process. No variations were observed visually among the acclimatized plantlets as shown in Fig (3). A lower survival rate of 65 % was obtained in C. microphyllum using different potting cul- ture mixture ( garden soil, vermiculite, and vermicom- post (1:1:1) (Singh et al., 2019) Plantlet needs an acclimatization period and this could be due to the effect of tissue differentiation, growth, and development (Quisen, 2013). The acclimatized plantlet may be affected by the change in environmental conditions during acclimatization, which may be due to the short acclimatization period evaluated in this study (Shatnawi., 2013). Different plants of the Fabaceae fam- ily have been successfully in vitro rooted and acclima- Rooting % Root length (mm) Number of roots / explant Shoot length (mm) Number of axillary shoots/explant Concentrations mg.l-1 20% 2.00 ± 0.70 ab 1.50 ± 0.48 b18.07 ± 7.46 ab 1.01 ± 0.23* b Control 0.0 IBA 40% 3.33 ± 0.90 a 4.06 ± 0.67 a 19.0 ± 9.5 a 1.16 ± 0.32 a 0.3 20% 1.44 ± 1.33b 3.17 ± 0.52 b 17.06 ± 7.06 ab 1.09 ± 0.18 a 0.6 30% 2.94 ± 1.86 a 2.67 ± 0.78 ab 12.28 ± 4.21 b 1.04 ± 0.15 a 1.2 30% 1.94 ± 2.83 b 2.06 ± 0.78 ab 13.33 ± 7.13 b 1.04 ± 0.16 a1.5 20% 3.17 ± 0.86 a 2.94 ± 0.81 ab 12.04 ± 5.29 b 1.06 ± 0.17 a 2.0 IAA 20% 0.89 ± 0.40 b0.56 ± 0.24 b13.78 ± 4.41 ab 1.00 ± 0.22 b 0.3 27% 2.61 ± 0.84 a 0.50 ± 0.20 b 12.61 ± 3.45 b 1.22 ± 0.18 a 0.6 30% 2.94 ± 1.50 a 2.14 ± 0.17 a 19.33 ± 4.02 a 1.06 ± 0.16 ab 1.2 26% 1.83 ± 1.54 ab 0.57 ± 0.60 b 12.28 ± 4.09 b 1.09 ± 0.12 ab 1.5 20% 2.39 ± 1.11 a 1.39 ± 0.41 ab 12.61 ± 3.38 b 1.02 ± 0.13 b 2.0 NAA 30% 0.89 ± 0.40 c 0.56 ± 0.24 a 17.00 ± 4.41 ab 1.00 ± 0.22 b 0.3 27% 1.61 ± 0.84 ab0.50 ± 0.20 a 18.01 ± 3.45 a 1.06 ± 0.18 ab 0.6 20% 1.94 ± 1.50 a 0.44 ± 0.17 b 16.03 ± 4.02 b 1.01 ± 0.16 b 1.2 26% 1.83 ± 1.54 a 0.17 ± 0.60 b 17.08 ± 4.09 ab 1.09 ± 0.12 ab 1.5 30% 1.39 ± 1.11 b 0.39 ± 0.41 ab 17.01 ± 3.38 ab 1.02 ± 0.13 b 2.0 Table 2: The effect of different auxin concentration on in vitro root formation of Tetragonolobus palaestinus after five weeks growth period. *Values represent means ± standard error. *Means within the column for each growth regulator having different letters are significantly different according to Tukey HSD at p ≤ 0.05 Figure 2: The effect of 0.3 mg l-1 indole-3-butyric acid (IBA) on in vitro growth of Tetragonolobus palaestinus after five weeks growth. The bar represents 1.0 cm Acta agriculturae Slovenica, 118/3 – 2022 7 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant tized. For example, the regenerated plantlets of licorice (Glycyrrhiza glabra L.; Fabaceae) were acclimatized, with a survival rate of 77.7  %, when transferred to ex vitro conditions and showed no morphological abnormalities (Shaheen, 2020). While, the in vitro seedlings of Caesal- pinia ferrea Mart. were acclimated without the presence of roots in different types of the substrate with 73.4 % surviving plantlets after 30 days of growth (Silva et al., 2018). On the other hand, in A. leiocarpa (L.A.S.Johnson ex G.J.Leach) K.R.Thiele & Ladiges plantlets the substrate composition did not affect the survival or growth of in vitro rooted plantlets during acclimatization (Haygert- Lencina it., 2017). 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Quang Trung DO 1, 2,The Anh LUU 1, 3, Minh Truong DAO 1, Quoc Nam HOANG 3, 4, Trong Tri NGUYEN 5 Received March 26, 2022; accepted July 07, 2022. Delo je prispelo 26. marca 2022, sprejeto 7. julija 2022 1 Central Institute for Natural Resources and Environmental Studies, Vietnam National University, Hanoi, Vietnam 2 Corresponding author: trungcnsinh@gmail.com 3 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam 4 Institute of Geography, Vietnam Academy of Science and Technology, Hamoi, Vietnam 5 Vietnam National University of Forestry, Hanoi, Vietnam Isolation of salt-tolerant Pseudomonas strains with potential for alleviation of salt stress in peanut plant (Arachis hypogaea L.) Abstract: Plant growth-promoting rhizobacteria (PGPR) is a promising solution to improve plant growth under salt stress. Among PGPR, Pseudomonas is a genus of bacteria that possesses a variety of mechanisms in promoting plant growth and inducing resistance to biological as well as non-biological stress. This study aimed to isolate the genus Pseudomonas from the salty-contaminated rhizosphere of plant root collecting at Nam Dinh, and also investigate their functions in promoting the growth of peanut seedlings under salty conditions. Nine Pseudomonas bacteria were isolated, but only seven of them were identified by Pseudomonas-specific primers. Two of those seven isolates, ND06 and ND09, were chosen based on their characteristics in promoting plant growth such as the produc- tion of indole-3-acetic acid (IAA), phosphate solubilization, and nitrogen fixation. In addition, both two strains also car- ried the coding gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase which plays an important role in support- ing plants to withstand various stress conditions. Especially, the ND09 strain improved the growth parameters of peanut seed- lings under normal and salty stress conditions; while the ND06 only presented the plant growth enhancement under salty stress but not in normal conditions. These results suggest the ND09 strain may be used as a biological agent for eco-friendly agricul- tural practices in the future. Key words: peanut plant; PGPR; Pseudomonas; salt stress resistance Izolacija na sol tolerantnih sevov bakterij iz rodu Pseudomo- nas s potencialom zmanševanja solnega stresa pri arašidu (Arachis hypogaea L.) Izvleček: Uporaba rast vzpodbujajočih rizobakterij (PGPR) je obetajoča rešitev za izboljšanje rasti rastlin v razme- rah solnega stresa. Med PGPR imajo bakterije iz rodu Pseudo- monas mehanizme, ki vzpodbujajo rast rastlin in povečujejo njihovo odpornost v razmerah biotičnega in abiotičnega stresa. Namen raziskave je bil izolirati bakterije iz rodu Pseudomonas iz rizosfere rastlin nabranih v zasoljenih tleh na območju Nam Dinh in preučiti njihove fukcije pri vzpodbujanju rasti sejank arašidov, rastočih v slanih tleh. Izoliranih je bilo devet vrst bak- terij iz rodu Pseudomonas, vendar je bilo samo sedem od teh potrjenih s specifičnimi primerji za rod Pseudomonas. Dva od teh sedmih izolatov, ND06 in ND09, sta bila izbrana na osnovi njunih lastnosti vzpodbujanja rasti rastlin s tvorbo indol-3-oce- tne kisline (IAA), raztaplanja fosfatov in fiksacije dušika. Doda- tno sta oba seva vsebovala gen za kodiranje 1-aminociklopro- pan-1-karboksilaze (ACC), deaminaze, ki ima pomembnmo vlogo pri podpori rastlinam za prenašanje različnih stresnih razmer. Še posebej je sev ND09 izboljšal rastne parametre se- jank arašidov v normalnih razmerah in ob solnem stresu. Med tem je sev ND06 izboljšal rast rastlin samo v razmerah solnega stresa, ne pa v normalnih razmerah. Razultati nakazujejo, da bi v prihodnosti sev ND09 lahko uporabili kot biotični aganes pri okolju prijaznem kmetovanju. Ključne besede: arašid; PGPR; Pseudomonas; odpornost na solni stres Acta agriculturae Slovenica, 118/3 – 20222 Q. T. DO et al. 1 INTRODUCTION Peanuts (Arachis hypogaea L.), a plant of high eco- nomic value in agriculture, is considered the most widely produced and consumed oilseed plant all over the world and also in Vietnam. However, peanuts are quite sensi- tive to salt stress, which causes inhibitory effects to plant development and subsequently a decrease in peanuts production (Goswami et al., 2014; Sharma et al., 2016; Zörb et al., 2019). Recently, many methods have been applied to reduce soil salinity and acidity (Upadhyay and Singh, 2015; El-Nahrawy and Yassin, 2020). Among those, the phytoremediation and bioremediation meth- ods are promising alternative approaches to retrieving salt-affected soils (Singh et al., 2015). Plant growth-promoting rhizobacteria (PGPR) are bacteria that support growth and control pathogens in plants. Especially, the PGPR inoculation presented the alleviation of salt stress in the development of various plants such as tomato, pepper, canola, bean, Arabidop- sis, and lettuce (Kang et al., 2009; Chu et al., 2019). The mechanism of plant growth stimulation of PGPR (such as Rhizobium, Azospirillum, Pseudomonas, Flavobacte- rium, Arthrobacter, and Bacillus) under saline conditions involves the biosynthesis of growth regulators such as indole-3-acetic acid (IAA); the enhancement of nutrients absorption for plants through the process of phosphate solubilization, nitrogen fixation; help plants maintain ionic balance; biosynthesis of 1-aminocyclopropane- 1-carboxylate (ACC) deaminase; inducing systemic tol- erance (IST) (Goswami et al., 2014; Chu et al., 2019). Among those, fluorescent Pseudomonas sp. is the most studied and exploited bacteria because of some advanced abilities such as an excellent root-colonizing capabil- ity and plant growth-promoting activity (Egamberdieva, 2011); is also salt tolerant and able to alleviate salt stress in plants (Shafi et al., 2017). For example, Egamberdieva (2011) reported a significant increase in shoot length (up to 50 %) of beans in salt stress (at 5.0, 7.5, and 10.0 dS m-1) when inoculated beans with P. extremorientalis TSAU20 and P. chlororaphis TSAU13. Hence, the selec- tion of native PGPRs with characteristics related to bac- terial suitability in the potential environment should be considered. In this study, we aimed to isolate the genus Pseu- domonas from the salt-contaminated rhizosphere of plants in Nam Dinh and to assess their abilities in stimu- lating peanuts’ growth under conditions of salty stress. The results suggest a promising PGPR to further exploit as a bioinoculant in the future. 2 MATERIAL AND METHODS 2.1 ISOLATION AND SCREENING OF PSEU- DOMONAS FOR SALT TOLERANCE FROM SALT-CONTAMINANT SOIL Bacteria were isolated from 18 samples of the rhizo- sphere of corn (Zea mays L.), rice (Oryza sativa L.), and peanuts (Arachis hypogaea) obtained from salt-contam- inated land in Quat Lam, Giao Thuy, Nam Dinh, Viet- nam. The 10-fold serial dilutions of the samples were plated on sterile LB agar plates supplemented with 10 % NaCl. After 48 hours of incubation (30 ± 1) oC, the fluo- rescent colonies under a 366 nm wavelength UV lamp were selected, purified, sub-cultured, and preserved by deep freezing techniques at -80 oC. Isolated strains were then reconfirmed by using PCR techniques to detect 16S rDNA sequences specifi- cally for Pseudomonas. The bacteria proliferated on the Tryptone Soya Broth (TSB) agar medium around 18 hours in (30 ± 1) oC. Use the PureLink™ Genomic DNA Mini Kit (Thermo Fisher) to extract the bacterial gDNA according to the manufacturer’s instructions. PCR reac- tion (25 µl) consists of 2.5 µl gDNA bacteria; 2.5 µl PCR reactive buffer solution (10X); 1 µl per primer (10 nM) and 1 U Phusion High-Fidelity DNA Polymerase (Ther- mo Fisher), and deionized water was added to get the desired volume. Forward primer (Psmn289: 5’–GGTCT- GAGAGGATGATGATCAGT–3’) and reverse primer (Psmn1258: 5’–TTAGCTCCACCTCGCGGC–3’) were used (Widmer et al., 1998). The PCR program included 5 minutes at 95 °C; 25 cycles (15 seconds at 94 °C, 30 seconds at 55 °C, and 1 minute at 72 °C); 10 minutes at 72 °C. PCR product (about 960 bp) was then detected by electrophoresis on the 1 % agarose gel. 2.2 CHARACTERIZE THE SALT-TOLERANT BACTERIA STRAINS FOR PLANT-GROWTH PROMOTING PROPERTY 2.2.1 Salt tolerance Strains of bacteria cultured on the TSB media sup- plemented with different NaCl concentrations ranging from 10 to 24 %. The culture was incubated on a shaker at 150 rpm at (30 ± 1) °C. The results were recorded after 1-4 days of incubation. 2.2.2 Phosphate solubilization Strains of bacteria were grown on Pikovskaya Acta agriculturae Slovenica, 118/3 – 2022 3 Isolation of salt-tolerant Pseudomonas strains with potential for alleviation of salt stress in peanut plant (Arachis hypogaea L.) (PVK) media agar plates (Pikovskaya, 1948). The plates were incubated at 30 °C for 7 days. Each treatment was done in triplicates. The bacterial colonies with clear halos in the PVK agar plate indicated solubilizing activity of the phosphate. These were sub-cultured on PVK media (Biobasic, Canada). The phosphate solubilization index (PSI) of bacteria grown on plates was measured as the following formula: Phosphate solubilizing index (PSI) = [(colony diameter + clearing zone)/ colony diameter] 2.2.3 IAA production The IAA content produced by isolates was deter- mined by the color reaction with the improved Salkowski reagent (Glickmann and Dessaux, 1995). Bacteria were grown in TSB media containing 5 % NaCl, with an ad- ditional 0.1 g l-1 tryptophan. After 5 days of incubation at 150 rpm, (30 ± 1) °C, 1 ml of bacteria was collected and centrifuged to remove biomass. The bacterial su- pernatant was then added Salkowski reagent (1:2 ratio). The reaction was kept for 1 hour at room temperature. The positive reaction with the color from pink to red has measured the absorption at a wavelength of 530 nm to determine the IAA content based on the IAA standard line.  2.2.4 Nitrogen fixation The bacterial isolates were cultured on a nitrogen- free mineral media containing 3  %  NaCl (Wright and Weaver, 1981). Bacteria with the ability in forming colo- nies, and change the color of the media after 5 days of culture were identified as nitrogen-fixed bacteria. 2.2.5 Biofilm formation The experiment was carried out in 96-well poly- styrene microtiter plates (Biobasic, Canada), using the method described by O’Toole and Kolter (1998). The iso- lates were grown overnight in LB and LB + 0.3 M NaCl (Costa-Gutierrez et al., 2020a). Then overnight culture was diluted to an OD600 = 0.1 before placing in the wells. The plate incubation was done at 30 °C without agitation. After the indicated times, the biofilm formation was ob- served by staining with crystal violet (0.4  %) and then using 30 % glacial acetic acid solution to solubilize the dye before qualifying the biofilm formation by measur- ing absorbance at 540 nm. 2.2.6 Identification of the ACC deaminase encoding genes PCR reaction (25  µl) consists of 2.5  µl bacterial g DNA; 2.5 µl PCR reactive buffer solution (10X), 1 µl per primer (10  nM), and 1 U Phusion High-Fidelity DNA Polymerase (Thermo Fisher) and deionized water was added to get the desired volume. Forward primer (5’–ATGAACCTGCTGCAACGATTC–3’) and reverse primer (5’–TCAGCCGTCGGAAGAT–3’) were applied (Saravanakumar and Samiyappan, 2007). The PCR pro- gram included 5 minutes at 95 °C; 25 cycles (15 seconds at 95 °C, 15 seconds at 58 °C, and 75 seconds at 72 °C); 5 minutes at 72 °C. PCR product (about 750 bp) was then detected by electrophoresis on the 1 % agarose gel. 2.3 EVALUATE THE PEANUT GROWTH PROMO- TION OF BACTERIA UNDER SALTY STRESS AND IN VITRO CONDITIONS Peanut seeds (Arachis hypogaea LDH12) were dis- infected and germinated on cotton wool that was im- pregnated with ½  MS media.  After 7-day incubation, the seedlings were transferred to ½  MS media with or without bacteria; and ½  MSmedia supplemented with 100 mM NaCl with or without bacteria (Sharma et al., 2016). The density of bacteria added to the media was 106 CFU ml-1. The seedling was grown in long-day condi- tions (day/night ratio was 16 hours/8 hours); room tem- perature ranged from 23 to 27 °C. The fresh biomass of the seedling was measured after 4 weeks of growth. 2.4 EVALUATE THE PEANUT GROWTH PROMO- TION OF BACTERIA UNDER SALTY STRESS AND GREENHOUSE CONDITION Peanut seeds (Arachis hypogaea LDH12) are disin- fected and germinated on cotton wool impregnated with ½ MS mineral media with or without bacterial supple- ments at a density of 106 CFU ml-1. After the seeds germi- nate, peanut seedlings were transferred to pots contain- ing soil not treated with salt or the soil is mixed with NaCl 75 mM (Goswami et al., 2014). The seedling was watered twice a week. In bacterial treatment experiments, bacte- rial suspension was added to the water reaching a density of 106 CFU ml-1, and watered every 2 weeks. With the salt treatment test, 2 weeks will be additionally watered with a 50  mM NaCl solution (Goswami et al., 2014).  After 40 days of sowing seeds, fresh biomass of seedlings was recorded. Temperature conditions (day: 34-38 °C; night: 29-32 °C) and relative humidity of 48-62 % in the nursery Acta agriculturae Slovenica, 118/3 – 20224 Q. T. DO et al. were recorded during the experiment. 2.5 DATA ANALYSIS All experiments were repeated three times the re- sults were presented as mean values with ± SD. Tukey’s honestly significant difference (HSD) method in SPSS (version 17) was applied to compare the means in all ex- periments. 3 RESULTS AND DISCUSSION 3.1 ISOLATION AND IDENTIFICATION OF BAC- TERIA From 18 root rhizosphere soil samples, we isolated 10 bacteria strains including 6 strains (ND01, ND02, ND03, ND04, ND05, and ND06) from corn rhizosphere; 2 strains (ND07 and ND08) from rice rhizosphere, and 2 strains (ND09 and ND10) from peanut rhizosphere. In fact, Pseudomonas is a genus of bacteria that is very com- mon in the soil and root rhizosphere of plants, but due to the diversity of specie composition along with the rela- tively low selective efficiency of the LB media limits the ability to isolate target bacteria, especially for those sam- ples with mold growth on agar plates. To confirm that selected strains belong to the genus of Pseudomonas, the PCR reaction was used to amplify a 16S rDNA sequence specific for the Pseudomonas (Kim et al., 2013; Yadav et al., 2014). The experiment results showed that only 7 isolates (ND01, ND03, ND04, ND06, ND07, ND09, and ND10) gave a band on the electrophoresis. These bacteria strains were evaluated for growth-promoting character- istics. 3.2 SCREENING THE BACTERIAL ISOLATES FOR SALT TOLERANCE AND PGPR TRAITS All 7 isolates were used to evaluate their possibili- ties of growing in high salt conditions. The isolates were cultured in the media containing a gradual increase in NaCl concentration from 10 % to 24 % (the gap between concentrations is 2 %). The results showed that isolated strains in Nam Dinh are likely to survive in media con- taining a quite high salt concentration, especially the two strains ND06 and ND09, which could grow in media supplemented with up to 18 % and 22 % NaCl, respec- tively (Table 1). The experimental results also presented in Table 1 showed that all isolates were capable of producing IAA. However, the amount of IAA produced by bacterial strains after 7 days of culture was relatively low in the range of 2.021 – 3.549 µg ml-1 (Malik and Sindhu, 2011). According to Egamberdieva (2015), three main factors affecting the IAA production of rhizobacteria were the bacterial strains; culture time, bacterial growth stage; and precursor to IAA synthesis. Many studies have shown that different strains of bacteria have different IAA pro- duction. Several strains of bacteria that are prominent for IAA production, such as Pseudomonas aureantiaca TSAU22 (Sheehy et al., 1991), Pseudomonas extremorien- talis TSAU6 and Pseudomonas extremorientalis TSAU20 (Egamberdieva, 2011), significantly increased root growth by up to 25 % under normal conditions and up to 52 % under 100 mM NaCl condition compared with control plants (Botelho and Mendonça-hagler, 2006; Egamberdieva, 2009). One of the important traits of PGPR is nitrogen fix- ation. Hence, the isolates were also investigated the nitro- gen fixation ability on media without nitrogen sources. The results were shown in Table 1, the majority of iso- Bacterial strain Highest NaCl concentration (%) IAA concentration (µg ml-1) Phosphate Solubility Index Nitrogen fixation ND01 12 3.521 ± 0.113a 1.557 ± 0.211b - ND03 12 3.327 ± 0.106ab 2.013 ± 0.131a + ND04 10 2.876 ± 0.132bc 1.252 ± 0.124c - ND06 18 3.021 ± 0.211b 1.239 ± 0.102c + ND07 10 2.417 ± 0.215c 1.532 ± 0.079b + ND09 22 3.549 ± 0.115a 1.635 ± 0.063ab + ND10 12 2.437 ± 0.102c 1.781 ± 0.023ab - Table 1: Characterization of plant growth-promoting bacteria isolated under salty stress conditions Data are means ± SD (n = 3). Values in the same column with the same letter(s) are not significantly different as determined by Tukey’s honestly significant difference test (p < 0.005). ‘-’ mean no media color; ‘+’ means media color changed Acta agriculturae Slovenica, 118/3 – 2022 5 Isolation of salt-tolerant Pseudomonas strains with potential for alleviation of salt stress in peanut plant (Arachis hypogaea L.) lated strains were capable of growth on the Nitrogen Free Mineral Medium (MNFM), excepted for ND01, ND04, and ND10. The MNFM is a media with no nitrogen sources, hence, the formation of colonies on this media demonstrates that bacterial strains were capable of using air nitrogen sources for cellular processes. In an MNFM, there was a supplement of blue bromophenol as a pH in- dicator, which is yellow when the pH < 7, green at pH = 7 and turns blue when the pH > 7. In this experiment, the environment changed from green to blue because nitro- gen-fixed bacteria created NH4 + products that increased the pH of the media.  All 7 isolated strains of bacteria showed the phos- phate solubilization capacity when produced a clear zone around colonies after 7 days (Table 1). The PSI ranged from 1.239 to 2.013. The mechanisms for dissolving phos- phate by bacteria vary widely, but according to Sharma et al. (2013), there are three main mechanisms: organic acid production, inorganic acid release, and extracellular polymeric substances (EPSs) (Fatima and Arora, 2021). 3.3 EVALUATION OF ACC DEAMINASE PRODUC- TION AND COLONIZATION OF BACTERIAL ISOLATES In order to identify the presence of ACC deami- nase in potential strains, the PCR to amplify the spe- cific DNA sequence of this gene in Pseudomonas was performed as the method described by Sheehy et al. (1991). In addition, Saravanakumar and Samiyap- pan (2007) proved that this pair of primers is specific to characterize Pseudomonas fluorescens (Flügge 1886) Migula, 1895. Electrophoresis results showed that the target product of about 750 bp appeared in all two se- lected strains. Moreover, the biofilm formation ability of isolates under salt stress conditions was also studied. The results were shown in Figure 1. As can be seen from Figure 1A, under normal con- ditions, the biofilm formation dynamics of bacterial iso- lates were different. The ND09 presented the late produc- tion of biofilm formation compared to the ND06 strain, showing a lower OD540nm value at the beginning and reaching a higher OD540nm value after 24 hours while the ND06 presented a decline of OD540nm value during the ex- periment. The results also indicated the delay effect of salt stress on the bacterial biofilm formation, which reached a maximum OD540nm value after 6 hours and higher than the maximum OD540nm value in normal conditions (Fig- ure 1B). These results are consistent with some previous studies on the formation of bacterial biofilm under salt stress (Costa-Gutierrez et al., 2020b; Costa-Gutierrezet al., 2021). 3.4 EVALUATE THE ABILITY TO STIMULATE PEANUT GROWTH UNDER SALT STRESS Based on the results of the growth-stimulating char- acteristics of the isolated strains, among the strains iso- lated from the soil rhizosphere, ND03, ND06, ND07, and Figure 1: Biofilm formation ability of bacterial isolates (ND06 and ND09) in LB (A) and LB + 0.3M NaCl (B) in different periods of time. CK: media only. Plotted data are means ± SD (n = 3). The same letter(s) are not significantly different as determined by Tukey’s honestly significant difference test (p < 0.05) Acta agriculturae Slovenica, 118/3 – 20226 Q. T. DO et al. ND09 are full of characteristics such as IAA production, nitrogen fixation, and phosphate solubilization. How- ever, when considering the results of IAA production, salt-resistance, and phosphate solubility index, 2 strains ND06 and ND09 were selected for further experiments. All two strains gave positive PCR results with primers specific for Pseudomonas. Pseudomonas strains isolated from rhizosphere and agricultural soil samples were as- sessed to be relatively safe for humans and animals. These two strains were selected for investigating their ability in promoting plant growth development under in vitro and in vivo conditions. In this experiment, the potential of bacterial strains in supporting plants to withstand salty stress will be eval- uated on agricultural plant models. Compared to corn, which is only sensitive to salty stress at an average of de- grees (Zörb et al., 2004), peanut plants are a very sensitive type to salty stress (Goswami et al., 2014; Sharma et al., 2016). Therefore, although 2 strains were selected from the root rhizosphere of corn and peanut, in this experi- ment peanut seedlings were selected as models to con- duct stress response tests. In in vitro experiments, the results were illustrated in Figure 2 and Table 2. The results indicated that un- der normal conditions, peanut seedlings treated with the ND09 strain showed growth stimulation expressed in an increase in total plant biomass (34.63 %), shoot biomass (35.22 %), and root biomass (32.87 %) compared to the control. In contrast, the ND06 strain presented no dif- ference from the control plant (Figure 2A and Table 2). It is notable that the IAA concentration reaching from 0.1 to 1 µg ml-1 could produce beneficial effects on plant growth (Bui, 2016). It implies that if co-inoculation of isolated strains with plants for a long time, these strains could provide enough exogenous IAA for plant growth by increasing root growth through root elongation and reducing ethylene. However, under salty stress, all seedlings treated with bacteria showed an increase in the biomass of the plants compared to the control. In particular, the seed- ling treated with the ND09 strain presented the highest efficiency (Figure 2B and Table 2). This might be because the bacterial isolates produced the ACC deaminase un- der salt stress to degrade ACC (the precursor of ethylene in all higher plants) and hence prevented the over-accu- mulation of ethylene in plants under salt stress condi- tions; subsequently enhancing plant development. This suggests that bacteria have the ability to reduce the effect of salty stress on the growth of peanut plants. The results of greenhouse experiments were consist- ent with the in vitro results and were illustrated in Figure 3 and presented in Table 3. As can be seen, under nor- mal conditions, seedlings treated with the ND09 strain showed an increase in shoot and root biomass respective- ly 30.37 % and 32.87 % compared to control seedlings. In contrast, seedlings treated with ND06 strain presented a slight decrease in biomass compared to control seedlings (Figure 3A and Table 3). Under salty stress conditions, all seedlings treated with bacteria had higher fresh biomass than control (Figure 3B and Table 3).  The results of the greenhouse experiments showed a match with ones under  the in vitro conditions. These results indicated that the ND09 strain not only effectively stimulated peanut seedling growth under normal condi- Figure 2: Bacteria isolates (ND06 and ND09) enhances the peanut seedling growth under normal condition (A) and 100 mM NaCl (B) after 4 weeks of sowing under in vitro conditions Acta agriculturae Slovenica, 118/3 – 2022 7 Isolation of salt-tolerant Pseudomonas strains with potential for alleviation of salt stress in peanut plant (Arachis hypogaea L.) tions but also had the potential to improve plant growth under salty tress conditions. Meanwhile, the remaining strain had only a positive effect under salty stresses but not under normal conditions. All of these results suggest the ND09 strain has shown to be a potential strain in the production of probiotic fertilizers that could improve crop yields, whether under normal conditions or salty stress conditions. These results were in agreement with previous stud- ies that also investigated the alleviation of salt stress by Pseudomonas in plant development. Cai et al. (2021) reported that Chenopodium quinoa Willd. inoculated with Pseudomonas sp. strain M30-35 significantly im- proved the dry mass of roots by 51.97 % at 150 mM NaCl treatments for 7 d. Another example is the report of Fa- tima and Arora (2021), who proved that P. entomophila PE3 in cobination with 2  % EPS enhanced the growth and resilience of sunflower in saline soil (increment in root and shoot length was 49 % and 85 % respectively in comparison to control). Although each plant species has different selective effects on rhizosphere diversity, P. fluorescens and P. putida Trevisan, 1889 are still the most dominant species (Egamberdieva, 2015). Many com- mercial preparations from these two species have been widely used. Several cases of P. putida causing disease in humans have been reported, however, these are rare and mostly occur in immunocompromised individuals (Fernández et al., 2015). Another member of this genus, P. aeruginosa (Schröter 1872) Migula 1900, has great po- tential for promoting plant growth and potent antago- nism against rhizosphere pathogens. Unlike P. fluorescens and P. putida, some strains of P. aeruginosa are opportun- istic pathogens in humans (Fernández et al., 2015). This bacterium is widely distributed in water, soil, and even in some foods. However, the level of risk posed by this bacterium is not large and is only classified as a Class II biosafety risk. Furthermore, not all strains of this species are pathogenic due to the absence of pathogenic genes in the genome. In summary, PGPR strains belonging to the genus Pseudomonas can be widely applied in agricultural practices with low risk and controllability. 4 CONCLUSIONS In this study, 7 bacteria strains belonging to the Pseudomonas genus and capable of living in salty con- ditions were isolated from soil in Nam Dinh. The two strains of bacteria ND06 and ND09 were selected based on phosphate solubilization, nitrogen fixation, and IAA production. The ND09 strain had the potential to stimu- late peanut seedling growth under both normal and salty stress conditions, indicating the potential of this strain in sustainable agricultural practices. However, before be- ing widely adopted or commercialized, the ND09 strain Experiments  Total plant biomass (mg) Shoot biomass (mg) Root biomass (mg) 0 mM NaCl 100 mM NaCl 0 mM NaCl 100 mM NaCl 0 mM NaCl 100 mM NaCl Peanut seedlings (control) 2213.3 ± 61.2b 1969.5  ± 91.3c 1634.5 ± 101.2b 1612.5 ± 132.1c 579.1 ± 125.0b 356.8 ± 82.3c Peanut seedlings + ND06 2268.2 ± 75.8b 2410.3 ± 151.3 b 1681.7 ± 87.2b 1897.5 ± 121.2b 586.2 ± 72.4b 512.7 ± 64.3b Peanut seedlings + ND09 2979.7 ± 135.5a 2707.7 ± 35.2a 2210.2 ± 112.3a 2102.3 ± 53.1a 769.5 ± 187.5a 605.3 ± 67.2a Table 2: Effect of selective bacteria on fresh biomass of peanut seedlings grown on different media after 4 weeks under in vit- ro conditions Data are means ± SD (n = 3). Values in the same column with the same letter(s) are not significantly different as determined by Tukey’s honestly significant difference test (p < 0.05) Figure 3: Illustration of a positive effect of bacteria isolates (ND06 and ND09) on the peanut seedling growth under nor- mal condition (A) and 75mM NaCl (B) after 40 days of sowing Acta agriculturae Slovenica, 118/3 – 20228 Q. T. DO et al. should be evaluated on the risk of disease in humans and animals, as well as the impact on the ecological environ- ment.  5 ACKNOWLEDGEMENTS This research has been done under the research project QG.21.59 “Study on phosphorus adsorption and proposing solutions to improve phosphorus fertilizer ef- ficiency in rice cultivation in the Red River delta” of Vi- etnam National University Hanoi. The Vietnam National University Hanoi provided funds for collecting samples and buying chemicals for experiments and supported ex- perimental facilities to carry out experiments. 6 REFERENCES Bui, T.V. (2016). 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Journal of Advances in Microbiology, 20, 59-79. https://doi. org/10.9734/jamb/2020/v20i130209 Experiments Total plant biomass (mg) Shoot biomass (mg) Root biomass (mg) 0 mM NaCl 75 mM NaCl 0 mM NaCl 75 mM NaCl 0 mM NaCl 75 mM NaCl Peanut seedlings (control) 2722.8 ± 31.7b 1559.8 ± 110.1c 2173.7 ± 102.3b 1231.2 ± 111.3c 548.7 ± 62.7b 328.2 ± 101.2b Peanut seedlings + ND06 2634.2 ± 56.2b 2302.5 ± 102.1b 2091.2 ± 35.7b 1497.3. ± 109.1b 542.6 ± 61.2b 803.7 ± 113.7a Peanut seedlings + ND09 5396.1 ± 52.1a 2688.9 ± 91.1a 3372.5 ± 115.7a 1895.5 ± 115.7a 2023.4 ± 94.5a 792.7 ± 82.3a Table 3: Bacterial isolate enhance peanut plant growth under salty stress condition after 40 days of Sowing under greenhouse conditions Data are means ± SD (n = 3). 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Isolation, sequence, and expression in Escherichia Coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase. Journal of Bacteriology, 173(17), 5260-5265. https://doi. org/10.1128/jb.173.17.5260-5265.1991 Upadhyay, S.K., Singh, D.P. (2015). Effect of salt-tolerant plant growth-promoting rhizobacteria on wheat plants and soil health in a saline environment. Plant Biology, 17(1), 288- 293. https://doi.org/10.1111/plb.12173 Widmer, F., Seidler, R.J., Gillevet, P.M., Watrud, L.S., Giovanni, G.D. (1998). A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples. Applied and Environmental Microbiology, 64(7), 2545-2553. https://doi.org/10.1128/ AEM.64.7.2545-2553.1998 Wright, S.F., Weaver, R.W. (1981). Enumeration and identifi- cation of nitrogen-fixing bacteria from forage grass roots. Applied and Environmental Microbiology, 42(1), 97-101. https://doi.org/10.1128/aem.42.1.97-101.1981 Yadav, S., Yadav, S., Kaushik, R., Saxena, A.K., Arora, D.K. (2014). Genetic and functional diversity of fluorescent Pseu- domonas from rhizospheric soils of wheat crop. Journal of Basic Microbiology, 54(5), 425-437. https://doi.org/10.1002/ jobm.201200384 Zörb, C., Schmitt, S., Neeb, A., Karl, S., Linder, M., Schubert, S. (2004). The biochemical reaction of maize (Zea mays L.) to salt stress is characterized by a mitigation of symptoms and not by a specific adaptation. Plant Science, 167(1), 91-100. https://doi.org/10.1016/j.plantsci.2004.03.004 Zörb, C., Geilfus, C.M., Dietz, K.J. (2019). Salinity and crop yield. Plant Biology, 21, 31-38. https://doi.org/10.1111/ plb.12884 Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2333 Original research article / izvirni znanstveni članek Quantification of apocarotenoids in commercial Indian (Kashmiri) saf- fron using UV-Vis spectroscopy and HPLC analysis Tahir ul Gani MIR 1, Jaskaran SINGH 2, Saurabh SHUKLA 1, 3 Received September 05, 2021; accepted July 10, 2022. Delo je prispelo 5. septembra 2021, sprejeto 10. julija 2022 1 Department of Forensic Science, School of Bioengineering & Biosciences, Lovely Professional University, Phagwara, India 2 Department of Forensic Science, Geeta University, Naultha, Panipat, India 3 Corresponding author, e-mail: sau47bt@gmail.com Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Abstract: Saffron is considered as the most expensive spice in the world. Due to low production, high demand and high cost, saffron is very prone to adulteration for economic benefits while putting public health at risk. The most significant characteristic for determining the quality of the saffron is color- ing strength (crocin content), which is determined by measur- ing UV-Vis absorption at 440 nm in the aqueous preparations of this spice. Picrocrocin and safranal are other key compo- nents used to determine saffron quality. This article aims to examine the quality of commercial saffron obtained from vari- ous geographical locations of Kashmir (India) by determining their apocarotenoid content using UV-Vis spectrophotometry followed by high-performance liquid chromatography (HPLC) to determine the concentration of saffron metabolites (crocin, picrocrocin and safranal). A total of 31 samples from different origins were used in this study. The UV-Vis spectrophotometric results showed that among 31, only 14 samples fell into grade I, while 9 samples fell in grade II and 5 samples fell in grade III of the ISO category. The remaining 3 samples could not satisfy ISO standards, which indicates that these samples were adulter- ated. The determination of apocarotenoid content using HPLC analysis varied significantly among samples. These variations may be due to different drying and storage conditions or adul- teration. Key words: saffron; adulteration; crocin; safranal; picro- crocin; UV-Vis spectroscopy; HPLC Količinsko ovrednotenje apokarotenoidov v komercialnih vzorcih indijskega (kašmirskega) žafrana z analizo UV-Vis spektroskopije in HPLC Izvleček: Žafran velja za najdražjo začimbo v svetovnem merilu. Zaradi majhne pridelave, velikega povpraševanja in visoke cene je zaradi ekonomskih koristi podvržen ponareja- nju, kar povzroča zdravstvena tveganja. Najznačilnejša lastnost za določanje kakovosti žafrana je njegova sposobnost obarvanja (vsebnost krocina), ki se določa z merjenjem UV-Vis absorb- cije pri 440 nm v vodnih pripravkih te začimbe. Pikrokrocin in safranal sta ostali klučni komponenti, ki se uporabljata za določanje kakovosti žafrana. V raziskavi smo preučevali kako- vost tržnega žafrana pridobljena iz različnih geografskih obmo- čij Kašmirja (Indija) z določanjem vsebnosti apokarotenoidov z UV-Vis spektroskopijo, ki ji je sledila analiza z visokotlačno tekočinsko kromatografijo (HPLC), kjer smo v vzorcih žafrana določali koncentracije metabolitov kot so krocin, pikrokrocin in safranal. V raziskavi je bilo analiziranih 31 vzorcev različ- nega izvora. Rezultati analize z UV-Vis spektroskopijo so po- kazali, da se je med 31 vzorci samo 14 uvrstilo v kvaliteto I, 9 vzorcev seje uvrstilo v kvaliteto II in 5 vzorcev v kvaliteto III, glede na ISO kategorije. Preostali 3 vzorci niso izpolnjevali ISO standardov, kar kaže, da so bili ponarejeni. Vsebnost apokaro- tenoidov v vzorcih se je pri analizi s HPLC značilno razlikovala, kar bi lahko bila posledica različnega sušenja, shranjevanja ali ponarejanja. Ključne besede: žafran; ponarejanje; krocin; safranal; pi- krokrocin; UV-Vis spektroskopija; HPLC Acta agriculturae Slovenica, 118/3 – 20222 T. G. MIR et al. 1 INTRODUCTION Saffron, often referred to as red gold, is obtained from the stigma of Crocus sativus L. (Saxena, 2010). Cro- cus sativus is an angiosperm plant, member of the As- paragales family. The flower of Crocus sativus is solitary, purple, with six petals, three stamens, one style, and three reddish-orange stigmas. Saffron crocus grows through- out the Mediterranean–Europe, and Western Asia. It is commonly cultivated in Iran, Greece, Spain, Italy, Af- ghanistan and India (Kashmir) (Kahriz, 2020). Saffron is quite costly because it is used mainly as a flavoring and aromatizing agent (Mir et al., 2022a; Mzabri et al., 2019). Saffron contains approximately 300 volatile and non- volatile metabolites, including crocin, safranal, picro- crocin, monoterpenes, aldehydes, and various other ca- rotenoids of therapeutic potential (Pandita, 2021). It is a well-known spice that is used to cure a variety of medical conditions, including depression (Siddiqui et al., 2018), cardiovascular illness (Kamalipour & Akhondzadeh, 2011), menstrual irregularities (Beiranvand et al., 2016), asthma (Zilaee et al., 2019)lipid profiles, basophils, eo- sinophils and clinical symptoms in patients with allergic asthma.\nSTUDY DESIGN: Our study was a clinical trial.\nMETHODS: Subjects (N = 80, 32 women and 48 men, 41.25 ± 9.87 years old, insomnia (Taherzadeh et al., 2020), and digestive problems (Khorasany & Hosseinza- deh, 2016). Crocin is a carotenoid chemical compound responsible for the golden yellow-orange color of saffron; picrocrocin gives bitter flavor, and safranal is responsible for the characteristic aroma of saffron. Saffron is current- ly known as a flavoring agent and a potent natural agent with many health advantages (Azami et al., 2021; Basker & Negbi, 1983; Bolhassani et al., 2014; Mir et al., 2022b). The potential of saffron and its constituents to protect against natural and artificial poisons has enhanced its significance. Due to the high price of saffron and its great demand in the pharmaceutical industry, illegal traffick- ing and adulteration are prevalent nowadays (Alonso et al., 1998). The common adulterants used in saffron in- clude maize silk, marigold floret, horsehair, wool, saffron stamens, red dried silk fiber, and safflower (Carthamus tinctorium L.). Mixing low-grade saffron with high-grade saffron or old stored saffron that has lost its quality with freshly harvested saffron is also a common method of adulteration in saffron (Kumari et al., 2021; Lowell, 1964; Marieschi et al., 2012; Sereshti et al., 2018). The sale and mixing of high-grade Kashmiri saffron with lower-cost Iranian imports is common in India; the resulting mixes are then sold as pure Kashmiri saffron. This trend has deprived saffron cultivators of Kashmir of a significant portion of their revenue (Hussain, 2005). Dyes such as erythrosine, tartrazine, amaranth, sunset yellow, carmoi- sine, picric acid, ponceau S, methyl orange, and Sudan red are also used as adulterants in saffron (Lozano et al., 1999; Patel et al., 2019; Petrakis et al., 2015). Internationally, the grading of saffron is based on the standards formulated by the International Organi- zation for Standardization (ISO). The ISO (ISO 3632- 1:2011) certification ensures customers that the saffron they purchase is authentic and safe to consume. ISO 3632 has classified saffron into three grades (Grade I, II and III) based on the concentration of crocin, picrocrocin and safranal present in saffron [Table 1]. A greater con- centration of these chemicals indicates a better quality of saffron. A quartz cell with a 1 cm pathway is used to measure E1% at 440, 330, and 257 nm wavelengths. The results are obtained by measuring the absorption at three wavelengths using the equation; E1 % 1 cm = (A × 10000]/ (M ×  (100-H)], where E1%  is the specific extinction co- efficient, 1  cm is the path length, A is the absorbance, M is the mass in grams of the saffron sample, H is the moisture and volatile sample material. The moisture and volatile content of the saffron is determined after drying the samples and represented as a mass fraction using the formula: [(initial mass-constant mass)/initial mass] × 100 (Hadizadeh et al., 2007; ISO - 3632-1:2011). Despite international standards, various methods have been reported to detect adulteration and determine the quality of saffron viz UV-Vis spectroscopy (Zalacain et al., 2005; Zougagh et al., 2005) HPLC (Haghighi et al., 2007; Hajimahmoodi et al., 2013; Lozano et al., 1999), mi- cellar liquid chromatography, FTIR (Karimi et al., 2016; Ordoudi et al., 2018), H-NMR (Petrakis et al., 2015), gel-electrophoresis (Paredi et al., 2016). Several factors, including geographical conditions, harvesting period, drying procedure employed, temperature and oxygen ex- posure during storage and adulteration, all have a signifi- cant impact on the quality of saffron (Caballero-Ortega et al., 2004). The primary objective of this research was to estimate the quality range and apocarotenoid content of commercial saffron in Kashmir using UV-Vis spectro- photometry and HPLC analysis. 2 MATERIAL AND METHODOLOGY Saffron in India is cultivated and commercialized in Kashmir. The main local commercial zones of saffron in Kashmir are Srinagar, Pampore and Budgam. Besides local markets, the saffron in Kashmir is also commercial- ized by government-operated commercial emporiums (e.g., Government Kashmir Art Emporiums). Twenty- four samples of saffron were collected from Kashmir, among which six samples were collected from Govern- ment operated commercial emporiums (KAE), six sam- Acta agriculturae Slovenica, 118/3 – 2022 3 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis ples were collected from the local market of Srinagar (SXR), six samples were collected from Pampore (PAM) district, and six samples were collected from Budgam (BUD). The samples collected were supposed to be yield- ed from the crop year 2019 and processed in 2020 as per their packing. Besides these samples, four samples were collected from Afghanistan (AFG), and two samples were collected from Iran (IRN). The samples collected from KAE, AFG and IRN had an origin certificate and were assured free from any adulteration. One sample was col- lected from Sigma Aldrich (SIG). A total of 31 samples were used for this study. Crocin and safranal standards were purchased from Sigma Aldrich. Picrocrocin was ob- tained from BioMall. HPLC-grade reagents (methanol, acetonitrile) were obtained from Loba Chemie. 2.1 DETERMINATION OF FLORAL WASTE CON- TENT About 1 g of each sample was taken, and each fila- ment was spread on the paper. With the help of forceps, different floral waste components were separated, and the samples were weighed again. The floral waste was taken in shoe glass and weighed. The floral waste content of the sample (wF) was expressed as per ISO guidelines as a percentage by mass, using the relation: wF = (m2 – m1) x 100/mo % Where m0 is the mass, in grams, of the test portion; m1 is the mass, in grams, of the shoe glass; m2 is the mass, in grams, of the shoe glass containing the floral waste. 2.2 DETERMINATION OF MOISTURE AND VOLATILE CONTENT The samples collected needed to be examined for their authenticity. For such purposes, ISO 3632 has pro- vided guidelines for conducting UV-Vis spectroscopy. To calculate E1 %, first, the moisture content of all the sam- ples was calculated. One gram of saffron from each sam- ple was placed in a Petri dish and kept in the oven for 18 hours at 70 0C. After that, samples were weighed again to measure the moisture and volatile matter content (wMV) and is expressed as: wMV = (m0 – m1) x 100/m0 % where m0 is the mass, in grams, of the test portion; m1 is the mass, in grams, of the dry residue. 2.3 UV-VIS SPECTROSCOPY The UV-Vis spectroscopy for samples was per- formed according to ISO guidelines with slight modifi- cations in order to get a greater yield of apocarotenoid compounds. Briefly, 100 mg mass of dried saffron sam- ples was extracted with 5 ml cold 50 % (v/v) ethanol in mortar and pastel. The extract was then transferred to a screw-capped 50 ml tube, and a total amount of 20 ml 50 % (v/v) ethanol was added. Tubes were sonicated for 20 minutes on ice, centrifuged for 15 minutes at 4000 rpm, and washed twice with 5 ml of 50 % (v/v) ethanol. Spectrophotometric technique was employed to analyze the supernatant. For analysis, the supernatant (1 ml) was diluted to 5 ml with 50 percent (v/v) ethanol. The ab- sorption of crocin, safranal, and picrocrocin at 440 nm, 330 nm, and 257 nm, respectively, was used to create a standard curve. The sample supernatants were diluted 100 times, and direct absorbance readings were obtained using a Shimadzu spectrophotometer (1  cm pathway quartz cell) at 440 nm, 330 nm, and 257 nm, respectively. A UV-Vis scan was also obtained to observe peaks in samples of different geographical locations. The results obtained were used to measure E1 % of aqueous saffron extract using the following relation: E1% 1 cm = (A × 10000/ [(M × (100-H)] 2.4 HPLC ANALYSIS For HPLC analysis, 50 mg of powdered saffron sam- ples were extracted with 10 ml of 50 % methanol-water (v/v) and magnetically stirred for 24 hours at 4 °C in the Component λmax Category I Category II Category III Crocin 440 nm ˃ 200 170-200 120-170 Safranal 257 nm 20-50 20-50 20-50 Picrocrocin 330 nm > 70 55-70 40-55 Moisture and volatile matter % (m/m), - 10 10-12 10-12 Table 1: Grades of saffron based on ISO 3632-1:2011 Acta agriculturae Slovenica, 118/3 – 20224 T. G. MIR et al. dark. The samples were then centrifuged for 30 minutes at 5000 rpm. The supernatant was collected and filtered through 0.2 µm syringe filters. For quantitative analysis of crocin, picrocrocin and safranal, 1 ml of 2-nitroaniline was added as an internal standard to each sample before analysis (Caballero-Ortega et al., 2007). The analysis was carried out in a Shimadzu HPLC equipped with quater- nary pumps; coupled to a photo-diode-array detector. Ethanol (50 %, v/v) and acetonitrile (15 %, v/v) were used as the mobile phase. Detection was carried out with an injection volume of 20 μl, a flow rate of 1 ml min−1 with 35-40 min of run time. Crocin was detected at 440 nm, picrocrocin at 250 nm and safranal at 330 nm. A calibra- tion curve was constructed for internal standard  using concentrations of 0.125, 0.25, 0.5, and 1.0 mg ml-1. Quan- titative analysis was carried out in accordance with the molecular absorption coefficient of each peak obtained at the wavelength of maximum absorbance of the compo- nents.  The R2 values ranged from 0.9722to 0.9890, and results were expressed in milligrams per gram of saffron stigmas. 2.5 STATISTICAL ANALYSIS One-way ANOVA was used to compare means and Duncan’s Multiple Range Test (DMRT) was used to assess significance using IBM SPSS (version-20). Two tailored Pearson correlations between apocarotenoid levels with floral waste content and moisture levels were done using IBM SPSS (v. 20). The results were also analyzed using the multivariate analysis technique principal component analysis (PCA) using Origin-2021b (version-9.8b). PCA is a dimensionality-reduction technique often used to decrease the dimensionality of big data sets by convert- ing a large collection of variables into a smaller one that still retains most of the information in the large set. 3 RESULTS AND DISCUSSION The determination of floral waste in the samples was performed by physical separation of floral waste and then measuring its weight. The floral waste in the samples var- ied in range, with samples obtained from SXR and BUD showing a high range of floral waste. KAE samples and sigma samples showed the lowest range of floral waste, while IRN and AFG samples showed a medium to low range of floral waste. Floral waste in the Sigma sample was not detected (Table 2). The moisture/volatile matter content was performed to analyze if the samples had been properly dried and processed. The average moisture level in KAE samples was found to be 6.26  %. Samples from SXR, BUD and PAM showed high levels of moisture and volatile content matter (12.45 %, 7.90 %, and 7.26 %, respectively). The average moisture and volatile content in the AFG and IRN samples was 6.35 % and 542 %, respectively, while in the SIG sample, it was found to be 4.49 % (Table 2). Apocarotenoid content (E1 %) was determined us- ing UV-Vis spectrophotometry. The main objective of this measure analysis was to analyze the quality range of commercial saffron sold in Kashmir. The saffron samples were evaluated in accordance with the ISO 3632-2:2010 guidelines. One-way ANOVA and DMRT were used to compare means and assess the level of significance. The results showed significant variation in all the sam- ples (Table 2). Results showed that average crocin con- tent varied from 198.5 in KAE samples, 135.16 in SXR samples, 184.5 in PAM samples, 166 in BUD samples, 197.25 in AFG samples, 200.5 in IRN samples and 203 in sigma samples. Based on crocin content, it was found that among thirty-one samples, fourteen samples fell in category I, nine fell into category II, five fell in category III and three were counterfeit or adulterated samples as they showed E1 % less than 110. Similarly, picrocrocin expressed as direct reading of the absorbance at 257 nm showed an average concentration of 36.5 in KAE sam- ples, 23.83 in SXR, 33 in PAM, 28.16 in BUD, 34 in AFG, 38.5 in IRN and 32 in SIG sample (Table 3). The safranal content in twenty-nine samples was found to be above 20, thus falling in the optimum range under ISO criteria. Three samples resulted in a safranal content range below 20, which is not optimal as per ISO guidelines. The floral waste and moisture/volatile content in saffron samples were negatively correlated with crocin content values (-0.87, -0.81, respectively). The results were analyzed us- ing PCA analysis. PC1 (76.23 %) and PC2 (18.06 %) ac- counted for 94.29 % of the total variance of the data. The coefficient for both the principal components is given in Table 5. A biplot of samples was obtained to distinguish between adulterated and pure saffron (Figure 1). HPLC analysis provides quick and simple measure- ment of the three major saffron components, with excel- lent linearity, selectivity, sensitivity, and accuracy. The crocetin, picrocrocin, and safranal were determined by HPLC at three wavelengths 440, 250, and 330 nm, re- spectively. The results were analyzed by one-way ANO- VA to compare means, and Duncan’s Multiple Range Test (DMRT) was used to assess significance. The con- centration of these metabolites varied significantly (Table 2). The variations may be attributable to the geographi- cal origin of samples, different drying procedures, stor- age conditions and adulteration (Biancolillo et al., 2020; Delgado et al., 2005; Maghsoodi et al., 2012). The aver- age concentration of crocin varied from 40.64 mg g-1 in Acta agriculturae Slovenica, 118/3 – 2022 5 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Sample wF % wMV % UV-Vis Analysis HPLC analysis Crocin (E1 %) 440 nm Picrocrocin (E1 %) 257 nm Safranal (E1 %) 330 nm Crocin (mg g-1) Picrocrocin (mg g-1) Safranal (mg g-1 ) KAE1 0.77 5.26 205a 85a 42a 39.32a 5.36a 0.26bc KAE2 0.41 6.10 212a 86a 44a 43.51a 5.89a 0.31a KAE3 0.66 5.15 178a 62b 28c 45.36a 6.21a 0.26bc KAE4 2.73 7.09 203a 74a 35a 39.95a 6.01a 0.29a KAE5 1.43 6.43 188a 69b 31ab 42.29a 4.03b 0.3a KAE6 6.17 7.53 205a 79a 39a 33.43ab 4.91a 0.27abc SXR1 11.51 9.34 134b 56c 24c 38.49a 4.02b 0.28ab SXR2 4.93 6.73 179a 59c 27c 26.36b 3.16c 0.17c SXR3 5.61 10.3 184a 62b 28c 32.41ab 3.98b 0.2c SXR4 20.49 14.68 80b 43c 23c ND ND 0.18c SXR5 9.49 11.35 162b 57c 25c 34.24ab 3.23c 0.29a SXR6 25.3 22.35 72b 40c 16c 18.26b 2.79c 0.23c PAM1 4.53 12.08 195a 69b 32ab 33.32ab 3.63b 0.29a PAM2 5.68 7.84 152b 65b 29b 38.43a 3.23c 0.26bc PAM3 7.85 7.63 201a 74a 34bc 42.45a 4.02b 0.28ab PAM4 0.60 6.26 163b 72ab 33bc 30.44ab 3.62bc 0.32a PAM5 1.93 8.23 189a 67b 30b 29.4ab 3.14c 0.26bc PAM6 6.25 5.39 207a 82a 40a 33.31ab 4.11b 0.27abc BUD1 2.48 6.19 202a 79a 38a 32.43ab 3.89b 0.29a BUD2 2.80 7.26 185a 60c 29b 26.38b 3.77b 0.27abc BUD3 3.91 8.15 149b 56c 25c 27.39b 3.56c 0.28ab BUD4 16.60 10.28 105b 53c 24c 20.2b 3.04c 0.21c BUD5 11.29 5.33 181a 59c 27c 30.3ab 3.69b 0.23c BUD6 10.24 6.35 176a 58c 26c 32.41ab 3.51c 0.25bc AFG1 1.56 5.68 201a 78a 37a 41.34a 5.1a 0.27abc AFG2 1.34 5.34 208a 81a 39a 30.49ab 4.25b 0.3a AFG3 1.97 6.63 192a 68b 31b 31.42ab 4.07b 0.29a AFG4 3.81 7.76 188a 64b 29bc 38.38a 3.81b 0.25bc IRN1 2.10 5.26 206a 84a 41a 35.42ab 5.33a 0.26bc IRN2 1.92 5.59 195a 75a 36a 35.12ab 5.1a 0.29a SIGMA ND* 4.49 203a 72ab 32ab 34.41ab 4.46ab 0.31a Table 2: Floral waste percentage (wF %), moisture and volatile percentage (wMV %), UV-Vis analysis, HPLC analysis of saffron samples Means followed by the same letter within the columns are not significantly different (p < 0.05) using DMRT *ND- Not Detected KAE samples, 29.952 mg/g in SXR samples, 34.55mg/g in PAM samples, 28.18 mg/g in BUD samples, 35.40 mg g-1 in AFG samples, 35.27 mg g-1 in IRN samples and 34.41 mg g-1 in sigma sample. Safranal, one of the main components responsible for the fragrance of the spice, is soluble in polar solvents and poorly soluble in nonpolar solvents. The safranal content as per the ISO 3632 (2011) method cannot be categorized in any grade as the ISO Acta agriculturae Slovenica, 118/3 – 20226 T. G. MIR et al. Sample Origin ISO Category Crocin (E1 %) 440 nm Safranal (E1 %) 257 nm Picrocrocin (E1 %) 257 nm KAE I (4) 203-212 28-31 62-69 II (2) 168-178 35-42 74-84 SXR II (2) 179-184 27-28 59-62 III (2) 134-162 24-25 56-57 IV* (2) 72-80 16-23 40-43 PAM I (3) 195-207 32-40 64-82 II (1) 189 30 67 III (2) 152-163 29-33 65-73 BUD I (1) 202 38 79 II (3) 176-185 26-29 58-60 III (1) 149 25 56 IV* (1) 105 24 53 AFG I (3) 192-208 31-39 68-81 II (1) 188 29 64 IRN I (2) 36-41 75-24 75-24 SIG I (1) 203 32 72 Table 3: Quality characteristics of saffron obtained from different geographical locations using the ISO-3632 method *Highly adulterated or counterfeit saffron samples Figure 1: PCA analysis (biplot) of saffron samples (UV-Vis analysis) Acta agriculturae Slovenica, 118/3 – 2022 7 Quantification of apocarotenoids in commercial Indian (Kashmiri) saffron using UV-Vis spectroscopy and HPLC analysis Figure 2: PCA analysis (biplot) of saffron samples (HPLC analysis) method doesn’t provide a precise classification of grades of saffron based on safranal content. The average safranal content differed significantly across KAE (0.28 mg g-1 ), SXR (0.22 mg g-1 ), PAM (0.28 mg g-1 ), BUD (0.25 mg g-1 ), AFG (0.27 mg g-1 ), IRN (0.27 mg g-1 ), and sigma samples (0.31 mg g-1 ). Meanwhile, average picrocrocin content ranged from 5.40 mg g-1 in KAE, 3.43 mg g-1 in SXR, 3.62 mg g-1 in PAM, and 3.57 mg g-1 in BUD sam- ples, 4.30 mg g-1 in AFG, 5.21 mg g-1 in IRN and 4.46 mg g-1 in SIG sample (Table 4). Crocin and picrocrocin were not detected in SXR4 sample. A biplot of samples was obtained to analyze the relation between metabolites in samples using PCA (Figure 2). PC1 (92.31 %) and PC2 (7.16 %) accounted for 99.47 % of the total variance of the data. The coefficient for both the principal compo- nents is given in Table 5. The results obtained from UV-Vis spectroscopy showed that saffron samples from KAE were of the high- est grade compared to other saffron obtained from other commercial sites. The saffron from PAM and BUD com- mercial sites showed a moderate range of quality. The samples from AFG and IRN fell in grade I and II as per ISO parameters. The saffron from SXR markets showed the lowest grade compared to other samples. The HPLC analysis showed a higher concentration of apocarotenoid content in KAE samples, followed by AFG and IRN sam- ples. The SXR samples showed the lowest quality and apocarotenoid content, which indicates an indication of adulteration. 4 CONCLUSION The evaluation of the quality of saffron selections was done by UV-Vis Spectroscopy according to the limit set by the ISO 3632, and the determination of apocarot- enoid content was analyzed by HPLC analysis. The UV- Vis spectrophotometric results categorized the sample into different grades as per standards formulated by ISO. Only fourteen samples were identified as Grade I, and 13 samples were either grade II or grade III. The remain- ing 3 samples were found to be highly adulterated. The results obtained from HPLC analysis showed significant variation. The highest concentration of apocarotenoids Acta agriculturae Slovenica, 118/3 – 20228 T. G. MIR et al. Sample Origin Crocin (mg g-1 ) Safranal (mg g-1 ) Picrocrocin (mg g-1 ) KAE 33.43-45.36 0.26-0.31 4.03-6.21 SXR 18.26-38.49 0.17-0.29 2.79-4.02 PAM 29.40-42.45 0.26-0.32 3.14-4.11 BUD 20.20-32.43 0.21-0.29 3.04-3.89 AFG 30.49-41.34 0.25-0.30 3.81-5.10 IRN 35.12-35.42 0.26-0.29 5.10-5.33 SIG 34.41 0.31 4.46 Table 4: HPLC-based concentration range of crocin, safranal and picrocrocin of saffron samples obtained from different geographical locations Variable Coefficients of PC1 Coefficients of PC2 UV-Vis analysis Crocin 0.5555 0.82244 Safranal 0.58313 -0.49034 Picrocrocin 0.59278 -0.28836 HPLC analysis Crocin 0.61407 -0.28918 Safranal 0.51423 0.85245 Picrocrocin 0.59875 -0.43554 Table 5: Loading of the first two principal components (PC’s) for concentration of metabolites was found in KAE samples, followed by AFG, IRN, PAM and BUD samples. Samples from SXR showed the least concentration of apocarotenoids, indicating a high level of adulteration. 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Analytica Chimica Acta, 535(1), 133–138. https:// doi.org/10.1016/j.aca.2004.11.060 Acta agriculturae Slovenica, 118/3, 1–15, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2529 Original research article / izvirni znanstveni članek Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits Reda Mohamed GAAFAR 1, 2, Mohamed Lotfi HALAWA 1, Adel Ramadan EL-SHANSHORY 1, Abdelhamid Abdelrahim EL-SHAER 3, Rana Hosny DIAB 1, Marwa Mahmoud HAMOUDA 1 Received January 18, 2022; accepted July 17, 2022. Delo je prispelo 18. januarja 2022, sprejeto 17. julija 2022 1 Botany Department, Faculty of Science, Tanta University, Egypt 2 Corresponding author, e-mail: redagaafar@science.tanta.edu.eg 3 Physics Department, Faculty of Science, Kafrelsheikh University, Kafrelsheikh, Egypt Seed priming with ZNPs reduced expression of salinity toler- ance genes in Glycine max L. and improved yield traits Abstract: Little has been done to evaluate the molecular role of ZnO nanoparticles (ZNPs) in regulating biochemical processes and plant yield in response to salt-induced stress. In this study, the molecular response of salt-stressed soybean (‘Giza111’) was assessed under different concentrations of ZNPs (25, 50, 100, and 200 mg l-1) by measuring some osmo- lytes, yield parameters, and Na+ and K+ content. The impact of salinity on the mRNA expression levels of three key salt-toler- ance related genes (GmCHX1, GmPAP3, and GmSALT3) using qRT-PCR was also determined. The high level of salinity (250 mM NaCl) led to a significant increase in Na+ content, total sol- uble proteins, and total soluble carbohydrates and significantly upregulated gene expression of GmCHX1, GmPAP3, and Gm- SALT3, while reducing K+ content, K+/Na+ ratio and all yield parameters compared to control plants. Soaking soybean seeds in various ZNP concentrations, on the other hand, increased K+ content and K+/Na+ ratio while decreasing Na+ content, to- tal soluble proteins, and total soluble carbohydrates in stressed plants, particularly at 50 mg l-1 ZNPs. Furthermore, GmCHX1, GmPAP3, and GmSALT3 expressions were all downregulated at 50 mg l-1 ZNPs, which ultimately improved soybean yield parameters. Accordingly, these results recommend the applica- tion of 50 mg l-1 ZNPs for improving the productivity of soy- bean cultivated in saline soils. Key words: ZnO; nanoparticles; salinity; soybean; gene expression; qRT-PCR ; productivity Predtretiranje semen s cinkovimi nano delci je zmanjšalo iz- ražanje genov tolerance na slanost pri soji (Glycine max L.) in izboljšalo lastnosti pridelka Izvleček: Malo je bilo narejenega za ovrednotenje mole- kularne vloge nano delcev ZnO (ZNPs) pri uravnavanju bio- kemičnih procesov in pridelka rastlin kot odziva na slanostni stres. V tej raziskavi je bil ocenjen molekularni odziv na sol- ni stres pri soji (‘Giza111’) pri uporabi različnih koncentracij ZNPs (25, 50, 100, in 200 mg l-1) z meritvami nekaterih osmoti- kov, parametrov pridelka in vsebnosti Na+ in K+. Vpliv slanosti na količino mRNK treh ključnih s toleranco na slanost poveza- nih genov (GmCHX1, GmPAP3, in GmSALT3) je bil določen z uporabo qRT-PCR metode. Velika slanost (250 mM NaCl) je vodila k znatnemu povečanju vsebnosti Na+, celokupnih topnih beljakovin, celokupnih topnih ogljikovih hidratov in značilno povečala izražanje genov GmCHX1, GmPAP3, in GmSALT3, med tem ko, je zmanjšala vsebnost K+, razmerja K+/Na+ in vse parameter pridelka v primerjavi s kontrolo. Namakanje semen soje v različnih koncentracijah ZNP je povečalo vsebnost K+ in razmerje K+/Na+ v rastlinah pod stresom in hkrati zmanjšalo vsebnost Na+, celokupnih topnih beljakovin in celokupnih to- pnih ogljikovih hidratov, še posebej pri uporabi 50 mg l-1 ZNPs. Dodatno je bilo pri tem obravnavanju zmanjšano izražanje ge- nov GmCHX1, GmPAP3,in GmSALT3, kar je na koncu izboljša- lo parametre pridelka soje. Skladno s temi rezultati priporoča- mo uporabo 50 mg l-1 ZNPs za izboljšanje pridelka soje, gojene na slanih tleh. Ključne besede: ZnO; nano delci; slanost; soja; izražanje genov; qRT-PCR; produktivnost Acta agriculturae Slovenica, 118/3 – 20222 R. M. GAAFAR et al. 1 INTRODUCTION Soybean (Glycine max L.) is one of the important food and industrial crops worldwide because of its con- tent of cholesterol-free oil (30  %) and proteins (40  %), which are similar in their nourishing value to animal proteins (Van Zanten et al., 2016). The fractions and de- rivatives of soybean seeds have major economic impor- tance in a wide range of industrial, food, pharmaceutical, and agricultural products (Chen et al., 2012). Salinity of the soil is a serious problem all over the world. It has been estimated that around 954 million hec- tares are already salinized (Qadir et al., 2014). It usually causes a reduction of water potential, ion imbalances or disturbances in ion homeostasis, resulting in a reduction of plant growth and crop productivity (Han et al., 2019). Mittler (2002) observed that the oxidative demolition of the cell (oxidative stress) occurs by injuring membranes (lipid peroxidation), proteins, RNA, and DNA molecules as a result of elevated ROS levels in the cells. DNA dam- age is caused by OH· and O2- radicals, and this damage results in heritable changes (Fatima et al., 2017). Moreo- ver, these signals play an important role in the adapta- tion process of plants to abiotic stress (Choudhury et al., 2017). Plant tolerance to salinity stress includes physiologi- cal and molecular changes such as accumulation of or- ganic solutes, antioxidant enzymes, and inorganic ions as well as gene expression responses (Ahanger et al., 2017). These alterations include either the induction of some polypeptides, the disappearance of others, or the overex- pression of other sets of proteins (El-Mashad et al., 2012). Therefore, linking the expression of a gene to a higher degree of tolerance within a genotype offers an impera- tive argument for a role in plant adaptation (Abreu et al., 2013). Numerous reports suggest that the harm- ful effect of salinity stress was manifested by relatively higher expression of salt-related genes in soybean, such as GmP5CS, GmDREB1a, GmGOLS, GmBADH and Gm- NCED1 (Liu et al., 2017), GmERF3 (Zhang et al., 2009), GmMYB genes, GmMYB76, GmMYB92 and GmMYB177 (Liao et al., 2008), GmPAP3 (Liao et al., 2003), GmCHX1 (Patil et al., 2016) and GmSALT 3 (Guan et al., 2014). Previous studies in soybean determined that a QTL on chromosome 3 is the major genomic region that dic- tates salinity tolerance in soybean (Patil et al., 2016; Chen et al., 2018). This gene locus carries the dominant func- tional sodium/hydrogen exchanger family gene in wild (GmCHX1) and cultivated soybean (GmNcl/GmSALT3), which explains more than 64 % of the phenotypic varia- tion (Qi et al., 2014). Normally, the GmCHX1 gene is ex- pressed under high salt conditions in root stellar cells and limits salt transport to shoot tissues (Guan et al., 2014). It has been described that the full-length GmSALT3 protein is closely correlated to the Arabidopsis thaliana AtCHX20 (a Cation/Proton Exchanger), which is a function- ally characterized member of the CPA2 (Cation/Proton Antiporter2) family of transporters (Padmanaban et al., 2007; Qu et al., 2020). Functional studies of AtCHXs have shown that they might play a role in modulating cation and pH homeostasis within the endomembrane system (Chanroj et al., 2011). The ER-localized AtCHX20 was suggested to be an endomembrane K+ transporter in- volved in the osmoregulation of guard cells (Padmana- ban et al., 2007). Purple acid phosphatases (PAPs) rep- resent a diverse group of acid phosphatases in animals, microorganisms, and plants (Vogel et al., 2001; Olczak et al., 2003). The primary biochemical reaction of PAPs is to catalyze the hydrolysis of phosphate esters and an- hydrides. The physiological role of GmPAP3 might be re- lated to the adaptation of soybean to NaCl stress, possibly through its involvement in reactive oxygen species (ROS) forming and/or scavenging or stress-responding signal transduction pathways (Liao et al., 2003; Soleimani et al., 2017). Zinc (Zn) is a metallic cofactor for more than 300 enzymes. The Zn-finger proteins that attach to deoxyrib- onucleic acid (DNA) are clear evidence of the usefulness of Zn in biological systems (Hezaveh et al., 2019). Zinc is a structural component of ribosomes and is essential for their structural integrity. On the other hand, it has other indirect effects on the control of stomatal opening and closing and ROS detoxification (Haliloglu et al., 2020). Currently, nanotechnology has broad perspectives in all fields of science (Dewdar et al., 2018). The application of nanoparticles to plants can be beneficial for growth and development due to their greater absorbance and high reactivity (Fraceto et al., 2016). ZnO nanoparticles (ZNPs) are one of the most frequently used nanoprod- ucts (Samei et al., 2019). Interestingly, priming of seeds with ZNPs positively affected the yield traits in salt- stressed plants, whereas ZNPs stimulated natural auxin (IAA), thus activating cell division and enlargement and also increasing K+ ion content, which increases storage of food in seeds (Ali and Mahmoud, 2013), maintain- ing the structural integrity of biomembranes (He et al., 2015), improving protein synthesis and DNA replication (Landa et al., 2015), scavenging free oxygen radicals and decreasing the uptake of excess Na+ and Cl− (Farhangi- Abriz and Torabian, 2018), as well as augmentation of photosynthesis, total soluble proteins, total soluble car- bohydrates, and total phenols in stressed plants (Abdel Latef et al., 2017). Transcription factors are the primary regulators of gene expression in a variety of genes that are involved in reducing and/or protecting against cellular stress damage Acta agriculturae Slovenica, 118/3 – 2022 3 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits (Linh et al., 2020). The catalytic activity of RNA polymer- ases, which is essential for gene expression, is well known to require Zn2+ ions. Zn stabilizes several structural mo- tifs in transcriptional regulatory proteins, such as Zn fin- ger domains (Albert et al., 1998). Zn has been shown to upregulate gene expression, particularly in Zn-controlled genes, in numerous studies. Plants treated with ZnO, for example, had the highest OsZIP1 expression in their roots after 7 days when compared to no-zinc controls (Selvaraj and Dananjeyan, 2016). Recently, ZNPs boost- ed the expression of the wheat drought-tolerance genes DREB2 and Wdhn13, catalase activity (CAT1), proline biosynthesis (P5CS), and proline biosynthesis (P5CS) genes (Raeisi Sadati et al., 2022). It was concluded that priming with ZNPs, particu- larly at 60 mg l-1, improved photosynthetic pigments, al- tered osmoregulation, and decreased MDA and Na con- centrations in lupine plants (Abdel Latef et al., 2017). So, the current study was conducted to investigate the effect of seed-priming using different concentrations of ZNPs on the expression of three salinity-tolerance genes. In addition, their impacts on alleviating salinity stress and improving productivity in soybean plants were assessed. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS Seeds of a soybean cultivar (‘Giza 111’) were pro- vided by the Food and Legumes Research Department, Field Crops Research Institute, Agricultural Research Center, Giza, Egypt. 2.2 SYNTHESIS AND CHARACTERIZATION OF ZNO NANOPARTICLES In this study, ZnO nanoparticles were synthesized using the chemical bath deposition (CBD) method as described by El-Shaer et al. (2018). The crystalline struc- ture and optical properties of the prepared ZnO nano- structures were examined with X-ray Diffraction (XRD, Shimadzu 6000), while the samples’ morphology was in- vestigated using a scanning electron microscope (SEM, JSM-651OLV). As shown in Fig. 1A, ZnO nanostruc- tures are formed as nano-rods with a hexagonal quartzite crystal structure. These nano-rods accumulate to form the surface morphology of grains, similar to flowers. The XRD pattern of ZnO nano-rods is shown in Fig. 1B. The diffraction peaks at 32°, 34.5°, 36.4°, 47.5°, 57°, 62.7°, 67.9°, and 69.3° correspond to the (100), (002), (101), (102), (110), (103), (112), and (201) lattice planes, re- spectively (Fig. 1B). 2.3 PLANT GROWTH CONDITIONS Priming and growing of the soybean seeds (‘Giza 111’) were performed as described by Gaafar et al. (2020). The seeds were sterilized with 70  % ethanol for 5 min and sodium hypochlorite (10 %) for 10 min, followed by washing several times with distilled water. Four concen- trations of ZnO nanoparticles (ZNPs) of 25 (ZNPs25), 50 (ZNPs50), 100 (ZNPs100), and 200 (ZNPs200) mg l-1 were used to prime the seeds for two hours at room tem- perature, and distilled water was used as a control (0). Previous studies have found that low concentrations of Fig. 1: (A) SEM image and (B) XRD chart of ZnO nanostructures prepared by CBD method Acta agriculturae Slovenica, 118/3 – 20224 R. M. GAAFAR et al. ZnO NPs are beneficial to plant growth, whereas concen- trations equal to or greater than 200 mg l-1 are detrimen- tal. Therefore, the used ZNP concentrations were chosen (Liu et al., 2015; Abdel Latef et al., 2017). After priming, the seeds were sown (20 seeds/pot) in plastic pots (45 cm x 40 cm) filled with 24 kg of 2:1 (clay: sandy) soil. Based on the preliminary experiment results, 250 mM NaCl (S) was chosen as a sub-lethal salinity level and used in this study. The pots were irrigated with tap water until seed germination (emergence), then with tap water and with 250 mM NaCl solution to 80 % field capacity for 21 days (seedling stage) and 90 days (yield stage). Three pots were used as replicates for each treatment. The germinated soybean seeds were let to grow in the green house under the following environmental conditions: 29 ± 2 oC/25 ± 2 oC day/night and 16h/8h light/dark regimes. The 21-day- old seedlings of all treatments were collected, washed, and used for further analyses, and the productivity of yielded seeds was determined on 90-day-old plants. 2.4 DETERMINATION OF SODIUM AND POTAS- SIUM CONTENT According to Allen et al. (1974), the mixed acid digestion method was used for element determination. The concentration of Na+ and K+ (mg g-1 d.m.) was deter- mined by using Inductively Coupled Plasma (ICP, STI) at the central laboratory of Tanta University. 2.5 QUANTITATIVE ESTIMATION OF TOTAL SOLUBLE PROTEINS AND TOTAL SOLUBLE CARBOHYDRATES The total soluble proteins were extracted according to the method described by Naguib et al. (1968). Then the protein content was determined as described by Bradford (1976), and the phenol-sulfuric acid method has been used for estimation of total soluble carbohy- drates according to Dubois et al. (1956). 2.6 QUANTITATIVE REAL TIME PCR (QRT-PCR) RNA EXTRACTION AND PURIFICATION For the extraction of total RNA, approximately 100 mg of ground plant fresh leaves were used, and RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Ger- many) according to the manufacturer’s protocol. The total RNA was then quantified and assessed for quality using a Nanodrop (ScanDrop, Analytik, Jena, Germany). Total RNA samples were kept at -80 °C until further analysis. 2.7 CDNA SYNTHESIS The cDNA synthesis was performed using the Sen- siFAST cDNA synthesis kit (Ameridian Life Science, USA) using the protocol of the manufacturer. The cDNA synthesis reaction contained the following components: 1 µg total RNA, 1 µl reverse transcriptase enzyme, 4 µl 5 × Trans Amp buffer, which was completed to a total volume of 20 µl. The conditions for cDNA synthesis were as follows: primer annealing for 10 min at 25 °C, reverse transcription for 15 min at 42  °C and finally 5 min at 85 °C for enzyme inactivation. After being diluted in 10 mM Tris-HCl (pH = 8) and 0.1 mM EDTA, the cDNA reaction products were stored at -20 °C. 2.8 GENE EXPRESSION ANALYSIS (QRT- PCR) In order to measure the gene expression of the three targeted genes, the reaction mix was prepared by mixing 10 µl of TOP real qPCR2x premix (SYBR Green with low ROX), 1 µl of each of the cDNA template, forward and Primer name Sequence (5’→3’) Length (bp) Annealing temp. (ºC) Reference GmPAP3F GTGGCCGGCAGTTGACATCC 20 55.5 Liao et al. (2003) GmPAP3R GCTGTGCCCTGGCTCTTCTGTG 22 55.5 GmCHX1F GATTTGTTTTCGGGCTAACG 20 49.5 Gutierrez Gonzalez et al. (2010) GmCHX1R ATCCACCACGCTTCGTAACT 20 49.5 GmSALT3F CGGTTGATGAAGGGAAAAC 19 48.5 Hu et al. (2009) GmSALT3R TCCTTGACGCTTGGAGTGTT 20 48.5 GmTublinF GAGAAGAGTATCCGGATAGG 20 50 Gutierrez Gonzalez et al. (2010) GmTublinR GTTTCCGAACACTCAAGCTC 20 50 Table 1: List of sequences of the primers used for gene expression study by qRT-PCR Acta agriculturae Slovenica, 118/3 – 2022 5 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits reverse primer (10 pmol μl-1) and was completed up to 20 µl. The Rotor-Gene Q5 plex (Qiagen, Germany) was used, and the PCR conditions were as follows: an initial denaturation step at 95 °C for 10 min; a denaturation step at 95 °C for 10 s; an annealing step at 60 °C for 15 s; and an elongation step at 72 °C for 15 s. The thermal cycler steps were repeated 35 times. The sequences of the prim- ers used for qRT-PCR analysis are shown in Table 1. The relative gene expression was calculated using the 2-∆∆CT method according to Livak and Schmittgen (2001). 2.9 YIELD TRAITS The yield parameters, including length of pods/ plant, mass of pods/plant, the mass of 1000 seeds, the number of pods/plant, the number of seeds/pod, the mass of seeds/pod, and the mass of seeds/plant, were determined at the end of the growing season. (approxi- mately 3 months from cultivation). The maturity (num- ber of viable - nonviable seeds in pods * 100) and the productivity of soybean (weight of the yielded seeds/pot in grams) were also calculated. 2.10 STATISTICAL ANALYSIS The statistical analyses were carried out according to a completely randomized design (CRD) using analysis of variance. The significance was determined using LSD values at p = 0.05 and 0.01 according to Bishop (1983). The results were analyzed using a one-way ANOVA test to determine the degree of significance. The statistical analyses were performed using CoStat Software version 6.311 (CoHort Software, CA, USA). The heatmap of the gene expression data and Pearson correlation were con- structed using R software (ver. 4.1.1). 3 RESULTS 3.1 SODIUM AND POTASSIUM CONTENT The results in Table 2 show the effect of salinity stress on mineral ion content (Na+, K+, and K+/Na+) in 21-day old soybean seedlings after soaking of soybean seeds in different concentrations of ZNPs (0, 25, 50, 100, and 200 mg l-1). The salinity stress (250 mM NaCl) se- verely decreased the content of potassium by 68 % com- pared to control. Similarly, it reduced the K+/Na+ ratio by 90 % compared to control. In contrast, the content of Na+ was highly increased by 2.16-fold compared to control. On the other hand, the combination of ZNPs50+S (50 mg l-1 + 250 mM NaCl) significantly increased the con- tent of potassium by 1.67-fold compared to salt-stressed seedlings and ameliorated the harmful effect of salinity Treatments K+ (mg g-1 d. m.) Na+ (mg g-1 d. m.) K+/Na+ ratio Salinity level (0 mM NaCl) Control 4.287 ± 0.04 a 2.346 ± 0.0008 f 1.827 ZNPs25 4.043 ± 0.04 b 2.160 ± 0.007 f 1.871 ZNPs50 4.242 ± 0.01 a 1.761 ± 0.022 g 2.409 ZNPs100 3.942 ± 0.04 b 2.247 ± 0.009 f 1.754 ZNPs200 3.756 ± 0.04 c 2.286 ± 0.048 b 1.642 Salinity level (S = 250 mM NaCl) Salinity (S) 1.336 ± 0.01 g 7.419 ± 0.08 a 0.180 ZNPs25+S 2.539 ± 0.14 e 5.425 ± 0.13 d 0.468 ZNPs50+S 3.569 ± 0.11 d 4.061 ± 0.03 e 0.878 ZNPs100+S 2.011 ± 0.008 f 6.629 ± 0.28 c 0.303 ZNPs200+S 1.118 ± 0.004 h 7.120 ± 0.06 b 0.157 F-value 689.8 894.8 - LSD (0.05) 0.138 0.226 - Significance * - Table 2: Effect of salinity (S = 250 mM NaCl) on the content of Na+, K+ and K+/Na+ ratio of 21-day old soybean (‘Giza 111’) seedlings grown in clay-sandy soil (2:1 w/w) after soaking of soybean seeds in four different concentrations of ZnO nanoparticles (ZNPs) (ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg/L) Values are the mean of three replicates ± SD. Values within the same column for each factor designated by different letters are significant at p ≤ 0.05, while values with identical letters are non-significant. *: significant at p ≤ 0.05 Acta agriculturae Slovenica, 118/3 – 20226 R. M. GAAFAR et al. stress. Also, the combination of ZNPs25 + S (25 mg l-1 + 250 mM NaCl) increased the content of potassium ions by only 1.07-fold. Moreover, results indicated that the combination of ZNPs200 + S (200 mg l-1 + 250 mM NaCl) exhibited a se- vere harmful effect compared to other treatments; thus, it reduced the potassium and K+/Na+ ratio content by 16 % and 12  %, respectively, compared to salt stressed seed- lings (Table 2). 3.2 TOTAL SOLUBLE PROTEINS AND TOTAL SOLUBLE CARBOHYDRATES The results in Figure 2 (A and B) indicated that high salinity stress (S = 250 mM NaCl) caused a highly signifi- cant increase in total soluble carbohydrates and protein content by 75.1 % and 76.1 %, respectively, compared to control. However, the results showed a general decrease in total soluble carbohydrates and protein content for all Fig. 2: Effect of NaCl (S = 250 mM) on the total soluble carbohydrates (A) and total soluble proteins (B) of 21-day old soybean (‘Giza 111’) seedlings grown in clay-sandy soil (2:1 w/w) after soaking of soybean seeds in four different concentrations of ZnO nanoparticles (ZNPs) (ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg l-1) Acta agriculturae Slovenica, 118/3 – 2022 7 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits ZNPs (25, 50, 100, and 200 mg l-1) combined with salin- ity, except for the combination of ZNPs25+S (25 mg l-1 + 250 mM NaCl), which exhibited the least reduction in total soluble carbohydrates and protein content with a percentage of 10 % and 23 %, respectively, compared to control plants, which were irrigated with water. The high- est reduction in total soluble carbohydrates and protein content was recorded in the case of ZNPs50+S (50 mg l-1 + 250 mM NaCl) with 29  % and 43  %, respectively, compared to control plants irrigated with salt only (Fig. 2A and B). 3.3 GENE EXPRESSION ANALYSIS (QRT- PCR) 3.3.1 GmCHX1 The results of qRT-PCR analysis showed that Gm- CHX1 expression was increased by ZNPs alone and the highest increase was with ZNPs100+S (100 mg l-1 + 250 mM NaCl) compared to control (no salinity) by about 1.2-fold (Fig. 3). Also, 250 mM NaCl (S) alone showed the highest increase in GmCHX1 gene expression by 1.9-fold compared to control (no salinity and no ZNPs). How- ever, ZNPs25+S (25 mg l-1 + 250 mM NaCl) decreased gene expression by 0.4-fold, and then it was increased with ZNPs50+S (50 mg l-1 + 250 mM NaCl). Also, Gm- CHX1 gene expression was decreased by 0.08-fold with ZNPs100+S (100 mg l-1 + 250 mM NaCl). In contrast, ZNPs200+S (200 mg l-1 + 250 mM NaCl) showed an in- crease of 1.8-fold, which was similar to that of salinity (S = 250 mM NaCl) (Fig. 3). 3.3.2 GmPAP3 GmPAP3 expression was slightly increased by ZNPs treatments, and the two concentrations (ZNPs100 and ZNPs200 mg l-1) showed the highest increases of about 0.73 and 0.70-fold, respectively, compared to control (no salinity and no ZNPs) (Fig. 3). In the case of salt treat- ment (S = 250 mM NaCl), GmPAP3 expression increased by 3-fold. ZNPs25+S (25 mg l-1 + 250 mM NaCl), ZNPs50 + S (50 mg l-1 + 250 mM NaCl), ZNPs100 + S (100 mg l-1 + 250 mM NaCl), and ZNPs200 + S (200 mg l-1 + 250 mM NaCl) all reduced GmPAP3 expression. The highest de- crease was by 0.46-fold and was recorded with ZNPs25+S (25 mg l-1 + 250 mM NaCl) treatment (Fig. 3). 3.3.3 GmSALT3 In the case of GmSALT3, gene expression was in- creased by ZNP treatment using a concentration of 100 mg l-1 compared to control (no salinity and no ZNPs) by about 2-fold, as shown in Fig. 3. However, salinity stress (250 mM NaCl) showed the highest increase in Gm- SALT3 gene expression by 7.7-fold. The concentration of 25 mg l-1 of ZNPs with salinity decreased gene expression by 1.56-fold, and then it was increased by the concentra- tion of 50 mg l-1 of ZNPs with salinity by 4.5-fold. Then, in comparison to the salinity stress alone (250 mM NaCl), gene expression decreased by 3.9-fold with ZNPs100 + S (100 mg l-1 + 250 mM NaCl) treatment and by 3.5-fold with ZNPs200 + S (200 mg l-1 + 250 mM NaCl). In con- trast to the salinity stress alone (250 mM NaCl), which increased gene expression by 8-fold (Fig. 3). Fig. 3: Heatmap of relative expression of three soybean salinity-linked genes (GmPAP3, GmCHX1, and GmSALT3) in 21-day old soybean (‘Giza 111’) seedlings after soaking of soybean seeds in four different concentrations of ZnO nanoparticles (ZNPs) (ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg l-1) Acta agriculturae Slovenica, 118/3 – 20228 R. M. GAAFAR et al. 3.4 YIELD PARAMETERS The results given in Table 3 show the effect of 250 mM NaCl and ZNPs (25, 50, 100, and 200 mg l-1) treat- ments on yield parameters. These results revealed an ob- servable increase in all measured yield parameters, spe- cifically in the case of ZNPs25 (25 mg l-1) and ZNPs50 (50 mg l-1) treatments without salinity, where these treat- ments increased the pod length, pod mass, number of pods/plants, and mass of pods/plant, number of seeds/ pods, mass of seeds/pods, and mass of seeds/plant. The most significant increase was with ZNPs50 (50 mg l-1) by 29 %, 27.8 %, 62.8 %, 39.9 %, 15.3 %, 47.8 %, and 78.8 %, respectively, compared to control. In contrast, results showed that the application of 200 mg l-1 ZNPs (ZNPs200) caused a highly significant decrease in all measured yield parameters: pod length, pod mass, number of pods/ plant, mass of pods/plant, number of seeds/pod, mass of seeds/pods, and mass of seeds/plant by 37.8 %, 16.1 %, 13.89 %, 20.9 %, 26.9 %, 11.5 %, and 18.1 %, respectively compared to control. Similarly, a remarkable increase in all measured yield parameters in the case of treatments ZNPs25 + S, ZNPs50 + S, and ZNPs100 + S was observed (Table 3). The most significant increases in pod length, pod mass, number of pods/plants, and mass of pods/plant, number of seeds/pods, mass of seeds/pods, and mass of seeds/ plant were with ZNPs50+S (50 mg l-1 + 250 mM NaCl) by 50.11 %, 85.4 %, 42.6 %, 47.7 %, 341.6 %, 100 %, and 119  %, respectively, compared to control. Whereas, a decrease in these yield parameters was observed with ZNPs200+S (200 mg l-1 + 250 mM NaCl) by 53.2  %, 15.2 %, 23.2 %, 14.5 %, 100 %, 15.7 %, and 4.7 %, respec- tively, compared to control. Similarly, the most significant increases in mass of the seeds (g/plant), mass of 1000 seeds, maturity per- centage, and productivity index (g/pot) were with treat- ment ZNPs50 + S (50 mg l-1 + 250 mM NaCl) by 14.8 %, 31.5 %, 32.6 %, and 118.9 %, respectively, compared to control. In contrast, treatment with ZNPs200 + S (200 mg l-1 + 250 mM NaCl) decreased these parameters by 3 %, 14.2 %, 30.3 %, and 17.8 %, respectively, compared to control. These results proved the efficiency of 50 mg l-1 (ZNPs50) for increasing the productivity of the soybean plant under high salinity levels (S = 250 mM NaCl). 3.5 PEARSON CORRELATION ANALYSIS As shown in Figure 4, the Na+ was only positively corrected with Zn (r = 1.0*), total proteins (r = 0.89*), and total carbohydrates (r = 92*), while it was negatively cor- rected with the rest of the studied characters, with dif- ferent correlation coefficients. The pod weight was posi- tively correlated with the weight of seeds/plant (r = 0.9*), K+ (r = 0.91*), K+/Na+ ratio (r = 0.88*), and the number of seeds/pod (r = 0.79 ns). 4 DISCUSSION As sessile organisms, plants have adapted a variety of signal perception mechanisms as well as pathways to control molecular responses in order to respond effec- tively to abiotic stress situations (Dudziak et al. 2019). Exposure of soybean seedlings to high salinity stress (250 mM NaCl) imposed a significant depletion in K+ content and in K+/Na+ ratio by 68 % and 90 %, respectively, com- pared to control. Similar findings were also found by Taf- fouo et al. (2009) and Khan et al. (2017) in cowpea and soybean, respectively. Contrarily, the amount of Na+ was 2.16-fold more than it was in the control plants. It is pos- sible that high salinity promoted the uptake of Na+ due to its adverse effects on membrane integrity. In this regard, a similar conclusion was also made by Abdel Latef et al. (2017) on lupine plants. According to reports, K+ is required for maintaining osmotic balance and is an essential co-factor for many enzymes. Therefore, K+ reduction negatively affects the growth and productivity of plants (Hauser and Horie, 2010). The results of this study indicated that K+ content in soybean seedlings showed a highly significant reduc- tion under acute salinity stress. However, treatment with ZNPs increased K+ by 1.67-fold and decreased Na+ by 45 % compared to salt-stressed seedlings, which is com- parable to the findings of Abdel Latef et al. (2017) on lu- pine plants (Lupine termis L.). This is due to the fact that Zn+2 helps in maintaining the structural and functional integrity of root cell membranes and therefore controls the influx and efflux of Na+ across the plasma membranes (Rezaie and Abbasi, 2014). The application of ZnO is associated with a remark- able increase in K+ uptake from soil to roots (Weisany et al., 2012; Soliman et al., 2015). As a consequence of enhanced K+ uptake, plants treated with ZnO had great- er K+/Na+ ratios than those under salinity stress alone. A high K+/Na+ ratio is often reported as a good indica- tor of a high tolerance to salt stress conditions (Khan et al., 2017). Thus, applications of ZNPs could be a useful strategy for achieving increased macronutrient uptake by plants (Dimkpa and Bindraban, 2016), which is similar to what was observed in this study, where ZNPs50+S (50 mg l-1+ 250 mM NaCl) treatment significantly increased the content of potassium by 1.67-fold compared to salt- stressed seedlings and ameliorated the harmful effect of salinity stress. Acta agriculturae Slovenica, 118/3 – 2022 9 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits Tr ea tm en ts Po d le ng th (c m ) Po d m as s (g ) N o. o f p od s/ pl an t M . o f p od s/ pl an t ( g) N o. o f s ee ds / po d M . o f s ee ds / po d (g ) M . o f s ee ds / pl an t ( g) M . o f 1 00 0 se ed (g ) Pe rc en ta ge o f m at ur ity (% ) Pr od uc tiv ity in de x (g /p ot ) Sa lin ity le ve l (0 m M N aC l) C on tr ol 6. 71 ± 0 .1 2 e 2. 41 ± 0 .0 5 d 5. 11 ± 0 .4 4 d 8. 42 ± 0 .1 7 d 5. 2 ± 0. 18 c 1. 38 ± 0 .1 2 e 13 4. 72 ± 0 .2 2 c 61 .1 2 ± 4. 5 h 36 ± 4 .1 f 5. 42 ± 0 .1 7 d ZN Ps 25 7. 34 ± 0 .2 6 c 2. 92 ± 0 .0 5 b 6. 16 ± 0 .4 4 c 10 .6 6 ± 0. 25 b 5. 8 ± 0. 16 ab 1. 58 ± 0 .0 3 d 15 4. 22 ± 0 .1 3 b 92 .3 3 ± 4. 4 b 55 .8 ± 1 .6 b 8. 37 ± 0 .1 1 b ZN Ps 50 8. 68 ± 0 .1 7 a 3. 08 ± 0 .1 3 a 8. 32 ± 0 .6 8 a 11 .7 8 ± 0. 16 a 6. 0 ± 0. 27 a 2. 04 ± 0 .1 2 a 17 5. 8 ± 0. 15 a 94 .6 5 ± 4. 8 a 69 .9 ± 3 .2 a 10 .4 8 ± 0. 14 a ZN Ps 10 0 7. 72 ± 0 .2 0 b 2. 12 ± 0 .1 5 e 4. 6 ± 0. 93 e 6. 30 ± 0 .3 2 f 4. 6 ± 0. 43 d 1. 68 ± 0 .0 6 c 12 4. 4 ± 0. 20 d 74 .2 4 ± 4. 8 d 29 .4 ± 3 .0 g 4. 35 ± 0 .1 1 g ZN Ps 20 0 4. 17 ± 0 .1 0 i 2. 02 ± 0 .0 6 f 4. 4 ± 0. 44 f 6. 66 ± 0 .4 1 e 3. 8 ± 0. 18 e 1. 22 ± 0 .0 6 f 11 7. 0 ± 0. 12 f 66 .3 4 ± 4. 4 f 27 .1 ± 3 .4 h 4. 64 ± 0 .1 7 f Sa lin ity le ve l (S =5 0 m M N aC l) Sa lin ity (S ) 4. 51 ± 0 .1 1 h 1. 31 ± 0 .1 h 4. 43 ± 0 .4 4 f 6. 62 ± 0 .1 1 e 1. 2 ± 0. 12 g 0. 95 ± 0 .1 1 h 10 6. 2 ± 0. 42 h 70 .1 2 ± 0. 63 e 41 .9 ± 2 .6 c 3. 65 ± 0 .3 6 h ZN Ps 25 +S 5. 22 ± 0 .1 2 g 1. 87 ± 0 .1 g 3. 46 ± 0 .4 4 h 8. 46 ± 0 .1 3 d 4. 3 ± 0. 15 de 1. 01 ± 0 .0 2 gh 11 3. 4 ± 0. 20 g 74 .2 3 ± 0. 48 d 39 .3 ± 3 .0 d 5. 35 ± 0 .4 1 e ZN Ps 50 +S 6. 77 ± 0 .1 5 d 2. 43 ± 0 .1 1 c 6. 32 ± 0 .6 8 b 9. 78 ± 0 .1 6 c 5. 3 ± 0. 11 bc 1. 9 ± 0. 15 b 12 2. 0 ± 0. 72 e 92 .2 2 ± 0. 40 c 55 .6 ± 3 .3 b 7. 99 ± 0 .4 6 c ZN Ps 10 0+ S 5. 55 ± 0 .1 1 f 1. 15 ± 0 .1 2 i 3. 6 ± 0. 93 g 4. 30 ± 0 .3 3 h 4. 6 ± 0. 13 d 1. 08 ± 0 .0 3 g 10 7. 0 ± 0. 30 h 66 .1 ± 1 .0 g 37 .6 ± 3 .4 e 4. 64 ± 0 .4 7 f ZN Ps 20 0+ S 2. 11 ± 0 .1 3 j 1. 11 ± 0 .1 2 j 3. 4 ± 0. 44 h 5. 66 ± 0 .2 3 g 2. 4 ± 0. 18 f 0. 80 ± 0 .0 4 i 10 3. 0 ± 0. 31 i 60 .1 ± 0 .3 1 i 29 .2 ± 0 .3 1 g 3. 0 ± 0. 31 i F- va lu e 11 46 55 .2 14 56 8. 5 17 94 .3 80 91 .5 62 .9 25 7. 5 40 36 .3 25 26 64 .1 53 84 .5 15 56 7. 3 LS D (0 .0 5) 0. 01 7 0. 01 7 0. 10 8 0. 07 7 0. 56 2 0. 07 7 1. 08 0. 07 7 0. 56 2 0. 05 6 Si gn ifi ca nc e * Ta bl e 3: E ffe ct o f s al in ity (S = 2 50 m M N aC l) on th e yi el d pa ra m et er s o f 9 0- da y ol d so yb ea n (c v. G iz a 11 1) p la nt s g ro w n in cl ay -s an dy so il (2 :1 w /w ) a fte r s oa ki ng o f s oy be an se ed s i n fo ur d iff er en t c on ce nt ra tio ns o f Z nO n an op ar tic le s ( ZN Ps ) ( ZN Ps 25 = 2 5, Z N Ps 50 = 5 0, Z N Ps 10 0 = 10 0, a nd Z N Ps 20 0 = 20 0 m g l-1 Va lu es ar e t he m ea n of th re e r ep lic at es ± S D . V al ue s w ith in th e s am e c ol um n fo r e ac h fa ct or d es ig na te d by d iff er en t l et te rs ar e s ig ni fic an t a t p ≤ 0 .0 5, w hi le v al ue s w ith id en tic al le tte rs ar e n on -s ig ni fic an t Acta agriculturae Slovenica, 118/3 – 202210 R. M. GAAFAR et al. Fig. 4: Correlogram based Pearson correlation analysis of Na+, K+, K+/Na+, Zn and yield parameters of 90-day old soybean (‘Giza 111’) plants grown in clay-sandy soil (2:1 w/w) after soaking of soybean seeds in different concentrations of ZNPs (25, 50, 100, and 200 mg l-1). On the right hand side of the correlogram, the legend color shows the correlation coef- ficients and the corresponding colors. The positive correla- tions are displayed in blue, while the negative correlations are shown in red. The color intensity and the size of the circle are proportional to the correlation coefficients In this study, high salinity stress (250 mM NaCl) caused a highly significant increase in total soluble pro- teins and carbohydrates content by 76 % and 75 %, re- spectively, in salt-stressed soybean seedlings. Similar results were reported by Sadeghipour (2017) in Vigna unguiculata (L.), Karimi et al. (2019) in Vitis vinifera (L.), and Cardoso et al. (2019) in two varieties of cowpea. It is well known that osmotic stress induced by salt stress leads to the synthesis of proteins, which play an impor- tant role in plant salt tolerance through cytosolic calcium signal. This signal activates the calcium sensor protein for activation of the protein kinase to regulate Na+/H+ antiporter in plasma membranes and tonoplasts, thus the osmo-sensory histidine kinase regulates osmotic ho- meostasis and ROS scavenging (Chinnusamy et al., 2005; Abdel Latef et al., 2017). In addition, total soluble carbo- hydrates are key osmolytes in the osmotic adjustment of all plants, ROS scavenging, and maintaining ion homeo- stasis under salinity stress (Chen and Jiang, 2010) and have a direct relationship with physiological processes in plants (Tombesi et al., 2019). In this study, treatment with ZNPs reduced total soluble proteins and total soluble carbohydrates content under salinity stress, and the highest reduction was re- corded in the case of ZNPs50+S (50 mg l-1 + 250 mM Na Cl) with 43  % and 36  %, respectively. This result indi- cates that ZNPs alleviated the harmful impacts of salin- ity stress. The ZNPs treatment might cause an inhibition of oxidative stress, decreasing the content of Na+ in the shoot tissues (Haidera et al., 2019). Indeed, ZNPs have been shown to increase CO2 fixation, photosynthetic pig- ments, photosynthetic efficiency, and plant growth res- toration in response to salt stress (Soliman et al., 2015; Kasim et al., 2017; Mathur et al., 2019). In addition, in this study, the expression levels of three key salt-tolerance related genes (GmCHX1, Gm- PAP3, and GmSALT3) were determined under 250 mM of NaCl salt alone. The gene expression was increased for all three genes (GmCHX1, GmPAP3, and GmSALT3) by 1.9-, 3-, and 7.7-fold, respectively. Generally, stress results in changes in the cellular program that involve significant transcriptional alterations aimed at increas- ing the chances of survival (Diédhiou et al., 2008). A study by Dang et al. (2014) proved that overexpression of GmPAP3 improved rice salt tolerance by increasing the ROS-scavenging ability and decreasing oxidative dam- age. Similarly, a possible tolerance role of GmPAP3 under oxidative stress was demonstrated in soybean, indicating that the GmPAP3 gene expression is regulated by salinity, osmotic, and oxidative stresses (Liao et al., 2003; Li et al., 2008b; Soleimani et al., 2017). It can be concluded that salinity induces the formation of ROS, which in turn ac- tivates GmPAP3, leading to an increase in ROS degrada- tion till reaching the proper level in the mitochondria, at which point the activity of the GmPAP3 gene is decreased (Francisca, 2005; Li et al., 2008a). As mentioned above, the results of this study re- vealed that GmCHX1 expression was increased under salinity stress by 1.9-fold, which is parallel to the results of Patil et al. (2016), who reported that salinity stress (200 mM NaCl) significantly induced the expression of the GmCHX1 gene in soybean, maintaining ion homeo- stasis by lowering the Na+/K+ ratio. This result is also con- sistent with data from this study, which showed a high reduction in K+/Na+ ratio by 90 % compared to control. Furthermore, the GmCHX1 gene was highly expressed in the leaves and roots of soybean seedlings in response to salinity stress (Do et al., 2016). It was reported that low Na+ accumulation in shoot tissues of soybean plants may be due to the powerful function of the GmCHX1 gene, which was highly expressed in salt-stressed soybean roots, forming Na+ exclusion proteins in root tissues and preventing Na+ entrance from soil to roots (Guan et al., 2014; Qu et al., 2020). This function of the GmCHX1 gene has been documented in other plant species such as cotton (Wu et al., 2004), rice (Ren et al., 2005), Arabidop- sis (Møller et al., 2009) and wheat (Munns et al., 2012). Acta agriculturae Slovenica, 118/3 – 2022 11 Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits Moreover, the results of this study indicated that GmSALT3 gene expression was significantly increased in response to salinity stress by 7.7-fold in salt-stressed soybean seedlings. It was reported that the GmSALT3 gene is the major salt tolerance gene in soybean belong- ing to the cation/H+ exchanger (CHX) family (Patil et al., 2016), which is mainly expressed in root cells associated with the phloem and xylem, leading to limiting the ac- cumulation of sodium ions in leaves (Pardo et al., 2006), which improved the physiological and morphological parameters and ultimately increased soybean yield under saline conditions (Do et al., 2016). As GmSALT3 is local- ized in the endoplasmic reticulum (ER), it plays a direct role in the retrieval of salt from the xylem (Padmanaban et al., 2007; Cao et al., 2019). It has been reported that GmSALT3 exerts a positive effect on soybean salt toler- ance by exclusion of Na+ in plant shoots and therefore prevents the toxic accumulation of Na+ in photosynthetic tissues (Maathuis et al., 2014). Furthermore, Do et al. (2016) suggest that CHX1/GmSALT3 controls Na+, K+, and Cl- accumulation and may function as a cation-chlo- ride co-transporter. Application of nanoparticles alters the levels of ex- pression of certain transcription factors, making it pos- sible to modify plant tolerance to salinity stress (Yamagu- chi et al., 2013). In particular, application of ZNPs could upregulate or downregulate the stress-tolerant genes de- pending on their function by cascade reactions, thereby enhancing salt tolerance (Jonak et al., 2002). The results of this study showed that application of ZNPs in combination with salt-stress downregulated the expression of the three studied salinity-tolerant genes in soybean seedlings compared to salt-stressed ones. The expression of GmCHX1, GmPAP3, and GmSALT3 was decreased by 0.4, 0.46, and 1.56-fold, respectively, par- ticularly with 25 mg l-1 ZNPs in combination with high salinity stress (250 mM NaCl). Interestingly, this finding confirms the ameliorative role of ZNPs in improving soy- bean plant tolerance in response to salinity, which was reflected in enhancement effects on mineral uptake, total soluble proteins, total soluble carbohydrates, and yield characteristics. This finding is in accordance with that of Almutairi (2019) and Alharby et al. (2016) in tomato plants, where ZNPs imposed a positive response on plant metabolism under salt stress. It was reported that the dif- ferential response of GmPAP3 expression in soybean to different ZNPs treatments under salinity stress could be as a result of reverted effects caused by NPs (Zhang et al., 2020) by excluding sodium ions from the roots, thus preventing the accumulation of toxic concentrations in the stem and leaves (Munns and Tester, 2008; Zhang et al., 2020). 5 CONCLUSIONS The results of the present study indicated the im- portance of Zn+2 in increasing soybean tolerance to salt stress. Soaking seeds of soybean cultivar Giza 111 in ZNPs at 50 mg l-1 reduced oxidative damage caused by salinity stress, downregulated salt-tolerant gene expres- sion, and increased soybean plant yield under high salin- ity stress (250 mM NaCl). Additionally, gene expression analysis of GmCHX1, GmPAP3, and GmSALT3 con- firmed their roles in salt tolerance in the soybean cultivar Giza 111. 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Plant Physiology and Biochemistry, 155,161-168. https://doi.org/10.1016/j. plaphy.2020.06.036 Acta agriculturae Slovenica, 118/3, 1–11, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2589 Original research article / izvirni znanstveni članek Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley Zohreh HAJIBARAT 1, Abbas SAIDI 1, 2 Received March 07, 2022; accepted July 26, 2022. Delo je prispelo 7. marca 2022, sprejeto 26. julija 2022 1 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 2 Corresponding author, e-mail: abbas.saidi@gmail.com Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley Abstract: AGC kinases are highly conserved regulators in a variety of cellular processes such as differentiation, prolifera- tion, and growth. They are known to play important roles in stress and hormonal responses, including ROS signaling. AGC kinases are the main class of protein kinases in plants, having central functions in different stages of plant growth. In the pres- ent study, the analysis of phylogenetic relationships, gene struc- tures, chromosomal locations, synteny analysis, gene ontology, subcellular localization, and gene expression of AGC kinase identified 28 AGC kinase genes in barley. Phylogenetic tree grouped them into seven subfamilies, as supported by exon- intron organization. Gene duplication and synteny indicated that tandom and block duplication events played an essential role in the expansion of AGC kinase gene families in barley. The Real-time quantitative reverse transcription PCR (qRT-PCR) analysis performed for  HvAGC kinase gene were largely ex- pressed in different tissues of roots, stems, and leaves in Azaran and Jolgeh cultivars under heat and cold stresses. The results of chromosomal localization showed that the AGC kinases were located on all chromosomes of barley except chromosome 1. Genome evolution of species was surveyed using identification of orthologous and paralogous genes. Identifying overlaps be- tween orthologous clusters can enable us to study the function and evolution of proteins in different species. To our knowl- edge, this is the first detailed report of using AGC kinases for bioinformatics analysis in barley. Results revealed a broad un- derstanding of the AGC kinase gene family in barley, which will be valuable for improving barley varieties’ response to heat and cold stresses. Also, HvNDR6.2 gene can utilized as molecular markers under cold stress in the three organs. Key words: AGC kinase protein; protein model; synteny; gene duplication Identifikacija genov na ravni celotnega genoma za kinaze AGC in njihovo izražanje kot odziv na vročinski in hladni stres pri ječmenu Izvleček: Kinaze AGC so v veliki meri ohranjeni regula- torji različnih celični procesov kot so diferenciacija, prolifera- cina in rast. Znano je, da imajo pomembne vloge pri stresnih in hormonskih odzivih, vključno s signalizacijo ROS. Kinaze AGC so glavna skupina proteinskih kinaz v rastlinah, ki imajo osrednjo vlogo v razlilčnih fazah rasti rastlin. V tej raziskavi je bilo pri ječmenu na osnovi analize filogenetskih odnosov, gen- skih struktur, kromosomskih lokacij, analize sintenije in genske ontologije, njihove subcelularne lokalizacije in izražanja genov kinaz AGC identificiranih 28 genov kinaz AGC. Filogenetsko drevo jih je na osnovi organizacije intronov in eksonov poraz- delilo v sedem poddružin. Podvojevanje genov in sintenija sta pokazali, da sta imela pri ječmenu tandemsko in bločno pod- vojevanje odločilno vlogo pri ekspanziji družin genov za kina- ze AGC. Analiza kvantitativne reverzne transkripcije PCR v realnem času (qRT-PCR) opravljene za gene kinase HvAGC je pokazala, da so se ti geni v veliki meri izrazili v različnih tkivih korenin, stebla in listov pri sortah Azaran in Jolgeh v razmerah vročinskega in hladnega stresa. Rezultati kromosomske loka- lizacije so pokazali, da so bili geni za kinase AGC pri ječmenu locirani na vseh kromosomih, razen na kromosomu 1. Evoluci- ja genoma ječmena je bila preučena z identifikacijo ortolognih in paralognih genov. Prepoznavanje prekrivanj med skupina- mi ortolognih genov omogoča preučevanje funkcije in razvoja proteinov pri različnih vrstah. Glede na vedenje avtorjev je to prvo podrobnejše poročanje o uporabi kinaz AGC z analizo bioinfomatskih pristopov pri ječmenu. Rezulati so odkrili ve- liki pomen družin genov za kinaze AGC pri ječmenu, kar bo pomembno za izboljšanje sort ječmena pri odzivu na vročinski in hladni stres. Gen HvNDR6.2 bi lahko uporabili kot mole- kularni marker odziva pri hladnem stresu v koreninah, steblu in listih. Ključne besede: AGC proteinske kinaze; nabor protei- nov; sintenija; podvajanje genov Acta agriculturae Slovenica, 118/3 – 20222 Z. HAJIBARAT and A. SAIDI 1 INTRODUCTION Protein kinases catalyze the transfer of phosphoryl group from adenosine triphosphate (ATP) to some ami- no acids such as (serine, threonine) in their substrate pro- teins. Most protein kinases, like AGC (cAMP-dependent, cGMP-dependent and protein kinase C) kinase family ) AGC kinase(, have seven subfamilies which have been reported in plants and bacteria. Protein phosphorylation is one of the most important post-translational modifica- tions (PTMs) for cellular signaling, mediated by a group of enzymes called protein kinases (Bradley and Beltrao, 2019). The activity of these enzymes lead to the regula- tion of almost all cellular processes. This is achieved at any time by a network of different kinases that are tran- siently active. Therefore, control of cellular systems re- quires that each kinase targets only a limited set of sub- strates. AGC kinases are divided into seven groups based on sequence similarity, evolutionary conservation, and known functions (Bradley and Beltrao, 2019). Seven sub- groups of AGC kinase (AGC1, AGC2, PDK-1, S6K, IRE, NDR, AGC2 related subfamilies) are common to eu- karyotic genomes of animals, plants, and diatoms. AGC kinases are termed after the cyclic AMP dependent ki- nases (PKA), cGMP-dependent kinases, and the diacyl- glycerol-activated/phospholipid-dependent kinase PKC. Details and biochemical properties of 28 AGC kinase genes in barley are given in Table 1. The member of the 3-phosphoinositide dependent protein kinase 1 (PDK1) genes are highly conserved among eukaryotes (Mora et al., 2004). Furthermore, orthologous of the p70 riboso- mal protein S6 kinase (S6K), the nuclear Dbf2-related (NDR) kinase subfamily identified in Arabidopsis (Gal- van-Ampudia and Offringa, 2007). Based on amino acid sequence homology, AGC1-4 were placed in AGCVIII kinases in Arabidopsis, implicated in the regulation of cell polarity, growth, and cell division. AGCVIII kinases have conserved domains and are most associated to animal PKA and PKC, playing a key role in developmental stag- es and stress responses. In AGC2 related subfamily, two phototropin genes (PHOT1 and PHOT2) were expressed in plant seeds (Galvan-Ampudia and Offringa, 2007). Most protein kinases regulate cell growth and division in embryo, cotyledons, floral organs, and stress signal- ing (Rentel et al., 2004). In AGC1 subfamily, PINOID (PID) gene has been revealed to regulate the polarity of auxin transport by phosphorylating the large central hydrophilic loop of auxin efflux carriers (Dhonukshe et al., 2010; Huang et al., 2010). AGC kinases have multiple functions in different biological processes such as pol- len germination and development and plant growth and development. Also, AGC kinase genes can be utilized in different abiotic and biotic stresses. Different function of proteins could be due to the presence of conserved do- mains or gene duplication (Saidi and Hajibarat 2020a). In this study, comprehensive analysis of phylogeny of AGC kinase genes, gene structures, gene duplications, synteny analysis, gene ontology, chromosomal distribution of AGC kinases were further performed. Gene expression of five AGC kinase genes were analyzed in response to heat and cold stresses in three tissues. 2 MATERIAL AND METHODS 2.1 PHYSICOCHEMICAL CHARACTERISTICS, PHYLOGENETIC ANALYSIS, AND GENE STRUCTURE OF AGC KINASE PROTEINS The ensemble plant database was utilized to down- load the sequences of AGC kinase family genes from barley (H. vulgare L), wild cabbage (B. oleraceae L.), rapeseed (B. napus L.), field mustard (B. rapa L.), maize (Z. mays L.), Arabidopsis, and rice (O. sativa L.). ExPASy server (https://www.expasy.org/) was used to predict the theoretical isoelectric point (pI) and the molecular mass (Mm) of each of the AGC kinase proteins. AGC kinase protein sequences were aligned in seven species using the MUSCLE and phylogenetic tree was drawn using MEGA 7, applying the Neighborjoining algorithm. The Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu. cn/) was used to obtain information on the exon – in- tron of AGC kinase proteins. The prediction subcellular localization of AGC kinase was performed using CELLO database. 2.2 CHROMOSOMAL DISTRIBUTION AND GENE ONTOLOGY (GO) ANALYSIS AND DETEC- TION OF ORTHOLOGOUS AND PARALO- GOUS OF AGC KINASE GENES For locating the AGC kinase genes on barley chro- mosome, AGC kinase genes were placed on each chro- mosome according to the physical location of the gene. The AGC kinase genes were distributed on all chromo- somes and depicted with MapChart (Voorrips, 2002). For functional annotation using default parameters, the nucleotide sequences of DEGs were submitted to the on- line annotation tool of Mapman (http://www.plabipd. de/portal/mercator-sequence-annotation) (Thimm et al., 2004). The orthologous and paralogous genes among species were identified using Blastp of proteins (Altschul et al., 1990). When the protein sequence identity exceed- ed 70 %, it was considered orthologous genes whereas, when the AGC kinase protein sequences identity exceed- Acta agriculturae Slovenica, 118/3 – 2022 3 Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley Protein Name Genomic Locus Duplication Chromosome location Number of amino acids Molecular weight Theoretical pI Subcellular localization HORVU2Hr1G072580 HvAGC1.8 chr2H : 520772898-520775368 384 42.4 5.61 Cytoplasmic HORVU3Hr1G082260 HvPDPK1 chr3H:598502260-598506773 481 53.7 6.17 Mitochondrial HORVU3Hr1G024030 HvAGC1.10 chr3H : 90576271-90577182 1315 145 6.36 Cytoplasmic HORVU4Hr1G062610 HvAGC1.11 chr4H : 523887114-523888691 417 47.7 6.03 Cytoplasmic HORVU3Hr1G031930 HvPHOT1 chr3H : 160765754-160767689 527 60.4 5.9 Cytoplasmic HORVU4Hr1G007540 HvIREH1 chr4H : 19756134-19766838 320 45 5.7 Mitochondrial HORVU4Hr1G062610 HvPK3 chr4H : 523887114-523888691 464 53.8 8.05 Cytoplasmic HORVU5Hr1G108690 HvAGC1.5 Block duplicate chr5H : 628434675-628436329 692 75.4 6.39 Cytoplasmic HORVU5Hr1G041960 HvAGC1.12 chr5H : 318743447-318747958 670 71.6 5.99 Cytoplasmic HORVU1Hr1G030190 HvNDR6 chr1H : 172906058-172908519 427 45.2 9.09 Cytoplasmic HORVU1Hr1G077430 HvNDR7 chr1H : 517244865-517251727 281 30 5.8 Cytoplasmic HORVU1Hr1G031420 HvAGC1.7 chr1H : 188829523-188831273 470 51.6 9.78 Cytoplasmic HORVU2Hr1G072580 HvKIPK chr2H : 520772898-520775368 465 50.6 9.64 Cytoplasmic HORVU2Hr1G093580 HvAGC1.3.1 chr2H : 658836435-658839125 123 13.7 9.99 Cytoplasmic HORVU5Hr1G035610 HvKIPK.1 chr5H : 248570403-248571724 337 37.3 9.19 Cytoplasmic HORVU5Hr1G072930 HvAGC2.4 chr5H : 537104020-537105417 512 55.2 9.27 Cytoplasmic HORVU0Hr1G005020 HvAGC2.2 chr6h : 28702476-28703321 337 37.3 9.19 Cytoplasmic HORVU6Hr1G062330 HvAGC1.3 chr6H : 417602550-417605458 340 38 9.2 Cytoplasmic HORVU5Hr1G009390 HvPID chr5H : 21969701-21971152 555 60.3 6.23 Cytoplasmic HORVU6Hr1G054770 HvAGC2.1 chr6H : 347811467-347812777 783 85.9 9.25 Cytoplasmic HORVU7Hr1G050240 HvAGC2.4 chr7H : 180272159-180273571 790 86 9.3 Cytoplasmic HORVU7Hr1G050240 HvAGC2.3 chr7H : 180272159-180273571 529 58.3 7.1 Cytoplasmic HORVU3Hr1G020020 HvNDR4 Block duplicate chr3H : 60417083-60425196 525 57 9.22 Cytoplasmic HORVU3Hr1G020020 HvNDR6.1 Block duplicate chr3H : 60417083-60425196 527 56.5 8.07 Cytoplasmic HORVU3Hr1G031930 HvAGC1.6 chr3H : 160765754-160767689 525 56.9 9.22 Cytoplasmic HORVU4Hr1G050660 HvS6K2 Chr3 : 2648625-2650407 483 52 9.46 Mitochondrial HORVU3Hr1G031930 HvAGC1.5.1 chr3H : 160765754-160767689 783 85.4 9.3 Cytoplasmic HORVU1Hr1G027770 HvNDR6.2 Block duplicate chr1H : 143842779-143854406 350 37.6 6.33 Cytoplasmic Table 1: Details and Biochemical properties of AGC kinase genes in barley ed 85 %, it was considered paralogous genes. The analysis of synteny of AGC kinase genes were performed using Circos program (http://mkweb.bcgsc.ca/tableviewer/ visualize/). PLAZZA was used to detect the duplica- tion patterns containing segmental/tandem duplications (Wang et al., 2009). Identification of orthologous cluster- ing between AGC kinase members of Arabidopsis thali- ana, (L.)  Heynh. rice, and barley was performed using OrthoVenn2 (https://orthovenn2.bioinfotoolkits.net/ home) webserver. 2.3 BARLEY GROWTH UNDER HEAT AND COLD TREATMENTS This study was done based on a randomized complete block design (RCBD) with three replications at the Seed and Plant Improvement Institute, Karaj (latitude 35 °, lon- gitude 50 ° and altitude 1313 m above sea level) during 2021. Azaran and Jolgeh cultivars were cultured in pots at 25 °C for two weeks. Young roots, stems, and leaves from 2-week-old seedlings were harvested for tissue-specific Acta agriculturae Slovenica, 118/3 – 20224 Z. HAJIBARAT and A. SAIDI expression analysis under cold or heat stress, treated for four hours at 4 °C or 42 °C, respectively. 2.4 RNA EXTRACTION AND QUANTITATIVE REAL-TIME PCR (QRT-PCR) ANALYSIS Total RNA was extracted from root, stem, and leaf under stress conditions using RNA-Plus kit (Sinaclone) based on the manufacturer’s instructions. For the prep- aration of tissue-specific RNA, the organ samples were collected separately two week-old seedlings under cold (4 °C) and heat (42 °C) stresses for 4 hrs. To remove re- sidual genomic DNA contamination in RNA samples, DNase I (Fermentase Company) was utilized. The pu- rity and concentration of RNA were determined by nan- odrop as well as the quality of which was validated using 1 % agarose gel analysis. Then, cDNA synthesis was per- formed according to Easy cDNA Synthesis Kit instruc- tions. Three replications were performed for the analysis of each gene, with the barley Actin gene utilized as the reference gene. All primers used in gene expression anal- ysis are listed in Table 2. Primers were designed using the Oligo program. Real time (qPCR) was done on ABI 7500 using SYBR Green Supermix as described in the manufacture’s guidelines. Relative expression was deter- mined via 2- ∆∆Ct technique after normalization of the Ct value for individual genes versus Actin as the reference gene. Analysis of gene expression was performed using the REST software (according to the Pfaffl method). RT- qPCR was conducted to determine the expression profile for the five AGC kinase genes using various tissues under heat and cold stresses. RT-qPCR expression analysis was carried out using our established protocol. 3 RESULTS AND DISCUSSION In the present study, AGC kinase genes in barley were surveyed using genome-wide identification, chro- mosomal distribution, evolutionary relationships, synte- ny analysis, and gene structure. Detailed information in- cluding the biochemical properties of the 28 AGC kinase genes are listed in Table 1. Sequence analysis showed that the lengths of the deduced AGC kinase proteins varied from 123 amino acids (HvAGC1.3.1) to 1315 amino acids (HvAGC1.10). The predicted molecular weights (MW) and isoelectric points (pI) ranged from 13.7 kDa (HvAGC1.3.1) to 85.9 kDa (HvAGC2.1) and from 5.61 (HvAGC1.8) to 9.99 (HvAGC1.3.1) (Table 1). AGC ki- nase proteins grouped into the same subfamily exhibited similar motif distributions, suggesting functional simi- larities for the members in the same subfamily. In addi- tion, same roles of AGC kinases in various plant species that showed differential aspects of AGC kinase function- ality in species. For instance, in contrast to the reported conserved functions of many AGC kinases, INCOM- PLETE ROOT HAIR ELONGATION (IRE), a kinase of the “AGC other” group, seems to have acquired a new function in Medicago Truncatula Gaertn.. In Arabidopsis, IRE kinase has been revealed to control root hair elonga- tion, while inMedicago a role in the formation of nodules has been indicated (Pislariu and Dickstein, 2007; Oyama et al., 2002; Saidi and Hajibarat, 2021a). Most of AGC ki- nase genes were located in the cytoplasm, but HvPDPK1, HvIREH1 and HvS6K2 genes were located in the mito- chondria. 3.1 PHYLOGENETIC ANALYSIS AND GENE STU- CRURE To assess the evolutionary relationships of AGC ki- anse proteins in H. vulgare, B.oleraceae, B.napus. B.rapa, Z.mays, Arabidopis, and O.sativa. used to discribe phy- logenetic analysis using MEGA7 based on protein se- quences (Figure 1). The phylogenetic relationships and exon/intron analysis of the barley were showed in Figure 1. The HvAGC from barley was distributed in all groups, proposing that the expansion of HvAGC occurred in barley genome. Many researchers have revealed that the AGC kinase genes from monocots, based on their do- mains, could be grouped into 7 subfamilies (Kong et al., 2021). The genes within each subfamily showed similar gene structures. Gene structure of AGC kinase genes in barley was grouped into 7 subfamilies, with the largest cluster related to AGC-1 subfamily. The smallest cluster was related to PDK-1 and SK6 subfamilies. The num- No. Primer Name Sequence 5’→ 3’ 1 HvNDR4F TGGCTTCGTCTGGACAACCTGCT HvNDR4R CTTGGTTGGAACACGCTACACC 2 HvNDR6F GAGGAGTTGTATACACCACC HvNDR6R ACGTCGCATCCCTTTGCTCA 3 HvNDR6.2F CCGATGAGTCCATAACCCGGCG HvNDR6.2R GGATTTCTACAAACTGCTCGCC 4 HvAGC1.5F AAGCAACACCCCTTCTTCGAG HvAGC1.5R GTTCTAGAAATACTCGAACTGGCC 5 HvAGC1.5.1F AGATCAAGCAGCACCCCTTC HvAGC1.5.1R CGCTGTGGTCTAAAAGAACTCG 6 Actin F: GGTCCATCCTAGCCTCACTC Actin R: GATAACAGCAGTGGAGCGCT Table 2: Primers used for AGC kinase genes in this study Acta agriculturae Slovenica, 118/3 – 2022 5 Figure 1: Phylogenetic tree of AGC kinase genes created by the neighbor-joining (NJ) method (a) in MEGA7.0 software in Arabi- dopsis, rice, barley, Brassica napus, Brassica rapa, and Brassica oleracea. The tree was constructed using the MEGA 6.0 software by the Neighbor joining method. Distributions of the exon- intron pattern in AGC kinase proteins in barley (b) Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley Acta agriculturae Slovenica, 118/3 – 20226 Z. HAJIBARAT and A. SAIDI MPKs although its mechanism is still unclear. PDK1 is a key factor involved in stress signaling (Petersen et al., 2009). PDK1 encodes a gene of the AGC protein kinase family and a significant regulator of AGC kinases. PDK1, detected in mammalian cells, has an essential role in re- lating lipid signaling to a comprehensive range of cellu- lar signaling and processes (Kyoko et al., 1989). Also, it is involved in the advancement of cell proliferation and survival and is overexpressed in many different tumors (Toda et al., 1988; Saidi and Hajibarat, 2021b). PDK1 contains an N-terminal kinase domain and a C-termi- nal pleckstrin homology (PH) domain through which it binds to phospholipids (Krupnick et al., 1998). 3.2 CHROMOSOMAL DISTRIBUTION AND DU- PLICATION OF AGC KINASE GENES Anslysis of physical locations on barley chromo- somes preseneted that 28 AGC kinase genes were drawn using Mapchart software (Figure 2). In barley, three AGC kinase genes were located on both chromosomes 2 and 6. Six and 10 HvAGC were located on the chromo- ber of exons in AGC kinase genes ranged from one to 17. The HvNDR6 and HvIREH1 genes had the highest number of exons. Some AGC kinases such as HvAGC2.1 and HvKIPK.1 genes contained only one exon, indicat- ing that these genes have conserved domains. The gene structure of the AGC kinase genes in the same subfamily is highly consistent (Figure 1b). Most of the AGC kinase genes were grouped in the same subfamily. Most genes had many exons with introns, indicating that patterns of exons and introns, which correlate well with the phyloge- netic tree, support their close evolutionary relationships between the AGC kinase genes within the same subfami- lies. The AGC2.1, also known as OXI1, was revealed to be a prerequisite for ROS-mediated responses in Arabi- dopsis like root hair elongation and for resistance to bio- trophic pathogens. The activity of OXI1 was enhanced through H2O2, wounding, and various elicitor treatments mimicking pathogen attack (Rentel et al., 2004; Petersen et al., 2009). Also, as oxi1 mutant plants are impaired in the activation of mitogen-activated protein kinase (MPK) and MPK6 in response to cellular injury and oxidative stress, OXI1 is a regulator of stress-responsive Figure 2: Chromosomal distribution and expansion patterns of AGC kinase genes in barley, drawn using Mapchart software Acta agriculturae Slovenica, 118/3 – 2022 7 Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley somes 2 and 3, respectively. Finally, nine HvAGC were distributed on chromosome 5. Our findings showed that the HvAGC genes are unevenly distributed on different chromosomes. Based on the research findings on rice, Arabidopsis and Brachypodium distachyon (L.) P.Beauv., it has been shown that AGC kinase gene families mainly expanded through whole-genome and chromosomal segment duplications (Xue et al., 2008; Yang et al., 2008). Genes located within a distance of less than 200 kb on the same chromosome are defined as tandem duplications, otherwise they are segmental duplications (Cheung et al., 2003). In barley, 11 pairs of HvAGC duplication genes were involved in tandem duplication events and no gene segmental duplication pairs were found (Figure 2). 3.3 ORTHOLOGOUS AND PARALOGOUS GENES STUDY IN AGC KINASE In this study, a comparative analysis was done to detect the orthologous of AGC kinase genes in barley genome (Figure 3). Based on the gene identity, ortholo- gous (exceeding 70 %) and paralogous (exceeding 85 %) gene pairs were revealed. Among 28 genes, one gene was paralogous. According to our results, high similarity in barley genes suggests genome duplication (polyploidy), playing a key role in the evolution of AGC kinase genes. The comparative analysis to identify orthologous of AGC kinase genes in barley genome showed that the HvNDR4 with HvNDR6 genes had high similarity (identity 70 %) was orthologous one gene pair and HvNDR6.2 with HvNDR4 genes was paralogous (Figure 3). The syntenic analysis indicated that duplications as main elements for the diversity in AGC kinase genes, suggesting the struc- tural and functional conservation of the genes underlying the origins of evolutionary with conserved domains (Al- tenhoff and Dessimoz, 2009). Often orthologous genes have similar expression among various species, while paralogous genes have the same basic but slightly differ- ent functions. It has been shown that gene duplication is collectively deemed from single-gene duplications (Saidi et al., 2020b). The structural conservation of NDR pro- teins indicates that they may had similar functions and regulatory mechanisms in various species. Surveys have suggested that NDRs are the main factors of the signaling mechanism in yeast and human (Hergovich, 2016). Figure 3: Orthologous and paralogous relationships of AGC kinase genes with three genomes visualized by Circos database in barley Acta agriculturae Slovenica, 118/3 – 20228 Z. HAJIBARAT and A. SAIDI 3.4 ORTHOLOGOUS GENE CLUSTERING OF AGC KINASE GENE FAMILY IN ARABIDOPSIS, RICE, AND BARLEY OrthoVenn2 web server was utilized to identify the evolutionary relationship of AGC kinases between dicot (A. thaliana) and monocot (O. sativa, and H.vulgare) plants using orthology analysis. In this study, 86 AGC kinase proteins from three species were clustered in 18 orthologous groups. One monocot-specific cluster was observed containing two AGC kinase proteins, whereas seven AGC kinase proteins were found to be present in two dicot-monocot orthologous clusters in Arabidopsis and barley (Fig 4). Also, 18 AGC kinase proteins were found to be present in nine dicot-monocot orthologous clusters in Arabidopsis and rice. One cluster containing two AGC kinase proteins has been identified between rice and barley (Figure 4). Interestingly, the OrthoVenn2 analysis also indicated the presence of an Arabidopsis- specific orthologous cluster containing two AGC kinases (Figure 7a). A singleton is a rare variant for which ge- netic difference is performed by a single chromosome in a genome. Among the three species, Arabidopsis had the highest number of singletons (54). Singletons usually are dispersed in the genome, indicating that they have been developed independently. Figure 4 a: Orthologous gene clustering analysis. The orthologous gene clusters among the AGC kinase gene families in A. thali- ana, O. sativa, and H. vulgare were identified and visualized using the OrthoVenn2 web platform. The e-value cut-off 1e-10 was used for the analysis. b. Number of singletons identified in A. thaliana, O. sativa, and H. vulgare Acta agriculturae Slovenica, 118/3 – 2022 9 Genome wide identification of AGC kinase genes and their expression in response to heat and cold stresses in barley 3.5 EXPRESSION PROFILES ANALYSIS OF AGC KIANASE GENES IN SPECIFIC TISSUES UN- DER COLD AND HEAT STRESS CONDITIONS In Azaran cultivar, most of AGC kinase genes showed a wide range of expression levels under heat and cold stress conditions. The expression profile of 5 AGC kinase genes under heat and cold stress conditions were performed using reverse transcription-PCR (qRT-PCR) quantitative analysis in three different tissues: root, stem, and leaf. Most of genes showed increased expression un- der heat stress as compared to cold stress and were ex- pressed in the leaves showed increased expression, indi- cating that the AGC kinase genes were involved in leaf development. AGC kinase genes were decreased in cold stress but were increased in response to heat stress. The HvNDR6.2 gene showed high expression in root, shoot, and leaf tissues under heat stress, while it was down-reg- ulated in response to cold (Figure 5a). In Jolgeh cultivar, AGC kinase genes showed differ- ent expressions in response to cold and heat stresses. The HvNDFR4 gene was up-regulated in roots under heat stress but was down-regulated under cold stress condi- tion. In Jolgeh cultivar, most genes were down-regulated in response to heat and cold stresses. Only the HvN- DR6.2 gene was up-regulated in leaves and stems under heat stress. The HvNDR6.2 gene showed up-regulated expression in roots, stems, and leaves under cold stress. Most genes showed high levels of relative expression un- der normal conditions in leaves. But AGC kinase genes was down-regulated under cold stress. The HvNDR4 gene had high expression in roots under heat condition. These results showed that the HvNDR4 gene can be used as a molecular marker in barley root improvement un- der heat and cold stresses. Also, the HvNDR6.2 gene can be utilized as a molecular marker under cold stress in all three tissues (Figure 5b). In our analysis, the expression profiles of AGC kinase genes under heat and cold stresses revealed differential and overlapping expression patterns. Various expression patterns of AGC kinase genes may suggest various roles in response to heat and cold stress conditions. Based on the synthetic analysis of the AGC kinase genes, the HvNDR4 and HvNDR6 genes were orthologous, indicating that the orthologous genes had similar expression. As a result, the HvNDR4 and HvN- DR6 genes can respond almost identically under heat and cold stress conditions. 3.6 GENE ONTOLOGY ANALYSIS In barley, the largest percentage of detected proteins was involved in protein metabolism (90.91 %) followed by hormonal metabolism (6.06 %) (Figure 6). Most of the AGC kinase genes in the barley were involved in protein metabolism (post-translational modification and pro- tein synthesis) and signaling, where many of the AGC2 kinases were involved in light signaling. Plants possess the same basic AGC kinase subfamilies (PDK1, S6K, and NDR) as other eukaryotes but they do not encode for the AGC kinases, such as PKA and PKC, implicated in the control of cell expansion, proliferation, and auxin polar- ity in fungi and animals (Zhang and Friml, 2020). One NDR protein, TaAGC1, has been indicated to have bio- Figure 5: Differential gene expression of Azaran (a) and Jolgeh (b) cultivars under heat and cold stress conditions. Green and red indicate up and down-regulated genes, respectively. Also, yellow color indicates low level expression under both stresses. Gene expression patterns in the three tissues (stem, root, leaf) Acta agriculturae Slovenica, 118/3 – 202210 Z. HAJIBARAT and A. SAIDI logical function, implicated in response to Rhizoctonia cerealis E.P. Hoeven used for controlling the induction of ROS-related and defense-related genes in wheat (Zhang and Friml, 2020). Plant responses to external or internal stimuli include rapid protein changes that ultimately lead to the activation of transcriptional processes. Phospho- rylation of proteins is commonly used in cellular signal- ing (protein kinase), where a phosphate group is added to the amino acid side chain. PID and phototropins are main factors in triggering and regulating growth by con- trolling auxin transport such as PIN. Phototropins have a key role in plant growth and were used directly in polar auxin transport (Galván-Ampudia and Offringa, 2007). The AGC kinase protein kinase family is involved in vari- ous signaling pathways, containing light blue and auxin signaling (Christensen et al., 2000; Robert and Offringa, 2008). 4 CONCLUSION In this study, 28 AGC kinase genes were detected in barley. We studied their phylogenetic relationships, gene structures, chromosomal locations, genes duplica- tion, gene ontology, and clustering of orthologous genes. Further, genome-wide identification of AGC kinase genes were performed. According to structure analysis results, various genes of the same subfamily had similar gene structure, proposing that they have the same evo- lutionary origin and probably the same functions. These results can provide insights into the functional differenc- es, evolutionary relationships, and comparative genom- ics analysis of AGC kinases in barley. The HvNDR4 and HvNDR6 genes were both orthologous and were found to serve as molecular signaling in different stresses. AGC kinase genes showed to have different functions, indicat- ing the presence of various conserved domains in these genes. Using this characteristic, candidate genes can be used to genetically improve plants in response to abiotic stresses. 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Journal of Experi- mental Botany, 66(21), 6591-6603. https://doi.org/10.1093/ jxb/erv367 Acta agriculturae Slovenica, 118/3, 1–17, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2399 Original research article / izvirni znanstveni članek Intercropping induces physiological and morphological plasticity in oil- seed rape and barley under drought stress Noushin SADEGHZADEH 1, Roghieh HAJIBOLAND 1, 2, and Charlotte POSCHENRIEDER 3 Received September 03, 2021; accepted August 03, 2022. Delo je prispelo 3. septembra 2021, sprejeto 3. avgusta 2022 1 Department of Plant, Cell and Molecular Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran 2 Corresponding author, e-mail: ehsan@tabrizu.ac.ir 3 Plant Physiology Laboratory, Bioscience Faculty, Universidad Autónoma de Barcelona, Spain Intercropping induces physiological and morphological plas- ticity in oilseed rape and barley under drought stress Abstract: Intercropping is an agricultural practice that can improve crop yield due to better availability of resources, including water. There are few studies, however, addressing the physiological mechanisms behind this phenomenon. In this work oilseed rape (Brassica napus L.) and barley (Hordeum vulgare L.) were cultivated either as monocrop (MC) or inter- crop (IC) under well-watered (WW) or drought stress (DS) conditions in a growth chamber. After eight weeks DS, the leaf relative water content was higher in the IC compared with the MC plants in both species and the DS-induced senescence of old leaves was considerably postponed in oilseed rape. Inter- cropped oilseed rape showed elevated levels of leaf photosyn- thesis rate, superior accumulation of organic osmolytes but higher water loss compared with the MC counterparts under DS conditions. In barley, less transpiration, an increased root : shoot ratio and osmolyte accumulation was observed in the IC compared with MC plants under DS conditions. The water use efficiency was higher in the IC compared to MC barley and the plants yield was higher in the IC than in the MC oilseed rape. Our data showed that intercropping is a reliable practice for cultivation of both species under arid and semi-arid regions or under rainfed conditions. Key words: drought stress; intercropping; osmotic adjust- ment; photosynthesis rate;, transpiration: water use efficiency This paper is part of PhD thesis of N.S under supervision of R.H; C.P was the advisor of this thesis. Medsetev vzpodbuja fiziološko in morfološko prilagodljivost oljne ogrščice in ječmena v sušnem stresu Izvleček: Medsetev je način kmetovanja, ki izboljšuje pri- delek poljščin zaradi zaradi boljše dostopnosti virov, vključno z vodo. Malo je raziskav, ki bi se ukvarjale s fiziološkimi me- hanizmi tega fenomena. V tej raziskavi sta bila v rastni komori gojena oljna ogrščica (Brassica napus L.) in ječmen (Hordeum vulgare L.) kot monokultura (MC) ali kot mešan posevek (IC) v razmerah dobre preskrbe z vodo (WW) ali v razmerah su- šnega stresa (DS). Po osmih tednih rasti v sušnem stresu je bila relativna vsebnost vode pri obeh vrstah večja pri medsetvi kot v monokulturi, pri oljni ogrščici je bilo odmiranje starih listov v razmerah sušnega stresa znatno kasnejše. Oljna ogrščica je imela v medsetvi v razmerah sušnega stresa večjo fotosintezo, večje kopičenje osmotikov, a večjo izgubo vode v primerjavi z gojenjem v monokulturi. Pri ječmenu je bila pri medsetvi v raz- merah sušnega stresa manjša transpiracija, povečano razmerje korenina : poganjek, povečana akumulacija osmotikov v pri- merjavi z rastjo v monokulturi. Učinkovitost izrabe vode je bila pri ječmenu večja v medsetvi kot v monokulturi, v medsetvi je bil večji tudi pridelek oljne ogrščice. Ti podatki kažejo, da je medsetev primeren način gojenja obeh vrst v sušnih in polsu- šnih območjih v razmerah preskrbe z vodo z deževjem. Ključne besede: sušni stres; medsetev; osmotska prila- goditev; velikost fotosinteze; transpiracija; učinkovitost izrabe vode Članek je del disertacije N.S. pod mentorstvom R.H. in svetovanjem C.P. Acta agriculturae Slovenica, 118/3 – 20222 N. SADEGHZADEH et al. 1 INTRODUCTION Drought stress is one of the most important envi- ronmental constrains limiting plants production world- wide (Tardieu et al., 2018). Photosynthesis, the key pro- cess responsible for growth and dry matter production of plants, decreases under water stress through both sto- matal and non-stomatal limitations (Zhou et al., 2013). Nonstomatal factors such as decreased leaf expansion and photosynthetic pigments concentration, leaf senes- cence and reduced electron transport activities, in com- bination with stomatal factors, reduce the overall pho- tosynthetic performance of plants under drought stress (Chaves et al., 2009). Since the CO2 assimilation is decreased simultane- ously with transpiration under water stress, the efficiency of plants for photosynthesis or biomass production at the expense of a given rate of water loss, i.e. water use efficiency (WUE), is an important parameter for plants drought tolerance (Tambussi et al., 2007). Plants adopt various strategies for confronting drought stress and survive under these conditions. The increased production of low molecular weight organic osmolytes such as free amino acids particularly proline and soluble carbohydrates is crucial for the regulation of cell water content under extreme osmotic environment (Singh et al., 2015). By decreasing the osmotic potential in the cytoplasm, these osmoprotectants help plants to prevent cell dehydration. Moreover, these organic os- molytes contribute to mitigate damage caused by reactive oxygen species (ROS), to prevent membrane injury and to stabilize proteins and enzymes (Krasensky and Jonak, 2012). Intercropping is establishing two or more crop spe- cies together at same field in the same time. Under in- tercropping conditions, both negative interaction (com- petition) and positive interaction (facilitation) can occur simultaneously (Brooker et al., 2015). However, by in- creasing facilitation and decreasing competition between crops, intercropping systems can use environmental re- sources more effectively. In fact, higher yield has been repeatedly recorded in many intercropping systems com- pared to monocultures (Martin-Guay et al., 2018). There are evidences showing that biomass and water use effi- ciency (WUE) of intercropping systems under drought stress are usually greater than that of monocultures (Daneshnia et al., 2015; Chimonyo et al., 2016). There are, however, studies that showed intercropping systems did not increase obviously WUE (Grema and Hess, 1994; Shackel and Hall, 1984), or sometime reduced it (Rees, 1986; Singh et al., 1988; Gao et al., 2009). Belowground interactions in the ecological and ag- ricultural systems are not restricted to the competition or facilitation mechanisms for nutrient acquisition (Mom- mer et al., 2016). Increasing evidences obtained from plants co-cultured under laboratory conditions showed considerable influence of both interspecific and conspe- cific interactions on plants development, metabolism and defense (Schmid et al., 2013; Chen et al., 2018). Almost all of these effects are independent from competition or complementary usage of resources (Semchenko et al., 2014; Kong et al., 2018). Almost all of previous works on the effect of crop- ping pattern on plants drought resistance have been un- dertaken under field conditions with little attention paid to explore the mechanisms behind the improvement of drought tolerance in the intercrop systems. In order to explore the physiological and biochemical effects of be- lowground root interactions, we cultivated oilseed rape plants and barley under well-watered or drought stress conditions either as monocrop or intercrop and analyzed plants for water content and osmotic parameters. Our working hypothesis is that, the interspecific interactions in the intercrop system may trigger some biochemical and physiological modifications in the co-cultured plants that influence their response to drought. 2 MATERIALS AND METHODS 2.1 PLANTS CULTURE AND TREATMENTS Seeds of oilseed rape (Brassica napus ‘Opera’) and barley (Hordeum vulgare ‘Makoui’) plants were provided by the Seed and Plant Improvement Institute (Karaj, Iran) and Dryland Agricultural Research Institute (DARI) (Maragheh, Iran), respectively. The seeds were surface sterilised using commercial bleach and germinated in the dark on perlite. After germination, the seedlings were transferred to the light. The 10-day old young oilseed rape seedlings were precultured in the 50 % Hoagland nutrient solution for two weeks before starting the ex- periment. Twenty five-day-old oilseed rape together with one- week-old barley seedlings were transferred to 0.8 l plastic pots filled with perlite and cultivated either as monocrop (MC) or intercrop (IC). Since the biomass and leaf area of one barley seedling was a quarter of oilseed rape, 4 bar- ley plants were cultivated with one oilseed rape in the IC pots. In the MC pots either two oilseed rape or 8 barley plants were cultivated. Two weeks after starting MC/IC treatments, two watering regimes including well-watered (WW) and drought stress (DS) were assigned randomly to the pots. The WW plants were continued to be irrigated to 100 % field capacity (FC) while watering was omitted from DS Acta agriculturae Slovenica, 118/3 – 2022 3 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress pots until they reached the 30 % FC. This was achieved one week after starting the DS treatment. Everyday throughout the experiment, after weigh- ing, the pots were irrigated with nutrient solution or wa- ter as interval. Control and water-stressed plants received the same amount of nutrient solution and the respective FC was achieved by different volumes of water used for irrigation. Water consumption (~water loss; the amount of water needed for adjustment of pots to the respective FC) was recorded daily. 2.2 HARVEST The plants were harvested eight weeks after reaching the 30 % FC (10 weeks after starting MC/IC treatments). The roots were separated from perlite and washed with distilled water and blotted dry on filter paper. After de- termination of fresh mass (FM), leaf and root samples were oven-dried at 70 °C for 48 h, and dry mass (DM) was determined. Because of almost complete intertwin- ing the roots in the IC pots under WW conditions, the root mass could not be determined for two species sepa- rately. 2.3 MEASUREMENT OF SPAD AND LEAF CHL CONCENTRATION Leaf greenness was measured daily as the Spectral Plant Analysis Diagnostic (SPAD) index in the second youngest, fully expanded leaf (young leaf) and in the second oldest leaf (old leaf) using a chlorophyll-meter (Minolta, 502). Leaf Chl concentration in the young and old leaves was spectrophotometrically determined after extraction in 70 % aceton for 24 h in the dark at 4 °C (Lichtenthaler and Wellburn, 1985). 2.4 DETERMINATION OF GAS EXCHANGE PA- RAMETERS AND WATER USE EFFICIENCY Net CO2 assimilation rate, transpiration and stoma- tal conductance to water vapour were measured in the attached young and old leaves with a calibrated portable gas exchange system (LCA-4, ADC Bioscientific Ltd., UK) between 10:00 and 13:00 a.m at a photosynthetic photon flux density of 350 μmol m–2 s–1. The instantaneous water use efficiency (iWUE) (µmol mmol–1) was defined at leaf scale as the net photo- synthesis rate divided by the water transpired in the same time period: The biomass WUE (bWUE) (g kg–1) was defined at whole plant scale as the ratio of biomass produced to the rate of water consumed (Tambussi et al., 2007): 2.5 MEASUREMENT OF RELATIVE WATER CON- TENT AND OSMOTIC POTENTIAL Relative water content (RWC %) was measured in the leaves harvested 1 h after starting the light period and calculated according to the following equation: For determination of turgid mass (TM), leaf disks (5 mm diameter) were submerged for 5 h in distilled water, thereafter, they were blotted dry gently on a paper towel and weighed. Osmotic potential was determined in the leaf and root samples harvested at 1 h after the lights were turned on in the growth chamber. Samples were homogenized in prechilled mortar and pestle and centrifuged at 4000 g for 20 min at 4 °C. The osmotic pressure of the samples was measured by an osmometer (Heman Roebling Messtechnik, Germany), and the miliosmol data were recalculated to MPa. 2.6 DETERMINATIONS OF BIOCHEMICALS For determination of soluble carbohydrates, leaf and root samples were homogenized in ethanol at 4 °C. After centrifugation at 12,000 g for 15 min, an aliquot of the supernatant was mixed with anthrone-sulfuric acid rea- gent and incubated for 10 min at 100 °C. After cooling, the absorbance was determined at 625 nm (Yemm and Willis, 1954). Glucose (Merck) was used to construct a standard curve. Total soluble proteins were determined using a commercial reagent (Bradford reagent, Sigma) and bovine albumin serum as standard. Proline was extracted and determined by the method of Bates et al. (1973). Leaf tissues were homogenized with 3 % sulfos- Acta agriculturae Slovenica, 118/3 – 20224 N. SADEGHZADEH et al. alicylic acid and the homogenate was centrifuged at 3000 g for 20 min. The supernatant was treated with acetic acid and acid ninhydrin, boiled for 1 h, and then absorbance at 520 nm was determined. Proline (Sigma) was used for production of a standard curve. Content of total free a- amino acids was assayed using a ninhydrin colorimetric method (Yemm and Cocking, 1955). Glycine was used for standard curve. 2.7 EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS The experimental design was a complete ran- domised block with four independent pots as four rep- lications. Pairwise comparison of means was performed by the Tukey’s test (p < 0.05) using Sigma stat (3.02). To assign different physiological parameters to distinct groups, principal component analysis (PCA) was con- ducted using Minitab 18. 3 RESULTS AND DISCUSSION 3.1 EFFECT OF DS AND IC ON THE BIOMASS AND LEAF AREA Drought stress (DS) decreased shoot biomass and leaf area of both species (Fig. 1). However, the effects of DS on the shoot biomass and leaf area under MC condi- tions were not significant in the barley and oilseed rape plants, respectively (Fig. 1). Leaf growth is accomplished through cell division and cell expansion which are both affected by water deficit (Koch et al., 2019). Cell expan- sion is one of the most drought-sensitive physiological processes because of its dependence on the turgor pres- sure. Impaired cell division and expansion results in re- duced plant height, leaf area and ultimately growth re- duction of plant under drought (Skirycz and Inze, 2010). Under long term water deficit as in our work, biomass of plants is also decreased due to the reduced CO2 assimila- tion rate (Tardieu and Granier, 2011). Similar to the shoot growth parameters, root bio- mass decreased under DS conditions in both species cultivated in the MC pots. The responses of root growth and elongation to drought largely depend on the plant species, the genotype and the severity of drought stress (Sánchez-Blanco et al., 2014). Under mild drought stress root : shoot ratio may increase as the result of a prefer- ential allocation of photosynthates to the roots allowing better water capture as an adaptation to drought (Faroog et al., 2009). Under severe drought, in contrast, root bio- mass and length is decreased likely because the limited photosynthesis reduces the sucrose export to the roots and ultimately inhibits root growth (Lemoine et al., 2013). Here in our work, root : shoot ratio was not modi- fied by DS in oilseed rape while decreased from 1.0 under WW to 0.71 under DS conditions in barley (Fig. 1). Intercropped (IC) oilseed rape plants showed high- er shoot biomass than the monocropped (MC) counter- parts under both well-watered (WW) and DS conditions. Barley, in contrast, produced less shoot biomass when cultivated in the IC pots irrespective the watering regime (Fig. 1). The leaf area also increased under IC conditions in oilseed rape under WW conditions, while this param- eter decreased in barley both under WW and DS condi- tions (Fig. 1). The improvement of shoot growth under intercropping conditions in oilseed rape, but its depres- sion in barley that was observed independent from wa- tering treatments will be discussed below. Root biomass was not influenced by the IC treat- ment in oilseed rape, while increased in barley under DS conditions (Fig. 1). The improvement of root biomass in IC barley grown under DS conditions contrasted with the effect of intercropping on shoot biomass and leaf area in this species. This led to an increase in root : shoot ratio from 0.71 in the MC to 3.12 in the IC barley plants, while this ratio was not influenced by intercropping in oilseed rape plants. The effect of IC on WW plants could not be detected because of lacking individual data for each species (see M & M). The total root biomass of plants in the IC pots (1.90 ± 0.22) was significantly higher (p < 0.05) than the sum of two MC pots (1.29 ± 0.37) (data not shown). 3.2 EFFECT OF DS AND IC ON THE CHL CON- CENTRATION, PHOTOSYNTHESIS AND TRANSPIRATION RATES The Chl a + b concentration was not influenced by DS in barley but decreased in the old leaves of oilseed rape plants (Table 1). Reduction of Chl under DS is likely the results of higher rates of degradation mainly due to the elevated levels of ROS under these conditions (Noc- tor et al., 2014). Loss of the balance between the pro- duction and scavenging of ROS induces oxidative stress and the accumulated ROS damages proteins, pigments, membrane lipids and other cellular components (Cruz de Carvalho, 2008). The photosynthesis and transpiration rates de- creased significantly by DS in the old leaves of both spe- cies and in the young leaves of oilseed rape (Table 1). Reduction of transpiration through stomatal control of water losses has been identified as an early event in plant response to water deficit leading in turn to limitation of Acta agriculturae Slovenica, 118/3 – 2022 5 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress Figure 1: Shoot and root dry biomass and leaf area in oilseed rape and barley cultivated either as monocrop (MC) or intercrop (IC) under well-watered (WW) or drought stress (DS) conditions for eight weeks. Bars indicated by the different letters are signifi- cantly different (p < 0.05) Acta agriculturae Slovenica, 118/3 – 20226 N. SADEGHZADEH et al. CO2 diffusion into the leaves (Zhou et al., 2013). Since the activity of the photosynthetic electron transport chain is finely tuned to the availability of CO2 in the chloroplast, restricted CO2 availability could lead to increased suscep- tibility to damage to photosynthetic apparatus (Chaves et al., 2002). In addition, reduction in photosynthesis arises by impaired activities of Calvin cycle enzymes and a de- cline in Rubisco activity (Chaves et al., 2009). Intercropping did not affect Chl a + b concentration in the young leaves of oilseed rape plants, but increased it in the old leaves of both WW and DS plants. In barley, concentration of Chl a + b was not influenced in either of the leaves under WW or DS conditions (Table 1). In oilseed rape, IC treatment increased photosynthesis rate in the young leaves of DS plants and in the old leaves of both WW and DS plants. In barley, in contrast, leaf photosynthesis rate was not influenced by intercropping (Table 1). Transpiration rate increased by IC treatment in oilseed rape that was significant for the old leaves, while decreased in the young leaves of barley (Table 1). 3.3 EFFECT OF IC ON THE PHENOTYPIC PLAS- TICITY OF BOTH SPECIES UNDER DS The cropping pattern influenced plants response to DS differently depending on species. In the DS oilseed rape, IC conditions resulted in a slight increase in the leaf area (Fig. 1), and photosynthesis and transpiration rates per surface area (Table 1). Despite the putatively higher water loss at whole plant level under IC conditions, this strategy may enable this species to keep higher ability for biomass production and synthesis of osmolytes (see be- low) compared with the MC counterparts. In barley, in contrast, reduction of leaf area and transpiration rate per surface area most likely led to lower water loss at whole plant level accompanied by an increased root biomass and higher root : shoot ratio. Such phenotypic plastic- ity in response to DS in barley that was observed only under IC conditions may enable this species to capture efficiently water from the dry substrate. Root growth and density, proliferation and size are key responses of plants to drought stress (Farooq et al., 2009). It is well plausi- ble that the belowground root interactions in the IC pots mediate some modifications in the phytohormone bal- ances in plants. In our barely plants, reduction of shoot growth and an increase in the root : shoot ratio under DS conditions are the well-known responses of plants to abscisic acid (Mc Adam et al., 2016). Modification in the levels of phytohormones through root interactions with the neighbor plants has been observed in tobacco (Chen et al., 2018). From an ecological point of view, the ability of plants to plastically adjust to environment play impor- tant role in the function of mixed cropping systems (Zhu, 2015). 3.4 EFFECT OF DS AND IC ON WATER STATUS OF THE YOUNG AN OLD LEAVES Drought stress expectedly decreased RWC in the young and old leaves of both species (Fig. 2). Intercrop- ping did not influence the leaf RWC in the WW plants while significantly increased this parameter in the young Oilseed rape Young leaf Chl a + b (mg g‒1 FM) Photosynthesis (µmol m‒1 s‒1) Transpiration rate (mmol m‒1 s‒1) Old leaf Young leaf Old leaf Young leaf Old leaf Well-watered MC 6.45 ± 0.85 a 2.17 ± 0.32 c 4.62 ± 0.12 ab 2.43 ± 0.39 b 1.41 ± 0.17 a 1.22 ± 0.20 a IC 7.08 ± 0.61 a 6.02 ± 0.74 a 5.35 ± 0.34 a 3.46 ± 0.42 a 1.58 ± 0.43 a 1.39 ± 0.14 a Drought stress MC 5.85 ± 0.87 a 1.67 ± 0.67 c 2.84 ± 0.28 c 1.48 ± 0.29 c 0.75 ± 0.15 b 0.30 ± 0.02 c IC 6.04 ± 0.37 a 4.12 ± 0.39 b 4.22 ± 0.60 b 2.71 ± 0.19 b 1.18 ± 0.08 ab 0.67 ± 0.01 b Barley Young leaf Chl a + b (mg g‒1 FM) Photosynthesis (µmol m‒1 s‒1) Transpiration rate (mmol m‒1 s‒1) Old leaf Young leaf Old leaf Young leaf Old leaf Well-watered MC 5.63 ± 0.65 a 3.45 ± 0.27 a 5.43 ± 0.72 a 3.97 ± 0.68 a 1.38 ± 0.07 a 0.85 ± 0.10 a IC 5.45 ± 0.31 a 3.04 ± 0.36 a 5.46 ± 0.44 a 3.64 ± 0.54 ab 1.30 ± 0.21 a 0.73 ± 0.19 a Drought stress MC 5.21 ± 0.78 a 3.26 ± 0.42 a 4.36 ± 0.17 a 2.71 ± 0.12 b 1.30 ± 0.08 a 0.35 ± 0.02 b IC 5.51 ± 0.64 a 3.00 ± 0.32 a 3.88 ± 0.69 a 2.94 ± 0.62 ab 0.66 ± 0.15 b 0.35 ± 0.05 b Table 1: Concentrations of chlorophyll (Chl) a + b , photosynthesis and transpiration rates in the young and old leaves of oilseed rape and barley cultivated either as monocrop (MC) or intercrop (IC) under well-watered or drought stress conditions for eight weeks. Data of each column indicated by the different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 118/3 – 2022 7 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress leaves of both species under DS conditions (Fig. 2). The leaf RWC is a reliable parameter to evaluate the water sta- tus of plants that reflects the balance between water sup- ply to the leaf tissue and transpiration rate (Lugojan and Ciulca, 2011). The improvement of RWC in the young leaves of both species upon intercropping in this work is an indication of an interspecific interaction occurred only under water deficit conditions being independent from the effect of IC on biomass production. 3.5 EFFECT OF DS AND IC ON THE WUE AND WATER CONSUMPTION Instant WUE (iWUE) increased under DS condi- tions in the old leaves of both species that was observed for both MC and IC plants (Fig. 3). Significant effect of IC on iWUE was observed in the young leaves of barley under DS conditions (Fig. 3). Drought stress increased the biomass WUE (bWUE) too (Fig. 3). This parameter differed also significantly among three culture pots; the lowest bWUE was observed in the MC barley pots both under WW and DS conditions (Fig. 3). Increases in WUE are commonly stated as a response of plants to moderate to severe water deficiency (Tambussi et al., 2007). There are evidences showing that the WUE of intercropping systems are usually greater than that of monoculture (Daneshnia et al., 2015; Chimonyo et al., 2016). There are, however, studies that showed intercropping systems did not increase obviously WUE (Grema and Hess, 1994; Shackel and Hall, 1984), or sometime reduced it (Rees, 1986; Singh et al., 1988; Gao et al., 2009). Here in our work, IC pots have higher bWUE than the MC barley pots both under WW and DS conditions. In oilseed rape, intercropping did not influence bWUE under DS condi- tions but decreased it under WW conditions (Fig. 3). Figure 2: Relative water content (RWC) in the young and old leaves of oilseed rape and barley cultivated either as monocrop (MC) or intercrop (IC) under well-watered (WW) or drought stress (DS) conditions for eight weeks. Bars indicated by the different let- ters are significantly different (p < 0.05) Acta agriculturae Slovenica, 118/3 – 20228 N. SADEGHZADEH et al. Figure 3: Instant water use efficiency (iWUE) in the young and old leaves of oilseed rape and barley and biomass water use effi- ciency (bWUE) in the monocrop (MC) or intercrop (IC) pots after eight weeks cultivation under well-watered (WW) and drought stress (DS) conditions. Bars within each culture mode indicated by the different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 118/3 – 2022 9 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress Figure 4: Daily water consumption of the monocrop (MC) pots of oilseed rape and barley and of the intercrop (IC) pots under well-watered (WW) (above) or drought stress (DS) (below) conditions for eight weeks Daily water consumption gradually increased dur- ing the two months experiment in both MC and IC pots under WW conditions (Fig. 4). Difference between MC and IC pots were obvious from 30 days after intercrop onward, and at the end of experiment, daily water con- sumption in the IC pots was considerably higher than that in MC pots (Fig. 4). Under DS conditions, the water consumption sharply decreased subsequent to omitting watering and remained lower throughout the experi- ment. Daily water consumption was consistently higher Acta agriculturae Slovenica, 118/3 – 202210 N. SADEGHZADEH et al. in the MC oilseed rape pots compared with IC and MC barley pots (Fig. 4). 3.6 EFFECT OF DS AND IC ON THE OSMOTIC HOMEOSTASIS OF LEAVES AND ROOTS Leaf osmotic potential decreased under DS condi- tions in both species. Effect of DS on the root osmotic potential, however, was significant only in oilseed rape (Table 2). Leaf concentration of organic osmolytes in- creased under DS conditions in oilseed rape. Significant effect of DS, however, was observed for proline in the young leaves and for soluble sugars in the old leaves while free amino acids contributed equally to the osmolytes concentration in the old and young leaves of this species (Table 2). In barley leaves, soluble sugars did not respond to the treatments. The effect of DS on the proline concen- tration was not significant but the free amino acids in- creased in the young leaves of this species in response to DS. In the roots, proline and soluble sugars accumulated in both species while free amino acids did not responds to DS in none of species (Table 2). One of the most common stress tolerance strategies in plants is the overproduction of different types of com- patible organic solutes including soluble sugars, free ami- no acids, proline and glycinebetaine (Singh et al., 2015). These osmolytes protect plants through contribution to osmotic adjustment, detoxification of ROS, and the sta- bilization of membranes, native structures of enzymes and proteins (Verbruggen and Hermans, 2008). Oilseed rape responded more to the DS than barley regarding the accumulation of osmolytes in the leaves. This may allow this species to have higher RWC despite higher transpira- tion that help also to produce biomass under DS condi- tions. Of particular importance was the proline accumu- lation particularly in the young leaves of oilseed rape that was much higher than that in barley. Proline accumula- tion is caused by a combination of increased biosynthesis and slow oxidation in mitochondria (Parida et al., 2008) and play important roles including stabilization of mac- romolecules, ROS scavenging, a sink for excess reductant Oilseed rape Young leaf Old leaf Roots Young leaf Old leaf Roots Osmotic potential (‒MPa) Proline (µmol g‒1 FM) Well-watered MC 0.564 ± 0.030 b 0.469 ± 0.079 b 0.101 ± 0.013 b 0.72 ± 0.18 c 0.25 ± 0.06 b 0.40 ± 0.06 b IC 0.524 ± 0.015 b 0.511 ± 0.003 b 0.118 ± 0.009 b 0.76 ± 0.09 c 0.35 ± 0.04 b 0.27 ± 0.06 b Drought stress MC 0.904 ± 0.039 a 0.832 ± 0.003 a 0.196 ± 0.011 a 3.10 ± 0.69 b 0.67 ± 0.22 b 1.50 ± 0.16 a IC 0.962 ± 0.032 a 0.909 ± 0.049 a 0.224 ± 0.031 a 5.47 ± 0.61 a 1.14 ± 0.35 a 1.63 ± 0.63 a Free amino acids (µmol g‒1 FW) Soluble sugars (mg g‒1 FM) Well-watered MC 5.22 ± 0.11 c 3.04 ± 0.78 d 4.09 ± 1.28 b 29.43 ± 3.37 b 25.91 ± 5.27 b 0.63 ± 0.07 c IC 7.48 ± 0.69 b 7.28 ± 1.14 b 4.78 ± 1.45 b 28.87 ± 0.99 b 24.88 ± 6.11 b 1.42 ± 0.08 c Drought stress MC 8.67 ± 1.69 b 5.23 ± 0.57 c 5.90 ± 0.97 b 35.91 ± 3.12 ab 48.79 ± 4.17 a 2.76 ± 0.28 b IC 17.0 ± 2.53 a 11.4 ± 1.09 a 9.09 ± 0.48 a 47.03 ± 11.7 a 49.89 ± 10.8 a 4.07 ± 0.78 a Barley Young leaf Old leaf Roots Young leaf Old leaf Roots Osmotic potential (‒MPa) Proline (µmol g‒1 FM) Well-watered MC 0.588 ± 0.014 b 0.543 ± 0.048 b 0.122 ± 0.008 b 0.25 ± 0.04 a 0.15 ± 0.02 b 0.22 ± 0.03 c IC 0.521 ± 0.057 b 0.511 ± 0.033 b 0.131 ± 0.008 b 0.24 ± 0.07 a 0.16 ± 0.04 b 0.23 ± 0.05 c Drought stress MC 0.856 ± 0.177 a 0.898 ± 0.071 a 0.141 ± 0.005 b 0.36 ± 0.04 a 0.31 ± 0.07 ab 0.66 ± 0.00 b IC 0.993 ± 0.022 a 0.985 ± 0.032 a 0.258 ± 0.028 a 0.41 ± 0.15 a 0.39 ± 0.15 a 1.48 ± 0.14 a Free amino acids (µmol g‒1 FW) Soluble sugars (mg g‒1 FM) Well-watered MC 2.79 ± 0.80 b 4.07 ± 0.07 a 1.82 ± 0.37 b 7.22 ± 1.52 a 7.25 ± 2.25 a 0.72 ± 0.03 c IC 2.85 ± 0.58 b 4.46 ± 0.58 a 1.63 ±0 .21 b 7.16 ± 1.29 a 7.98 ± 2.96 a 0.94 ± 0.09 c Drought stress MC 5.49 ± 1.53 a 4.28 ± 0.45 a 2.03 ± 0.13 b 7.93 ± 1.88 a 7.32 ± 2.31 a 1.99 ± 0.27 b IC 4.94 ± 0.43 a 4.45 ± 0.40 a 2.86±0.24 a 7.58 ± 2.96 a 11.0 ± 2.51 a 3.94 ± 0.15 a Table 2: Osmotic potential and concentrations of proline, free amino acids and soluble sugars in the young and old leaves and roots of oilseed rape and barley cultivated either as monocrop (MC) or intercrop (IC) under well-watered or drought stress condi- tions for eight weeks. Data of each column indicated by the different letters are significantly different (p<0.05) Acta agriculturae Slovenica, 118/3 – 2022 11 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress and a source for carbon and nitrogen for use after relief of water deficit (Verbruggen and Hermans, 2008; Szabados and Savoure, 2009). Intercropping did not influence the leaf or root os- motic potential in oilseed rape but decreased it in the roots of barley grown under DS conditions (Table 2). In the oilseed rape plants grown under DS conditions, IC plants showed higher concentration of proline and free amino acids in the leaves and higher free amino acids and soluble sugars in the roots compared to the MC plants. In the WW oilseed rape plants, only the leaf concentration of free amino acids was altered by the IC treatment. In barley, leaves did not respond to the IC treatment either in the DS or WW plants, while the roots accumulated all three osmolytes under DS conditions and proline and soluble sugars under WW conditions (Table 2). The mechanisms behind the influence of the crop- ping pattern on the osmolyte accumulation are obscure. Intensification of water deficit following an increased competition for water and a faster depletion from the substrate in the IC pots could not be the mechanism for higher osmolytes accumulation. Indeed, the severity of DS could not be affected by cropping pattern because of daily irrigation up to the desired FC in our experiment. In addition, the total water consumption was rather lower in the IC pots compared to the MC oilseed rape (Fig. 1). A modification in the metabolism of plants under the ef- fects of belowground root interactions is not restricted to the influence on the concentrations of organic osmolytes observed in this work and seems to be rather common in intercropping systems. In a proteomics analysis in the millet/peanut intercrop system, the expression of several proteins that are mainly involved in carbon and nitrogen metabolism are upregulated by interspecific root interac- tions (Zou et al., 2019). To evaluate the relevance of the different osmotic adjustment parameters in the plants responses to the ap- plied treatments, data were subjected to PCA (Fig. 5). The result showed that the photosynthesis and transpi- ration rates and the RWC were clustered with biomass data and, thus, were likely the most important parame- ters determining the plants response to the applied treat- ments (Fig. 5). Contrastingly, the osmotic adjustment parameters were separately clustered from the biomass data in both species. This was unexpected because the os- molytes contribute undoubtedly to sustaining leaf turgor required for photosynthesis and growth. Nevertheless, results of this analysis may highlight the negative effect of osmolytes synthesis on plants biomass production due to its high carbon and energy costs. Collectively, these data may suggest that, different patterns of osmolytes ac- cumulation could not explain the biomass response of plants to the IC or DS conditions in our experiment. 3.7 EFFECT OF INTERCROPPING INDEPENDENT FROM WATERING TREATMENT An improvement in the shoot growth of oilseed rape but reduction of it in barley under IC conditions was observed irrespective the watering treatment in this work (Fig. 1). Response of dry matter production to the neighboring plants has been observed for several inter- crop systems. Quite different effects have been found: improvement in both crops (Xue et al., 2016), increase of growth only in one of the crops (Zuo et al., 2003), re- duction in both (Inal et al., 2007) or even without bio- mass response (Zuo et al., 2004). Here, higher biomass production in oilseed rape after 10 weeks intercrop may be partly related to the competition for nutrients with barley favoring growth of oilseed rape. Nevertheless, an improved shoot biomass in oilseed rape upon intercrop- ping with barley has also been observed in the hydro- ponically grown plants supplied with adequate nutrients provided through repeated replacement of nutrient solu- tion (Sadeghzadeh et al., 2021). This may suggest addi- tional mechanisms for the benefit of oilseed rape from an intercropping system. Similarly, reduction of biomass in barley under IC conditions cannot only be explained by competition for nutrients. Growth impairment in intercropped plants may be mediated by chemical factors released from the roots of neighboring plants including, but not restricted to, allelochemicals. In an oilseed rape/barley intercrop system, we have observed activation of defense pathways, including phenylpropanoid- and salicylic acid-mediated pathways in barley but not in oilseed rape (Hajiboland, unpublished data). Activation of defense that was also observed in other mixed cropping systems (Schmid et al., 2013; Fu et al., 2015), may divert carbon resources from the growth and is likely the mechanism for reduction of dry matter production in barley under IC conditions. In- terspecific relations independent from nutrient acquisi- tion capacity in intercropped systems has attracted much less attention and our knowledge about the underlying mechanisms of belowground interactions is largely lim- ited compared to other types of biotic interactions (Sub- rahmaniam et al., 2013). The measured physiological parameters subjected to PCA (Fig. 6) showed a distinct clustering of four treat- ment combinations in oilseed rape. In barley, in contrast, the physiological parameters relevant to the cropping pattern were not clustered separately under WW condi- tions. This confirmed again the prominent effect of inter- cropping in oilseed rape irrespective the watering treat- ment and suggested that, barley may benefit from IC only under DS conditions. Acta agriculturae Slovenica, 118/3 – 202212 N. SADEGHZADEH et al. Figure 5: Principal component analysis of various physiological parameters in the young (YL) and old leaves (OL) and roots (R) of oilseed rape and barley cultivated either as monocrop or intercrop under well-watered or drought stress conditions for eight weeks. Abbreviations: Chl (chlorophyll), A: photosynthesis, E: transpiration, FM: fresh mass, DM: dry mass; RWC: relative eater content, AA: concentration of free amino acids, Sug: concentration of soluble sugars, Prol: concentration of proline, Osm: osmotic potential Acta agriculturae Slovenica, 118/3 – 2022 13 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress Figure 6: Principal component analysis of various physiological parameters in oilseed rape and barley cultivated either as mono- crop (MC) or intercrop (IC) under well-watered (WW) or drought stress (DS) conditions for eight weeks Acta agriculturae Slovenica, 118/3 – 202214 N. SADEGHZADEH et al. Fig. 7: Difference in the greenness of the old leaves in oilseed rape cultivated either as monocrop (above) or intercrop (below) with barley under drought stress conditions for eight weeks Acta agriculturae Slovenica, 118/3 – 2022 15 Intercropping induces physiological and morphological plasticity in oilseed rape and barley under drought stress 3.8 DIFFERENCE BETWEEN THE YOUNG AND OLD LEAVES The separate analysis of young and old leaves in this work showed differences between these leaves in the two species. The IC-mediated increase in the RWC was ob- served only in the young leaves of both species. There are evidences on the different response of leaves to drought stress depending on the leaf ontogenetic stage (Chastain et al., 2016). It has been stated that the leaves which de- velop after imposition of drought stress are more toler- ant to water deficit than the old leaves; both in primary photochemistry and carbon reactions (Hajiboland et al., 2014; Chastain et al., 2016). In oilseed rape, further dif- ferences in the response to DS and IC between the young and old leaves were observed. The accumulation of pro- line under DS conditions was much higher in the young than in the old leaves (Table 2). 3.9 LEAF SENESCENCE AS AFFECTED BY INTER- CROPPING The old leaves of intercropped oilseed rape plants retained much better their green colour than the MC plants. This was particularly found under DS conditions (Fig. 7) and was also obvious from the Chl a + b data associated with a higher photosynthesis rate (Table 1). Drought-induced leaf senescence that is characterized by reduction of Chl and photosynthesis rate is an intricate process resulting in remobilization of nutrients to young- er leaves thereby contributing to plant fitness (Jan et al., 2019). A direct role in the regulation of drought-induced leaf senescence has been demonstrated for cytokinins and ABA operating at opposite manner (Munné-Bosch and Alegre, 2004). Cytokinin levels that show a positive correlation with the photosynthetic rate and Chl con- tent decrease under drought stress (Munné-Bosch and Alegre, 2004). The mechanism for the IC-mediated pre- vention of leaf senescence in oilseed rape plants was not addressed here, but could likely be related to an elevated level of cytokinin as was also observed in other below- ground root interactions (Chen et al., 2018). Similar to our work on the improvement of Chl and photosynthesis in the oilseed rape, in the peanut/maize intercrop sys- tem, a proteomics study showed a three-fold increase in the expression of Rubisco small and large subunits, Ru- bisco activase and Chl a/b binding proteins compared to monocrop peanut young leaves (Xiong et al., 2013). Our data on the postponing of senescence in the old leaves of oilseed rape by intercropping will putatively increase the leaf area duration in this species and may contribute significantly to the higher biomass production under IC conditions in this species. 4 CONCLUSIONS Cropping pattern considerably influenced the plants water and osmotic homeostasis under drought stress conditions. Elevated RWC, WUE and an improved osmotic adjustment in both species showed a conspicu- ous effect of belowground root interaction on plants re- sponse to water deficit conditions. Further benefits of IC were higher biomass production and leaf area duration in oilseed rape plants and higher root : shoot ratio in barley. Such plasticity in plant morphological and physiological traits is expected to increase plant performance, canopy photosynthesis and productivity and enhance water cap- ture under intercropping conditions in the field. 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Frontiers in Plant Science, 6, 726. https://doi.org/10.3389/ fpls.2015.00726 Acta agriculturae Slovenica, 118/3, 1–6, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2363 Original research article / izvirni znanstveni članek Viability of seeds of two varieties of Coffea arabica L. using different pre- treatments in the tetrazolium test José Duván BUENDÍA CONTRERAS 1, Laura Yolima MORENO ROZO 2, Seir Antonio SALAZAR MER- CADO 2, 3 Received October 06, 2021; accepted August 04, 2022. Delo je prispelo 6. oktobra 2021, sprejeto 4. avgusta 2022 1 Master of Biological Sciences, Universidad Francisco de Paula Santander, San José de Cúcuta, Colombia 2 Department of Biology, Universidad Francisco de Paula Santander, San José de Cúcuta, Colombia 3 Corresponding author, e-mail: seirantoniosm@ufps.edu.co Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test Abstract: This research attempted to determine the effica- cy of the tetrazolium test in the evaluation of the seed viability of two varieties of Coffea arabica L. (‘Castillo’ and ‘Cenicafé’). The fruits were obtained from crops located in the municipali- ties of Salazar de las Palmas and Arboledas (Norte de Santander - Colombia). The test was carried out with embryos manually extracted from the seeds using tweezers. Three pretreatments were established: distilled water, sodium hypochlorite (2.5 %), sucrose (10  %), and a control (no pretreatment). Embryos were placed in a cysteine solution (0.5 %) to prevent oxidation, then immersed in tetrazolium solutions with concentrations of 0.035 %, 0.075 %, and 0.1 % for a period of 6, 9, and 12 hours in darkness. The results of the viability test were validated with seed germination, using the wet paper towel method in dark- ness. The best viability percentages were found with the ap- plication of sodium hypochlorite (NaClO 2.5 %), with a high correlation with the germination percentage. The use of pre- treatments improved the efficiency of the viability test and al- lowed the use of low concentrations of the reagent (0.035 %), giving the farmer a quick and less expensive alternative to de- termine germination capacity. Key words: coffee; germination; optimization; pretreat- ments Viabilnost semen dveh sort kavovca (Coffea arabica L.) z upo- rabo različnih predobravnavanj pred tetrazolijevim testom Izvleček: V raziskavi smo poskušali določiti učinkovitost tetrazolijevega testa za ovrednotenje viabilnosti semen dveh sort kavovca, Coffea arabica L. (‘Castillo’ in ‘Cenicafé’). Plodovi kavovca so bili pridobljeni z območij Salazar de las Palmas in Arboledas (Norte de Santander - Colombia). Test je bil izve- den na embrijih, ki smo jih s pinceto izolirali iz semen. Upo- rabljena so bila tri predobravnavanja in sicer: distilirana voda, natrijev hipoklorit (2,5 %), saharoza (10 %) in kontrola (brez predobravnavanja). Embriji so bili najprej vstavljeni v raztopi- no cisteina (0,5 %) za preprečitev oksidacije in nato potopljeni v različne koncentracije raztopin tetrazolija (0,035 %; 0,075 % in 0,1 %) za 6, 9, za 12 ur v temi. Rezulti viabilnosti embrijev so bili ovrednoteni s kalitvijo semen v temi na vlažnem filtrirnem papirju. Največji odstotek viabilnih semen je bil ugotovljen pri uporabi natrijevega hipoklorita (NaClO 2,5 %), z največjo korelacijo z odstotkom kalitve. Uporaba predobravnavanj je iz- boljšala učinkovitost testa viabilnosti in je dopuščala uporabo majnih koncentracij reagenta (0,035 %), kar daje kmetom hitro in manj drago alternativo določanja kalivosti. Ključne besede: kavovec; kalitev; optimizacija; predo- bravnavanja Acta agriculturae Slovenica, 118/3 – 20222 J. D. BUENDÍA CONTRERAS et al. 1 INTRODUCTION Coffea arabica represents one of the most important crops in the world (RAMÍREZ et al., 2016; Selmar et al., 2008). Global coffee consumption has been increasing due to its pleasant aroma and flavor, as well as its benefi- cial health effects, leading to a large amount of by-prod- uct generation (Khochapong et al., 2021). Coffee is one of the most traded beverages in the world and in 2016/17 more than 9 million tons were consumed (da Silva et al., 2021). The main coffee producers are Brazil, Colombia, Vietnam, India, and Indonesia which represents relevant economic importance for these countries (da Silva et al., 2021). 12.5 million households worldwide receive in- come from coffee cultivation (Montagnon et al., 2021). Commercially cultivated coffee species are Coffea ara- bica L. and C. canephora Pierre ex A.Froehner (Mishra & Slater, 2012). Tropical African countries such as Ethiopia, Sudan, Kenya, Guinea, or Mozambique are usually point- ed out as possible centers of origin, although the most accepted is Ethiopia (Rojo, 2014). The coffee plant belongs to the Rubiaceae family and there are more than 70 species of the Coffea L. genus (da Silva et al., 2021), it is a tropical evergreen tree, grow- ing at altitudes between 700 and 2000 m.a.s.l. (Tinoco & Peña, 2019). The green seeds of Coffea arabica are very rich in secondary metabolites. These metabolites have antioxidant properties that reduce the incidence of can- cer and diabetes (Aissaoui et al., 2020). The coffee seed is a nut, oblong, flat-convex, of variable size (10-18 mm long and 6.5-9.5 mm wide) and mostly constituted by a horny endosperm where, at one of its ends and very superficially, lies an embryo of 3.5 to 4.5 mm long, with a conical radicle and cordate cotyledons (Arcila et al., 2007). An undesirable feature of coffee seeds is that they have slow and asynchronous germination, which pre- vents obtaining seedlings of desirable quality; this type of germination hinders rapid evaluations of viability and/or vigor due to the excessive time required to obtain results (Da Rosa et al., 2010). The use of quick methods to know the viability is important, to accelerate decision-making regarding the management of seed lots, (Medeiros et al., 2015). For this reason, the tetrazolium salt test has assumed a prominent place for some crops, mainly due to the large amount of information that the test provides. Tetrazolium (TZ) can be used regardless of the degree of seed dormancy, be- coming very important for species with this problem. It is also a very useful tool for seed producers, graders, and traders as it can help in decisions that need to be made quickly. (França-Neto and Krzyzanowski, 2019). The tetrazolium test is based on the activity of dehydrogenase enzymes that reduce the tetrazolium salt in living seed tissues by generating triphenyl formazan, a non-diffus- ible red-colored compound, which indicates respiratory activity and viability of cells and tissues; in contrast, dead tissues show no coloration (Salazar et al., 2018; Salazar et al., 2020a). It is important to indicate that the effective- ness of the tetrazolium test is mediated by the develop- ment of preconditioning procedures, which facilitate the entry of the tetrazolium solution into the seed (Hosomi et al., 2017; Salazar et al., 2019). Decreasing the concentration of tetrazolium and verifying its efficacy allows the evaluator a reasonable and optimal use of this reagent, reducing the total cost of the test. According to the above, this work aims to deter- mine the efficacy of the tetrazolium test in the evaluation of seed viability of two varieties of Coffea arabica using different pretreatments. 2 MATERIAL AND METHODS 2.1 PLANT MATERIAL The ripe fruits of the Castillo coffee variety (C. ar- abica) were collected in village Alto del Angulo in the municipality of Salazar de las Palmas. The Cenicafé va- riety drupes were collected in plantations in the village of San Onofre in the municipality of Arboledas, both belonging to Norte de Santander (Colombia). The mate- rial was placed in plastic containers with lids, lined with newspaper to avoid deterioration, and transported to the Biology laboratories of the Faculty of Basic Sciences at the Universidad Francisco de Paula Santander where the research study was carried out. 2.2 PRETREATMENT AND VIABILITY OF SEEDS Solutions of 2,3,5-triphenyl tetrazolium chloride were prepared with concentrations of 0.035 %, 0.075 % and 0.1  %. To avoid embryo oxidation, a 50 mg l-1 cysteine solution was prepared (Azofeifa, 2009). The so- lutions were kept in dark bottles and stored in the refrig- erator at 5 °C (Salazar et al., 2020b; Salazar et al., 2020c). Three pretreatments were established: 6-hour immersion in 2.5  % sodium hypochlorite (NaClO), 10  % sucrose (C12H22O11), and water (H2O). The fourth group of seeds corresponded to the control group. The fleshy layers (protective skin, pulp, parchment) and the integument were removed from the fruits to ob- tain the endosperm containing the embryos. The embry- os were extracted manually, using a dissecting case and forceps. One hundred embryos were placed in each Petri dish immersed in 5 ml of tetrazolium solution. Each test Acta agriculturae Slovenica, 118/3 – 2022 3 Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test had 5 replicates for a total of 500 embryos for each expo- sure time established (6 h, 9 h, 12 h) in complete dark- ness. An intense reddish coloration of the embryo was determined as positive for the tetrazolium test. 2.3 GERMINATION TEST The germination test was performed to corroborate the data obtained in the viability test. Five replicates of 100 seeds were used for 500 seeds (Salazar et al., 2020b). For the test, the seeds were removed from the endosperm, washed with distilled water, and placed to germinate for 30 days in total darkness on paper towels moistened with distilled water in previously disinfected plastic contain- ers. Germinated seeds were considered to be those that presented a root growth at least 4 mm long or that pre- sented geotropic curvature and the result was expressed as germination percentage. 2.4 STATISTICAL ANALYSIS For the viability test and germination test, data were randomly distributed with 5 replicates and 100 seeds per replicate. The experimental design consisted of a com- pletely randomized block analysis. Data were analyzed using Infostat statistical software by analysis of variance (ANOVA), followed by Tukey’s multiple range HSD (Honest Significant Difference) test, to compare aver- ages and determine significant differences at a level of p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 PRETREATMENT AND VIABILITY OF SEEDS The tetrazolium test evaluates the physical and physiological conditions of the embryonic structure of each seed (França-Neto & Krzyzanowski, 2019). Moreo- ver, viable seeds are easily identified by their carmine red coloration (Figure 1), due to the reductive reaction of the tetrazolium solution under the action of dehydrogenase enzymes in cellular respiration (Salazar et al., 2020a). In the results shown in Table 1, it can be observed that the pretreatment with chlorine (2.5 % 6 h) was the one that obtained the highest viability in all concentra- tions and exposure times with tetrazolium, values that ranged between 92 % and 100 % viability in C. arabica ‘Castillo’. However, no significant differences were found between the control and sucrose pretreatment at the fol- lowing concentrations and exposure time with tetrazo- lium: 0.035 % 6, 9, 12 h; 0.075 % 9, 12 h; 0.1 % 6, 9, 12 h (Table 1). The lowest viability values were found in the pretreatment with distilled water in most of the concen- trations and exposure time with tetrazolium, unlike the pretreatments with sucrose (0.035 % 12 h, 0.075 % 6 h, and 0.1 % 6 h) where the viability using the tetrazolium test were lower. The findings in the Cenicafe variety showed few significant differences among pretreatments, concentra- tions, and exposure times with tetrazolium (Table 2). However, the chlorine treatment had a better effect on seed viability. The lowest viability value (88 %) was found in the sucrose pretreatment, at the tetrazolium concen- tration and exposure time of 0.035 % 9 h (Table 2). In ad- dition, the preconditioning with distilled water identified statistically homogeneous means with the chlorine pre- treatment data, unlike the concentration and exposure period of 0.075 % 12 h of tetrazolium. According to Lamarca and Barbedo (2014), the higher the concentration and exposure period to tetra- zolium, the greater the intensity of embryo coloration, which facilitates the analysis and evaluation of viable and non-viable seeds. According to Tola et al. (2019), expo- sure times of 5, 16, and 24 h and concentrations of 0.1 %, 0.5 %, and 1 % tetrazolium are suitable for viability analy- sis in coffee seeds. However, in this research, it was pos- sible to obtain reliable viability levels thanks to the use of pre-treatments with chlorine (2.5 % 6 h), in concentra- tions of 0.035 %, 0.075 %, 0.1 %, and exposure times of 6, 9, 12 h of tetrazolium. These parameters are decisive in the tetrazolium test since they improve the efficiency of the viability test. Likewise, Salazar et al. (2020b) in- creased the efficiency of the tetrazolium test on S. lyco- persicum L. seeds, using 1  % sodium hypochlorite and Figure 1: Viability of Coffea arabica seeds using the tetrazo- lium test. (A, B, C) non-viable seeds C. arabica Castilla’ (D, E) non-viable seeds C. arabica ‘Cenicafe’. (F, G, H): viable seed C. arabica ’Castilla’ (I, K) viable seed C. arabica ‘Cenicafe’ Acta agriculturae Slovenica, 118/3 – 20224 J. D. BUENDÍA CONTRERAS et al. tetrazolium concentrations of 0.25% and 0.15% in 24h. However, in a study by Clemente et al. (2011) and Clem- ente et al. (2012), subjecting sodium hypochlorite (5 %, 6  %) for 6  h negatively impairs tetrazolium test results in C. arabica seeds with a moisture content below 25 %. Strobel et al. (2016) state that chlorine doses can generate embryo damage, leading to decreased seed viability. In addition, Salazar and Maldona, (2020) express, that chlo- rine generates toxic effects on cells at low doses, because it is a strong oxidant compound (Jiang et al., 2017). In several studies sucrose solution (10 %) has had a positive effect on seed viability because sucrose is linked to the activation of metabolic processes related to de- hydrogenase enzymes in cellular respiration, helping to maintain an osmotic balance in the seed, which prevents damage to the embryo (Mercado and Jaimes, 2022; Sa- lazar and Botello, 2020; Salazar et al., 2019; Hosomi et al., 2017; Hosomi et al., 2012). Moreover, distilled water treatments resulted in low viability in the Castillo variety at several periods and tetrazolium concentrations. These results differ with Mercado et al. (2020), where pretreat- ment with distilled water on Epidendrum microtum Lindl seeds had 100% viability. According to Clemente et al. (2012), an imbibition time of 48 hours facilitates embryo extraction and does not affect the results of the tetrazo- lium test. According to Carvalho et al. (2014), hydration favors the absorption of tetrazolium and provides the ac- tivation of enzymatic metabolism. 3.2 COMPARISON OF GERMINATION AND VI- ABILITY The viability test should be validated with the ger- mination test, indicating the consistency of the findings concerning the behavior of the physiological quality of the seeds (Tola et al., 2019). In the Castillo variety, the average germination percentage was 95  %, comparing with the viability results, the same trend was maintained, where no statistically significant differences were found with the hypochlorite treatments. Similarly, in the ceni- cafe variety, the germination percentage was 96 %, which is positively correlated with the viability test. This reflects the efficacy of the pretreatments in improving seed vi- ability. According to Salazar et al. (2020c), the germina- tion test indicates whether the seeds did not germinate because they were dormant or because they showed embryo deterioration, being used to corroborate the vi- Tetrazolium concentration and exposure time Pretreatments 0.035 % 6 h 0.035 % 9 h 0.035 % 12 h 0.075 % 6 h 0.075 % 9 h 0.075 % 12 h 0.1 % 6 h 0.1 % 9 h 0.1 % 12 h Control 90 ± 5.4 a 98 ± 4.4 a 96 ± 4.4 a 94 ± 8.9 a 98 ± 4.4 a 100 a 96 ± 5.4 ab 98 ± 4.4 a 100 a H2O 6h 72 ± 8.3 b 84 ± 8.9 b 96 ± 5 a 96 ± 5.4 a 88 ± 4.0 b 98 ± 4.4 a 92 ± 8.3 ab 98 ± 3.4 a 92 ± 5.4 b Sucrose 10% 6h 92 ± 8.3 a 96 ± 54 a 94 ± 5.4 a 78 ± 7.8 b 98 ± 4.1 a 100 a 82 ± 7.8 a 98 ± 4.2 a 98 ± 1.9 a Chlorine 2.5% 6h 92 ± 7.5 a 98 ± 4.4 a 96 ± 8.9 a 98 ± 4.4 a 98 ± 3.9 a 100 a 100 b 100 a 100 a Table 1: Viability of Coffea arabica ’Castilla’ seeds Different letters indicate significant differences (p ≤ 0.05) ± Standard deviation Tetrazolium concentration and exposure time Pretreatments 0.035 % 6 h 0.035 % 9 h 0.035 % 12 h 0.075 % 6 h 0.075 % 9 h 0.075 % 12 h 0.1 % 6 h 0.1 % 9 h 0.1 % 12 h Control 89 ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 96 ± 5.4 a 100 a 98 ± 4.4 a 98 ± 4.4 a 98 ± 3.5 a H2O 6h 96 ± 5.4 a 98 ± 4.2 a 94 ± 8.9 a 96 ± 5.4 a 98 ± 4.4 a 92 ± 8.3 b 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.1 a Sucrose 10% 6h 96 ± 5.4 a 88 ± 4.1 b 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 100 a 92 ± 8.3 a 92 ± 8.4 a 98 ± 3 a Chlorine 2.5% 6h 96 ± 5.2 a 98 ± 4.2 a 98 ± 4.2 a 98 ± 4.4 a 98 ± 4.4 a 100 a 98 ± 5.4 a 98 ± 4.4 a 100 ± 5 a Table 2: Viability of Coffea arabica ‘Cenicafe’ seeds Different letters indicate significant differences (p ≤ 0.05) ± Standard deviation Acta agriculturae Slovenica, 118/3 – 2022 5 Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test ability results generated by the tetrazolium test. However, in most cases the viability test generates higher percent- ages concerning the germination test, agreeing with the studies conducted by Tola et al. (2019) on Coffea arabica seeds. According to the results reported by Fantazzini et al. (2020), the tetrazolium test is not suitable for seeds with germination percentages lower than 60 %. Likewise, these authors report that for seeds with a germination value greater than 60 %, the results of the tetrazolium test were similar to those of the germination test on C. ara- bica seeds. Likewise, Figueiredo et al. (2017) point out, that several investigations have shown the discrepancy between the results of the tetrazolium and germination test in coffee seeds, specifically in those of lower quality. Germination of C. arabica seeds occurs slowly and inconsistently. Therefore, the tetrazolium test is present- ed as a quick alternative to evaluate viability, provided that seed preparation and imbibition are established (Clemente 2012). In this research, germination percent- ages were higher than 93 % in the Castillo and Cenicafe varieties and indicated a close relationship with viability percentages with tetrazolium. According to the above, the use of pretreatments can improve the efficacy of the tetrazolium test on C. arabica seeds. In this case, the seeds exposed to sodium hypochlorite (2.5 % 6 h) gen- erated high viability percentages, which allows reducing the concentrations and exposure periods of tetrazolium to 0.035 % and 6 h, respectively, which leads to a reduc- tion in the cost of the reagent triphenyl tetrazolium chlo- ride to determine the viability of C. arabica seeds. 4 CONCLUSIONS Rapid identification of viability in a seed group is important because it allows decisions to be made about it. Coffee seed gives germination results in 25 to 30 days, but this can be extended up to 40 days. 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Apthapi, 5(3), 1671-1682 Acta agriculturae Slovenica, 118/3, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2194 Original research article / izvirni znanstveni članek Evaluation of forage maize yield and soil organic matter content under green manure cultivation Elias ARAZMJOO 1, 2, Alireza MOGHRI FERIZ 3, MohammadAli BEHDANI 4, Sohrab MAHMOODI 4 Received April 29, 2021; accepted August 08, 2022. Delo je prispelo 29. Aprila 2021, sprejeto 8. avgusta 2022 1 Horticulture Crops Research Department, South Khorasan Agricultural and Natural Resources Research and Education Centre, AREEO, Birjand, Iran 2 Corresponding author, e-mail: e.arazmjo@areeo.ac.ir 3 Soil and Water Research Department, South Khorasan Agricultural and Natural Resources, Research and Education Centre, AREEO, Birjand, Iran 4 Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Birjand, Birjand, Iran Evaluation of forage maize yield and soil organic matter con- tent under green manure cultivation Abstract: To investigate the effect of different green ma- nures from Gramineae and Brassicaceae families on yield, some agronomic traits of forage maize, overgrowth with weeds and soil organic matter, an experiment was conducted based on a randomized complete block design with three replications for three consecutive years (2017-2020) at the Agricultural and Natural Resources Research and Education Centre of Southern Khorasan. Experimental treatments included control (without application of green manure) and application of green ma- nures from the cultivation of barley, triticale, canola, arugula with their optimum and twice optimum densities. The results showed that barley and triticale at twice optimum density with 865.7 and 802.9 g m-2, respectively, had a higher green manure dry mass at the time of returning to the soil. Just before maize cultivation, soil organic matter with an average of 0.73 % was higher in barley green manure at twice optimum density com- pared to other treatments. Based on the results, the highest maize forage yield with 45.7 and 44.9 t ha-1 were achieved after treatment with barley green manure in twice optimum and op- timum density (22.8 and 20.7 percent more than control treat- ment) and after that triticale in both densities, and canola and arugula at twice optimum density had the highest yield. Key words: barley; triticale; canola; arugula; forage maize; production; weeds Ovrednotenje pridelka silažne koruze in vsebnosti organske snovi v tleh v razmerah zelenega podora Izvleček: Za ovrednotenje učinka različnega zelenega po- dora iz družin trav in križnic na pridelek in nekatere kompo- nente pridelka silažne koruze, poraslosti s pleveli in vsebnosti organske snovi v tleh je bil izveden popolni naključni bločni poskus s tremi ponovitvami v treh zaporednih rastnih sezonah (2017-2020) na posestvu Agricultural and Natural Resources Research and Education Centre of Southern Khorasan. Obrav- navanja v poskusu so obsegala kontrolo (brez uporabe zelene- ga podora) in uporabo zelenega podora z ječmenom, trtikalo, oljno ogrščico in rukvico, z optimalno in dvakrat optimalno gostoto setve. Rezultati so pokazali, da je imelo zeleno gnoje- nje z ječmenom in tritikalo pri dvakratniku optimalne setve, 865,7 in 802,9 g m-2, večjo suho maso zelenega podora. Pred začetkom setve koruze je bila suha masa organske snovi v tleh, poprečno 0,73 g, večja pri zelenem gnojenju z ječmenom pri dvakratni optimalni gostoti kot pri drugih obravnavanjih. Naj- večji pridelek silažne koruze, 45,7 in 44,9 t ha-1, je bil dosežen pri zelenem gnojenju z ječmenom pri dvakratniku optimalne gostote setve in optimalni gostoti setve (22,8 in 20,7 odstotkov več kot pri kontrolnem obravnavanju) in potem pri tritikali pri obeh gostotah setve ter pri dvakratniku optimalne gostote setve pri oljni ogrščici in rukvici. Ključne besede: ječmen; tritikala; oljna ogrščica; rukvi- ca; silažna korura; pridelek; pleveli Acta agriculturae Slovenica, 118/3 – 20222 E. ARAZMJOO et al. 1 INTRODUCTION Maize (Zea mays L.) is the third most important ce- real grain in the world, after wheat and rice, providing nutrients for humans and animals and serving as a basic raw material for the production of starch, oil, protein, al- coholic beverages, food sweeteners, and fuel (Bouis and Welch, 2010). With the increasing population and increasing hu- man need for chicken and eggs, the area under maize cultivation is increasing. According to the World Food Program, the annual production of maize is about 1.06 billion tons, and the largest producers in the world, the United States and China, together account for 58  % of this amount (FAO, 2020). In 2020, the area under maize cultivation in Iran was 200,000 hectares with a produc- tion of 1,400,000 tons, while its domestic consumption in the country was equal to 8,900,000 tons in this year and the gap between annual production and demand was provided with an import of 7,500,000 tons (USDA, 2020). One of the important limiting factors for the de- velopment of animal husbandry and the production of livestock materials is the provision of fodder to feed the country’s livestock. In such a way that the import of fod- der and fodder grains constitute considerable several imported items of the country. In this regard, the impor- tance of forage production is increasingly felt. The use of chemical fertilizers to produce crops around the world is also increasing (Abril et al., 2007), the continued use of which poses serious risks to the environment and human health (Graham and Vanca, 2000). In Iran, the indiscriminate use of chemical fertiliz- ers, especially nitrogen fertilizers, and the lack of appli- cation of organic fertilizers in recent years has been the cause of a significant reduction in the amount of organic matter in agricultural soils (Malakouti, 1999). Soils with more than 3 % organic matter are needed to make suit- able soils for plant growth (Pramanik et al., 2004). Ad- ditionally, the use of chemical fertilizers does not have a beneficial effect on physical soil properties. Adverse effects of fertilizers and pesticides on the environment have led to more attention and the use of methods with- out the use of chemicals, and the issue of sustainability in agriculture to be considered. One practical way to meet this goal is to use green manures that can reduce the use of chemical fertilizers. The application of green manures is one of the man- agement methods of choice in many agricultural produc- tions systems because these fertilizers can reduce soil erosion and improve the physical properties of the soil, increase organic matter and soil fertility, increase nutri- ent circulation and reduce global warming potentials and finally increase the system stability (Dinnes et al., 2002). Plants used as green manure increase soil water storage in arid lands by increasing water infiltration, reducing evaporation, and improving soil structure. Return of green manures to the soil as a result of microbiological processes increase soil organic matter and release nutri- ents in plants for plants (Talgre et al., 2009). In the study of Abdi et al. (2012) the plants of Gramineae (sorghum, millet, and oat), Brassicaceae (aru- gula), and Leguminosae (white clover, red clover, bersim clover, sainfoin, and vetch) were used as green manure, and the evaluation of soil nutrient changes, and nitrogen mineralization were studied and the highest amount of organic carbon was obtained by returning sorghum for- age residues to the soil (1.59 %). The amount of total soil nitrogen in all tested plants increased during different sampling times. The highest amounts of total nitrogen (0.23  %) were obtained by white clover in five months after the return of the remains. Clement et al. (1995) examined different types of green manures and found that the ratio of lignin and poly- phenols to nitrogen and tannins to nitrogen controlled the amount of nitrogen released. On the other hand, the decomposition of green manure and the release of its nu- trients depends on soil physical (moisture, temperature, texture, minerals, and pH), chemical (carbon/nitrogen ratio, soil nutrient content), and biological properties (the rate of soil biological activity) (Myers et al., 1994), among which the ratio of carbon/nitrogen has a great- er impact on the mineralization of organic matter than other factors. Regarding the effect of red clover, common alfalfa, vetch, and oats as green manure on bioavailable nitrogen, it was observed that the amount of soil nitrogen increased significantly under common alfalfa use and the wheat grain protein content in the next crop was the highest (Maikstenien and Arlauskiene, 2004). It has been reported that in dry winters, nitrate accumulates in top- soil and the cover plant controls nitrate leaching during the early stages of growth, and that vetch is less efficient in leaching control in contrast to barley but increased soil nitrogen storage (Gabriel et al., 2012). Additionally, the combined application of vetch winter cover plant and a small amount of fertilizer can significantly improve the sustainability of low-input maize-based conservation ag- riculture (Dubeab et al., 2013). Green manure in Iran is used only in some areas and to a very limited extent. Animal manures are also not stored and used properly. Besides, the high cost of live- stock manure and the lack of common use of them have caused organic fertilizers to play a negligible role in in- creasing fertility and improving Iran soils. This can cause serious problems in agricultural planning and operations, especially in large-scale agriculture. Thus, the purpose of this study was to investigate the effect of Gramineae (bar- Acta agriculturae Slovenica, 118/3 – 2022 3 Evaluation of forage maize yield and soil organic matter content under green manure cultivation ley and triticale) and Brassicaceae (canola and arugula) as green manure in two different densities on the maize yield, weeds growth and percentage of soil organic matter during the periods after adding green manure residues to the soil and finally introducing the desired plant or plants as green manure in the studied conditions. 2 MATERIALS AND METHODS A field experiment was conducted between 2017 and 2020 at the Agricultural and Natural Resources Re- search and Education Centre of South Khorasan Prov- ince, Birjand, Iran (32̊ 52ʹN, 58̊ 59ʹE). Before cultivation, a pre-planting composite sample of the soil was taken from a depth of 0-30 cm for determination of particle size distribution, pH, EC and soil organic matter. The re- sults of soil analysis during the years of the experiment are presented in Table 1 and meteorological information during the years of the experiment is also presented in Figure 1. There were nine treatments in the trial, laid out in a randomized complete block design in three repli- cations. Plants used as green manure included barley (Hordeum vulgaris L.), triticosecale (Triticosecale spp.), canola (Brassica napus L.), and arugula (Eruca sativa L.). Experimental treatments included control (none applica- tion of green manure) and application of green manures from the cultivation of barley (with optimum density), barley (with twice optimum density), triticale (with op- timum density), triticale (with twice optimum density), canola (with optimum density), canola (with twice opti- mum density), arugula (with optimum density), arugula (with twice optimum density). Cultivation of barley with two densities of 400 and 800, triticale with two densities of 400 and 800, canola with two densities of 70 and 140, and arugula with two densities of 80 and 160 plants per square meter and based on their 1000-grain mass on five 60 cm rows with a length of five meters was done in November of 2017, 2018 and 2019. The distance between the plots was one meter and between the replication was two meters. After planting, irrigation was done and a total of two irrigations were carried out in autumn and one irrigation in spring. In April, the green manures were cut into small pieces using a disk and returned to the soil with plowing. Considering that weed control by green manure as well as the contribution of green manure in increas- ing soil organic matter is directly related to the dry mass produced, the biomass of each green manure at the time of return to the soil was evaluated. To estimate the dry mass of green manures, sampling was performed before returning them to the soil. For this purpose, one square meter was randomly taken from each plot and the sam- ples were dried in an oven at 70 °C for 48 hours and then weighed. After returning the green manure the land was left uncultivated and then maize was planted in early summer. Maize cultivar SC647 was cultivated in the first half of July in 2018, 2019, and 2020 in furrows with a distance of 75 cm and a length of five meters, and a density of 20 plants per square meter. Before planting maize, samples were taken from each plot to measure the amount of soil organic matter. Fertilizers used include 300 kg ha-1 urea (50 kg ha-1 before planting, 150 kg ha-1 at 6-7 leaves stage, and 100 kg ha-1 before the emergence of male inflores- cence), 100 kg ha-1 triple superphosphate (before sowing) and 100 kg ha-1 of potassium sulfate (before sowing) were used during the maize growing period based on the di- mensions of the plots. During the maize growing season, irrigation was carried out in the first months with an in- terval of eight days and in the last month with an interval of 10 days, and a total of nine irrigations was carried out. During the growing season, traits such as number of days to emergence of tassels, number of days to emergence of silk, number of rows per ear, number of grain per row, ear length, ear diameter, plant height, and forage yield of maize, as well as weed biomass and weed density, were measured or weighted and recorded. To measure ear length, ear diameter, and plant height by observing the margin effect, five plants were randomly selected and the average of five plants for each of these traits was record- ed. The number of rows per ear, the number of grain per row, and the number of grain per ear were counted and recorded in five ears after harvest. A sampling of weeds in maize cultivation was done in 4-6 leaf stage in a quadrate of one square meter in each plot and after determining the density of weeds, these samples were dried at a tem- perature of 70 degrees in an oven for 48 hours and then weighed. To measure the maize forage yield, the area of one square meter was harvested and the fresh mass of the forage was weighed by observing the margin effect. After ensuring the homogeneity of variance of ex- perimental error with Bartlett test, the data were analyzed using SAS statistical software based on a randomized complete block design and means comparison based on Duncan test at 5 % probability level. 3 RESULTS AND DISCUSSION The results of combined analysis of variance for three years of the experiment showed that year had a significant effect at the level of 1 % on the dry mass of green manure, the number of grain per row and weeds dry mass and a significant effect at the level of 5 % on ear Acta agriculturae Slovenica, 118/3 – 20224 E. ARAZMJOO et al. length (Table 2). Also, except for the number of days to emergence, the number of days to the emergence of silk and the diameter of the ear, the year had a significant ef- fect on the other studied traits, whereas the interaction of year in green manure was significant only on the dry mass of green manure at the time of return to soil (Table 2). The results of mean comparing for the year effect showed that the highest dry mass of green manure with an average of 564.4 g m-2 and the highest weeds dry mass with an average of 175.6 g m-2 were observed in the sec- ond year of the experiment (Table 3). The highest num- ber of grain per row with 36.7 grains and an ear diameter of 48.8 mm was also found in the third year whereas the difference in ear length between the third and the sec- ond year was not significant (Table 3). According to the meteorological statistics presented in Figure 1, one of the reasons for the better growth of green manures and their higher dry mass when returning to the soil in the spring in the second and third years of the experiment, was more precipitation during the autumn, winter, and spring during these years compared to the first year when the amount of precipitation was lower. The results of mean comparison of the traits showed that at the time of return to the soil, barley and triticale green manure at twice optimum density treatment with 865.7 and 802.9 g m-2, respectively, had higher dry mass compared with other treatments (Table 3). After control or none green manure cultivation, canola, and arugula treatments had the lowest dry mass at the optimum den- sity at the time of return to the soil, so that their dry mass was about 55 percent lower compared to barley green Figure 1: Mean temperature and rainfall during 2017-2020 (Climate data: Iran, Birjand - Tutiempo.net) Year Sand (%) Silt (%) Clay (%) Soil texture pH EC (dS.m-1) Organic matter (%) 2017 38 42 20 Loam 7.9 4.79 0.36 2018 40.5 36 23.5 Loam 7.82 4.27 0.49 2019 46.5 36 17.5 Loam 7.85 5.67 0.44 Table 1: The results of soil physiochemical properties Acta agriculturae Slovenica, 118/3 – 2022 5 Evaluation of forage maize yield and soil organic matter content under green manure cultivation manure at twice optimum density (Table 3). Means com- parison for the interaction effect of year in green manure also showed that barley green manure treatment with twice optimum density in the second year with an aver- age of 937 g m-2 had the highest dry mass among oth- er treatments, but not statistically different with barley green manure treatments with twice optimum density in the third year and triticale with twice optimum density in the second and third year. In contrast, canola, and aru- gula green manures with averages of 291.1 and 314.7 g m-2 in the first year had the lowest dry mass among other treatments, respectively (Table 4). Naturally, the more residues returned to the soil, the more organic will be added to the soil. Abdi et al. (2012) also reported in their study that compared to the green manures of sorghum, millet, oats, arugula, and several types of clover, sorghum produced the highest fresh and dry mass of shoots and consequently it has also led to the production of higher organic carbon in the soil. In terms of the number of grain rows per ear, bar- ley green manure had the highest rate at the optimum density with an average of 16.6 rows and there was no statistically significant difference between this treatment with barley and arugula treatments with twice optimum density, whereas control treatment with 14.1 rows had the lowest amount (Table 3). There was no significant difference in the number of grain per row between barley, triticale, canola, and aru- gula green manures, but these treatments were signifi- cantly superior to the control (Table 3). The number of grains per ear had the lowest values in control and canola with optimum density treatments, while barley with optimum density and then barley and arugula with double optimum density was in the supe- rior statistical group in this regard (Table 3). Maize ear length was higher in barley green manure treatments at both twice and optimum densities and also arugula at twice optimum density (Table 3). Barley green manure in twice optimum density with an average of 212.5 cm had the highest plant height among other treatments and resulted in a 16.5 % increase in maize plant height compared to the control treatment and its difference with barley treatments with optimal density, triticale and canola with twice optimum density and arugula with both densities and twice the density were not statistically significant (Table 3). It seems that these treatments provided more access to nutrients to the plant than other treatments and an increased in ear length and plant height are observed. Evaluation of leg- ume winter cover crops in maize cultivation also showed a 37 % increase in maize plant height (Miguez and Bol- lero, 2005). Increasing the ear length in the application of green manure is consistent with the results of Ghasemi et al. (2016). According to the results, the lowest amount of maize forage with 37.2 t ha-1 was related to the control treatment or no green manure cultivation, and barley green manure at twice optimum density with 45.7 t ha-1 had the highest maize forage which had no significant difference with barley in optimum density treatment with 44.9 t ha-1. Also, after them, triticale green manure in both density and canola and arugula at twice optimum density had higher forage yield and there was no statisti- cally significant difference between them (Table 3). Bar- ley at optimum and twice optimum density leads to an increase of 20.7, 22.8 %, triticale at optimum and twice optimum density leads to an increase of 14.5 and 14.8 %, canola at optimum and twice optimum density leads to an increase of 10.2 and 14.8 % and finally arugula at opti- mum and twice optimum density led to an increase of 9.7 and 18.3 % in the yield of maize forage compared to the control treatment (Table 3). The increase in maize for- age yield as a result of the return of the mentioned green fertilizers can be justified by increasing the soil organic matter and the availability of nutrients for the next crop as well as improving the biological and physical proper- ties of the soil. In addition to improving soil structure and nutrient accumulation in soil surface layers (Cherr et al., 2006), green manure has been reported to be the most important source for bacterial activity, and bacteria are more efficient in these conditions (Orhana et al., 2006). The use of organic fertilizers by increasing soil organic matter strengthens the properties of aggregates, micro- bial activity, soil quality, crop fertility, and storage capac- ity of nutrients such as nitrogen, phosphorus, potassium, zinc, and iron in the soil (Wei and Liu, 2005). The predominant weeds in the maize field included Amaranthus retroflexus L., Portulaca oleracea L., Convol- vulus arvensis L., and Alhaji camelorum L. The results of the experiment showed the superiority of barley and triticale green manures in controlling weeds in the maize field. Namely, the barley treatment at twice optimum density with 54.2 g m-2 and triticale in twice optimum density with 83.8 g m-2 had the lowest weeds dry mass, respectively (Table 3). Barley and triticale at twice opti- mum density resulted in a reduction of 85.3 % and 77.2 % in weeds dry mass compared to the control (Table 3). The highest weeds density and weeds dry mass of 155.9 plants m-2 and 368.8 g m-2 were obtained from control treatment or no cultivation of green manure, followed by canola and arugula at the optimum density. Plants with high biomass and more shading can control weeds well. One of the reasons for the decrease in the weeds density and their dry mass in green manure treatments compared to fallow has been reported to be the sharp reduction of Acta agriculturae Slovenica, 118/3 – 20226 E. ARAZMJOO et al. M ea n sq ua re s So ur ce o f V ar ia tio n df . G re en m an ur e dr y m as s N o. o f d ay s to e m er ge nc e of ta ss el s N o. o f d ay s to e m er ge nc e of si lk s N o. ro w pe r e ar N o. g ra in pe r r ow N o. g ra in pe r e ar Ea r le ng th Ea r di am et er Pl an t he ig ht Fo ra ge yi el d W ee ds dr y m as s W ee ds de ns ity So il or ga ni c m at te r Ye ar 2 17 96 42 ** 23 .1 9 ns 43 .9 0 ns 37 .1 6 ns 15 5. 85 ** 22 69 4 ns 10 5. 92 * 37 6. 9 ns 22 18 .5 n s 24 6. 2 ns 11 21 9. 9* * 39 1. 66 n s 0. 13 8 ns Bl oc k (Y ea r) 6 37 19 .4 4. 88 11 .0 7 0. 74 2. 92 25 57 .6 9 9. 11 35 .7 9 66 8. 71 0. 68 42 0. 82 40 0. 91 0. 00 2 G re en m an ur e 8 58 33 58 ** 4. 73 n s 10 .0 6 ns 4. 55 ** 21 .6 2* * 17 75 1. 2* * 28 .8 1* * 28 .8 7 ns 12 80 .6 ** 58 .6 4* * 86 12 9. 5* * 10 24 3. 9* * 0. 05 2* * G re en m an ur e* Ye ar 16 59 70 .1 * 0. 92 n s 2. 46 n s 0. 22 n s 0. 41 n s 49 1. 7 ns 1. 87 n s 0. 69 n s 42 .8 2 ns 7. 05 n s 54 4. 29 n s 27 5. 65 n s 0. 00 06 n s Re sid ua l e rr or 48 29 51 .5 25 .0 8 25 .8 6 0. 73 4. 85 28 83 .0 8 4. 76 27 .0 2 15 0. 87 6. 41 53 3. 59 20 6. 70 0. 00 2 C oe ffi ci en t o f v ar ia tio n - 10 .7 8 8. 03 7. 25 5. 62 6. 39 10 .5 3 7. 94 11 .3 4 5. 86 5. 97 14 .2 0 21 .3 9 7. 93 Ta bl e 2: Th e re su lts o f a na ly sis o f v ar ia nc e fo r i nv es tig at ed tr ai ts * a nd ** a re si gn ifi ca nt ly d iff er en t a t α = 0 .0 5 an d α = 0. 01 , r es pe ct iv el y an d ns is n on -s ig ni fic an t Ye ar G re en m an ur e dr y m as s ( g m -2 ) N o. o f d ay s to e m er ge nc e of ta ss el s N o. o f d ay s to e m er ge nc e of si lk s N o. ro w pe r e ar N o. g ra in pe r r ow N o. g ra in pe r e ar Ea r le ng th (c m ) Ea r di am et er (c m ) Pl an t he ig ht (c m ) Fo ra ge yi el d (t ha -1 ) W ee ds d ry m as s ( g m -2 ) W ee ds de ns ity (p la nt m -2 ) So il or ga ni c m at te r ( % ) 20 17 -2 01 8 41 1. 3 b 61 .4 a 68 .7 a 15 .6 a 31 .9 c 49 8. 7 ab 25 .3 b 41 .6 b 20 4. 9 a 38 .9 b 13 9. 1 b 65 .6 a 0. 51 b 20 18 -2 01 9 56 4. 4 a 63 .3 a 71 .2 a 14 .0 b 34 .8 b 48 7. 8 b 27 .8 a b 47 .1 a b 22 0. 2 a 44 .5 a 17 5. 6 a 64 .5 a 0. 64 a 20 19 -2 02 0 53 6. 7 a 62 .3 a 70 .6 a 16 .2 a 36 .7 a 54 2. 5 a 29 .3 a 48 .8 a 20 4. 0 a 43 .8 a 17 3. 1 a 71 .5 a 0. 63 a G re en M an ur e C on tr ol 0. 0 f 62 .9 a 71 .4 a 14 .1 c 31 .0 b 42 4. 3 c 25 .0 c 42 .7 a 18 5. 7 c 37 .2 c 36 8. 8 a 15 5. 9 a 0. 47 e Ba rle y op tim um d en sit y 59 9. 7 b 62 .6 a 69 .9 a 16 .6 a 36 .5 a 58 4. 6 a 29 .9 a 46 .3 a 21 6. 3 a 44 .9 a 90 .5 d 58 .0 b 0. 63 b c Ba rle y tw ic e op tim um d en sit y 86 5. 7 a 63 .3 a 71 .7 a 15 .6 a b 35 .2 a 53 0. 5 ab 30 .0 a 49 .1 a 22 1. 5 a 45 .7 a 54 .2 e 57 .0 b 0. 73 a Tr iti ca le o pt im um de ns ity 55 2. 5 bc 62 .8 a 70 .7 a 14 .9 b c 34 .2 a 49 4. 5 bc 26 .6 b c 45 .8 a 19 5. 3 bc 42 .6 a b 10 1. 3 d 55 .6 b 0. 59 cd Tr iti ca le tw ic e op tim um d en sit y 80 2. 9 a 61 .3 a 68 .6 a 14 .9 b c 35 .2 a 50 8. 9 b 27 .5 a bc 47 .3 a 21 6. 7 a 42 .7 a b 83 .8 d 52 .7 b 0. 68 a b C an ol a op tim um d en sit y 37 9. 6 e 61 .3 a 68 .9 a 14 .9 b c 33 .6 a b 48 2. 9 bc 26 .1 b c 45 .7 a 20 6. 3 ab 41 .0 b 22 8. 5 b 57 .8 b 0. 54 d C an ol a tw ic e op tim um d en sit y 46 7. 6 d 62 .8 a 70 .7 a 15 .1 b c 34 .8 a 50 8. 1 b 26 .7 b c 46 .0 a 21 5. 5 a 42 .7 a b 17 4. 7 c 48 .2 b 0. 58 cd A ru gu la o pt im um d en sit y 37 8. 2 e 62 .4 a 70 .1 a 15 .2 b c 34 .4 a 50 5. 2 b 26 .3 b c 44 .4 a 21 1. 8 a 40 .8 b 21 2. 2 b 68 .1 b 0. 54 d A ru gu la tw ic e op tim um d en sit y 49 1. 0 cd 61 .7 a 69 .7 a 15 .9 a b 35 .5 a 54 8. 1 ab 29 .1 a b 45 .1 a 21 8. 4 a 44 .0 a b 14 9. 7 c 51 .5 b 0. 59 cd Ta bl e 3: Th e re su lts o f m ea n co m pa ris on fo r e ffe ct s o f y ea r a nd g re en m an ur e on in ve st ig at ed tr ai ts M ea ns fo llo w ed b y sim ila r l et te rs in e ac h co lu m n ar e no t s ig ni fic an tly d iff er en t a t p = 5  % b as ed o n D un ca n te st . Ba rle y op tim um a nd tw ic e op tim um d en sit ie s: 40 0 an d 80 0 pl an ts m -2 , r es pe ct iv el y Tr iti ca le o pt im um a nd tw ic e op tim um d en sit ie s: 40 0 an d 80 0 pl an ts m -2 , r es pe ct iv el y C an ol a op tim um a nd tw ic e op tim um d en sit ie s: 70 a nd 1 40 p la nt s m -2 , r es pe ct iv el y Acta agriculturae Slovenica, 118/3 – 2022 7 Evaluation of forage maize yield and soil organic matter content under green manure cultivation light reaching the lower parts of the plant canopy in these treatments, reducing the weeds photosynthetic activity of and thus reducing their density (Bilalis et al., 2009). Resi- dues mixed with the soil of green plants with allelopathic effects (Ohno et al., 2000), stimulation of soil pathogens (Conklin et al., 2002), impact on nutrients access (Gal- landt et al., 1999), increase crop growth, and improving its competitiveness with weeds (Boquet et al., 2004) can reduce weed density and growth. A report states that non-legume species such as canola and rye are suitable if the main purpose of using cover crops is to control weeds (Campiglia et al., 2009). The amount of soil organic matter in barley green manure treatment with twice optimum density with an average of 0.73 was higher than other treatments. Since the highest biomass produced among green manures was related to this treatment, the higher amount of soil or- ganic matter in this treatment can be attributed to this factor. After this treatment, triticale with twice opti- mum density and barley with optimum density was in the second and third ranks in terms of soil organic mat- ter (Table 3). The high percentage of organic carbon in these treatments is probably due to the larger volume of soil-derived residues in these treatments. In the study of Ghaffari et al. (2013), rye, barley and triticale treatments with three times planting density and rye with normal density increased 26, 25, 21, and 25  % of soil organic carbon content, respectively, compared to the control treatment. In some studies, an increase in soil organic carbon content due to the application of green manure compared to the conventional low-input system (without fertilizer) has been reported (Clark et al., 1998). It has been reported that the return of green manure plants to the soil increases carbon, organic matter, total nitrogen, and soil fertility, which occurs as a result of microbiologi- cal processes and causes the release of nutrients for plants (Talgre et al., 2009). 4 CONCLUSIONS Based on the results, the highest maize forage yield with 45.7 and 44.9 t ha-1 was obtained from barley green manure at twice optimum density and its optimum den- sity, followed by triticale at both density and canola and arugula at twice optimum density. Due to severe organic matter deficiency in many soils of South Khorasan prov- ince, cultivation of green manure plants before maize cultivation, depending on the type of green manure se- lected and its density can increase maize forage yield by 9.6 to 20.7 % compared to not cultivating them. Reduc- ing weeds in the soil will be another advantage of grow- ing green manure before maize cultivation. 5 REFERENCES Abdi, S., Tajbakhsh, M., Abdollahi Mandulakani, B. & Rasouli Sadaghiani, M. (2012). Effect of green manure on the soil organic matter and nitrogen. Journal of Agricultural Knowl- edge, 5(7), 41-52. Abril, A., Baleani, D., Casado-Murillo, N. & Noe, L. (2007). Effect of wheat crop fertilization on nitrogen dynamics and balance in the Humid Pampas, Argentina. Agricul- ture, Ecosystems & Environment, 119, 171-176. https://doi. org/10.1016/j.agee.2006.07.005 Bilalis, D., Karkanis, A. & Efthimiadou, A. (2009). 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Agron- Green Manure 2017-18 2018-19 2019-20 Control 0 k 0 k 0 k Barley optimum density 482.4 egf 678.3 bc 638.4 c Barley twice optimum density 738.6 b 937.0 a 921.6 a Triticale optimum density 430.4 efgh 638.9 c 588.1 cd Triticale twice optimum density 632.4 c 921.7 a 854.7 a Canola optimum density 291.1 j 443.6 efgh 404.1 ghi Canola twice optimum density 375.9 hij 529.2 de 497.8 defg Arugula optimum density 314.7 ij 417.2 fgh 402.6 ghi Arugula twice optimum density 436.4 efgh 513.9 def 522.7 de Table 4: The results of mean comparison for interaction effect on the dry mass of investigated green manures (g m-2) Means followed by similar letters in each column are not significantly different at p = 5 % based on Duncan test. 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Chemosphere, 59, 1257-65. https://doi.org/10.1016/j.chem- osphere.2004.11.052 Acta agriculturae Slovenica, 118/3, 1–13, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2485 Original research article / izvirni znanstveni članek Fruit collapse incidence and quality of pineapple as affected by biopesti- cides based on Pseudomonas fluorescens and Trichoderma harzianum Diego Mauricio CANO-REINOSO 1, Loekas SOESANTO 1, 2, KHARISUN 1, Condro WIBOWO 3 Received December 28, 2021; accepted August 10, 2022. Delo je prispelo 28. decembra 2021, sprejeto 10. avgusta 2022 1 Department of Agrotechnology, Faculty of Agriculture, Jenderal Soedirman University, Purwokerto, Indonesia 2 Corresponding author, e-mail: lukassusanto26@gmail.com 3 Department of Food Science and Technology, Faculty of Agriculture, Jenderal Soedirman University, Purwokerto, Indonesia Fruit collapse incidence and quality of pineapple as affected by biopesticides based on Pseudomonas fluorescens and Trich- oderma harzianum Abstract: In this study the effect of Pseudomonas fluore- scens and Trichoderma harzianum based biopesticides on fruit collapse disease incidence and pineapple quality was investigat- ed. The experiment was implemented in a split-plot design with two factors, one involving two inoculation methods (spray and inject), and a second factor involving four treatments, A (con- trol: no biopesticides used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest) and D (Bio P32 + Bio T10 from 13 weeks before harvest). The inoculated pathogen was Dickeya zeae. The incidence of fruit collapse, to- tal soluble solids, total acidity, sucrose, ascorbic acid, mineral content, and electrolyte leakage were determined. The inject method caused more fruit collapse incidence than the spray method. Treatments C and D provided the best results hav- ing a low incidence of fruit collapse (spray: 5 and 1.7 %, inject: 20 % in both cases), high antioxidant capacity (regarding ascor- bic acid), high mineral nutrient content (in terms of Ca and Mg), and low electrolyte leakage content (< 70 % in average), with a healthier cell wall characteristic. Meanwhile, treatments A and B were less efficient in these aspects and promoted the incidence of fruit collapse, especially when the inject method was used, as this was more harmful regarding the fruit physiol- ogy. In conclusion, the biopesticides employed can reduce the incidence of fruit collapse and positively affect the fruit quality. Key words: biopesticide; Dickeya zeae; fruit quality; dis- ease incidence; Pseudomonas fluorescens; Trichoderma harzia- num Vpliv uporabe biopesticidov na osnovi bakterije Pseudomo- nas fluorescens in glive Trichoderma harzianum na propad in kakovost plodov ananasa Izvleček: V raziskavi je bil preučevan učinek uporabe biopesticidov na osnovi vrst Pseudomonas fluorescens in Tri- choderma harzianum na propad in kakovost plodov ananasa. Poskus je bil izveden kot faktorski poskus z deljenkami, kjer je prvi dejavnik obsegal dva načina vnosa patogena (pršenje in injeciranje), drugi pa naslednja štiri obravnavana: A (kon- trola: brez uporabe biopesticidov), B (uporaba Bio P32 13 te- dnov pred pobiranjem), C (uporaba Bio T10 13 tednov pred pobiranjem) in D (uporaba Bio P32 + Bio T10 13 tednov pred pobiranjem). Inokuliran patogen je bila bakterija Dickeya zeae. Po obravnavanjih so bili določeni naslednji parametri: pojav propada plodov, vsebnost topnih snovi v plodovih in njihova celukopna kislost, vsebnost saharoze, askorbinske kisline in mi- neralov ter puščanje elktrolitov iz plodov. Injeciranje patogena je povzročilo večji propad plodov kot pršenje. Obravnavanji C in D sta dali najbojše rezultate z najmanjšim propadanjem plodov (pri pršenju 5 in 1,7 %, pri injeciranju 20 % v obeh pri- merih), veliko vsebnostjo antioksidantov (vsebnost askorbinske kisline), mineralov (kot vsebnost Ca in Mg), manjšo vsebnost elektrolitov v iztoku (v poprečju manj kot 70 % ) in bolj zdrave celične stene. Obravnavanji A in B sta bili glede na prej naštete parametre manj učinkoviti in sta pospešili propadanje plodov, posebej še pri injeciranju patogena, kar je bilo tudi bolj ško- dljivo glede na fiziološke lastnosti plodov. Zaključimo lahko, da uporaba biopesticidov zmanjša propadanje plodov in pozitivno vpliva na njihovo kakovost. Ključne besede: biopesticidi; Dickeya zeae; kakovost plodov; pojav bolezni; Pseudomonas fluorescens; Trichoderma harzianum Acta agriculturae Slovenica, 118/3 – 20222 D. M. CANO-REINOSO et al. 1 INTRODUCTION Pineapple diseases are common problem affecting fruit quality, with infections usually beginning in the field and before harvest (Rohrbach and Johnson, 2003; Sipes and Pires de Matos, 2018). Fruit collapse is caused by the bacterium Dickeya zeae (formerly Erwinia chrysan- themi (Peckham et al., 2010; Sueno et al., 2014), which is characterized by exudation of sap and gas in the form of bubbles, an olive-green skin color and cavities within the skeletal fibers that show up in the flesh of the fruit (Aeny et al., 2020; Cano-Reinoso et al., 2021). D. zeae can in- fect the plant via infection vectors coming from the field, such as already infected plants, ants, beetles, and flies that attack during flower induction, or directly affecting the developed fruit when high temperatures weeks before harvest increase transpiration and allow the bacterium to penetrate directly through the stomata of the skin (Pires de Matos, 2019; Cano-reinoso et al., 2021). For this rea- son, fruit collapse symptoms usually occur just before harvest or during postharvest handling, as D. zeae can remain latent for a long time (Rohrbach and Johnson, 2003; Pires de Matos, 2019). Recently, low acidity pineapple hybrids have been reported to be more susceptible to this disease (Soteriou et al., 2014; Cano-Reinoso et al., 2021). Currently, these hybrids are the most commonly exported by the indus- try as fresh fruit because they are attractive to consumers (Chen et al., 2009; Kleemann, 2016). Therefore, a solu- tion must be found to address this problem. In addition, the solution should preserve the quality of the fruit and protect the environment, as the use of chemical pesticides is expected to be reduced worldwide in the near future. In this context, the use of biopesticides presents itself as an alternative, as they are environmentally friendly and easier to apply. These can interact with the plant and fruit during development through the plant stomata, lenticels, and natural cracks, and can also be applied after harvest (Soesanto et al., 2011, 2018). Bio P32 is a biopesticide derived from Pseudomonas fluorescens, a strain of P. fluorescens isolated from the rhizosphere of wheat. Bio T10 is another biopesticide base on Trichoderma harzianum, a soil-borne fungus used for biological control of plant pathogens (Soesanto et al., 2011, 2018). These biopesticides have been widely used on various crops, both preharvest and postharvest, to reduce the incidence of bacterial diseases and improve crop characteristics, such as dragon fruit (Hamarawati et al., 2017), eggplant (Soesanto et al., 2011), and cucumber (Soesanto et al., 2020). However, since few studies has been reported on the effect of these products on fruit col- lapse and pineapple quality, this experiment aims to eval- uate the effect of Pseudomonas fluorescens and Tricho- derma harzianum based biopesticides on fruit collapse disease incidence and pineapple quality. This will involve a comparison of different inoculation methods that rep- resent how D.zeae infect the plant during flowering or in the weeks just prior to harvest, and investigating different variables that could characterize the optimal fruit traits for future consumption in a low acid hybrid. 2 MATERIAL AND METHODS 2.1 EXPERIMENT DESIGN AND TREATMENTS The research was set in pineapple fields located in Lampung, Sumatra island of Indonesia, between Febru- ary and June of 2020. A pineapple low acid hybrid (MD2) was used for this experiment. MD2 is known for its ex- ceptional sweetness, consistency and uniformed size at harvest; currently is one of the most exported fresh culti- vars, with a price tree times higher than any acid hybrid (Bin Thalip et al., 2015). The fruits were harvested in 148 days after flowering, considered an optimal time in MD2 to obtain the best physico-chemical characteristics for a future consumption (Bin Thalip et al., 2015; Ding and Syazwani, 2016). The soil was previously fertilized with 200 kg ha-1 of di-ammonium phosphate, 1000 kg ha-1 K2SO4, and 200 kg ha-1 kieserite crystal. Several foliar applications were carried out after the planting using 700 kg ha-1 urea, 700 kg ha-1 (NH₄)₂SO₄, 1000 kg ha-1 K2SO4, 170 kg ha -1 MgSO4, 60 kg ha -1 FeSO4, 60 kg ha -1 ZnSO4, in intervals of 30 days during three months; besides, after flowering borax was sprayed on the plant in doses of 30 kg ha-1. The pedological and mineral characteristic of the soil where the experiment was implemented are presented in Table 1. Furthermore, during the experiment, a weather sta- tion (LSI Lastem; equipped with a CR6 data logger from Campbell Scientific; Italy) measured an average relative humidity (RH) of 89.34  %, an ambient temperature of 26.8  °C, solar radiation of 16.83 w m-1, and a monthly average rainfall of 133.77 mm. The experiment was arranged in a split-plot design, with two factors. One factor concerning two methods of inoculation of D. zeae bacterium and a second factor about four treatments implemented. Each treatment had three replications with 44 fruits. Field rows where the treatments were administrated consisted of 0.4 m width and 3.75 m length. Pineapple plants were arranged in two lines of 22 plants in the rows with a separation of 0.25 m. Observations were carried out once every two weeks, from six weeks before harvest. The arrangement of the experiment factors used with their respective character- istics are presented in Table 2. Acta agriculturae Slovenica, 118/3 – 2022 3 Fruit collapse incidence and quality of pineapple as affected by biopesticides ... The control had only inoculated the bacterium for each of the methods used (sprayed or injected, respec- tively). For both inoculation methods, juice of previously infected fruits extracted from the flesh (including D. zeae) was employed. For the spray method, doses of 20 ml juice/plant-fruit were employed using a hand sprayer. These doses were selected after field trails before the be- ginning of this experiment demonstrated that with their employment the fruits exposed symptoms of fruit col- lapse just after flowering. Also, those trials proved that sprays applications during flowering were more effective to cause fruit collapse than injections. The plants were sprayed at night, in two and one week before flowering and one week thereafter (13, 12 and 11 weeks before har- vest). The sprayings moment tried to represent the typi- cal field infection during flower induction, where a latent bacterium in the environment enters the plant through the nectarthodes (Wang et al., 2011; Sipes and Pires de Matos, 2018). On the other hand, for the inject method were ad- ministrated doses of 0.2 ml juice/fruit with a syringe. These doses were implemented after previous field trials exposed that with these doses a fruit can present fruit collapse symptoms during advance stage of development, close to harvest. Also, these doses were employed by Bar- ral et al. (2017). They demonstrated that injections with these doses in pineapple are enough to inoculate a disease before harvest. Moreover, these trials demonstrated that for an advance fruit development, D. zeae inoculations with injections were more effective than sprayings. The sprayed inoculations on the plant were administrated in six, four, and two weeks before harvest. For this method, four eyes of the pineapple shell were inoculated by push- ing a syringe through them. Two eyes were inoculated on the upper part and two on the lower part of the shell, similar to the technic described in Barral et al. (2017). The inoculation time selected for the inject method in- tend to replicate another typical moment of infection by D. zeae, in this case close to harvest, entering through the shell stomata, as described in Sipes and Pires de Matos (2018). Concerning the biopesticides applications, from 13 to 10 weeks before harvest, those were employed weekly. Later after ten weeks, those were applied one time every two weeks until harvest. Bio P32 [in 1 l of product solu- tion: 10 % of snail meat, 2 g of fermented Shrimp, and 10 ml of P. fluorescens - (1012 cell ml-1) - strain 32] and Bio T10 [in 1 l of product solution: 10 g of rice flour and white sugar, and 10 ml of T. harzianum (108 conidia ml- 1) - strain 10] were used in doses 20 ml/per plant-fruit (v/v: 20 ml l-1) during night time. Furthermore, where the fruits had an advance maturation, the biopesticides were not only sprayed in the leaves, also directly into the shell and crown, understanding that the stomata and lenticels available in those structures could permit their absorption and assimilation, as recommend by Soesanto et al. (2011) and (2020). 2.2 DETECTION OF THE TOTAL SOLUBLE SOL- IDS (TSS), TOTAL ACIDITY (TA) AND FRUIT COLLAPSE INCIDENCE The TSS and TA were calculated following the pro- cedures described in Shamsudin et al. (2020), in a com- position of four fruits per replication of each treatment arranged. TSS was measured by implementing a hand- held refractometer (MASTER-53 α; Atago: Japan), while the TA was detected by titration to pH 8.1 with 0.1 M NaOH using phenolphthalein as an indicator and re- Texture Clay (%) 18.56 Loam (%) 13.01 Sand (%) 68.43 Chemical composition pH (H2O) 6.8 C (%) 0.7 N (mg kg-1) 800 P (mg kg-1) 43.75 K (mg kg-1) 319.8 Ca (mg kg-1) 638 Mg (mg kg-1) 235.2 Na (mg kg-1) 4.6 Table 1: Pedological and mineral nutrients characteristics of the soil in the experiment *The N, P, K, Ca, Mg and Na represent the available mineral nutrients content in the soil Factor one (Inoculation method) 1. Spraying before the open-heart stage. 2. Injection into the fruit flesh from six weeks before harvest. Factor two (Treatments) A. Control (No biopesticides used) B. Bio P32 from 13 weeks before harvest C. Bio T10 from 13 weeks before harvest D. Bio P32 + Bio T10 from 13 weeks before harvest Table 2: The organization of the experiment design employed in the research Acta agriculturae Slovenica, 118/3 – 20224 D. M. CANO-REINOSO et al. vealed as a percentage of citric acid. The incidence of fruit collapse was measured by detecting and collecting the percentage of fruits presenting the disease symptoms described in Cano-Reinoso et al. (2021). 2.3 ASCORBIC ACID (ASA) AND SUCROSE CON- TENT DETERMINATION The AsA and sucrose content of the fruits was meas- ured by the method reported in Siti Roha et al. (2013), using a High-Performance Liquid Chromatography (model L-2000 instrument; Hitachi: USA) with a Refrac- tive Index detector model L-2490. A juice extracted from the fruit flesh adjacent to the core was used. The samples were obtained from a composition of four fruits per rep- lication in each of the treatments arranged. Standard so- lutions of AsA and sucrose were dissolved in distilled wa- ter and filtered through a Millipore 0.45 µm membrane filter. The AsA and sucrose content were quantified, com- paring the peak area by a chromatographic procedure. 2.4 MINERAL NUTRIENTS DETERMINATION The calcium and magnesium content of the fruits was calculated using atomic absorption spectrometry (AAS 932 Plus; GBC scientific equipment: USA), em- ploying a composition of four fruits per replication in each of the field treatments. The method applied was the one described in Benton-Jones (2001). Juice samples were put in a digestion tube with 5 ml of 65 % nitric acid and left overnight. Later, the samples were heated with a block digester at 125 °C for one hour. After that, 3 ml of 30 % hydrogen peroxide (H2O2) were added and reheated for one hour; thereafter, HNO3 was used (1 ml residue) and 5 ml of nitric acid with distillate water (1:10) were added and shaken. Finally, the samples were move to a 25/50 ml flask quantitatively and pitched with distillate water, with the goal of creating an extract ready to deter- mine the calcium and magnesium content. As the water content of the samples were previously detected, the re- sults are expressed in a dry basis content. 2.5 DETECTION OF THE ELECTROLYTE LEAK- AGE (EL) Following the EL calculation in pineapple fruit re- ported in Chen and Paull (2001), the EL of the fruit flesh was obtained from the composition of four fruits per replication of the treatments implemented. Plugs were taken with a cork borer applying a longitudinal cut and then slides into a disk of 2 mm of thickness. Around 6 g of the disk were washed three times to remove any ly- sed material from the cell. For two hours, the disks were shaken and incubated in 60 ml of 0.3 M mannitol solu- tion. Later on, the conductivity of the previous solution was obtained with a radiometer. After that, the samples were boiled for two hours to release all the electrolytes, and the conductivity was determined. The EL is shown as the percentage of the total conductivity. 2.6 SCANNING ELECTRON MICROSCOPE (SEM) EVALUATION SEM analysis was performed using a similar meth- od reported in Hu et al. (2012). A piece of tissue adjacent to the core (5 × 5 × 2 mm3) was split from the middle of the flesh with a tweezer. Before scanning, the slices were dehydrated in a series of ethanol solutions and dried at a critical point of liquid CO2 using a desiccator. The sam- ples were mounted onto aluminum specimen stubs em- ploying conductive silver glue and sputter-coated with gold. SEM was executed with a scanning electron micro- scope (ZEISS/EVO MA 10: German) equipped with an energy dispersive spectroscopy (EDS) at 15.00 kV. 2.7 STATISTICAL ANALYSIS Statistical analyses were performed using SPSS Ver- sion 22.0 software (SPSS Inc.; Chicago, IL: USA). All data were analyzed by a two-way ANOVA. Mean significant differences at p < 0.05 were determined by Duncan’s mul- tiple range tests and Kruskal-Wallis test (in case of the fruit collapse incidence data). 3 RESULTS AND DISCUSSION 3.1 TOTAL SOLUBLE SOLIDS (TSS), TOTAL ACID- ITY (TA), AND SUCROSE CONTENT IN THE FRUIT The TSS presented significant differences in the in- teraction results. The treatment D obtained the highest value (14.87 %), when the spray method was employed; however, the same treatment in the case of the inject method delivered the lowest outcome (12.33 %). Lower TSS content was associated with a higher fruit collapse incidence (Table 3). Previous studies reported that the value of the TSS for commercial consumption of pine- apple low acid hybrids should be at least close to 12 % (Lu et al., 2014; Bartholomew and Sanewski, 2018; Ca- Acta agriculturae Slovenica, 118/3 – 2022 5 Fruit collapse incidence and quality of pineapple as affected by biopesticides ... no-Reinoso et al., 2022a); this requirement was assessed in the treatment results of both inoculation methods; also, this circumstance could have been promoted by the treatments used as previous authors have reported a positive effect on the TSS content by the administration of biopesticides based on of P. fluorescens and T. harzi- anum (Jiang et al., 2019; Carillo et al., 2020). Besides, it has been demonstrated that pathogens interfere with the metabolism of the host by increasing their sugar uptake, especially at the phloem level, decreasing the final TSS content in sink organs like the fruit (Morkunas and Rata- jczak, 2014; Naseem et al., 2017). This fact explains why the TSS treatment results of the inject method were lower than the spray one, due to the most critical case of infec- tion in this method causing fruit collapse. In the case of the TA, there were no significant dif- ferences delivered in the interaction outcomes; However, The TA values were higher in the inject method while lower in the spray method (0.69 % and 0.51 %, respec- tively) (Table 3). This more superior TA content was linked to a higher fruit collapse incidence. In pineap- ple, TA mainly is a measuring of the citric acid level of the fruit (Saradhuldhat and Paull, 2007; Paull and Chen, 2018). In MD2, the total TA value range between 0.4–0.7 % (Lu et al., 2014; Paull and Chen, 2018). Values inside this range were represented in the interaction results at harvest. However, the higher content of TA in the inject method could have been provoked by a more superior citric acid accumulation. Nevertheless, further studies should be done on this matter. Concerning the sucrose content, in the interaction results there were significant differences evidenced. The treatment D with inject method had the most reduced outcome (4.31  %); on the contrary, the highest result was observed in the same treatment but when the spray method was employed (9.85 %) (Table 3). A higher con- tent of sucrose was noticeably associated with a more re- duced incidence of fruit collapse. The most crucial sugar in pineapple is sucrose. Previous research reported that in low acid hybrids the sucrose should be between 7–9 % at harvest (Nadzirah et al., 2013; Lu et al., 2014). Val- ues among that range were reflected in this research out- comes. It has been proved that cell wall invertase (CWI) is one of the enzymes highly correlated with the sucrose ac- cumulation in pineapple (Saradhuldhat and Paull, 2007; Paull and Chen, 2018). Recently evidence indicated that pathogens generated the induction of CWI activity, pro- ducing more hexose as sugars to support their metabolic activities, interfering the normal sugar accumulation in the fruit (Yamada et al., 2016; Naseem et al, 2017). These previous facts inferred that D.zeae influencing the CWI activities affected the sucrose accumulation, especially with the inject method, causing a more superior fruit col- lapse incidence. However, despite reports explaining the increase of sucrose under biopesticides applications of P. fluorescens and T. harzianum in several fruits (Jiang et al., 2019; Carillo et al., 2020); this phenomenon was not fully evidenced in the inject method, as this was more harm- ful to the fruit, nullifying this positive characteristic, es- pecially in treatment D. More studies could be elaborated to determine the relation of the biopesticides used in this experiment with the inoculation methods influencing sugar enzymes activities. Treatments*Inoculation methods TSS (%) TA (%) Sucrose (%) AsA (mg kg-1)  Fruit collapse Incidence (%) A*Spray 13.87 ± 0.18 ab 0.52 ± 0.02 a 9.59 ± 0.21 ab 188.61 ± 28.09 a 3.33 bc B*Spray 13.93 ± 0.18 ab 0.50 ± 0.02 a 9.22 ± 0.25 abc 91.35 ± 69.37 b 0.00 c C*Spray 14.13 ± 0.24 ab 0.53 ± 0.01 a 9.11 ± 0.11 abc 53.07 ± 1.39 b 5.00 bc D*Spray 14.87 ± 0.35 a 0.50 ± 0.01 a 9.85 ± 0.06 a 78.96 ± 15.37 ab 1.67 c A*Inject 12.80 ± 0.61 ab 0.71 ± 0.13 a 7.93 ± 0.25 c 98.99 ± 5.75 ab 20.0 ab B*Inject 12.80 ± 1.44 ab 0.65 ± 0.12 a 8.43 ± 1.05 bc 233.42 ± 72.41 a 23.3 a C*Inject 12.33 ± 0.71 b 0.70 ± 0.16 a 8.77 ± 0.19 abc 132.10 ± 1.05 ab 20.0 ab D*Inject 12.33 ± 0.27 b 0.70 ± 0.12 a 4.31 ± 0.06 d 181.75 ± 21.63 a 20.0 ab Table 3: Effects of the interaction between the treatments and the inoculation methods implemented on pineapple quality and fruit collapse incidence **Each value represents a mean ± standard error. Mean values in each column followed by the same lower-case letters are not statistically different by Duncan’s multiple range test and Kruskal-Wallis test (for the fruit collapse incidence and severity data) (p < 0.05) ***A (Control: No biopesticide used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest), D (Bio P32 + Bio T10 from 13 weeks before harvest). TSS (Total Soluble Solids), TA (Total Acidity), AsA (Ascorbic Acid) Acta agriculturae Slovenica, 118/3 – 20226 D. M. CANO-REINOSO et al. 3.2 ASCORBIC ACID (ASA) CONTENT IN THE FRUIT Observations of the AsA results exposed significant differences in the interaction outcomes. The highest value was obtained in treatment B with the inject method (233 mg kg-1), and the lowest outcome in treatment C with the spray one (53.07 mg kg-1) (Table 3). Overall, higher val- Figure 1: Trend of the ascorbic acid (AsA) content during the experiment for the treatments applied in both inoculation methods. A (Control: No biopesticide used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest), D (Bio P32 + Bio T10 from 13 weeks before harvest). Values are the mean of three replicates, and error bars represent the standard error Acta agriculturae Slovenica, 118/3 – 2022 7 Fruit collapse incidence and quality of pineapple as affected by biopesticides ... ues of AsA were linked to a more superior incidence of fruit collapse. The trend of the AsA content trough the experiment is presented in Figure (1). This figure shows that in four weeks before harvest there is a remarkable change in the trend of AsA in both inoculation methods, particularly in the inject one, which could have caused a physiological impact generating the final content at har- vest. The AsA in MD2 pineapple usually range between 300–600 mg kg-1 at harvest (Lu et al., 2014; Paull and Chen, 2018; Cano-Reinoso et al., 2022a). Besides, pre- vious researches reported a positive correlation between the AsA content in pineapple and its antioxidant activity. The AsA values obtained in this research were lower than the range formerly determined; however, this could be ascribed to the plant’s environmental conditions through the experiment time. It is possible to identify that from four weeks before harvest, when the organic acids accu- mulations start to happen, the AsA content never reach values close to 300 mg kg-1 (Figure 2). Seemingly, the ir- radiation, rainfall and resulting temperature could have affected the AsA accumulation in the fruit, as described in Ferreira et al. (2016) and Paull and Chen (2018). As has been proved to encourage the activities of several scavenger enzymes like catalase (CAT), peroxi- dase (POD) and ascorbic peroxidase (APX) (Akram et al., 2017; Noichinda et al., 2017). Pathogen infections cause an increase in the reactive oxygen species (ROS); this circumstance creates a rise of AsA and subsequent scavenging activities to cope with these ROS generation in fruits (Lu et al., 2014; Noichinda et al., 2017). Further- more, T. harzianum and P. fluorescens in different liquid culture applications have proved to enhance the anti- oxidant capacity, scavenger enzyme activities, and resist- ance mechanisms like hypersensitive responses (HR), in fruits and vegetables (Garcia-Seco et al., 2015; Sood et al., 2020). The past information demonstrated why the treat- ments having biopesticides applications in the inject method increase substantially their AsA content. Be- sides, due to its more harmful impact, this method could have promoted a higher activity of scavenger enzymes, AsA accumulation, and HR to mitigated the fruit injure; a phenomenon that could have occurred also in treat- ment A without biopesticides used. However, the ex- hibition of this situation was not enough to reduce the damage created by the pathogen infection, which is evi- denced in the higher fruit collapse incidence associated with the more superior AsA content, also in the inject method. Moreover, the insufficient AsA content detected weeks before harvest could have made more difficult to generate an optimal physiological respond of the fruit on these circumstances. 3.3 MINERAL NUTRIENTS CONTENT AND ELECTROLYTE LEAKAGE (EL) Mineral nutrients interaction outcomes exposed significant differences at harvest. Nonetheless, the obser- vation of the results exposed that the method of inocula- tion impacted these variables. For the calcium, the most elevated value was detected in treatment C with the inject method (2522.27 mg kg-1); meanwhile, the lowest one was observed in the same treatment but using the spray method (1575.63 mg kg-1). In the case of magnesium, the most elevated result was determined in treatment C using the inject method (2526.31 mg kg-1) and the most reduced in treatment D with the spray one (1837.48 mg kg-1) (Table 4). For the result of both mineral nutrients, the higher content was associated with a more superior incidence of the fruit collapse. Calcium has been proved to rise the resistance of fruits and vegetables to pathogens attacks by increasing the cellular responses to biotic signals and reducing the cell wall breakdown (Madani et al., 2016; De Freitas and Resender Nassur, 2017). Concerning magnesium, this is a component of the middle lamella and also has been reported to activate calcium-dependent protein kinases (CPDKs) (Waraich et al., 2011; Huber and Jones, 2013); proteins that translated the Ca2+ signature into specific phosphorylation events generating signaling responses as part of plant defense mechanisms (Gao et al., 2014; De Freitas and Resender Nassur, 2017; Cano-Reinoso et al., 2022b). Evidently, due to the more severe infection generated by the inject method, the fruit as a protection mechanism could have promoted the increase in the up- take of calcium and magnesium to maintain the cell wall structure, encouraging more molecular ions assimilation and enzyme activities (Ca+2, CDPKs, respectively). Be- sides, T. harzianum and P. fluorescens have been associat- ed with a more remarkable assimilation of mineral nutri- ents content in terms of N, P, K, Ca and Mg in plants and fruits (Pérez-Rodriguez et al., 2020; Sood et al., 2020). These facts make clearer that the biopesticides may have an influence on the plant and fruit defense mechanism when a certain high degree of affectation is reached, in this case, triggering the respective calcium and magnesi- um increase. However, like the situation observed in the AsA results, these effects were not enough to decrease the incidence of fruit collapse in the inject method. The interaction results for the EL content at harvest presented significant differences. The most elevated value was observed in treatment D using spray method of inoc- ulation (72.10 %), while the most reduced value was ob- tained in treatment A with the inject method (54.19 %) (Table 4). A trend of the EL content trough the experi- Acta agriculturae Slovenica, 118/3 – 20228 D. M. CANO-REINOSO et al. ment is presented in Figure (2). In this figure it is possible to observe that the EL had a noticeable increase in both methods of inoculation between four and two weeks be- fore harvest, more remarkable in the treatment B of the inject method (around 80 %), which had a EL content much higher than those of the spray method. This out- come can provide a broader understanding concerning the relation of EL with the fruit collapse incidence at har- vest, especially for treatment B. The EL reflects a loss of integrity in cell membranes, common during a pathogen infection (Demidchik et al., 2014). In pineapple fruit, the EL speeds up from six weeks before harvest in concomitance with the sucrose accu- mulation (Paull and Chen, 2003, 2018). This research ex- posed that treatment using the spray method had higher EL, which should be correlated with a more superior in- cidence of fruit collapse; however, this only happened in the treatments employing the inject method. The differ- ences in EL percentage between the two methods were more related to the experiment design and unique status of the sample analyzed. In the Figure (2) it is possible to observe that fruits of the inject method two weeks be- fore harvest had an EL percentage almost like those of the spray method at harvest, especially in treatment B. This situation means that at this time, the fruits gathered from the inject method were predominantly affected by fruit collapse, while at harvest, the number of fruits with disease symptoms were highly reduced. Therefore, it is possible to infer that the EL in the inject method can be correlated to a more superior fruit collapse incidence, analyzing the results from two weeks before harvest. Fur- thermore, the higher EL percentages of the spray method can be more associated with the normal process of sugar accumulation in pineapple than a physiological response to the stress induced by the bacterium attack; because of that, the lower fruit collapse incidence. Concerning T. harzianum and P. fluorescens, there is still insufficient information of their influence on the EL in plants and fruits; however, their recognized beneficial impact on calcium uptake could suggest that these biopesticides would help to decrease the percentage of EL under a dis- ease infection. High calcium accumulation has been re- lated to a leakage reduce when a plant is subjected to an abiotic or biotic factor (Demidchik et al., 2014; De Freitas and Resender Nassur, 2017). More experiments could be done on this aspect. 3.4 SEM ANALYSIS SEM analysis was conducted at harvest time in the treatments A, B, C, and D of the inject inoculation method (Figure 3). The sample of the treatments A and B showed characteristics of a low cell wall integrity, identi- fied by arrows with lack of significant thickness, and an undulated shape not attached to the vascular bundles of the cell. On the contrary, in treatments C and D it was possible to observe symptoms of membrane well-func- tion, with arrows presenting more significant thickness and turgor. During infections bacteria can cause an increase in the activities of pectolytic and polygalacturonases (PG) enzymes, which are known for their the cell wall de- grading properties (Hocking et al., 2016; De Freitas and Resender Nassur, 2017). The activities of these enzymes can be mitigated by minerals like calcium, which binds to the cell wall and increases its strength, making the cell wall matrix less accessible to them (De Freitas and Resender Nassur, 2017). This information suggested the treatments C and D of the inject method caused a lower Treatments*Inoculation methods Ca (mg kg-1) Mg (mg kg-1) EL (%) A*Spray 1853.55 ± 106.34 bc 2077.48 ± 106.54 abc 65.27 ± 5.48 abcd B*Spray 1736.12 ± 107.81 c 1993.41 ± 102.55 bc 67.99 ± 6.61 abc C*Spray 1575.63 ± 90.16 c 1875.93 ± 94.90 c 70.81 ± 1.39 ab D*Spray 1780.47 ± 29.35 c 1837.48 ± 81.18 c 72.10 ± 3.92 a A*Inject 2335.41 ± 306.44 ab 2335.25 ± 237.64 ab 54.19 ± 1.70 d B*Inject 2474.59 ± 233.43 a 2442.74 ± 195.10 ab 58.92 ± 2.59 bcd C*Inject 2522.27 ± 188.09 a 2526.31 ± 96.49 ab 57.47 ± 2.63 cd D*Inject 2399.15 ± 52.38 a 2451.99 ± 42.28 ab 57.69 ± 2.70 cd Table 4: Effects of the interaction between the treatments and the inoculation methods implemented on pineapple mineral nutri- ents content, and the electrolyte leakage (EL) ** Each value represents a mean ± standard error. Mean values in each column followed by the same lower-case letters are not statistically different by Duncan’s multiple range test (p < 0.05) ***A (Control: No biopesticide used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest), D (Bio P32 + Bio T10 from 13 weeks before harvest). EL (Electrolyte Leakage) Acta agriculturae Slovenica, 118/3 – 2022 9 Fruit collapse incidence and quality of pineapple as affected by biopesticides ... Figure 2: Trend of the Electrolyte leakage (EL) content during the experiment for the treatments applied in both inoculation methods. A (Control: No biopesticide used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest), D (Bio P32 + Bio T10 from 13 weeks before harvest). Values are the mean of three replicates, and error bars represent the standard error activity of these enzymes, generating a healthier cell wall status, opposite to treatments A and B, as exposed in the SEM analysis. Despite of the high concentration con- tent of calcium in the treatments A and B, their cell wall primary layer displayed unhealthy characteristics. This situation could be attributed to the lower assimilation Acta agriculturae Slovenica, 118/3 – 202210 D. M. CANO-REINOSO et al. of calcium ions (Ca2+) into the cell wall matrix produced by the harmful impact of the inject method when treat- ments A and B were implemented. Besides, the calcium ions of these treatments could have also been employed in other calcium-influenced-process like sugar produc- tion and fruit respiration (Hocking et al., 2016; Meeteren and Aliniaeifard, 2016); decreasing its sensing activity into the cell wall. 3.5 FRUIT COLLAPSE INCIDENCE There were significant differences evidenced in the interaction outcomes of the fruit collapse incidence. The treatment B in the spray method obtained the lowest incidence (0 %), while the same treatment but in the in the inject method had the highest one (23.7 %) (Table 4). Moreover, the inject method delivered in average a higher incidence than the spray one for all the treatments (20.93 and 2.50 %, respectively). This evidence finally proves that the inject method was more effective in caus- ing symptoms of this disease. On the other hand, the ob- servation of the significant differences and the mean val- ues of the interaction outcomes can provide the insight that C and D can be considered as the best options to control fruit collapse disease; meanwhile, A and B could be taken as less effective in this aspect. This affirmation can be supported by the examination of the influences of these treatments on the quality variables studied (spe- cially the mineral nutrients content, AsA content, and EL), the cell wall status by the SEM analysis previously described, and their relation with the fruit collapse in- cidence in both inoculation methods. C and D despite not exposing always the highest outcomes, those deliv- ered mostly optimal results in the previous parameters mentioned, primordially a healthy cell wall, which could help to predict that under a more harmful conditions of infection than the implemented in this experiment, these treatments could satisfactorily mitigate the fruit collapse occurrence. On the contrary, A and B, although dis- played high outcomes, regarding antioxidants and resist- ance parameters, like AsA and Ca content, especially in the case of B, their high EL from weeks prior to harvest, together with their unhealthy cell wall status, suggested that under elevated infections these treatments could not provide enough protection to the fruit. Furthermore, due to the characteristics of both in- oculation methods used in this research, where the juice had to be extracted from previously infected fruits, it was complicated to determine in every juice concentra- tion the number of colonies forming unit (CFU) existed. Previous laboratory trials before the beginning of this experiment demonstrated that the minimal number of CFU required to inoculate D. zeae in pineapple should be around 107–109 CFU ml-1, which was in agreement with former experiments described by Sueno et al. (2014) and Aeny et al. (2020)HI, on a pineapple cultivar (Ananas comosus ‘PRI 73-114’. This information could help to support the explanation about why the spray method was less effective in showing fruit collapse symptoms. Because of the characteristic of this method, the number of CFU ml-1 required to cause a D. zeae infection could be higher than the inject method. Moreover, because of the number of colonies necessary to produce an infection in Figure 3: Effects of the treatments A, B, C, and D in the cell walls of the inject method of inoculation detected by SEM (20 and 10 µm size, respectively; with 2000 x of magnification). The smaller thickness and undulated arrows of the cell wall (red square) and more significant thickness and non-undulated arrows (green square) were examined. Treatments, A (Control: No biopesticide used), B (Bio P32 from 13 weeks before harvest), C (Bio T10 from 13 weeks before harvest), D (Bio P32 + Bio T10 from 13 weeks before harvest) Acta agriculturae Slovenica, 118/3 – 2022 11 Fruit collapse incidence and quality of pineapple as affected by biopesticides ... the inject method, the doses of biopesticides employed (20 ml/plant-fruit), together with the concentration number of cell ml-1 and conidia ml-1 in those products (P. fluorescens and T. harzianum, respectively), could not be enough to mitigate the impact of fruit collapse. For future experiments, the doses and the concentration number of cells and conidia per ml of P. fluorescens and T. harzianum should be increased in the case that this experiment wants to be replicated in pineapple. On top of that, those future trials could also implement a chemi- cal pesticide treatment as positive control. These future arrangements could help to stablish the differences be- tween the biopesticides administrated in this research and any conventional pesticide, essentially concerning pineapple quality and fruit collapse occurrence. As the employment of chemical agents were outside of the scope of this experiment, this should be a point to be observed eventually. 4 CONCLUSIONS The biopesticides based on Pseudomonas fluores- cens and Trichoderma harzianum affected the fruit col- lapse disease incidence and pineapple quality. Treatment C (Bio T10 from 13 weeks before harvest), and D (Bio P32 + Bio T10 from 13 weeks before harvest) delivered the best results having an ideal AsA, EL, mineral nutri- ents content, healthier cell wall characteristics, and a low fruit collapse incidence, essentially after analyzing their outcomes in both inoculation methods. Meanwhile, treatments A (Control: No biopesticide used), and B (Bio P32 from 13 weeks before harvest) were less effective in these aspects. Finally, the inject inoculation method caused more fruit collapse incidence than the spray one. The number of CFU of D. zeae were considered as the reasons why the inject method was more severe affect- ing the fruit physiology and effective in generating the higher incidence. 5 ACKNOWLEDGEMENTS Sincere thanks want to be expressed by the authors of this article to PT Great Giant Pineapple in Lampung Indonesia, their research department, and the laboratory staff for the vital and logistic support with all the activi- ties carried out during this experiment. 6 REFERENCES Aeny, T. N., Suharjo, R., Ginting, C., Hapsoro, D. W. I., & Ni- swati, A. (2020). Characterization and host range assess- ment of Dickeya zeae associated with pineapple soft rot disease in east Lampung, Indonesia. Biodiversitas, 21(2), 587–595. https://doi.org/10.13057/biodiv/d210221 Akram, N. A., Shafiq, F., & Ashraf, M. (2017). 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Science, 354(6318), 1427–1430. https://doi. org/10.1126/science.aah5692 Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2454 Original research article / izvirni znanstveni članek Enhance the phytoremediation efficiency of Echinochloa colona (L.) Link for Pb-contaminated soil by phosphorus solubilizing bacteria Quang Trung DO 1, 2 Received December 05, 2021; accepted August 22, 2022. Delo je prispelo 5. decembra 2021, sprejeto 22. avgusta 2022 1 Central Institute for Natural Resources and Environmental Studies, Vietnam National University Hanoi, Ha Noi, Vietnam 2 Corresponding author, e-mail: trungcnsinh@gmail.com Enhance the phytoremediation efficiency of Echinochloa colo- na (L.) Link for Pb-contaminated soil by phosphorus solubi- lizing bacteria Abstract: A promising solution for phytoremediation of metal-contaminated soils is to use plants in combination with phosphate-solubilizing bacteria (PSB). In this study, we subjected to isolate PSB from paddy soil and investigate their ability in improving the phytoremediation of lead (Pb2+) by a weed plant (Echinochloa colona (L.) Link) as well as in promot- ing the growth of E. colona under Pb stress condition. Total 06 PSB (labeled from TB01 to TB06) were isolated and the TB04 showed the strongest phosphate-solubilizing activity with the highest values of phosphorus solubilization index (PSI = 7.13) obtained from Ca3(PO4)2. Especially, the phosphorus solubi- lizing ability of the TB04 strain was not affected by the high Pb2+concentration. The TB04 strain was identified as Pseudo- monas putida Trevisan, 1889 (accession number FJ976601.1). Furthermore, E. colona inoculated with TB04 strain signifi- cantly increased the phytoremediation efficiency of Pb from Pb-contaminated soil and the growth was enhanced clearly. These results suggest that the TB04 strain could potentially use as an inoculant in combination with E. colona to construct novel constructed wetlands for phytoremediation of metal- contaminated soil. Key words: lead immobilization; Pseudomonas putida; soil fertility; phytoremediation; metal-contaminated soil Povečanje fitoremediacijske učinkovitosti vrste Echinochloa colona (L.) Link z bakterijami, ki sproščajo fosfor v tleh, one- snaženih s svincem Izvleček: Obetajoča rešitev za fitoremediacijo s kovinami onesnaženih tal je uporaba rastlin v kombinaciji z bakterijami, ki sproščajo fosfor (PSB). V raziskavi so bili preučevani izola- ti teh bakterij iz riževih polj in njihova sposobnost izboljšanja fitoremediacije svinca (Pb2+) s plevelno vrsto kostrebe (Echino- chloa colona (L.) Link) kot tudi izboljšanje rasti te rastline v raz- merah kovinskega stresa zaradi onesnaženja s svincem. Celo- kupno je bilo izoliranih 6 izolatov PSB (označenih kot TB01 do TB06), pri čemer je imel izolat TB04 največjo sposobnost spro- ščanja fosforja (z indeksom PSI = 7,13) iz kalcijevega fosfata (Ca3(PO4)2). Na sposobnost sproščanja fosforja pri sevu TB04 niso vplivale velike koncentracije Pb2+. Sev TB04 je bil identi- ficiran kot vrsta bakterije Pseudomonas putida Trevisan, 1889 (številka akcesije FJ976601.1). Inokulacija kostrebe s sevom TB04 je značilno povečala njeno fitoremediacijsko učinkovitost za svinec v s svincem onesnaženih tleh, pri čemer je bila njena rast značilno pvečana. Rezultati nakazujejo, da bi sev TB04 lah- ko potencialno uporabili kot inokulant kostrebe kot nov način fitoremediacije s kovinami onesnaženih močvirnih tal. Ključne besede: imobilizacija svinca; Pseudomonas puti- da; rodovitnost tal; fitoremediacija; s kovinami onesnažena tla Acta agriculturae Slovenica, 118/3 – 20222 Q. T. DO 1 INTRODUCTION It is a fact that industrial development, agricultural practices, and human activities caused a quick increase in areas of soil contaminated with heavy metals (Xiao et al., 2021). Importantly, pollution with lead (Pb) was the most concern because it has no function in biology or physiology for the living cells, but was determined as a toxic chemical for living cells (Yahaghi et al., 2018; Aran- siola et al., 2019). Especially, the metal chemicals were not biodegraded leading to their accumulation in the soil, which can increase the risk of these metals enter- ing the food chain by uptake activity of crops (Noble et al., 2018; Xiao et al., 2021). Hence, the removal of metal pollutants from the soil is very important and necessary. Although there are several methods have been applied to remediate the metal pollution in soil, phytoremediation of heavy metals is a promising one that uses plants to up- take the metal pollutants from soil accumulating them in the above-ground part of the plant for disposal. Hence, phytoremediation is environmentally friendly, low-cost, and easy to set up (Noble et al., 2018; Xiao et al., 2021). In agricultural practices, the application of PGPR, particularly phosphate solubilizing bacteria (PSB), to im- prove crop yield is becoming more and more frequent. Besides assisting plants in nutrient uptakes and disease protection, PSB also presented its ability to enhance plant growth in harsh conditions caused by contaminants in the soil such as metal pollutants (Noble et al., 2018; Adhi- kari et al., 2020). Therefore, the inoculation of PSB in the phytoremediation of metal pollutants from the soil is very potential. It was reported that a weed named Echi- nochloa colona (L.) Link that has a wide distribution in an agroecosystem and has played role in the uptake of heavy metals from metal-contaminated soil (Subhashini and Swamy, 2016; Noble et al., 2018). It demonstrated their efficiency in the phytoremediation of lead, nickel, zinc, cadmium, and chromium from contaminated soils (Subhashini and Swamy, 2016). In addition, Noble et al. (2018) reported that with the assistance of plantain peels the phytoremediation of Pd and Cd in soil by E. colona was significantly enhanced. Therefore, the application of E. colona for phytoremediation of metal-contaminated soils is very promising. However, it is a fact that phytore- mediation presents some limitations such as being time- consuming, and the removal efficiency of metals depends strongly on the plants vegetated in that system. Interestingly, the combination of plants and plant growth-promoting rhizobacteria (PGPR) could improve the phytoremediation efficiency (Noble et al., 2018; Xiao et al., 2021). However, the study using PSB to enhance the removal efficiency of metal pollutants from the soil by E. colona are scarce. Hence, this study’s aims were (1) to isolate PSB from Thai Binh paddy soil and (2) to inves- tigate their ability in improving the phytoremediation of lead (Pb2+) by a weed plant (Echinochloa colona) as well as in promoting the growth of E. colona under Pb stress condition. 2 MATERIAL AND METHODS 2.1 ISOLATION OF PHOSPHORUS-SOLUBILIZING BACTERIA Samples of soil were collected from different loca- tions at agricultural fields in Thai Binh Province, Viet- nam for isolation of PSB. About 2 g of each soil sample adhered to the rice roots was collected and carefully transferred into sterile tubes containing sterile deion- ized (DI) water (about 2 ml). Then, each test tube was vortexed thoroughly and let set for 5 minutes at room temperature. The 10-fold dilutions in the same buffer were applied. After that, it took 100 μl of diluted samples to plate onto Pikovskaya (PVK) media agar plates (Piko- vskaya, 1948). The bacterial colonies with clear halos in the PVK agar plate indicated solubilizing activity of the phosphate. These were sub-cultured on PVK (Biobasic, Canada). Similar methods were applied to screen for micro- organisms that could solubilize aluminum phosphate (AlPO4) and iron phosphate (FePO4). In this experiment, the medium was modified from the PVK medium, in which the Ca3(PO4)2 was altered by either 5 g l -1 of AlPO4 or 5 g l-1 of FePO4. The PVK medium used in study include (g l-1): glu- cose, 10; (NH4)2SO4, 0.5; MgSO4.7H2O, 0.1; yeast extract, 0.5; KCl, 0.2; NaCl, 0.2; FeSO4.7H2O, 0.002; MnSO4.7H2O, 0.002; Ca3(PO4)2, 5; pH 6.5 (for agar plate, 15 g of agar was added). The plate incubation was carried out at 30 °C for 7 days. All media and glassware used were sterilized in an autoclave before use. 2.2 MOLECULAR IDENTIFICATION OF TB04 STRAIN The total DNA of strain TB04 was extracted using a Rapid Bacteria Genomic DNA Isolation Kit (Biobasic, Canada) as per the kit instructions. The PCR amplifi- cation of 16S rDNA was done with the extracted DNA by using the universal primers 27F and 1492R. The se- quence of 16S rDNA sequences obtained was blasted on NCBI to identify the species. The sequences with high similarity were used for multiple cluster alignment and phylogenetic analysis on MEGA software (v.7.2). Acta agriculturae Slovenica, 118/3 – 2022 3 Enhance the phytoremediation efficiency of Echinochloa colona (L.) Link for Pb-contaminated soil by phosphorus solubilizing bacteria 2.3 DETERMINE PHOSPHATE SOLUBILIZING EF- FICIENCY OF THE ISOLATES Single colonies were cultured separately in liquid LB media at 30 °C for 24 h on the shaker (150 rpm). Then, bacterial cells of each strain were collected by applying a described procedure. The bacterial suspension of iso- lates (106 CFU ml-1) was determined for their ability to solubilize different insoluble phosphorus compounds (Ca3(PO4)2, sodium phytate, FePO4, or AlPO4) on either solid or liquid PVK medium. The condition for plate in- cubation was at 30 °C for seven days. The medium with no bacteria was used as the control. After seven days of incubation, the determination of soluble P concentration in bacterial culture was done using the molybdenum blue method (Waterlot, 2018), and the phosphate solubilization index (PSI) of bacteria grown on plates was measured as the method described by Liu et al. (2015). The pH measurement of the bacterial culture was carried out by using the pH meter. In addition, the isolated PSB were also character- ized their P solubilizing efficacy in soil conditions by a method adapted from Wan et al. (2020). Different treat- ments have been done in triplicates: (T1) 100 g sterilized soil + 10 ml bacterial solution; (T2) 95 g sterilized soil + 10 ml bacterial solution + 5 g Ca3(PO4)2, (T3) 95 g steri- lized soil + 10 ml bacterial cultures + 5 g Ca3(PO4)2 + 10 ml nutrient solution (PVK liquid medium removed Ca3(PO4)2). Soil moisture in the experiments was adjust- ed to 80 % by sterile water and kept for 30 days at 25 °C. After that, the amount of available P (AP) in treated soils was determined by the molybdenum blue method (Wa- terlot, 2018). 2.4 INDOLE-3ACETIC ACID (IAA) PRODUCTION OF PSB The isolates were also screened for IAA production by using LB medium supplemented 0.1 % L-tryptophan. The colorimetric method using ferric chloride-perchlo- ric acid reagent (FeCl3–HClO4) as described by Luu et al. (2021) was applied to measure the amount of IAA pro- duced. 2.5 PHOSPHORUS SOLUBILIZATION ABILITY OF TB04 ISOLATE UNDER LEAD STRESS The isolates were then investigated for the solubili- zation of Ca3(PO4)2 under Pb 2+ stress. The Pb-contami- nated soil was artificially produced by mixing the steri- lized soil with Pb(NO3)2 (0, 200, 400, 800, 1600, or 2400 mg Pb/ kg soil) and Ca3(PO4)2 (as the P source). Then, 100 ml of the culture of isolated PSB were added to the prepared soil and were kept for four months at room temperature. For control experiments, soil with only Ca3(PO4)2. The moisture in all experiments was kept at 80 % by watering with sterile water every five days. After four months of incubation, the soil sample was collected, air-dried, ground, sieved through a 0.2-mm sieve, and subsequently extracted at room temperature for 30 min by a mixed solution of 0.025M HCl and 0.03 M NH4F (1:10 soil:water ratios). The amount of the available P in the treated soil was determined by the molybdenum blue method (Waterlot, 2018). 2.6 EFFECT OF TB04 STRAIN INOCULATION TO THE DEVELOPMENT AND PB UPTAKE OF WEED PLANT (Echinochloa colona) The pot experiments were prepared as the method described in Luu et al. (2021). Briefly, seeds of E. colona were sterilized on their surface by using ethanol 70 % for 30 s and sodium hypochlorite solution 2  % for 5 min- utes. Then these seeds were washed three times with sterile water and dried on autoclaved filter papers. The TB04 strain with the highest efficiency of Pb uptake and IAA production was overnight grown, centrifugated, and washed with sterile water before being resuspended with sterile water to make a bacterial solution with OD = 1. The sterilized seeds were covered with selected PSB by soaking in the bacterial solution for 30 minutes before sowing. For the control, sterile water was used instead of the bacterial solution. The treatment was done in triplicates by sowing ten bacterized seeds of E. colona per plastic pot filled with about 1 kg of lead-contaminated soil (600 mg kg-1 of Pb(NO3)2). After plant establishment, one plant per pot was done. The pots were kept in the nursery garden and soil moisture was held at 60 % of water holding capac- ity during the experiment by adding a specific amount of sterile water as the method described by Steadman et al. (2004). After one month, 100 ml of the bacterial culture (OD = 1) were added to the treated pot as biofertilizer while sterile water was used for the control. The experiments were carried out in 3 months. The measured parameters for plant growth were plant height, shoot and root dry mass. The plant height was measured from the aboveground to the tip of the upper-most leaf of the plant. The root was cut from the plant and removed Pb ions bounding to its surface by washing with 1 mM Ca(NO3)2.4H2O and sterile water. The dry mass of root and shoot were determined after dried in an oven at 70 ˚C for 72 h. The Pb in the oven-dried shoot and root Acta agriculturae Slovenica, 118/3 – 20224 Q. T. DO was extracted by using a solution of HNO3-HCl (70 %) and H2O2 (30 %) (Jones et al., 1990) and were measured by FAAS. All measurements were done in triplicates. 2.7 DATA ANALYSIS All experiments were repeated three times the re- sults were presented as mean values with ± SD. Tukey’s honestly significant difference (HSD) method in SPSS (version 17) was applied to compare the means in all ex- periments. 3 RESULTS AND DISCUSSION 3.1 ISOLATION AND CHARACTERIZATION OF PHOSPHATE-SOLUBILIZING BACTERIA Bacteria isolates that produced a transparent zone around colonies in the Pikovskaya (PVK) medium were determined as phosphate-solubilizing bacteria and were selected. There were six single colonies were observed and further transferred into new PVK plates for purifica- tion (Table 1). All isolates showed different efficiency in solubiliz- ing phosphorus after 7 days of incubation at 30 oC, which was illustrated by different values of PSI ranging from 1.53 to 7.13 (Table 1). A further characteristic of isolates indicated their ability in IAA production, in which the highest amount of IAA (7.86 mg l−1) was observed for the TB04 strain. Furthermore, the results also presented the differ- ent capabilities in solubilizing phosphorus compounds of all isolates from different phosphate sources. All iso- lated strains could solubilize multiple insoluble phos- phorus compounds (Ca3(PO4)2, AlPO4, and FePO4) but only TB03 and TB04 presented the phytate solubi- lization (Table 2). For inorganic P, the results indicated that Ca3(PO4)2 was the most favorable compound for all strains demonstrated by the highest amount of soluble P (173.11-572.13 mg l−1) released from this compound; and the TB04 also presented the highest efficiency. In addition, approximately 10-fold less of solubilization efficiency was observed for the remaining complexed phosphate sources including AlPO4 (21.17 to 72.13 mg l−1) and FePO4 (10.51 to 29.73 mg l −1) by most of the iso- lates (Table 2). Furthermore, only two strains, TB01 and TB04, showed the ability in solubilizing organic phytate supplemented with a modified PVK broth medium (1.53 and 3.61 mg l−1, respectively). These results indicated that TB04 could solubilize multiple P sources and might be used to reverse insoluble phosphate to soluble form in agriculture. It was a fact that the solubilization of AlPO4 and FePO4 was lower than the one of (Ca3(PO4)2. It can be explained by two possible reasons. Firstly, it was reported that the interaction of aluminum (Al3+) and iron (Fe3+) with phosphate ion (PO4 3-) is a reversible reaction. Hence, it could be that the acids produced by PSB during the solubilization might force the reversible reaction of alu- minum (Al3+) and iron (Fe3+) with phosphate ion (PO4 3-) to form insoluble complexes (Sánchez-Cruz, 2020) lead- ing to an inefficient in solubilizing FePO4 and AlPO4. Secondly, it could be differences in affinity among cati- ons and anions in the solution, in which the anions gen- erated by PSB such as carboxylic and hydroxylic groups preferred calcium (Ca2+) to aluminum (Al3+) and iron (Fe3+) and subsequently enhanced the phosphorus solu- bilization (Sánchez-Cruz, 2020). Moreover, the results indicated the pH reduction and production of a phytase of strains TB01 and TB04 played significant roles in solu- bilizing inorganic phosphate. These were demonstrated by some previous research (Kumar and Rai., 2015; Wan et al., 2020). All of these suggest the organic acids and/or phosphate solubilizing enzymes produced by PSBs play important roles in mineralizing phosphorus compounds (Walpola et al., 2013). Moreover, the correlation analysis showed a low correlation between the values of PSI and the amount of soluble P released (r = 0.442), and between pH of super- natant and the amount of soluble P released (r = 0.501). These could be related to P solubilizing mechanisms, in which the PSB produced external metabolites such as hy- drolytic enzymes, and/or organic acids that enhanced the solubilization of mineral phosphates and could reduce the pH of bacteria culture. Some reports demonstrated a positive correlation between the pH of culture and the solubilized amount of PSB isolates Phosphate solubilization index (Agar) IAA production (mg l-1) TB01 4.13 ± 0.11b 1.87 ± 1.21c TB02 4.12 ± 0.12b 2.02 ± 1.02c TB03 3.37 ± 0.31bc 3.25 ± 1.01bc TB04 7.13 ± 0.15a 7.86 ± 1.01a TB05 1.53 ± 0.23c 4.52 ± 1.12b TB06 1.67 ± 0.12c 1.91 ± 1.12c Table 1: Characteristics of isolated phosphate-solubilizing bacteria (PSB) Data are means ± SE of three independent biological replicates. Data with the same letters in the same column are not significantly different from each other according to the honestly significant difference (HSD) test (p < 0.05) Acta agriculturae Slovenica, 118/3 – 2022 5 Enhance the phytoremediation efficiency of Echinochloa colona (L.) Link for Pb-contaminated soil by phosphorus solubilizing bacteria phosphorus complexes (Ca3(PO4)2) (Marra et al., 2019). However, the results showed an uncorrelation between the soluble P release and pH reduction. This might be chelation between metal cations (Ca2+, Al3+, Fe3+) and an- ion groups of produced organic acids (Stevenson, 2005) leading to pH decrease and subsequently the increase of soluble P. Therefore, it could be said that the solubiliza- tion of phosphorus compounds is simultaneously affect- ed by pH decrease and acid production in the solution (Fankem et al., 2006). 3.2 MOLECULAR IDENTIFICATION OF STRAIN TBO4 The molecular identification of TB04 indicated that this strain was Pseudomonas putida (accession number FJ976601.1) (Figure 1). The sequence of 16S rDNA of TB04 was deposited in GenBank with an accession num- ber OP141766. This strain showed a significant efficiency of Ca3(PO4)2 solubilization compared to reported Pseu- domonas sp. (such as Pseudomonas fluorescens (Flügge 1886) Migula, 1895 (184 mg l-1) (Katiyar and Goel, 2003), Pseudomonas putida (247 mg l-1) (Pandey et al., 2006). These differences can be explained due to the difference in isolated strains that were grown and developed under specific conditions. 3.3 EFFECT OF PSB AND TRICALCIUM PHOS- PHATE IN UNCULTIVATED SOIL Next steps, we investigated the phosphate solubiliz- ing ability of the isolates in Ca3(PO4)2 -rich soil condi- tions. As we expected, all isolated PSB could solubilize the Ca3(PO4)2 incubated in soil (Figure 2). After 30 days of incubation, the AP content in soil supplemented with TB04 was significantly higher in all experiments than the one in the control. Notably, the soil added with TB04 showed the highest amount of AP in the same soil treatments. Particularly, the AP amount in TB04-incubated soils, in T1 (soil + PSB), T2 (Soil + PSB + Ca3(PO4)2 ), and T3 (Soil + PSB + Ca3(PO4)2 + Nutri- ent) treatments were 0.55, 0.87, and 1.72 mg g-1, respec- tively. Especially, the significantly higher values of AP in T3 treatment compared to those in T1 and T2 treat- ments (p < 0.05) were observed. This might be because of the results of the addition of sufficient nutrients for bacterial growth (Figure 2). As can be seen from Figure 2, the positive correlation between the AP amount in PSB-inoculated soil and added amount of Ca3(PO4)2 in the presence of TB04 strain. These results were consistent with previous reports, which demonstrated the potential application of PSB in improving soil quality, particular- ly by increasing the amount of available P that directly influences the plant development and plant uptake and subsequently the yield (Himani and Reddy, 2011; Teng et al., 2019; Wan et al., 2020). These improvements might be due to the inoculated PSB in treatment solubilized the Ca3(PO4)2 fertilizer to release soluble P that was partially used for the development of PSB, subsequently enhanc- ing the phosphorus’s efficiency. These explanations were demonstrated by studies that reported a positive correla- tion between the change in the amount of soil organic carbon and the change in bacterial development in soil (Nakhro and Dkhar, 2010; Wan et al., 2020). In addi- tion, another contributor to the improvement of solu- ble P amount in treated soil might be the difference in hydrolytic enzymes (such as phosphatase, and phytase) PSB isolates PVK with Ca3(PO4)2 PVK with AlPO4 PVK with FePO4 PVK with sodium phytate Soluble P (mg l-1) pH Soluble P (mg l-1) pH Soluble P (mg l-1) pH Soluble P (mg l-1) pH TB01 251.15 ± 10.71b 4.95 ± 0.21b 54.31 ± 4.71b 3.55 ± 0.12c 10.51 ± 1.53d 3.47 ± 0.23d ND 4.35 ± 0.21b TB02 176.15 ± 7.12c 3.93 ± 0.11c 72.13 ± 2.35a 3.30 ± 0.17bc 13.25 ± 1.31c 3.64 ± 0.17c ND 3.71 ± 0.18c TB03 248.12 ± 12.72b 3.78 ± 0.14c 44.16 ± 2.31c 3.25 ± 0.31bc 29.73 ± 2.42a 3.82 ± 0.16c 1.53 ± 0.19b 3.92 ± 0.13bc TB04 572.13 ± 12.41a 4.22 ± 0.15c 51.34 ± 3.17b 3.27 ± 0.12bc 23.15 ± 1.27ab 3.53 ± 0.12cd 3.61 ± 0.71a 3.53 ± 0.51b TB05 182.13 ± 11.10c 5.21 ± 0.13b 21.17 ± 2.73e 4.31 ± 0.15b 20.53 ± 1.17b 4.05 ± 0.13b ND 3.37 ± 0.33d TB06 173.11 ± 9.13c 4.57 ± 0.21bc 33.17 ± 2.23d 3.78 ± 0.17bc 19.56 ± 1.15b 3.77 ± 0.32c ND 4.17 ± 0.27b Control media ND 6.51 ± 0.11a ND 6.52 ± 0.13a ND 6.47 ± 0.15a ND 6.53 ± 0.31a Table 2: Determination of phosphate solubilization ability in PVK broth medium with Ca3(PO4)2, AlPO4, FePO4, and sodium phytate by isolated PSB Data are means ± SE of three independent biological replicates. Value with the same letter in the same row is not significantly different from each other according to the honestly significant difference (HSD) test (p < 0.05). ND: not detected Acta agriculturae Slovenica, 118/3 – 20226 Q. T. DO Figure 1: A neighbor-joining tree shows the phylogenetic relationships among 16S rDNA sequences of TB04 and their closely related sequences from NCBI Figure 2: Evaluation of available P in soil incubation (mg l-1). The presenting results are the mean value of three replicates. PSB: phosphorus solubilizing bacteria; TB01 to TB06 are phosphorus solubilizing bacteria Acta agriculturae Slovenica, 118/3 – 2022 7 Enhance the phytoremediation efficiency of Echinochloa colona (L.) Link for Pb-contaminated soil by phosphorus solubilizing bacteria presented in soil (Teng et al., 2019). Presumably, the re- sults indicated that TB04 showed a promising application in solubilizing insoluble phosphorus compounds in soil that increase soil health. 3.4 EVALUATION OF THE PHOSPHORUS SOLU- BILIZATION ABILITY OF TB04 ISOLATES UNDER LEAD STRESS In fact, the agricultural soil was contaminated with metals caused by the overuse of chemical fertilizer. Hence, we investigated the ability of strain TB04 to sol- ubilize phosphorus compounds in the presence of lead with different concentrations. As shown in Figure 3, the amount of available P in treated soil was higher than that of initiated soil (about 0.19 mg g-1). These results indi- cated that the TB04 strain could solubilize phosphorus compounds in soil and this ability was not affected by an increasing amount of Pb concentration. 3.5 INOCULATION OF TB04 STRAIN IMPROVES THE DEVELOPMENT AND PB UPTAKE OF WEED PLANT (ECHINOCHLOA COLONA) The effect of TB04 inoculation on the growth prop- erties of weed (Echinochloa colona) under greenhouse conditions was studied. The obtained results of green- house experiments were shown in Table 3. As can be seen, the TB04 strain significantly improved the plant growth parameters of E. colona compared to the control experiment, which used sterile water instead. Particu- larly, the length of TB04 inoculated plants was increased approximately 1.5 times compared to non-bacterized plants. Similarly, the increase in shoot and root dry mass observed for the plants bacterized with TB04 with 1.5 times higher than the control. These data were not in agreement with some previ- ous studies, which reported that plant development was inhibited when grown on heavy metal-contaminated soil (Tangahu et al., 2011). This might be due to the TB04 strain produced IAA (a plant up-regulator) and solubi- lized phosphorus compounds increasing the amount of available P in soil, and subsequently enhancing the plant development. Lin et al. (2018) demonstrated the growth of Wedelia trilobata (L.) H.Rob. & Cuatrec. cultivated in Cu2+-contaminated soil was significantly upregulated when inoculated with Paenibacillus polymyxa (Praz- mowski, 1880) Ash et al., 1994, a phosphate-solubilizing bacterium. Another example is the study of Yahaghi et al. (2018) who showed the inoculation of a bacterial mix- ture (Brevibacterium frigoritolerans Delaporte and Sas- son, 1967 YSP40 and Bacillus paralicheniformis sp. nov. YSP151) improved the development of Brassica juncea (L.) Czern that grown in a soil contaminated with heavy metal by producing IAA, siderophores, and solubilizing inorganic phosphate. The data in Table 3 also indicated that the Pb con- centration in the shoot of bacterized E. colona plant was dramatically increased in the comparison with one of non-bacterized plants. The result also showed that the inoculation of TB04 was not clearly influenced by the amount of Pb in the root. In addition, the result also presented that the TB04-treated plants contained more amount of Pb uptake in the shoot than the control did. Table 3: Enhanced effect of TB04 strain on the development and Pb uptake of Echinochloa colona Phosphorus solubilizing bacteria Plant length (cm) Shoot dry mass (g/plant) Root dry mass (g/plant) Pb concentration in shoot (mg kg-1) Pb concentration in root (mg kg-1) Pb uptake by shoot (µg/pot) SDWa 52.37 ± 1.79 ab 18.32 ± 2.73 a 14.05 ± 2.27 a 40.27 ± 3.02 a 94.17 ± 2.79 a 71.28 ± 11.53 a TB04 73.51 ± 3.73 b 29.21 ± 3.72 b 22.07 ± 1.17 b 73.17 ± 5.27 b 84.92 ± 2.76 a 223.72 ± 18.74 b Table 3: Enhanced effect of TB04 strain on the development and Pb uptake of Echinochloa colona a TB04: selected phosphorus solubilizing bacteria; SDW: Sterile distill water b Presenting values the mean ± standard deviation. Values with a different letter in the same column indicated a significant difference according to HSD (p < 0.05) Acta agriculturae Slovenica, 118/3 – 20228 Q. T. DO The data showed that the bacterial inoculation increased the Pb concentration in the shoot of bacterized E. colona plant and was not clearly influenced by the amount of Pb in the root. The increase in Pb2+ absorption could be due to the inoculated PSB produced metabolites (such as organic acids) that enhanced the bioavailability of Pb2+ in the root rhizosphere, and subsequently improved the Pb2+ absorption of root (Aransiola et al., 2019; Xiao et al., 2021). In addition, the result also indicated a higher amount of Pb uptake in the shoot than the control did. This might be the result of the improvement in shoot biomass and the Pb2+ translocation caused by the TB04 inoculation. Yahaghi et al. (2018) reported that the Pb2+ uptake in the shoot of B. juncea inoculated with Brevi- bacterium frigoritolerans YSP40 and Bacillus paralicheni- formis YSP151 strains was increased 3 and 4 times, re- spectively. 4 CONCLUSIONS This study demonstrated the potential application of PSB isolated from paddy soil collected from Thai Binh province for enhancing the removal efficiency of Pb2+ pollutants from metal-contaminated soil by E. colona. The inoculation of PSB isolated into the Pb-contaminat- ed soil not only promoted the plant growth of E. colona but also enhanced the Pb2+ uptake by the root of E. colo- na. These data suggest a potential application of isolated PSB combined with a phytoremediator for improving the phytoremediation of metal pollutants from metal-pollut- ed soil. 5 REFERENCES Aransiola, S.A., Ijah, U.J.J., Abioye, O.P. and Bala, J.D. (2019). Microbial-aided phytoremediation of heavy metals con- taminated soil: a review. 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Journal of Microbiology and Biotechnology, 28(7), 1156-1167. https://doi.org/10.4014/ jmb.1712.12038 Acta agriculturae Slovenica, 118/3, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.3.1873 Original research article / izvirni znanstveni članek Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. Nima MOSAVAT 1, 2, 3, Maryam YOUSEFIFARD 4, Pooran GOLKAR 5, 6, Rabia JAVED 7 Received September 12, 2020; accepted August 31, 2022. Delo je prispelo 12. septembra 2020, sprejeto 31. avgusta 2022 1 Department of Agricultural Biotechnology, Payame Noor University (PNU), Tehran, Iran 2 Institute of Biotechnology, School of Agriculture, Shiraz University, Shiraz, Iran 3 Corresponding author, e-mail: nima.mosavat@gmail.com 4 Department of Engineering and Technology, Payame Noor University (PNU), Tehran, Iran 5 Department of Natural Resources, Isfahan University of Technology, Isfahan, Iran 6 Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan, Iran 7 Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan Influence of Ag nanoparticles on physiological and biochemi- cal aspects of callus of Thymus species and Zataria multiflora Boiss. Abstract: Thymus species have found remarkable im- portance in food and medicine industries. The present study investigates the potential effect of Ag nanoparticle elicitors on proliferation of callus, and production of carvacrol and thymol in Zataria multiflora and three Thymus species. Firstly, callus was induced on Murashige and Skoog (MS) medium contain- ing 2 mg l−1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg l−1 of kinetin (Kin)). Secondly, the effects of two differ- ent concentrations of Ag nanoparticles (4 and 8 mg l-1) were studied on callus growth and its secondary metabolites produc- tion. Results elucidated that after elicitation by 8 mg l-1 of Ag NPs, significantly the highest callus growth rate (CGR) (0.02 mm day-1), callus fresh mass (CFM) (0.99 g), and carvacrol (0.68 mg l-1) and thymol (11.09 mg l-1) content was achieved. Comparing different Thymus species, notably the greatest car- vacrol and thymol amount was obtained in .kotschyanus Boiss. & Hohen. and T. Daenesis Čelak. at 8 mg l-1 concentration of Ag NPs. Hence, it is evident that the stimulation by NPs is dose- dependent. This study has potential to be commercially applied for the enhancement of pharmaceutical compounds in different species of Thymus. Key words: Ag nanoparticles; Thymus species; Zataria multiflora; callus; carvacrol thymol Abbreviations: 2,4-D, 2, 4-dichlorophenoxyacetic acid; Kin, kinetin; PGRs, plant growth regulators; HPLC, high per- formance liquid chromatography; NPs, nanoparticles Vpliv nanodelcev srebra (Ag) na fiziološke in biokemične la- stnosti kalusa dveh vrst materine dušice (Thymus sp.) in vrste Zataria multiflora Boiss. Izvleček: Vrste iz rodu materine dušice (Thymus sp.) ima- jo velik pomen v prozivodnji hrane in zdravil. V raziskavi je bil preučevan potencialni učinek nanodelcev srebra kot eliciator- ja na rast kalusa, tvorbo karvakrola in timola pri vrsti Zataria multiflora in treh vrstah materine dušice. Najprej je bil na Mu- rashige in Skoog (MS) gojišču, ki je vsebovalo 2 mg l-1 2,4-D in 1 mg l-1 kinetina, vzgojen kalus. Potem je bil preučevan učinek dveh različnih koncentracij srebrovih nanodelcev (4 in 8 mg l-1) na rast kalusa in in tvorbo sekundarnih metabolitov. Rezultati, pridobljeni z visokotlačno tekočinsko kromatografijo (HPLC) so pokazali, da je bila po uporabi 8 mg l-1 srebrovih nanodelcev kot iliciatorjev dosežena značilno največja rast kalusa (CGR) (0,02 mm dan-1), največja sveža masa kalusa (CM) (0,99 g) in največja vsebnost karvakrola (0,68 mg l-1) in timola (11,09 mg l-1). V primerjavi različnih vrst materine dušice je bila dosežena največja vsebnost karvakrola in timola pri vrstah T. kotschyanus Boiss. & Hohen in T. daenesis Čelak pri koncentraciji srebrovih nanodelcev 8 mg l-1. Očitno je, da je stimulacijski učinek nano- delcev odvisen od doze. Izsledke raziskave bi lahko komercial- no uporabili za povečanje tvorbe zdravilnih spojin pri različl- nih vrstah materine dušice. Ključne besede: Ag nanodelci; vrste iz rodu Thymus; Za- taria multiflora; kalus; karvakrol; timol Okrajšave: 2,4-D: 2, 4-diklorfenoksi ocetna kislina; Kin: kinetin; PGRs: rastlinski rastni regulatorji; HPLC: visokotlačna tekočinska kromatografija; NPs: nanodelci Acta agriculturae Slovenica, 118/3 – 20222 N. MOSAVAT et al. 1 INTRODUCTION Nanobiotechnology has enormous applications in diverse fields including agriculture, cosmetics, pharma- ceutics, and food industry) Kim et al., 2017; Rastogi et al., 2017). Nanoparticles (NPs) are employed as elicitors of various cell signaling pathways in metabolism of plants (Kim et al., 2017; Marslin, et al., 2017). NPs have been found to play crucial role in enhancement of plant sec- ondary metabolites (SMs) by imposing oxidative stress and increasing cell membrane permeability (Jasim et al., 2017; Marslin et al., 2017; Ahmad et al., 2020). The effects of NPs on growth rate of plants (Sharma et al., 2012), ger- mination of seeds (Zaka et al., 2016), production of SMs (Marslin et al., 2017; Mosavat et al., 2019; Zaka et al., 2016; Golkar et al., 2021) and plant physiology (Jasim et al., 2017; Sharma et al., 2012) have been studied recently. Although there are few studies related to effect of NPs on callus culture development, physiology and second- ary metabolism (Dykman & Shchyogolev, 2017; Kokina et al., 2013; Marslin et al., 2017; Sanzari et al., 2019; Zu- verza-Mena et al., 2017), still this research domain needs to be explored further. Silver (Ag) NPs possess unique properties in terms of toxicity and alteration of yield, de- velopment, antioxidant activities, and SMs production of plants due to their high catalysis and reactivity (Rastogi et al., 2017; Sadak 2019). Furthermore, the influence of Ag NPs on the callus cultures of Solanum nigrum L. (Ewais et al., 2015), Prunella vulgaris L. (Fazal et al., 2019) and Caralluma tuberculata N.E. Brown (Ali et al., 2019) has recently been studied. Thymus L., belonging to Lamiaceae family, has world-wide distribution (Sajed et al., 2013). However, it is dominantly present in Asia, Europe and North Africa (Zarshenas & Krenn, 2015). The essential oils and SMs enhance the commercial value of flowers and leaves of Thymus making it a valuable crop in cosmetics, pharma- ceutics, and food and agriculture industry (Miraj & Ki- ani, 2016). A thyme-like plant, Zataria multiflora Boiss., belonging to Lamiaceae family, is wild plant found in only southern and central Pakistan, Afghanistan, and Iran (Sajed et al., 2013). Tissue culture propagation of thyme plant is well-known because it is a wide source of ingredi- ents of pharmacology. Both Thymus sp. and Z. multiflora possess anti-cancerous, anti-inflammatory, anti-oxidant, anti-bacterial, anti-fungal, and anti-spasmodic proper- ties (Mathela et al., 2010; Sajed et al., 2013). Naturally occurring terpenoid thymol (2-isopropyl-5-methyl phe- nol) and its phenol isomer, carvacrol/cymophenol are the phenolic compounds that play an important role in inducing these properties to Thymus sp. and Z. multiflora (Kianersi et al., 2021; Mathela et al., 2010). Some other properties like their use as additive in perfumes, deodor- ant, toothpastes, soaps, etc. and as important flavoring agent in foods are also attributed to these compounds (Sajed et al., 2013). The defense system of plants is activated by chemi- cal, biological or physical elicitors (Asadollahei et al., 2022; Zhao et al., 2005). The gene expression is then modulated that transcribes the formation of SMs (Ajun- gla et al., 2009). A more efficient method could be the use of callus cultures for extracting SMs naturally from Thymus sp. and Z. multiflora in a sustained manner (Ramakrishna & Ravishankar, 2011). Previously, there is no report concerning the carvacrol and thymol produc- tion by imposing Ag NPs in cell cultures of Thymus sp. and Z. multiflora. Hence, the current study investigates proliferation of callus and SMs production in three Thy- mus species, i.e., T. vulgaris L., T. daenensis Čelak, and T. kotschyanus Boiss. & Hohen as well as Z. multiflora Boiss. after Ag NPs exposure. 2 MATERIALS AND METHODS The seeds of Thymus species, i.e., T. vulgaris, T. dae- nensis, T. Kotschyanus and Zataria multiflora (two acces- sions) (Table 1) were deposited at Botanic Herbarium of Research Institute for Biotechnology and Bioengineer- ing, Isfahan University of Technology (IUT), Isfahan, Iran, after collection from different geographical regions and identification by using Flora Iranica (Rechinger, 1982). Their characteristics is shown in Table 1. The seeds of four different species were surface sterilized with 70 % (v/v) ethanol for 1 min, followed by the addition of 3 % (v/v) sodium hypochlorite for 20 min, and then rinsing in sterile distilled water thrice. After surface steriliza- tion, the seeds were grown in Murashige and Skoog (MS) (1962) medium (Duchefa, Netherland). These were incu- bated for germination and growth of plantlets. The leaflet explants from about 1-month old plantlets were cultured in MS medium containing 2,4-D (2 mg l-1) and Kin (1 mg l-1) supplemented with 3  % (w/v) sucrose (Sigma- Aldrich, USA), 0.8 % (w/v) agar (Sigma-Aldrich, USA) and 0.1 mg l-1 myoinositol for callus induction. The pH was adjusted at 5.7. The samples were exposed to 16h/8h (light/dark) photoperiod for a period of 2 months at 23 ± 2 ℃. Silver (Ag) nano-powder was purchased from US Research Nanomaterials Inc., Houston, TX, USA hav- ing an average size of 30–50 nm and purity of 99.99 %. The nanoparticles were characterized by x-ray diffraction (XRD) and scanning electron microscopy (SEM) tech- niques by following the protocols of Javed et al. ( 2016). XRD was performed using Carlo ERBA Model EA 1108 analyzer and the instrument for getting SEM image was Acta agriculturae Slovenica, 118/3 – 2022 3 Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. Hitachi S4800 (Japan). These NPs were added to MS me- dium after filter sterilization. The 2-months old friable callus (0.25 g) was transferred to MS containing 2,4-D (2 mg l-1) and Kin (1 mg l-1) under Ag NPs stress of 4 and 8 mg l-1. This callus material was placed at 23 ± 2 ℃ under a photoperiod of 16h/8h. After incubation for 21 days of callus with Ag NPs, the callus growth rate (CGR) and callus fresh mass (CFM) was calculated. CGR was measured according to Afshar et al. (2016) every 7 days in 21 days period. Later on, the quantity of carvacrol and thymol was obtained by high performance liquid chromatography (HPLC). The method of Castro et al. (2016) was utilized for preparing callus extracts. The process involved dry- ing of 200 mg of callus from each treatment in an oven at 50 ℃ for 24 h, and then soaking it in 5 ml of diethyl ether for a period of 24 h. In order to prevent the evapo- ration of diethyl ether, the vials were kept closed and ex- traction was performed in a cold room. After adding the 80 % of methanol (1 ml) to remaining solid material, the extracts were filtered (0.22 μm pore size) into clean vi- als and prepared for injection to HPLC instrument. The HPLC (SY-8100 series, Beijing Beifan-Ruili Analytical Instrument, China) was performed by UV-VIS detec- tor, a flow rate of 0.9 ml min-1, injection volume of 20 μl at 28 °C, C18 column (25 cm × 4.6 mm, partial size 5 μm), mobile phase methanol-water (80:20; v/v), and flow rate of 0.9 ml min-1. The detection was carried out at 280 nm of wavelength and a pressure of 12 atm. The UV spectra of phenolic compounds were recorded at 280 and 320 nm. The content of carvacrol and thymol were determined based on the calibration curve of standard compounds, including carvacrol (Sigma-Aldrich, USA) and thymol (Sigma-Aldrich, USA). For this purpose, 4 concentrations (10, 25, 50, and 100 mg l-1) of carvacrol and 3 concentrations (25, 100, and 400 mg l-1) of thymol were examined by HPLC. The retention time for car- vacrol and thymol were appeared at 3-4 min and 14-15 min, respectively. After calibration of the standards with HPLC, the quantities of carvacrol and thymol in different samples were calculated. 2.1 STATISTICAL ANALYSIS The experimentation was conducted with three rep- lications in completely randomized design and the statis- tics was determined using two-way analysis of variance (ANOVA). LSD test (p ≤ 0.05) in SAS software (SAS 9.1 Inc. USA) was applied to determine significant difference among the treatments. 3 RESULTS AND DISCUSSION Callus growth and development as well as forma- tion of secondary metabolic compounds is positively or negatively influenced by supplementing the growth medium with abiotic or biotic stress elicitors (Ajungla et al., 2009; Zaka et al., 2016). The contents of SMs show significant changes under elicitation of callus by different stresses (Fazal et al., 2016; Mosavat et al., 2019; Sanzari et al., 2019). NPs, specifically the metallic oxide NPs like ZnO, CuO, TiO2 act as oxidative abiotic stress elicitors (Lala 2021). According to Al-jibouri et al. (2012), thymol amount was increased by proline in Origanum vulgare L. Similarly, the production of significantly the highest con- tent of hyperforin in Hypericum perforatum L. (Sharafi et al., 2013), rebaudioside A and stevioside in Stevia rebau- diana Bertoni (Javed, et al., 2018), and proline in Triti- cum aestivum L. (Barbasz, et al., 2016) under ZnO NPs elicitation has been previously documented. Ag NPs show significant effect on the production of SMs in callus culture, resulting in their ultimate increase (Fazal et al., 2016). For instance, Ali et al. (2019) reported that various concentrations of Ag NPs significantly af- fected the callus proliferation and substantially increased the callus biomass and SMs in Caralluma tuberculata. In another study, Fazal et al. (2019) reported the positive ef- fects of Ag NPs and Au NPs on the production of bio- mass and SMs in the cell culture of Prunella vulgaris L. XRD of Ag NPs is given in Figure 1 which shows 100  % phase purity by the sharpness of peaks. Similar XRD pattern was obtained by Kim et al. (2006). The Species Abbreviation Origin Genotype code Latitude (m) Longitude (m) Altitude (m) Zataria multiflora (1) Zm (1) Dehbala, Yazd, Iran RIBB/ZM01/2016 31°59’ N 54°11’ E 2600 Zataria multiflora (2) Zm (2) Abadeh, Fars, Iran RIBB/ZM02/2016 31°45’ N 51°21’ E 2030 Thymus vulgaris Tv Marvdasht, Fars, Iran RIBB/TV01/2016 35°56’ N 52°10’ E 1620 Thymus daenensis Td Aligoodarz, Lorestan, Iran RIBB/TD01/2016 33°24’ E 49°41’ E 2022 Thymus kotschyanus Tk Lahijan, Gilan, Iran RIBB/TK01/2016 37°12’ N 50°14’ E 396 Table 1: The geographical origins of Thymus sp. and Z. multiflora with their geographical traits collected from Iran Acta agriculturae Slovenica, 118/3 – 20224 N. MOSAVAT et al. spherical shape of Ag NPs was illustrated by SEM image given in Figure 2 which is coinciding with the results of Elumalai et al. (2010). The size, shape, surface, concentration and chemi- cal composition of NPs cause stimulatory or inhibitory effects on the growth of callus cells (Al-Jibouri et al., 2012). The synthesis and accumulation of SMs in cells is enhanced by an increased surface area of NPs as a result of reduced size and transport of NPs in to the cells apo- plastically which increases electrostatic interactions be- tween living cell membranes (Javed et al., 2017). Table 2 and Figure 3 shows the effect of Ag NPs on callus growth of Thymus sp. and Zataria multiflora. Compact calli with white and greenish colour were obtained after 10 days upon control culture (no AgNPs), whereas friable watery calli with white, greenish or yel- lowish colour were observed after 10-13 days upon cul- ture supplemented with AgNPs (Figure 4.) A significant effect is produced on callus traits by different concentrations of Ag NPs in this study that is coherent with the reports about effects of Ag NPs on Figure 1: X-ray diffractogram (XRD) of Ag nanoparticles Figure 2: Scanning electron micrograph (SEM) of Ag nano- particles Nanoparticle Concentration (mg l-1) Color and Texture CGRІ (mm day-1) CFM II (g)7 14 21 Mean Ag 8 Green, friable 0.045 0.06 0.015 0.022 a 0.99 a Ag 4 Green, friable 0.07 0.06 0.032 0.015 b 0.85 ab Control - White to yellow, soft 0.02 0.01 0.006 0.012 c 0.61 b Table 2: Effect of different concentrations of Ag NPs on color, texture, growth rate, and fresh mass of callus cultures of Thymus sp. and Zataria multiflora Mean values followed by the same letter in each column are not significantly different at p<0.05 (Least Significant Difference Test). І: CGR: Callus growth rate, II: CFM: Callus fresh mass Fig 3: Effect of Ag nanoparticles on callus growth rate (A) and callus fresh mass (B) content of different Thymus species and Z. multiflora under callus culture Acta agriculturae Slovenica, 118/3 – 2022 5 Influence of Ag nanoparticles on physiological and biochemical aspects of callus of Thymus species and Zataria multiflora Boiss. callus culture of Solanum nigrum L. (Ewais et al., 2015) which gain friable watery calli with greenish or yellow- ish colour were observed after 10-13 days upon culture supplemented with AgNPs and effects of TiO2 NPs on the callus of Hordeum vulgare L. (Mandeh et al., 2012). The higher TiO2 NPs concentration influenced callogen- esis of Hordeum vulgare explants in this study. Also ac- cording to Kokina et al. (2013) elicitation with Ag and Au NPs shows positive effects on callus width and length in Linum usitatissimum L. The positive effects of different NPs on callus growth of Prunella vulgaris L. is reported by Fazal et al. (2019). Callus growth traits affected by ZnO NPs in Solanum lycopersicum Mill. have also been reported (Alharby et al., 2016). The effects of elicitation by Ag NPs on production of carvacrol and thymol under in vitro conditions are presented in Figure 5. The production of thymol and car- vacrol was determined at 8 mg l-1 concentration of Ag NPs and control treatment. The carvacrol (0.68 mg l-1) and thymol (11.09 mg l-1) quantity was enhanced under 8 mg l-1 of Ag NPs. The chromatographic separation of the methanolic extracts in Zataria multiflora for carvacrol and thymol by HPLC is given (Figure 6). Asadollahei et al. (2022) employed different concentrations of CuNPs in in vitro culture medium and observed significant rise in thymol and carvacrol content compared to control in Za- taria multiflora. This study elucidated that the selection of appropriate plant species and suitable elicitor is crucial for increasing the production of bioactive compounds as well as antioxidants of Zataria multiflora. This can be done by inducing expression changes in the biosynthetic pathways of thymol and carvacrol. In fact, the gene ex- pression patterns of the pathways of formation of thymol and carvacrol were greatly influenced by the Ag NPs in our study which is the phenomenon well explained by the studies of Kianersi et al. (2021). The interactive effects of NPs and genotypes/species of Thymus and Zataria multiflora for production of SMs Fig 4: Friable calli with greenish or yellowish colour of Thymus species and Z. multiflora after culture treatment with AgNPs (a) and under control culture (b) Fig 5: Effect of 8 mg l-1 Ag NPs on in vitro production of carvacrol and thymol in Thymus sp. and Zataria multiflora Fig 6: Representative HPLC chromatograms of thymol and carcacrol of Z. multiflora. Peak identifications were performed by matching retention time and UV spectra against commer- cially available reference compounds Acta agriculturae Slovenica, 118/3 – 20226 N. MOSAVAT et al. have been presented in Table 3. The exposure of Ag NPs has significantly increased the content of two SMs of cal- lus compared to control. Moreover, in vitro synthesis of thymol was notably greater than that of carvacrol which is also evident from the molecular studies of Kianersi et al. (2021). The highest content of carvacrol (1.06 mg l-1) was observed at 8 mg l-1 concentration of Ag NPs in T. kotschyanus, whereas the least amount (0.10 mg l-1) was observed in control treatment of T. daenensis. Further- more, the highest concentration of thymol was obtained at 8 mg l-1 of Ag NPs in callus of T. daenesis (19.75 mg l-1), while the least thymol content (3.95 mg l-1) was achieved in T. daenesis under control condition. This result can be well supported by the phenom- enon that NPs trigger thymol synthetic pathways and/or transcription factors more than the carvacrol pathways (Mosavat et al., 2019). Taking into account of concur- rent studies, the phenolics and flavonoids production is activated by ZnO NPs in seedlings of Brassica nigra L. (Zafar et al., 2016) Additionally, the significant rise in hy- perforin content in cell suspension culture of Hypericum perfolatum L. under ZnO NPs stress is reported (Sharafi et al., 2013). A complex variety of elicitation effects on in vitro synthesis of SMs is obtained using different types of elicitors (Goswami et al., 2017; Marslin et al., 2017; Syu et al., 2014), plant tissues (Ajungla et al., 2009), and physi- ochemical environment of various species (Shakya et al., 2019). 4 CONCLUSION The formation of callus from Thymus species and Zataria multiflora was performed in the presence of Ag NPs elicitors. Addition of abiotic elicitors, i.e., Ag NPs (8 mg l-1) to the MS medium played a vital role in enhanc- ing the thymol and carvacrol content in the callus cul- tures of different Thymus species and Zataria multiflora. In other words, Ag nano-elicitors applied to the in vitro callus cultures of Thymus species and Zataria multiflora in our study resulted in increase in SMs production at a concentration of 8 mg l-1. Our finding opens the way for studies involving relationship between chemical elicitors and formation & accumulation of thymol/carvacrol. 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Plant Physiology and Biochemistry, 110, 236–264. https://doi.org/10.1016/j. plaphy.2016.05.037 Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2279 Original research article / izvirni znanstveni članek Field resistance phenotyping of durum wheat to fusarium head blight in Algeria Salah HADJOUT 1, 2, 3, Zouaoui BOUZNAD 2, Leila MEKLICHE 2, Mohamed ZOUIDI 1 Received July 21, 2021; accepted September 06, 2022. Delo je prispelo 21. julija 2021, sprejeto 6. septembra 2022 1 Centre de Recherche en Aménagement du Territoire (CRAT), Campus Universitaire Zouaghi Slimane, Constantine, Algérie 2 Ecole Nationale Supérieure Agronomique (ENSA), El-Harrach, Algérie 3 Corresponding author, e-mail: hadjout.salah@gmail.com Field resistance phenotyping of durum wheat to fusarium head blight in Algeria Abstract: In Algeria, several research studies point to the importance of the causative agents of fusarium head blight. Indeed, our research aims to study the phenotyping of the re- sistance of some durum wheat genotypes for their behavior to fusarium head blight, caused by four isolates of Fusarium culmorum (Wm.G.Sm.) Sacc.. For this purpose, the disease assessment is carried out in the field. The different evaluation criteria are: incubation period, measurement of the mass of a thousand grains and AUDPC (Area Under the Disease Progres- sion Curve). The results obtained revealed that the varieties and lines resulting from crosses had a quite different level of suscep- tibility with regard to the four isolates studied and no genotype showed complete resistance (immunity) under our growing conditions. Among the tested material, the lines showed higher resistance than their parents. The reasons for this phenomenon is that crosses between genotypes implicated cultivars from Europe and Western Asia (Syria), where wheat domestication has occurred very early (between 12 000 and 10 000 years BP), which may be promising sources of resistance to fusarium head blight. The results also show a slight variability in behavior, also linked to the aggressiveness of the Fusarium species studied in this work. Key words: durum wheat; phenotyping; fusarium head blight; resistance; susceptibility; aggressiveness Ugotavljanje odpornosti trde pšenice na fuzariozo klasov na prostem v Alžiriji Izvleček: Številne raziskave so poudarile pomen fuzarioz pšeničnih klasov v Alžiriji. Namen te raziskave je bil ugotoviti fenotipsko odpornost nekaterih genotipov trde pšenice na fu- zarioze, ki jih povzročajo štiri izolati glive Fusarium culmorum (Wm.G.Sm.) Sacc.. Za oceno bolezni je bil izveden poljski po- skus. Za oceno okužbe so bili uporabljeni naslednji kriteriji: in- kubacijsko obdobje, meritev mase tisočih zrn in AUDPC (Ob- močje pod naraščajočo krivuljo bolezni). Rezultati so pokazali, da so imele sorte in linije, ki so nastale s križanji zelo različno občutljivost na štiri v raziskavi uporabljene isolate glive, ven- dar ni imel noben genotip popolne odpornosti (imunosti) v razmerah potekanja poskusa. Med testiranimi vzorci pšenice so imele linije večjo odpornost kot njihovi starši. Razlog za ta fenomen je ta, da so bila križanja med genotipi sort iz Evrope in Zahodne Azije (Sirija), kjer je bila trda pšenica udomačena že zelo zgodaj (med 12 000 in 10 000 let pred sedanjostjo, t.j od začetka datiranja starosti na osnovi radioaktivnega ogljika), kar bi lahko bil obetajoč vir odpornosti na fuzarioze klasov. Izsledki so pokazali tudi manjšo variabilnost v odzivnosti med genotipi analizirane pšenice, kar je lahko povezano z različno ogresiv- nostjo v raziskavi uporabljenih sevov glive iz rodu Fusarium. Ključne besede: trda pšenica; fenotipsko določanje; fuza- rioza klasov; odpornost; občutljivost; agresivnost Acta agriculturae Slovenica, 118/3 – 20222 S. HADJOUT et al. 1 INTRODUCTION Durum wheat (Triticum durum Desf.) is one of the oldest and the most important cultivated cereal species in the world (Royo et al., 2009; Tidiane et al., 2019; Boua- naka et al., 2021). It is of great importance in the cereal- growing areas of the Mediterranean basin and North America, where most of the world production of this crop is concentrated (USDA, 2005; Xynias et al., 2020). However, durum wheat is no longer just a staple crop for food security, but has also become a major cash crop. Africa as a whole spends more than 4 billion euros per year for import of durum wheat to provide the raw mate- rial for its food industry (Tidiane et al., 2019). In Algeria, wheat consumption (both durum wheat and soft wheat) is far greater than its real production capacity. Conse- quently, the domestic market has been dependent on a significant level of imports in recent years. In addition, yields are quite low for locally grown wheat and should be improved (Touati-Hattab et al., 2016). Various reasons are at the origin of this situation such as precipitation and biotic (pests and diseases) and abiotic stresses (drought, sunshine, cold and salinity) (Xynias et al., 2020). Among the biotic constraints to wheat production, fusarium head blight (FHB) (Ghimire et al., 2020). Fusarium head blight, reported by several species of the genus Fusarium (Bouanaka et al., 2020; Saharan, 2020), is one of the most destructive diseases of wheat (Dweba et al., 2017; Wachowska et al., 2020), particularly affecting durum wheat (Moreno-Amores et al., 2020) and thus leading to significant reductions in yield and quality throughout the world (Touati-Hattab et al., 2016; Dweba et al., 2017; Saharan, 2020). In addition, FHB poses additional food and animal safety concerns due to the contamination of grains with mycotoxins (Ghimire et al., 2020). Among the most important species associated with the disease worldwide is Fusarium culmorum. The cereal pathogen Fusarium culmorum (Wm.G.Sm.) Sacc. is a ubiquitous soil fungus (ascomy- cete) (Bilska et al., 2018), considered a chronic fungus of economic interest worldwide, including in African countries from the North like Algeria. This pathogen produces a wide range of mycotoxins, including the trichothecene-B type deoxynivalenol (DON) (Yekkour et al., 2015), which constitutes a potential health hazard (Bilska et al., 2018). Previous studies carried out in Alge- ria have shown that Fusarium culmorum appears to be the major pathogen associated with fusarium head blight (Yekkour et al., 2015; Touati-Hattab et al., 2016; Laraba et al., 2017). The use of various methods to limit the development of Fusarium cereal ear diseases and their contamination with mycotoxins, before and after harvest, is an impor- tant part of sustainable agriculture and the production of healthy foods (Mielniczuk & Skwaryło-Bednarz, 2020). Genetic resistance is the most effective and sustainable approach to manage diseases in wheat (Ghimire et al., 2020), in particular, reducing the problem of mycotoxins in farmers’ fields affected by fusarium head blight (Saha- ran, 2020). Today, FHB phenotyping performed by breeders is performed by visual examination (Serre et al., 2015). In this context, the main objective of our present study is to compare the phenotypic resistance to fusarium wilt of two new durum wheat lines of Algerian origin selected against those of three parental commercial varieties. 2 MATERIALS AND METHODS 2.1 VEGETAL MATERIAL In our experience, five durum wheat genotypes were chosen. To this end, two pedigree lines (G1 and G4) selected in Algeria and three commercialized varieties (G9, G10 and G11) were tested in the field. These lines are composed of F15 seeds resulting from simple crosses between 4 parental varieties: Saadi, Siméto, Ardente and Waha (Mekliche et al., 2013). The main characteristics of these genotypes are shown in Table 1. The aim is to com- pare their levels of resistance. 2.2 FUNGAL MATERIAL During our study, four isolates of Fusarium culmo- rum (F.C.T5, F.C.T7, F.C10.11 and F.C1.12) were used (Table 2). These isolates were obtained from the ears and crowns of the ’Vitron’ variety of durum wheat, show- ing typical symptoms of the disease. The ears and col- lars were harvested in the area of Oued Semar (Algeria) in northern Algeria. The preliminary identification was made on the basis of the conidial morphology accord- ing to Leslie and Summerell (2006) then confirmed by Codes Genotypes Origin Precocity G1 Saadi × Waha ENSA, Algeria Early G4 Ardente × Siméto ENSA, Algeria Early G9 Siméto Italy Semi- Early G10 Ardente France Early to very early G11 Waha ICARDA, Syria Early Table 1: F15 pedigree lines and parental varieties used during the experiment Acta agriculturae Slovenica, 118/3 – 2022 3 Field resistance phenotyping of durum wheat to fusarium head blight in Algeria molecular tools thus using classical PCR (Touati-Hattab et al., 2016; Hadjout et al., 2022). 2.3 INOCULUM PREPARATION Fusarium isolates are cultured in Petri dishes con- taining PDA medium. They are then incubated in the dark and at a temperature of 25 °C until sporulation. Af- ter 20 days of incubation, a layer of sterile distilled water of 1 to 2 mm is placed on the colony contained in each Petri dish and then poured into a container. After count- ing in the Malassez cell, the inoculum is prepared from a suspension of conidia in water, adjusted to 5.104 spores per milliliter, prepared extemporaneously (Hadjout et al., 2017). 2.4 ARTIFICIAL FIELD INOCULATION METHOD Inoculation in the field was done by spraying the ears of each genotype until the inoculum begins to run- off, approximately 200 ml m-2. The inoculations were car- ried out at the flowering stage corresponding to a mini- mum of 10 % of the ears from which the stamens have emerged. The controls consist of plots where no artificial inoculation was carried out. In the field, the inoculations were carried out in the evening, after sprinkling irriga- tion for about 20 min before inoculation and then 10 min after, in order to maintain sufficient humidity on the plants during the night, but also to promote adhesion and the germination of conidia. Depending on climatic conditions, the plots are then irrigated regularly in the evening. 2.5 FIELD EXPERIMENTAL SET-UP The experiment was carried out in the field, with the installation of five tests: a control test and four tests inoculated with the four isolates of Fusarium culmorum mentioned above. The experimental set-up was of the complete random block type, with three repetitions (Fig. 1). The spacing between the blocks was 1 m. The area of each microplot was 1 m2, consisting of 5 lines of 1 meter (linear meter) 20 cm apart. The distance between each microplot was 50 cm. Lines of triticale were sown be- tween trials to avoid cross-contamination. 2.6 FIELD DISEASE ASSESSMENT 2.6.1 Incubation period The incubation period corresponds to the period between artificial inoculation and the appearance of a fusarium blighted spikelet in the plot. 2.6.2 Symptom scoring In the case of our study, disease severity scoring was performed 21, 26 and 31 days after inoculation. The observation unit consisted of 25 ears selected at random from each microplot. On these spikes, the total number of spikelets per spike and the number of Fusarium colo- nized spikelets were counted. The proportion of spikelets showing symptoms is assessed using a logarithmic rat- ing scale described by Michel (2001), ranging from 0 (no symptoms) to 9 (completely dead ear, generalized drying out). 2.6.3 Calculation of the area under the disease pro- gression curve (AUDPC) The AUDPC is calculated on the number of fused spikelets for all scoring dates according to the formula described by Shaner and Finney (1977): Where: n: total number of observations; xi: number of fusarium infected spikelets in 25 heads at each obser- vation; (ti - ti-1): time separating two consecutive observa- tions. Code Species Origin Isolation organ Variety F.C.T5 F. culmorum Oued Smar Ear Durum wheat (‘Vitron’) F.C.T7 F. culmorum Oued Smar Ear Durum wheat (‘Vitron’) F.C10.11 F. culmorum Oued Smar Ear Durum wheat (‘Vitron’) F.C1.12 F. culmorum Oued Smar Collar Durum wheat (‘Vitron’) Table 2: Fusarium culmorum isolates used in the study Acta agriculturae Slovenica, 118/3 – 20224 S. HADJOUT et al. 2.6.4 Evaluation of the Thousand Grains Mass (TGM) at harvest TGM was measured to assess the impact of the dis- ease on yield, all 25 heads were threshed using a thresh- ing machine with poor ventilation. 2.7 STATISTICAL ANALYSIS OF DATA The statistical analysis of the results in the field is carried out using statgraphics software version 15.1.0. Next, a multiple comparison of the means was performed using the ppds (least significant difference) test to deter- mine the groups homogeneous at the 5  % significance level. Fig. 1: Diagram of the open-field experimental set-up for each of the five trials Acta agriculturae Slovenica, 118/3 – 2022 5 Field resistance phenotyping of durum wheat to fusarium head blight in Algeria 3 RESULTS AND DISCUSSION 3.1 MANIFESTATION OF THE DISEASE Fusarium head blight of wheat was observed in the field. In fact, the inoculated plots showed symptoms of the disease, the attacks of which on wheat ears by this disease most often result in the scalding of certain groups of spikelets, part or all of the ear. Symptoms are mani- fested by the presence of one or more discolored spikelets on the green spikes (Fig. 2. a, b, c, d). The ripe kernels harvested were scalded, light, chalky white or sometimes pink (this is referred to as mummified or damaged ker- nels, fusarious kernels) (Fig. 2.e). It should be noted that the amount of symptoms depends on the stage of the plant at the time of inoculation; the peak of sensitivity Fig. 2: Characteristic symptoms of fusarium head blight in durum wheat (personal photos) a, b and c: fusarious ears, the orange tint denotes the presence of the pathogenic fungus d: Hard wheat field almost completely fused; e: fusarium grains Acta agriculturae Slovenica, 118/3 – 20226 S. HADJOUT et al. corresponds to the flowering of the varieties. Burrows et al. (2008) report that the initial infection is characterized as a discolored lesion at the base of the glume and the rachis which then spreads in both directions of the ear. Previous data were obtained using a spray inoculation method, frequently used to screen for resistance to fusar- ium head blight in wheat (Prat et al. 2014). According to Miedaner et al. (2003), spray inoculation, compared to single flower inoculation, is more adequate to reproduce the natural conditions of infection. According to Touati-Hattab et al. (2016) and Laraba et al. (2017), F. culmorum is the main fungal pathogen associated with fusarium head blight in Algeria. In addi- tion, F. culmorum, the causative agent of various diseases of the ear and crown of cereals, is considered a chronic fungus of economic concern worldwide, including North African countries such as Algeria. (Yekkour et al., 2015). 3.2 SSESSMENT OF GENOTYPE BEHAVIOUR BY INCUBATION PERIOD The results obtained show that the appearance of the first symptoms on the ears estimated by the incubation time varies according to the genotype / isolate interaction (Fig. 3). Analysis of variance for all four trials revealed a significant difference for genotypes (p < 0.001) and for treatments (isolates) (p < 0.001); on the other hand, the interaction (genotypes / treatments) has no significant effect (p > 0.05) (Fig. 3.a, b). In our trials, we were able to characterize the behavior of genotypes with respect to the incubation period. This criterion allowed us to observe that the G1 line ranked well compared to other genotypes, due to the long incubation period recorded, 18 days after contamination. This reflects a good level of type I resistance for this line, linked to a cellular mecha- nism that slows the expression of the first symptom and therefore the onset of the disease. In contrast, varieties G9 and G11 recorded a shorter incubation period, ap- proximately 10 and 11 days respectively after contami- nation. They are considered to be the sensitive controls chosen during our experiments; G4 and G10 genotypes had a very comparable average incubation period, an av- erage of 13 days after contamination. It should be not- ed that the resistant behavior of G1 line expressed by a longer incubation period is in agreement with the work of Trottet and Saur (1994) who also used this parameter. From these results, we can say that the period of onset of symptom onset is directly related to the level of resist- ance of the genotypes, but also to the aggressiveness of the isolates. The mechanisms of resistance in plants to fusarium wilt are very complex (Mesterhazy et al., 1999). It is generally accepted that resistance to fusarium wilt is controlled by a polygenic system, which is known to slow the development of individual infections, the spread of the disease in fields, and the rate of spread of the fungus in adjacent plant tissues (Qi et al., 1999; Lindhout, 2002). 3.3 EVALUATION OF GENOTYPE BEHAVIOUR BY AUDPC VALUE OF THE NUMBER OF FUSARI- UM INFECTED SPIKELETS AUDCP analysis of variance for the number of Fusarium infected spikelets showed a significant differ- ence for genotypes (p < 0.001) (Fig. 4.a) and trials (p < 0.001) (Fig. 4. b). On the other hand, the interaction be- tween genotypes and trials is not significant (p > 0.05). Our results show that the AUDPC of the number of Fusarium infected spikelets for the G1 line is very low (9.75), while the two susceptible varieties (G9 and G11) Fig. 3: Behavior of genotypes towards isolates according to incubation time Acta agriculturae Slovenica, 118/3 – 2022 7 Field resistance phenotyping of durum wheat to fusarium head blight in Algeria recorded very high AUDPC values (67.57 and 63.49); the G4 and G10 genotypes marked AUDPCs intermediate between the resistant and the susceptible, namely 28.23 and 28.64 respectively. It is therefore clearly established that the G1 line behaves resistant to the progression of symptoms after inoculation. This variability in the behav- ior of durum wheat genotypes is most likely the result of the presence or absence of genes for resistance to this pathogen, but also the presence or absence of virulence or non-virulence genes in the pathogen. On the patho- genic side, the four isolates show different aggressiveness indicating that they differ in their pathogenicity. In many studies, the assessment of the severity of the disease in the field is essentially based on the calculation of the val- ues of the AUDPC (Hadjout, 2013, Hadjout et al., 2017). 3.4 EFFECT OF DIFFERENT FUSARIUM ISOLATES AND SPECIES ON THOUSAND GRAIN MASS (TGM) Analysis of variance integrating all trials showed the effects of genotypes and trials to be statistically signifi- cant (p < 0.001), while the genotypes / trials interaction showed a non-significant effect (p > 0.05) (Fig. 5 a, b). Analysis of the losses of the main component of yield showed that the different isolates affect all genotypes by decreasing TGW. According to our results, it is the sus- ceptible varieties G9 and G11 which recorded the great- est losses in TGM (44.37 g and 45.30 g) followed just af- ter by the moderately resistant variety G10 (47.41 g). The treatments affected the G1 (resistant) and G4 (moderate- ly resistant) lines with relatively very low losses, namely 48.68 g and 54.82 g respectively, this reflects their good level of resistance, probably those of type II linked to the progression of the pathogen in the ear. The fact remains that the G10 variety showed more losses (47.41 g) than the two lines, something which was observed in previ- ous work by Hadjout (2013). In addition, the symptoms observed explained part of the losses in TGM, this is in agreement with current knowledge on the epidemiology of fusarium head blight. The fact that the pathogen devel- ops after the flowering stage, at the onset of the disease, the number of kernels per ear is already fixed, while the kernel filling has only just begun. The disease therefore affects this parameter and results in a large drop in TGM, especially in susceptible varieties (G9 and G11). The work of Gate et al. (1991) showed that a low TGM can be the result of end-of-life diseases (fusarium wilt), or late rains associated with high heat, and to a lesser extent with lodging. Fusarium head blight reduces grain yield and quality at the end of the crop’s growth cycle, when non-diseased wheat kernels normally develop into fleshy, healthy kernels (McMullen et al., 2012). 4 CONCLUSIONS The growing interest of the cereal sector for the sanitary quality of grains and particularly for mycotoxin contamination, strongly increases the demand for pro- ductive genotypes that accumulate few mycotoxins in their grains. In the absence of reliable information on the ability of genotypes to limit the accumulation of these Fig. 4: Average AUDPC values of the number of Fusarium infected spikelets Acta agriculturae Slovenica, 118/3 – 20228 S. HADJOUT et al. molecules, attention is focused on finding varieties with a good level of resistance to fusarium head blight. Indeed, the use of resistant genotypes linked to good agronomic practices remains the most satisfactory solution for farm- ers. Therefore, our study falls within the overall frame- work of the genetic control against fusarium head blight and this by the selection of genotypes resistant to the disease. To this end, the behavior of tested durum wheat genotypes with respect to fusarium head blight is evalu- ated under open field conditions. This behavior indicates that the G1 line exhibits longer incubation times, lower AUDPC values and thus exhibiting low disease yield losses compared to other genotypes and therefore it is of interest from a standpoint seen resistance to the appear- ance of the first symptoms and to the rate of spread of the fungus inside the ear. 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World Journal of Microbiology and Biotechnology, 31(6), 875-881. https://doi. org/10.1007/s11274-015-1841-2 Acta agriculturae Slovenica, 118/3, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.3.2599 Original research article / izvirni znanstveni članek Flight activity of Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae) infesting two Algerian olive varieties in north-west Algeria Zineb BOURAKNA 1, 2, Kada RIGHI 1, Fatiha ASSIA 1, Abdelkader ELOUISSI 1 Received March 08, 2022; accepted September 16, 2022. Delo je prispelo 8. marca 2022, sprejeto 16. september 2022 1 Biology Systems and Geomatics Laboratory, Department of Agronomy, Faculty of Natural Sciences and Life, Mascara University, Algeria 2 Corresponding author, e–mail: Zineb.bourakna@univ-mascara.dz Flight activity of Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae) infesting two Algerian olive varieties in north- west Algeria Abstract: Bactrocera oleae (Rossi, 1790) (Diptera: Tephri- tidae) is the most dangerous insect pest of the olive tree in the Mediterranean region. This study was conducted in the Mas- cara region (North-West Algeria) during 2019-2020 season, in order to monitoring the flight activity of B. oleae by using Mc Phail type traps and evaluating the infestation rate on two ol- ive varieties (Sigoise and Chemlal) by fruits sampling. The data obtained indicated that the flight activity of B. oleae developed five peaks of the abundance. The General Linear Model (GLM) showed that infestation rate and fruit caliber varied consider- ably among varieties and across the sampling date, which grad- ually increased with time. ‘Sigoise’ having the highest caliber and was more infested than ‘Chemlal’. The northern cardinal orientation of the tree was the least attacked by this pest. The GLM function showed that there was relationship between the infestation rate and fruit size. Key words: Bactrocera oleae; flight activity; infestation; caliber; ‘Sigoise’; ‘Chemlal’ Let oljčne muhe (Bactrocera oleae (Rossi, 1790), Diptera: Tephritidae) na dveh alžirskih sortah oljke v severozahodni Alžiriji Izvleček: Oljčna muha (Bactrocera oleae (Rossi, 1790), Diptera: Tephritidae) je najškodljivejša žuželčja vrsta na oljkah v Sredozemlju. Raziskava je bila izvedena na območju Mascare (severozahodna Alžirija) v rastni dobi 2019-2020, z namenom načrtnega spremljanja leta oljčne muhe z uporabo Mc Phailo- vih pasti in ovrednotenja stopnje napada dveh sort oljke (‘Sigo- ise’ in ‘Chemlal’) z vzorčenjem plodov. Pridobljeni podatki na- kazujejo, da je imela oljčna muha pet vrhov pojavljanja. Splošni linearni model je pokazal, da sta se stopnja napada in debelina plodov znatno spreminjala glede na sorto in datum vzorčenja in sta s časom naraščali. Sorta Sigoise je imela najdebelejše plodove in je bila bolj napadena kot sorta Chemlal. Na sever orientirani deli krošenj so bili najmanj napadeni. Splošni line- arni model je pokazal, da obstaja povezava med stopnjo napada oljčne muhe in debelino plodov oljk. Ključne besede: Bactrocera oleae; aktivnost izletov; napa- di; debelina plodov; ‘Sigoise’; ‘Chemlal’ Acta agriculturae Slovenica, 118/3 – 20222 Z. BOURAKNA et al. 1 INTRODUCTION  Algeria is one of the main olive (Olea europaea L., Oleaceae) producing countries. In 2019 it took the ninth class in world olive production with a produc- tion of 868,754 tons on an area of 431,634 ha (FAO Stat, 2021). Algerian olive oil production was 90,000 tons in the 2020/2021 campaign. This crop is attacked by various pests and diseases. The olive fly Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae), is the most serious and eco- nomically harmful insect pest of commercial olive pro- duction worldwide (Ras et al., 2017; Torrini et al., 2020). This fly is multivoltine and homodynamic, i.e. their population dynamics, number of generations and the length of their life cycles depend mainly on the climate (temperature and humidity), but also vary according to other factors: geographic regions , availability and quality of olive fruits (Daane & Johnson, 2010; Malheiro et al., 2015; Pertíñez & Vélez, 2020). This pest causes the severe qualitative and quantitative damage, where economic losses can reach 100% due to uncontrolled infestation and oil losses of up to 80% (Rice, 2000; Genç & Nation, 2008; Zalom et al., 2009). Also, the formation of tunnels inside mesocarp and exit holes allowing the introduction of bacteria and fungi that rot the fruit and increase the acidity of the oil (Athar, 2005; Zalom et al., 2009). The infestation of olives caused by B. oleae varies greatly be- tween years, regions and olive varieties (Goncalves et al., 2012). Gaouar and Debouzi (1991) found that the level of infestation was quite high near 100% in orchards close to the coast, in the province of Tlemcen (North West Alge- ria) on two local varieties (Sigoise and Chemlal). Other authors have also shown a fruit infestation level of up to almost 100% in Portugal (Bento et al., 2009) and in Cali- fornia (Burrak et al., 2011). The preference and sensitivity of olive cultivars by the B. oleae vary by three factors: physical, chemical and molecular. The physical factor remains the most influ- encing, which includes size, mass, volume, fruit color and hardness of the exocarp (Malheiro et al., 2015). The female of B. oleae prefers to oviposite on cultivars with large, unripe olives (Neuenschwander et al., 1985). Sev- eral studies (Burrack & Zalom, 2008; Goncalves et al., 2012; Garantonakis et al., 2017; Medjkouh et al., 2018) have confirmed that the oviposition preference by the fe- male B. oleae was positively correlated with the maturity index, mass and volume on the other hand oviposition was negatively correlated with the hardness of the exo- carp. The olive varieties studied ‘Sigoise’ and ‘Chemlal’ are two Algerian varieties renowned for their excellence in quality and productivity. In order to preserve the quantity and the marketable quality of these two varie- ties against the attacks of such a pest, it was imperative to determine its population dynamics and its infestation rates in relation to the size of the fruits than with the four cardinal orientations of the tree in the region of Mascara (North-West Algeria). 2 MATERIAL AND METHODS 2.1 STUDY AREA This study was carried out in Oued Taghia region at an altitude of 471 m (35 ° 6 ‘35 “N, 0 ° 5’ 19” E) in the province of Mascara (North-West of Algeria), during the Figure 1: The geographical localization of study area (Mascara: Algeria) and the situation of study orchard Acta agriculturae Slovenica, 118/3 – 2022 3 Flight activity of Bactrocera oleae ... infesting two Algerian olive varieties in north-west Algeria period that extends from June 2019 to April 2020. The study region is characterized by a semi-arid, dry and cold climate, far from the sea by a distance of about 120 km (Figure 1). The olive orchard has been planted with two varieties, Sigoise (intended for the production of table ol- ives) and Chemlal (intended for the production of oil). The trees were medium in size, about 16 years old, and were spaced about 10 m × 8 m, being irrigated artificially by gravity and pruned bi-annually. The olive orchard has not received any treatment against diseases and pests for the past three years, but chemical fertilizers are applied every winter (Figure 2). 2.2 SAMPLING METHODOLOGY The flight activity of adults of B. oleae was monitored using 4 plastic Mc Phail traps with a transparent upper half and a yellow lower half, baited with a 3 % aqueous solution of di ammonium phosphate which is attractive to both sexes. The traps are installed at the beginning of June 2019. The solution has been renewed every 10 to 20 days. The traps were tied under the shade of the branches inside the foliar crown in the southwest direction of the tree, at a human height. The traps were distributed ran- domly in the olive orchard with 50 m distance between them. Which were checked every 10 days and the olive flies were counted, sexed and removed. The total number of individuals captured in the McPhail traps was used to estimate the population index (Pi) which was expressed as the total number of captures per trap per day in each date (Goncalves et al., 2012). Sex ratio was estimated by the ratio (male / total and female / total). Every 10 days, from the appearance of the first stings (beginning of September which corresponds to the slight drop in temperature and after the setting of the olives) until the harvest (end of December), fruit samples were taken from 5 trees of each variety of olive tree, to assess the infestation rate of the olive tree and the size of the fruit. 40 olives per tree were harvested at head height from 4 cardinal orientations of each tree (north, south, east, and west), due to 10 olives for each orienta- tion. The olives collected were brought to the laboratory and were observed under a binocular stereo-microscope (EUROMEX, The Netherlands) to check for the pres- ence of oviposition stings and exit holes insect. The B. oleae infestation rate was expressed as a percentage of the infested olives relative to the total number of olives collected. According to Burrack et al. (2011), olives with oviposition stings were considered infested. To estimate the caliber of the fruit, 50 olives were chosen by chance for each variety (10 olives per tree). Us- ing a digital caliper (OEM, China), the widest dimension was measured in mm. 2.3 DATA ANALYSIS The statistical software SPSS (version 21) was used to analysis the data on infestation rate and fruit caliber with General Linear Model (GLM): Repeated Measures with “variety” and “sampling date” as effects. ANCOVA was used to study the effect of cardinal orientation on infestation rate in both varieties. Tukey post-hoc test was applied to compare the infestation rate of different cardi- nal orientations. The GLM function in R environment (R Core Team, 2021) was used to build the relation between the infestation rate and the fruits caliber. The significance level for all analyses was 0.05. 3 RESULTS 3.1 POPULATION DYNAMICS OF THE OLIVE FLY AND ENVIRONMENTAL CONDITIONS Olive fly flight activity was distributed throughout the year (Figure 3). The dynamic of adult flights was showed five major peaks, which correspond to the num- ber of generations. The first flies in our study area were captured on 23/06/2019 with a Pi population index of 0.12 flies / trap /day. So that, the first peak appeared on 13/07/2019. From this date, the number of individuals de- creased and coincided with the increasing in temperature and the falling in humidity (summer period). In Septem- ber, the population returns to increase relatively with the decreasing in temperature and the increasing in humidi- ty, forming a succession of 3 autumn-winter generations; September 03 (0.70 flies / traps / day), November 03 (2.4 Figure 2: The study orchard (Oued Taghia, Mascara) Acta agriculturae Slovenica, 118/3 – 20224 Z. BOURAKNA et al. than that of ‘Chemlal’ (Figure 5). At the beginning of September, the infestation rate was low (11.50 ± 1.66 %, 7.00 ± 1.27 %) respectively for the two varieties Sigoise and Chemlal. As of October 03, the infestation increased for ‘Sigoise’ variety, while for ‘Chemlal’, the increase of the infestation was moderate. In December, the infesta- tion in ‘Chemlal’ was intensified and reached high value at the time of harvest (78 %), which is near to ‘Sigoise’ infestation rate (84 %) (Figure 5). Fruit calibers differed significantly among varieties, the sampling date and the interaction between the two factors (Table 1). The fruit caliber was higher in ‘Sigoise’ than ‘Chemlal’ throughout the study period. We noted a rapid increase of caliber in ‘Sigoise’ variety and a slight increase in ‘Chemlal’ variety (Figure 6). flies / traps / day), December 23 (0.70 flies / traps / day) respectively. The value of the population index decreased from the end of December to February, where the tem- perature in this period is ≤ 10 °C which corresponds as a limiting factor (Fig 3 and 4). The 5th generation is spring generation that appeared at 03/03/2020 (0.80 flies / traps / day). The sex ratio of captured flies was constantly in favor of males (0.63 males and 0.37 females). 3.2 INFESTATION RATE Infestation rate differed significantly between varie- ties and across the sampling date as well as the interaction of them (Table 1). Infestation rate of ‘Sigoise’ was higher Figure 3: Population indexes Pi (total, male and female), during the study period Figure 4: Average daily data of the temperature and relative humidity from June 23, 2019 to April 23, 2020 in the Mascara region Acta agriculturae Slovenica, 118/3 – 2022 5 Flight activity of Bactrocera oleae ... infesting two Algerian olive varieties in north-west Algeria In order to study the influence of the fruit size factor on both varieties susceptibility to B. oleae, a relationship was estimated between the infestation rate and the calib- er of the fruits by the GLM function in R (R Core Team, 2021). The results showed that size coefficient is highly significant (p <0.001), while the variety coefficient is not significant (p = 0.14). This mean that the infestation rate is not linked to the variety but to the size. The relation is written in the following form: Infestation rate = -15.414 + -2.32 * Variety + 5.17 * Size (1) On the other hand, ANCOVA analysis revealed that there is a significant difference of the infestation between the cardinal orientations of the tree throughout the study period (F = 44.03, df = 3, p = 0.006) and between varieties (F = 111.28, df = 1, p = 0.002), while their interaction did not found significant for infestation (F = 0.29, df = 3, p = 0.83). Posthoc tests of Tukey’s confirmed that the North direction is the least infested by the olive fly in both vari- eties, but there is no significant difference between other orientations (East, South and West) (Figure 7). 4 DISCUSSION 4.1 STUDY OF THE POPULATION DYNAMICS OF THE OLIVE FLY The presence and fluctuation of the fly throughout the year are well demonstrated by our results with an important number of generations (five peaks per year), this latter depends on several factors, mainly the climate which is closely linked to the longevity of this pest, fruits Variable Factor df F p Infestation rate Sampling date 6.46 238.02 <0.0001 Variety 1 26.28 <0.0001 Interaction 6.46 7.79 <0.0001 Fruit caliber Sampling date 10.16 295.62 <0.0001 Variety 1 1743.06 <0.0001 Interaction 10.16 49.87 <0.0001 Table 1: Effects of variety and sampling date on infestation rate and fruits caliber for the year 2019 (GLM: Repeated measures) Figure 5: Infestation rate (mean ± S.E) of the two varieties by the olive flies (September to December 2019) Figure 6: Mean fruit caliber (mean ± S.E) of the two varieties by the olive flies (September to December 2019) Figure 7: Variations in the infestation rate (mean ± E.S) of olives in relation to cardinal orientation of the tree throughout the study period Acta agriculturae Slovenica, 118/3 – 20226 Z. BOURAKNA et al. damaged which remain after harvest which ensures the maintaining and continuity of the species in the orchards (Jimenez et al., 1994). Daane & Johnson (2010) claim that even if the olive tree is unsuitable for oviposition, adults have the ability to reproduce and survive when their nu- trition is available, which constitutes a true danger to ol- ive orchard. In the study area, the first generation was reported in July. Also, Gaouar (1996) in the Tlemcen region in Algeria, Yokoyama et al. (2006) in southern California, Goncalves et al. (2012) in Portugal, Ait Mansour et al. (2015) in Morocco and Pertíñez & Vélez (2020) in Ma- drid (Spain) found a generation was marked at the end of June or the beginning of July in the olive orchards close to the sea (fresh and humid), however in inland areas which far of coastline, the summer generation was absent. This can be explained by the hot, arid conditions and the unavailability of fruit. We reported the second genera- tion on September 03, 2019 where the temperature and humidity conditions become ambient also the receptive olives are available and premature. It was followed by the third at the beginning of November and the fourth at the end of December. This succession of three generations in the autumn was similar to the results of Goncalves et al. (2012) and Ait Mansour et al. (2015). However, Gaouar (1996) and Yokoyama et al. (2006) found two generations in this period and Pertíñez & Vélez (2020) found only one generation in the fall. This overlap of generations was explained by the contribution of each generation to the coexistence of the future generation. The fourth gen- eration in our result was absent in most of the studies, this can be explained by the late harvest of the fruits un- til the end of December. Generally according to several researchers, in most regions, autumn is the season best suited to the development of the olive fly, when its lar- val food is available (Daane & Johnson, 2010). Besides, Yokoyama et al. (2006) explained that the unusually large number of adults captured from March to April is due to the presence of fruits in the orchard of the previous year, which provides oviposition sites and food for the devel- opment of this pest. This ascertainment explains well and justifies the appearance of the fifth generation (March 03, 2020) in our study region. Also, the population density is closely related to climatic conditions (temperature and humidity). According to Marchi et al. (2016), interannual variations of the population are explained by tempera- ture and according to Broufas et al. (2009) Relative hu- midity can lead to increased longevity of the fly and the fertility of their females. Concerning the study of the sex ratio, it was noticed from the results that the number of catches of males was greater than that of females (0.63 males and 0.37 females), this can be justified by the color yellow traps and nature of bait. This ascertainment is similar to that of Katsoyannos & Kouloussis (2001) who explain that catches are strongly influenced by the color of the traps, where he reported that males of olive flies are attracted to the yellow, orange and white color traps, while females by the colors red and black. Rice et al. (2003) revealed that traps baited only with ammonium bicarbonate, more male than female flies were collected. 4.2 INFESTATION RATE The fruit infestation started on September 03, 2019 where the olives reached the fruit enlargement and stone hardening stage, which is considered the receptive stage for oviposition olive fly (Civantos, 1999), thus coincid- ing with the period of ovarian maturation of females (Tzanakakis, 2003). Our results are similar to those of (Ibnsouda et al., 2004; Goncalves et al., 2012). The sig- nificant increase of the infestation over time was justi- fied by the increase in the number of captures. Pertíñez & Vélez (2020) mentioned that the proportional increase of damage was caused by the increase of the population size, while all reductions in population size maintained the total amount of damaged olives. The study of the in- fluence of the cardinal orientation of the tree on the level of infestation revealed that the northern cardinal orien- tation of the tree is less attacked by the fly, according to Goncalves et al. (2012) the olive fly prefers to oviposite on the coldest areas of the tree. While Gaouar & Debouzi (1991) indicated that the cardinal orientation in olive trees did not influence the infestation. Not only the number of captures by olive flies is re- sponsible for of the damage importance to the olive tree, but also the different aspects of cultivars play an impor- tant role in their susceptibility to oviposition. The olive flies preference for oviposition appears to lie in the inter- action and correlation of three aspects: physical, chemi- cal and molecular (Malheiro et al., 2015). Certain physi- cal characteristics of fruits including color, elongation, hardness and volume affect their susceptibility to this pest (Rizzo et al., 2012). Several studies have evaluated a good correlation between fruit size and olive tree in- festation (Neuenschwander & Michelakis, 1979; Burrak & Zaloum, 2008; Rizzo et al., 2012; Garantonakis et al., 2017; Medjkouh et al., 2018). We have reported in our case that there is an important relationship between fruit size and infestation rate. The difference between the in- festations of the two varieties was justified by the sensi- tivity of the table variety (Sigoise) which has a larger size than that of the ‘Chemlal’ variety with small fruits and high oil content, where this latter is less infested. Our re- sults are in agreement with those of Jerraya et al. (1982), Arambourg (1984) and of Gaouar & Debouzi (1991). A Acta agriculturae Slovenica, 118/3 – 2022 7 Flight activity of Bactrocera oleae ... infesting two Algerian olive varieties in north-west Algeria similar infestation rate in the two varieties studied was observed at the end of the season (84.00 ± 2.65 % ‘Sigoise’, 78.00 ± 2.57 % ‘Chemlal’), despite that the fruits calibers are different ( 18.48 ± 0.24 mm for the ‘Sigoise’ and 13.24 ± 0.19 mm for ‘Chemlal’). These rates which seem the same important mark the third generation with a num- ber of adults which reaches its maximum (2.4 flies / traps / day), we can say that the adults of this generation could oviposite their eggs in almost all the fruits not infested (regardless of caliber) (Gaouar & Debouzi, 1991). 5 CONCLUSION The trapping of the olive fly adults allowed us to determine their population dynamic and to evaluate the number of generations in Mascara region, this pest is present throughout the year with five generations. The overlapping of the autumn generations causes important damage to the olives. The evaluation of the infestation rate showed that at the beginning of the season, the olives of the Sigoise variety are more attacked by B. oleae than the olives of ‘Chemlal’. This difference remain linked to the size of the fruits, where the Sigoise variety had large caliber olives (table variety) compared to the small-fruit- ed Chemlal variety (olive intended for oil). However, at the end of the season, despite the two varieties were dif- ferent in the size of their olives, but the infestation rate is high for both. The knowledge the dynamics of B. oleae populations and the determination the level of infestation that can in- flict on different olive varieties remains the key to obtain better integrated control strategy against such parasites in an area as important as Mascara (Algeria) recognized by its olive vocation and its national and international fame. 6 ACKNOWLEDGEMENTS The authors are grateful to the olive orchard owner who permitted access for this study, and to Professor A. Hammimed (Mascara University) who provided us with climatic data for the study region. My thanks also to my colleague Elhouacine Houcine for his invaluable help during the experiment. 7 REFERENCES Ait-Mansour, A., Kahime, K., Chemseddine, M. & Boumez- zough, A. (2015). Study of the population dynamics of the olive fly Bactrocera oleae Rossi. 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Delo je prispelo 25. novembra 2021, sprejeto 17. september 2022 1 Scientific and Technical Research Center on Arid Regions (CRSTRA), Biskra, Algeria 2 Laboratory of Ecosystems Diversity and Dynamics of Agricultural Production Systems in Arid Zones, Mohamed Khider University, Biskra, Algeria 3 Corresponding author, e-mail: lahmadisalwa@yahoo.fr 4 Biotechnology Research Center, Constantine, Algeria Phytochemical analysis, antioxidant and photoprotective ac- tivities of aqueous extract of Euphorbia retusa Forssk. differ- ent parts from Algeria Abstract: Euphorbia retusa is an endemic medicinal plant of Sahara. This study aimed to determine the total phenolic and flavonoid contents of Euphorbia retusa seed, capsule and leaves aqueous extracts as well as to evaluate the antioxidant and pho- toprotective activities. The correlations between these activities and the different contents were also performed. The antioxidant activity was estimated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis-3-ethyl benzthiazoline-6-sulfonic (ABTS) scavenging, β-carotene bleaching, cupric-reducing ac- tivity (CUPRAC) and reducing power essays. In addition, the sun protection factor (SPF) was reported for the first time and measured according to the Mansur equation. Results showed that, seeds exhibit a higher total phenolics and flavonoids con- tents. This organ showed the highest capacity in DPPH (IC50 = 50.79 ± 1.87 µg ml-1), ABTS (IC50 < 6.25 µg ml -1), β-carotene bleaching (IC50 < 6.25 µg ml -1), reducing power (A0.50 = 6.97 ± 0.75 µg ml-1) and CUPRAC (A0.50 = 7.64 ± 0.30 µg ml-1) essays. Accordingly, seed extracts characterized by a high sun protec- tion factor (SPF = 38.26 ± 0.07). Nevertheless, the Pearson cor- relation coefficients calculated show the highest positive cor- relation between total phenolic and flavonoids contents and photoprotective activity, while no correlations were found be- tween SPF and other antioxidant activity. This plant could be used as alternative adjuncts in sunscreen product preparation. Key words: Euphorbia retusa Forssk.; polyphenols; anti- oxidant activity; sun protection factor; Pearson correlation Kemična analiza, antioksidacijska in fotoprotektivna aktiv- nost vodnih izvlečkov iz različnih delov vrste mlečka Euphor- bia retusa Forssk. iz Alžirije Izvleček: Vrsta Euphorbia retusa Forssk. je endemična zdravilna rastlina iz Sahare. Namen raziskave je bil določiti vsebnost celokupnih fenolov in flavonoidov v vodnih izvlečkih semen, glavic in listov te rastline kot tudi ovrednotiti njihovo antioksidacijsko in fotoprotektivno aktivnost. Pokazale so se povezave med različnimi aktivnostmi in vsebnostmi analizi- ranih sestavin. Antioksidacijska aktivnost je bila ocenjena na osnovi redukcijske moči snovi kot so DPPH, ABTS, bledenja β-karotena in redukcijske aktivnosti bakra (CUPRAC). Dodat- no je bil prvič izmerjen zaščitni faktor pred soncem po Man- surjevi enačbi. Rezultati so pokazali, da imajo semena veliko vsebnost celokupnih fenolov in flavonoidov. Izvlečki iz semen so pokazali tudi največjo sposobnost pri uporabi DPPH (IC50 = 50,79 ± 1,87 µg ml-1), ABTS (IC50 < 6,25 µg ml -1), bledenju β-karotena (IC50 < 6,25 µg ml -1), redukcijski moči (A0.50 = 6,97 ± 0,75 µg ml-1) in pri preiskusu CUPRAC (A0.50 = 7,64 ± 0,30 µg  ml-1). Sorazmerno temu je bil za izvlečke semen značilen velik zaščitni faktor pred soncem. (SPF = 38,26 ± 0,07). Kljub temu, da je izračunani Pearsonov koeficient korelacije pokazal največjo pozitivno korelacijo med vsebnostjo celokupnih feno- lov in flavonoidov ter aktivnostjo zaščite pred soncem ni bilo nobene korelacije med zaščito pred soncem in drugimi antio- ksidacijskimi aktivnostmi. Iz izledkov sledi, da bi se ta rastlina lahko uporabljala kot alternativni dodatek pri izdelavi zaščitnih pripravkov pred soncem. Ključne besede: Euphorbia retusa Forssk.; polifenoli; an- tioksidacijska aktivnost; zaščitni faktor pred soncem; Pearso- nova korelacija Acta agriculturae Slovenica, 118/3 – 20222 S. LAHMADI et al. 1 INTRODUCTION Recently, a high demand of natural antioxidants has increased to replace synthetic antioxidants that are known by their undesirable side effects on human health (Megdiche et al., 2013). In addition, the demand for herbal cosmetics used in sunscreens has grown rap- idly also to give better protection against UV radiations which can provoke more damages and develop a number of skin diseases (Napagoda et al., 2016). However, plants are an important source for the development of new chemotherapeutic including an- tioxidant agents, which can be protect cell constituents against oxidative damage and withstand the risk of vari- ous diseases associated with oxidative stress (Herlina et al., 2018). Furthermore, phenolic compounds are known for their potential antioxidant to eliminate toxic reactive oxygen species (ROS) as well as flavonoids, which were characterized by a strong potential protection against UV radiations (Hopkins, 2003; Macheix et al., 2005). The usage of plant species in the traditional medicine for the treatment of a variety of diseases (El-haj et al., 2014; Nematy et al., 2015) may be an important way to facili- tate research on the sources of natural additives. Besides, medicinal plants of Sahara have higher secondary metab- olites contents including phenolic compounds (Trabelsi et al., 2010; Gasmi et al., 2019). Euphorbia retusa Forssk. is an endemic species of northern and central Sahara. It is an annual plant which grows naturally up to 30 cm high in hard climatic condi- tions of Sahara (Quezel and Santa, 1962; Ozenda, 2004). This plant is known for its use in folk medicine particu- larly as a treatment of dermatosis in the central Algerian Sahara (Ghareeb et al., 2018; Abdallah, 2014; Sdayria et al., 2019; Hammiche and Maiza, 2005). Thus, the aim of this study was to investigate the antioxidant and photoprotective activities of the selected plant and to provide also the relationship between these activities and total phenolics and flavonoids contents of the aqueous extracts of E. retusa seed, capsule and leaves. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL COLLECTION Euphorbia retusa Forssk. plant parts were col- lected from the South-East arid region of Algeria (34°54’21.751’’N, 005°38’27’’E) in June 2016. The plant samples were identified based on the flora of Ozenda (2004). The plant samples were separated into different parts: seeds, capsules and leaves. Then, samples were cleaned, dried in shade and grounded to powder. 2.2 EXTRACTION METHODS In order to extract the phenolic compounds present in our plant, 10 g of each part of plant (seeds, capsules or leaves) were extracted separately with 100 ml of distilled water using Soxhlet apparatus at 40 °C for 8 hours. After extraction, the solvent of each part extracts was evapo- rated using a rotary vacuum evaporator until dryness. 2.3 TOTAL PHENOLICS AND FLAVONOIDS CON- TENTS DETERMINATION The total phenolics content of aqueous extracts was determined using Folin-Ciocalteu method following the protocol of Singleton et al. (1999) with slight modifica- tion. Briefly, 20 μl of each sample was mixed with 100 μl Folin–Ciocalteu reagent (10 fold diluted) and 75 μl of 7.5 % sodium carbonate solution. This mixture was incubated for 2 h at room temperature and the absorb- ance was measured at 765 nm using a 96-well microplate multimode plate reader (En Spire, PerkinElmer, MA, USA). The phenolic compounds concentrations were expressed as gallic acid equivalents/mg solid dry extract (µg GAE/mg DE) and calibration equation was found as: y = 0.002× + 0.010, (R2 = 0.989). The total flavonoids content of aqueous extracts was quantified according to Moreno et al. (2000) method. 20 μl of each diluted extract solution was mixed with 10 μl of 10 % aluminium nitrate, 10 μl of potassium acetate (1 M) and 130 μl of methanol. After 40 min incubation at room temperature, the absorbance was measured at 415 nm. The total flavonoid content concentrations were ex- pressed as Quercetin equivalents/ mg solid dry extract (µg QE/mg DE) and calibration equation was deter- mined as: y 0.006× - 0.006, (r² = 0.998). 2.4 ANTIOXIDANT ACTIVITY EVALUATION 2.4.1 Antiradical activity The free radical scavenging activity of the aqueous extracts of each part was evaluated using DPPH assay described by Bloi (1958). 40 µl of the each extract con- centrations (6.25, 12.5, 25, 50,100, 200 and 400 µgml-1) was mixed with 160 µl of a methanolic DPPH solution. The mixture was incubated at room temperature for 30 min. Then, the absorbance was measured at 517 nm us- ing 96 well microplate reader. Results were expressed as % inhibition and as IC50 values in µg ml -1. Butylhydroxy- toluène (BHT) was used as a positive control. The inhibi- Acta agriculturae Slovenica, 118/3 – 2022 3 Phytochemical analysis, antioxidant and photoprotective activities of aqueous extract of Euphorbia retusa ... from Algeria tion percentage was calculated according to the following formula; % Inhibition = [(A0 – A1 / A0)] × 100 Where; A0 is the absorbance of the negative control, and A1 is the absorbance of the sample at 30 min. 2.4.2 ABTS•+ scavenging activity ABTS radical-scavenging activity of aqueous ex- tracts was assessed according to the method developed by Re et al. (1999). 40 µl of extract at different concentra- tions (6.25, 12.5, 25, 50,100, 200 and 400 µg ml-1) were mixed with to 160 µl of ABTS•+ solution in micro plate 96 wells. After 10 min of incubation, the absorbance was re- corded at 734 nm. Butylhydroxytoluène (BHT) was used as a positive control and the inhibition percentage was calculated. % Inhibition = [(A0 – A1 / A0)] × 100 Where; A0 is the absorbance of the negative control, and A1 is the absorbance of the sample at 10 min. 2.4.3 β-carotene–linoleic acid bleaching assay β-carotene–linoleic acid bleaching assay of seeds, capsules and leaves aqueous extracts of E. retusa Forssk was measured following the method of Marco (1968). 40 µl of each sample at seven different concentrations was added to 160 μl of the β-carotene–linoleic acid emulsion. The first absorbance was measured in the zero-time at 470 nm and the second absorbance was recorded after 120 min of incubation on the same wavelength. Butyl- hydroxytoluène (BHT) was used as a positive control and the inhibition percentage as measured as following: % Inhibition=[1-(A0Extract–AtExtract)/(A0Control-AtControl)]×100 Where; A0 Control is the absorbance of the negative control at 0 min. At Control is the absorbance of the nega- tive control at 120 min. A0 Extract is the absorbance of the sample at0 min. At Extract is the absorbance of the sample at 0 min. 2.4.4 Reducing power assay The reducing power of studied extracts was deter- mined following the method of Bouratoua et al. (2017). 10 µl of extract were added to 40 μl of phosphate buffer (0.2 M, pH 6.6) and 50 μl of potassium ferricyanide (1%). The plate was incubated at 50 °C for 20 min. Then, 50 μl of tricarboxylic acid (10 %), 40 μl of distilled water and 10 μl of ferric chloride (0.1%) were added to mixture. Butylhydroxytoluène (BHT) was used as a positive con- trol and the absorbance was measured at 700 nm. Results were expressed as absorbance against reagent blank and as A0.50 values (µg ml -1) corresponding the concentration indicating 0.50 absorbance intensity. 2.4.5 Cupric reducing antioxidant capacity (CU- PRAC) The cupric reducing antioxidant capacity of aque- ous extracts was determined according to the method of Apak et al. (2004). 40 µl of the extracts were added to 50 µl of copper (II) chloride (10 mM), 50 µl of neocuproine at 7.5 mM, and 60 µl of ammonium acetate (NH4Ac) buffer (1 M, pH = 7.0) solutions. After 1 hour of incuba- tion, the absorbance was measured at 450 nm and bu- tylhydroxytoluène (BHT) was used as a positive control. 2.5 IN VITRO SUN PROTECTION FACTOR (SPF) DETERMINATION In order to evaluate ultraviolet (UV) absorption ability of the aqueous extracts, the in vitro SPF is deter- mined according to the spectrophotometric method of Mansur et al. (1986). The aliquots prepared were scanned between 290 and 320 nm, and the obtained absorbance values were multiplied with the respective EE (λ) values. Then, their summation was taken and multiplied with the correction factor. Methanol was taking as blank. Where; EE: erythemal effect spectrum, I: solar in- tensity spectrum, Abs: absorbance of sunscreen product, CF: correction factor (= 10). The value of (EE × I) is con- stant and determined by Sayre et al. (1979) (Table 1). 2.6 STATISTICAL ANALYSIS All values were expressed as the mean ± SD (stan- dard deviation). Analysis of variance (ANOVA) test fol- lowed by Newman–Keuls test were performed to check significant differences between the studied samples using the statistical software Statistica version 6.0. p < 0.05 compared to control was considered to be statistically significant. Acta agriculturae Slovenica, 118/3 – 20224 S. LAHMADI et al. 3 RESULTS AND DISCUSSION 3.1 TOTAL PHENOLICS AND FLAVONOIDS CON- TENTS Total phenolics and flavonoids contents of E. retusa different parts were described in Table 2. The results re- vealed that the seeds aqueous extract exhibited the high- est amount of total phenolic contents (356.83 ± 3.69 µg GAE/mg DE) followed by the capsules (114.25 ± 0.35 µg GAE/mg DE) and the leaves (75.83 ± 8.96 µg GAE/mg DE) extracts. According to Öztürk et al. (2006) and Ka- roune et al. (2015), secondary metabolites inter-organs as well as phenolic compounds are more variable in plant organs. Moreover, this variability may be dependent on the endogenous and exogenous factors (Oueslati et al., 2012). Furthermore, the aqueous extracts of E. retusa capsule registered the highest content on total phenolics than the methanolic extracts (105.33 ± 7.75 µg GAE/mg DE) reported by Lahmadi et al. (2020). Thus, the phe- nolic compounds of E. retusa capsules are very soluble in water in distilled water than methanol, which means that this organ is rich in polar polyphenol (Baldosano et al., 2015). Flavonoids contents were higher in seeds aque- ous extract followed by capsules and leaves extracts (194.38 ± 8.31, 30.7 ± 0.4 and 44.25 ± 5.9 µg QE/mg DE respectively). However, flavonoids contents in leaves reported in the present work were higher than that re- ported by Sdayria et al. (2019) (20.50 ± 0.107 µg QE/mg DE) extracted by the maceration method which suggest that extraction with water was more effective than with 96 % ethanol. 3.2 DPPH SCAVENGING ACTIVITY DPPH free radical scavenging activity of aqueous extracts of E. retusa seed, capsule and leaves is shown in Table 3. The results were expressed as inhibition percent- age at different concentrations (6.25, 12.5, 25, 50, 100, 200 and 400 µg ml-1) and as IC50 values in µg ml -1. Data revealed that DPPH scavenging capacity increases with the raise in concentration of each extract. Furthermore, our findings showed that seeds aqueous extract exhibited a high activity competing with the both other extracts at all concentrations. Likewise, research reports found that seeds phenolic compounds are capable more for donat- ing hydrogen to a free radical to scavenge the potential damage (Ksouri et al., 2009; Saeed et al., 2012). Ashraf et al. (2015) reported also the DPPH free radical scaveng- ing activity of roots aqueous extract of E. royleana. The comparing of our results with this study showed that our samples (seeds, capsule and leaves) at 100 µg ml-1present a higher effective scavenger of hydroxyl radical (79.67 % ± 1.44, 57.87 % ± 0.76 and 36.09 % ± 1.11 respectively) than their samples (20.18 % ± 0.96). 3.3 ABTS SCAVENGING ACTIVITY For the ABTS radical-scavenging activity, seed ex- tracts have a stronger capacity to quench ABTS•+ at con- centrations ≥ 50 µg ml-1as well as BHT (Table 4). The inhibition percentage of seed, capsule and leaves aque- ous extracts was significantly important (92.01 % ± 1.64, 92.44 % ± 0.25 and 52.63 % ± 0.09 respectively) com- pared to those reported by Alaklabi et al. (2018) for root aqueous extracts of Saururus chinensis (Lour.) Baill. with 19.07 % ± 0.12 at same concentration (100 µg ml-1). Wavelength (λ nm) EExI (λ) (Normalized) 290 0.0150 295 0.0817 300 0.2874 305 0.3278 310 0.1864 315 0.0837 320 0.0180 Table 1: Correlation between the erythemogenic effect (EE) and the radiation intensity at each wavelength (I) Extract Seeds Capsules Leaves Total phenolics (µg GAE/mg DE) 356.83 ± 3.69a 114.25 ± 0.35b 75.83 ± 8.96c Total flavonoids (µg QE/mg DE) 194.38 ± 8.31a 30.7 ± 0.4b 44.25 ± 5.9c Table 2: Total phenolics and flavonoids contents of the aqueous extracts of E. retusa different parts Values expressed as mean ± SD (n = 3). Values in the same line followed by a different letter (a-c) are significantly different (p < 0.05). µg GAE/mg DE: microgram gallic acid equivalent per milligram of dry plant extract. µg QE/mg DE: microgram quercetin equivalent per milligram of dry plant extract Acta agriculturae Slovenica, 118/3 – 2022 5 Phytochemical analysis, antioxidant and photoprotective activities of aqueous extract of Euphorbia retusa ... from Algeria Concentrations µg ml-1 % Inhibition in DPPH scavenging assay Seed Capsule Leaves BHT 6.25 9.21 ± 1.25 1.44 ± 1.29 1.94 ± 1.80 18.55 ± 2.46 12.5 16.14 ± 2.34 4.60 ± 1.37 5.16 ± 0.35 32.60 ± 3.72 25 27.12 ± 1.10 11.47 ± 1.03 8.88 ± 1.15 53.80 ± 2.58 50 49.99 ± 1.62 28.19 ± 1.11 19.77 ± 0.35 74.97 ± 2.14 100 79.67 ± 1.44 57.87 ± 0.76 36.09 ± 1.11 83.41 ± 0.86 200 84.12 ± 0.34 82.20 ± 0.68 73.69 ± 0.95 84.59 ± 0.46 400 92.82 ± 0.41 92.83 ± 0.17 81.79 ± 0.42 85.76 ± 0.91 IC50 µg ml -1 50.79 ± 1.87b 87.38 ± 1.53c 158.49 ± 3.24d 23.54 ± 1.83a Table 3: Antioxidant activity of the aqueous extracts of E. retusa different parts by DPPH assay Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-d) are significantly different (p < 0.05). BHT: butyl hydroxytoluene. IC50: half maximal inhibitory concentration expressed as the necessary concentration to decrease the initial absorbance of DPPH by 50 % Concentrations µgml-1 % Inhibition in ABTS assay Seeds Capsules Leaves BHT 6.25 69.10 ± 2.97 18.38 ± 1.51 - 61.38 ± 0.57 12.5 88.06 ± 2.60 33.88 ± 1.99 9.68 ± 2.78 62.02 ± 3.82 25 88.93 ± 1.51 56.27 ± 4.35 12.92 ± 3.96 76.50 ± 1.40 50 91.14 ± 0.37 86.05 ± 2.87 25.83 ± 1.78 82.55 ± 1.04 100 92.01 ± 1.64 92.44 ± 0.25 52.63 ± 0.09 88.60 ± 2.66 200 92.82 ± 0.41 92.83 ± 0.17 73.69 ± 0.95 90.38 ± 0.67 400 - 92.89 ± 0.19 90.33 ± 0.25 - IC50 µg ml -1 < 6.25a 21.12 ± 0.76b 95.92 ± 1.20c < 6.25a Table 4: Antioxidant activity of the aqueous extract of E. retusa different parts by ABTS assay Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-c) are significantly different (p < 0.05). BHT: butylhy- droxytoluene. IC50: half maximal inhibitory concentration expressed as the necessary concentration to decrease the initial absorbance of DPPH by 50 % Concentrations µg ml-1 % Inhibition in β-carotene–linoleic acid bleaching assay Seeds Capsules Leaves BHT 6.25 97.83 ± 0.38 96.14 ± 0.43 92.94 ± 0.50 57.25 ± 3.1 12.5 97.42 ± 0.22 96.02 ± 0.37 89.37 ± 0.95 82.39 ± 2.79 25 96.29 ± 0.20 94.21 ± 0.41 82.29 ± 0.30 83.12 ± 2.82 50 94.67 ± 0.39 93.77 ± 1.52 72.53 ± 1.72 92.99 ± 3.26 100 93.07 ± 0.18 92.01 ± 0.14 57.08 ± 2.74 92.65 ± 3.19 200 89.6 ± 0.04 89.28 ± 0.23 30.34 ± 1.36 93.52 ± 0.00 400 79.83 ± 0.32 84.44 ± 0.18 14.24 ± 0.91 94.22 ± 0.30 IC50 µg ml -1 < 6.25 ± 0.00a < 6.25 ± 0.00a 23.82 ± 0.95b < 6.250 ± 00 a Table 5: Antioxidant activity of the aqueous extract of E. retusa different parts by β-carotene–linoleic acid bleaching assay Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-b) are significantly different (p < 0.05). BHT: butylhy- droxytoluene. IC50: half maximal inhibitory concentration expressed as the necessary concentration to decrease the initial absorbance of DPPH by 50 % Acta agriculturae Slovenica, 118/3 – 20226 S. LAHMADI et al. ance of samples and BHT as a standard were increased by the rising of concentrations. However, the results showed that seeds aqueous extract had the strongest capacity to reduce ion at all concentrations compared with BHT or stems and leaves aqueous extracts. Moreover, seeds aque- ous extract (A0.50 = 6.97 ± 0.75 µg ml -1) indicates a high A0.50 value as compared with seeds methanolic extract (A0.50 = 11.84 ± 1.72 µg ml -1) reported by Lahmadi et al. (2019). 3.6 CUPRIC REDUCING ANTIOXIDANT CAPAC- ITY (CUPRAC) The CUPRIC reducing antioxidant capacity method described by Apak et al.(2004) measures the absorbance of Cu(II)- neocuproine (Nc) chelate formed by the re- dox reaction of chain-breaking antioxidants with the CUPRAC reagent. Cupric reducing antioxidant capacity 3.4 Β-CAROTENE–LINOLEIC ACID BLEACHING ASSAY The bleaching of β-carotene assay was used to evaluate the ability of the antioxidants to inhibit lipid peroxidation (Moualek et al., 2016). Furthermore, up to our knowledge, there are no reports on the bleaching of β-carotene assay of E. retusa organs. So this is the first report which deals with this effect. The results of this activity were expressed as inhibition percentage and as IC50 µg ml -1 (Table 5). Results showed that seed, capsule aqueous extracts and BHT as a standard have a stronger capacity to inhibit the coupled oxidation of β-carotene and linoleic acid (IC50 < 6.25 µg ml -1). 3.5 REDUCING POWER ASSAY For reducing power activity (Table 6), the absorb- Concentrations µg ml-1 Absorbance in reducing power assay Seeds Capsules Leaves BHT 6.25 0.22 ± 0.01 0.08 ± 0.01 - 0.05 ± 0.02 12.5 0.31 ± 0.02 0.1 ± 0.00 - 0.07 ± 0.02 25 0.46 ± 0.03 0.14 ± 0.00 - 0.11 ± 0.03 50 0.7 ± 0.04 0.19 ± 0.02 - 0.19 ± 0.02 100 0.85 ± 0.11 0.29 ± 0.01 0.05 ± 0.00 0.30 ± 0.03 200 1.29 ± 0.09 0.35 ± 0.01 0.05 ± 0.00 0.74 ± 0.18 400 2.14 ± 0.00 0.57 ± 0.01 0.06 ± 0.02 1.07 ± 0.17 A0.50 µg ml -1 6.97 ± 0.75 a 84.49 ± 2.38c > 100 37.41 ± 3.89b Table 6: Antioxidant activity of the aqueous extracts of E. retusa different parts by reducing power assay Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-c) are significantly different (p < 0.05). BHT: butyl- 4-methylphenol ou butylhydroxytoluene. A0.50: corresponding the concentration indicating 0.50 absorbance intensity Concentrations µg ml-1 Absorbance in CUPRAC assay Seeds Capsules Leaves BHT 6.25 0.44 ± 0.01 0.14 ± 0.01 - 0.44 ± 0.03 12.5 0.72 ± 0.03 0.20 ± 0.02 0.07 ± 0.00 1.32 ± 0.07 25 1.15 ± 0.05 0.30 ± 0.02 0.08 ± 0.00 1.80 ± 0.09 50 2.00 ± 0.19 0.53 ± 0.03 0.08 ± 0.00 1.82 ± 0.22 100 3.28 ± 0.03 0.81 ± 0.02 0.09 ± 0.00 2.39 ± 0.39 200 3.69 ± 0.12 0.98 ± 0.08 0.10 ± 0.00 2.71 ± 0.46 400 4.06 ± 0.02 1.45 ± 0.07 0.18 ± 0.05 2.76 ± 0.46 A0.50 µg ml -1 7.64 ± 0.30b 49.50 ± 1.51c > 400 6.64 ± 0.18a Table 7: Antioxidant activity of the aqueous extract of E. retusa different parts by CUPRAC assay Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-c) are significantly different (p < 0.05). BHT: butyl- 4-methylphenol ou butylhydroxytoluene. A0.50: A0.5 (µg ml -1) corresponding the concentration indicating 0.50 absorbance intensity Acta agriculturae Slovenica, 118/3 – 2022 7 Phytochemical analysis, antioxidant and photoprotective activities of aqueous extract of Euphorbia retusa ... from Algeria (CUPRAC) of the organ extracts and the BHT are shown in Table 7. The ranking order for CUPRAC test was BHT > seeds > capsules > leaves at all concentrations. Accord- ingly, the results of CUPRAC test showed that BHT have a higher activity (A0.50 = 6.64 ± 0.18 µg ml -1) followed by seeds (A0.50 = 7.64 ± 0.30 µg ml -1), capsule (A0.50 = 49.50 ± 1.51 µg ml-1) and leaves (A0.50 = > 400 µg ml-1) extracts. 3.7 IN VITRO SUN PROTECTION FACTOR (SPF) DETERMINATION The sun protection factor (SPF) values of different part samples were shown in the table 8. The seeds ex- tract has the higher SPF values (38.26 ± 0.07) followed by leaves and capsules extracts (SPF = 22.21 ± 0.56, 15.57 ± 0.24 respectively). In literature, the data about photoprotective activity of this plant is not available. Ac- cording to Afssaps (2011), SPF is generally divided into four protection classes; low (SPF values: 6-15), medium or moderate (SPF values: 15-30), high (SPF values: 30 - 50) and very high (SPF values > 50). Thus, seed aqueous extract belongs to the range of good sunscreen activity while leaves and capsules aqueous extracts were charac- terized by moderate sunscreen activity. In comparison to other works on SPF values, seed extracts of E. retusa pre- sented a higher SPF than Mentha spicata L. aerial parts methanolic extract reported by El Aanachi et al. (2021) with SPF = 35.76 ± 0.21 and the aerial parts methanolic extract of Capnophyllum peregrinum (L.) Lange reported by Lefahal et al. (2018) with SPF = 35.21 ± 0.18. In fact, SPF result shows that the aqueous extracts of seed have a good sun protection activity against ultraviolet radiation. In order to analyze the relationship between to- tal phenolics and flavonoids contents, antioxidant and photoprotective activities of E. retusa seed, capsule and leaves aqueous extracts, Pearson’s correlations were ap- plied (Table 9). A statistically significant positive correla- tion between total phenolic and flavonoid contents and photoprotective activity with Pearson’s correlation coeffi- cients r > 0.90, suggesting that the SPF was dependent not only on the total phenolic but also on the total flavonoid contents, which may be attributed to their synergistic ac- tion. These results support the hypothesis that flavonoid contents contribute to photoprotective activity (Macheix et al., 2005). However, total phenolics contents showed a significant negative correlation with antioxidant ac- tivities (DPPH and ABTS radical-scavenging capacity, β -carotene bleaching and cupric-reducing antioxidant capacity (CUPRAC) and reducing power) with r > - 0.59. Similarity, a statistically significant negative correlation between total phenolic contents, DPPH free radical scav- enging capacity and β carotene bleaching was support- ed by Terpinc et al. (2012). Also, Kainama et al. (2020) found the negative correlation between total phenolic content and ABTS scavenging activity in Garcinia stem and bark ethyl acetate extracts (r = – 0.91), indicating that the antioxidant activity may be linked to the struc- ture and the nature of the phenolic compounds (Oueslati, 2013). However, negative correlation between flavonoid contents and antioxidant activities (DPPH and ABTS cation radical scavenging, cupric-reducing antioxidant capacity (CUPRAC) and reducing power) was shown. On the other hand, reducing power showed a significant negative correlation with β carotene bleaching inhibition and SPF values with Pearson’s correlation coefficients of -0.56 and -0.67 respectively. These results indicated the antagonist effects of reducing power with β carotene bleaching inhibition and with SPF. Furthermore, no cor- relations were found between SPF and other antioxidant activity. Similar results were found by Ebrahimzadeh et Absorbance CF× EE(λ)×I(λ)× Abs (λ) Seeds Capsules Leaves Seeds Capsules Leaves 3.85 2.21 2.84 0.58 0.33 0 .43 3.86 1.85 2.48 3.15 1.51 2.02 3.85 1.61 2.26 11.07 4.62 6.48 3.82 1.50 2.17 12.53 4.90 7.10 3.80 1.46 2.15 7.08 2.71 4.00 3.78 1.46 2.14 3.17 1.22 1.79 3.75 1.50 2.12 0.68 0.27 0.38 Sun Protection Factor (SPF) 38.26 ± 0.07a 15.57 ± 0.24b 22.21 ± 0.56c Table 8: SPF values of the aqueous extract of E. retusa different parts Values expressed as mean ± SD (n = 3). Values in the last line followed by a different letter (a-c) are significantly different (p < 0.05). EE: erythemal effect spectrum, I: solar intensity spectrum, Abs: absorbance of sunscreen product, CF: correction factor Acta agriculturae Slovenica, 118/3 – 20228 S. LAHMADI et al. al. (2014). Hence, our results indicated no correlations between SPF and DPPH radical-scavenging activity. 4 CONCLUSIONS Our results revealed that seed extract demonstrates the best total phenolics and flavonoids contents and SPF value. Also, this extract showed a great potential for an- tioxidant activity. Furthermore, the correlation analysis revealed that SPF is positively correlated with total phe- nolics and flavonoids contents. But, generally no correla- tions were found between SPF and antioxidant activity. According to the obtained results, E. retusa may be con- sidered as a remarkable antioxidant and pharmaceutical source. 5 ACKNOWLEDGEMENT This work was supported by MESRS-DGRSDT (Al- geria). 6 REFERENCES Abdallah, E.M. (2014). Antimicrobial properties and phyto- chemical constituents of the methanol extracts of Euphor- bia retusa Forssk. and Euphorbia terracina L. from Saudi Arabia. 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Delo je prispelo 14. maja 2022, sprejeto 23. septembra 2022 1 University of Djelfa, Faculty of Nature and Life Sciences, Djelfa, Algeria 2 University of Blida, Faculty of Nature and Life Sciences, Medicinal and Aromatic Plants Laboratory, Blida, Algeria 3 Corresponding author, e-mail: mekious.sch.uni.djelfa@gmail.com 4 Catholic University of Louvain, Beekeeping Research and Information Center (CARI), Louvain-la-Neuve, Belgium 5 Faculty of Medicine, Badji Mokhtar University, Annaba, Algeria 6 Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria 7 Department of Agricultural, Forest and Food Sciences (DISAFA), University of Torino, Torino, Italy Pollen quality and sensory attributes of Algerian jujube (Ziziphus lotus (L.) Lam.) honeys Abstract: Honey bees and beekeeping activity are of huge importance for the crop production and biodiversity conser- vation as well as for the economic impact due to ecosystem services. In the south of Algeria, the jujube blooming is an essential forage source for honey bees. The aim of this study was to determine the melissopalynological and sensory char- acteristics of Algerian jujube honey. Nineteen samples of jujube honey collected in south Algeria over the period from 2016 to 2018 were analyzed. The unifloral designation attributed to the honey was confirmed by a pollen analysis following the estab- lished standard methods. Sensory analysis is carried out test- ing the color, the odor and aromas. The results showed that Ziziphus lotus pollen was predominant in all samples, and in terms of sensory analysis, color ranged from amber yellow to light brown; the determined scent classes were warm, floral and woody with medium intensity; the aroma was represented by medium intensity with the warm caramelized, floral fruity and woody classes. Sweet flavor is perceived at medium intensity, acidic flavor is weak, astringent sensation is average and the pi- quant note is perceived with a low intensity. This work proves to be important for improving the knowledge in typical honeys. Key words: jujube; Ziziphus lotus; honey; melissopalynol- ogy; sensory analysis; pollen Kakovost peloda in senzorične lastnosti medu iz alžirske vrste čičimaka (Ziziphus lotus (L.) Lam.) Izvleček: Medonosne čebele in njihova reja imajo velik pomen za pridelavo gojenih rastlin in ohranjanje biodiverzitete kot tudi velik ekonomski pomen pri ekosistemskih storitvah. Na jugu Alžirije je cvetenje alžirskega čičimaka (žižole) najpo- membnejša paša za medonosne čebele. Namen raziskave je bil določiti sestavo in senzorične lastnosti medu, nabranega na tej rastlini. Analiziranih je bilo 19 vzorcev čičimakovega medu, na- branih na jugu Alžirije v obdobju 2016-2018. Pripadnost medu tej medonosni vrsti je bila potrjena s pelodno analizo in in drugimi uveljavljenimi standardnimi metodami. Pri senzorič- ni analizi so bili preiskušeni barva, vonj in aroma. Rezultati so pokazali, da je v vseh vzorcih medu prevladoval pelod te vrste čičimaka. Barva medu je bila od oranžnorumene do svetlorja- ve, vonj je bil srednje močan, določen kot topel, cvetlični do lesni. Aroma medu je bila srednje močna, po toplih karamelah, cvetno-sadna z odtenki po lesu.Sladkost je bila srednja, kislost šibka, trpkost srednja, pikantnost je bila zaznana kot šibke jako- sti. Raziskava je pomembna, ker prispeva k poznavanju tipičnih medov. Ključne besede: čičimak; Ziziphus lotus; med; melisopali- nologija; senzorična analiza; pelod Acta agriculturae Slovenica, 118/3 – 20222 S. MEKIOUS et al. 1 INTRODUCTION Bees are the primary animal pollinators in most ecosystems (Neff & Simpson, 1993) and honey bees (Apis mellifera L., 1758) in particular play a key role as provid- ers of pollination services essential for agricultural pro- ductivity and biodiversity conservation (Potts et al., 2003; Klein et al., 2007; Salami et al., 2016; Ferrazzi et al., 2017). An economic study demonstrated that the total econom- ic value of pollination services globally amounts to ap- proximately €153 billion annually (Gallai et al., 2009), corresponding to about 9 % of the total economic value of agricultural crops grown for human consumption. Moreover, beekeeping supplies market beehive products (honey, pollen, royal jelly, propolis and wax) and live- stock (artificial swarms, packed bees, queens). Currently, a total of 92.3 million hives have been recorded around the world and the honey production amounts to around 1.8 million tons (Vercelli et al., 2021). Mediterranean basin represents an important area for beekeeping activity and honey production character- ized by typical unifloral and multifloral honeys (Battesti, 1990). However, in Mediterranean areas, climate change is a severe threat to honey bees (Le Conte &Navajas, 2008; IPBES, 2016; Novelli et al., 2021; Vercelli et al., 2021), influencing significantly other strictly related fac- tors such as diseases, parasites, predators, parasitoids, vi- ruses and pesticide use (Goulson et al., 2015; Zawislak et al. 2019).In arid and semi-arid areas of southern Algeria, the jujube (Ziziphus lotus L.) blooming is essential for- age sources (nectar and pollen) for honey bees usually between May and June as well as other flowering present during the year (Mekious et al.,2015). Due to this im- portant source, it is possible to produce unifloral honey that is highly sought and appreciated by beekeepers and consumers. As Jujube honey is exposed to a lot of fraud, particularly with regard to its unifloral appellation, the identification and characterization of this honey is essen- tial to preserve the quality as well as geographical and botanical origins. Some studies have been targeted the characterization of this unifloral honey but there is an in- sufficient knowledge about its sensory properties (Song et al., 2012; Zhou et al., 2013; Mekious et al., 2015; Cha- kir, et al., 2016; Zerrouk et al., 2017; Mekious et al., 2020; Zerrouk et al., 2021). Melissopalynological and sensory properties are one of the main ways used to identify unifloral and mul- tifloral honeys (Ferrazzi&Medrzycki, 2002; Piana et al., 2004; Ferrazzi& Vercelli, 2014; Prđun et al., 2020). The aim of this study was to determine pollen characteristics and sensory properties in several of honey samples of the same floral origin. The study of the pollen profile is im- portant to verify the unifloral attributed designation and to know the botanical origin of pollen collected by honey bees. This analysis provides valuable information on the foraging activity of honey bee (Battesti, 1992; Floris et al., 2020). When pollen of one species is highly present in a honey, it is very likely that that species gives an im- portant contribution in terms of nectar in the process of elaboration of this honey except for some honeys derived from plants that do not provide much pollen (pollen un- der-represented, e.g. lavender honeys) or from plants that produce much more pollen than nectar (pollen over-rep- resented, e.g. eucalyptus honey) (Louveaux et al., 1978; Von Der Ohe et al., 2004). Furthermore, sensory properties describe the gen- eral physical characteristics of honey perceptible by our senses. Taste and aroma vary and depend on the plant origin. Usually, sensory evaluation is commonly used to complete physico-chemical and pollen analyses. It is used to confirm quality, to verify the absence of defects, to establish sensory profiles of unifloral honeys, and also to understand consumer preferences (Piana et al., 2004; Marcazzan, 2018). Organoleptic qualities are also consid- ered as markers of floral origin (Amiot et al., 1989). In the absence of sensory standards specific to jujube honey, this study was based on the evaluation of the intensity of organoleptic attributes in 19 honey samples of the same floral origin in order to define the majority aromatic notes. 2 MATERIALS AND METHODS 2.1 STUDY AREA AND HONEY COLLECTION During the years 2016-2018, in the regions of Djelfa (34° 40′ 0″ N, 3° 15′ 0″ E) and Laghouat (33° 48′ 23″ N, 2° 52′ 56″ E) located in the south of Algeria, character- ized by a hot and dry climate with pastoral vegetation well adapted to pedoclimatic conditions of these regions. According to the information collected by the profes- sional climatological stations of the national metrological office of Djelfa and Laghouat, in the years of our study, the maximum average temperature reached 33.82 °C in Djelfa and 41.09 °C in Laghouat  Regarding the precipi- tation, 275 mmyear-1 and163 mmyear-1were registered in Djelfa and in Laghouat, respectively.19 unifloral declared as Z. lotus honey samples were collected from hives be- longing to professional beekeepers and placed in jujube blossom area. Acta agriculturae Slovenica, 118/3 – 2022 3 Pollen quality and sensory attributes of Algerian jujube (Ziziphus lotus (L.) Lam.) honeys 2.2 QUALITATIVE MELISSOPALYNOLOGICAL ANALYSIS In order to complete a qualitative melissopalyno- logical analysis, the extraction and analysis of the pol- len spectra were carried out using methods established by the International Commission of Apicultural Botany, described by Louveaux et al. (1978), Ferrazzi (1992) and Von der Ohe et al. (2004). Ten grams of honey was dis- solved in 20 ml of distilled hot water (20-40  °C). The solution was centrifuged once for 10 min (30,000 rpm), and then another centrifugation was done for 5 min under the same conditions. The sediment was put on a slide with an area of 24× 24 mm. 500 pollen grains were counted and their relative frequency classes were determined according to the in- ternational melissopalynological nomenclature: predom- inant pollen for pollen occurring for more than 45 % of the total pollen count, accompanying pollen or second- ary pollen (16-45 %), important minor pollen (3-15 %) and minor pollen, occurring < 3 %. The identification of pollen types was carried out by comparing the morphology and dimensions of the pol- len grains present in our samples observed under a light microscope with those of microphotographs of refer- ence pollens established by Ricciardelli d’Albore (1998), by our database (Laboratory of aromatic and medicinal plants, University of Blida1) and other pollen atlas. The pollen type includes species and/or genera present in the area, which have the same or similar pollen morphology microscopically. 2.3 SENSORY ANALYSIS The sensory analysis, visual, olfactory, and gustatory characteristics was performed using the technique de- scribed by Piana et al. (2004). The samples were tested by a panel of three assessors from the CARI (Center for Bee- keeping Research and Information) trained to identify a sensory stimulus on the basis of odor and aroma wheel developed by CARI laboratory (Bruneau et al., 2000). The wheel synthesizes a common lexicon of refer- ence and defines a list of descriptors with a precise mean- ing and the same meaning for all, and on the other hand to approve standardized aromatic references correspond- ing to each descriptor. Assessors recorded the color using the Pfund method. The intensity of odor and aroma is carried by a system of quantified evaluation on a scale of 1 to 3. 1: low intensity, 2: medium intensity, 3: high intensity. 3 RESULTS AND DISCUSSION 3.1 QUALITATIVE MELISSOPALYNOLOGICAL ANALYSIS The unifloral declaration attributed to the honey samples was confirmed by a qualitative pollen analysis. The types of pollen present in honey samples divided into four classes of pollen frequencies are illustrated in Table 1. The pollen grains of nectarless species from the families such as Poaceae, Chenopodiaceae, Pinaceae, Cistaceae, Plantaginaceae and Oleaceae were calculated separately (Louveaux et al., 1978 and Von Der Ohe et al., 2004). The taxa Ziziphuslotus,Thapsia garganica, Centau- rea, Carduus, Euphorbia bupleuroides, Retama retam and Peganum harmala have a large distribution (>50  %) in the honey samples. Usually, these pollens are markers of the floristic environment, having a high pollen frequency Figure1:The jujube tree in the steppe region of Djelfa and the pollen grain of Ziziphus lotus (photo credit: Scherazad Mekious) Acta agriculturae Slovenica, 118/3 – 20224 S. MEKIOUS et al. Family Taxon (Pollen type) F (%) M (<3 %) I (3-15 %) S (16-45 %) P (>45 %) Max Anacardiaceae Pistacia lentiscus L.* 10.53 10.53 - - - 1.1 Apiaceae Thapsia garganica L. 89.47 42.11 36.84 10.53 - 17.3 Apiaceae A Daucus carota L. 36.84 36.84 - - - 7.6 Arecaceae Arecaceae 21.05 21.05 - - - 1.6 Asteraceae Echinops 47.37 47.37 - - - 2.5 Centaurea 68.42 52.63 15.79 - - 6.1 Carthamus 10.53 10.53 - - - 1.1 Asteraceae S Carduus 57.89 31.58 26.32 - - 15.2 Calendula 42.11 42.11 - - - 2.7 Scolymus 15.79 15.79 - - - 1.2 Asteraceae T Taraxacum 26.32 26.32 - - - 1.2 Boraginaceae Echium 15.79 15.79 - - - 2.7 Brassicaceae Sinapis form 21.05 21.05 - - - 1.7 Brassicaceae Brassica form 21.05 21.05 - - - 1.2 Caprifoliaceae Caprifoliaceae 31.58 31.58 - - - 1.1 Chenopodiaceae Chenopodiaceae* 15.79 15.79 - - - 2.8 Cistaceae Cistus* 21.05 21.05 - - - 1.4 Ericaceae Erica 10.53 10.53 - - - 1.2 Euphorbiaceae Euphorbia bupleuroides (Desf.) Soják, 57.89 42.11 - 15.79 - 23.7 Fabaceae         Acacia 10.53 10.53 - - - 1.7 Vicia 21.05 21.05 - - - 1.1 Medicago 10.53 10.53 - - - 2.5 Ononis 31.58 31.58 - - - 1.7 Retama retam (Forssk.) Webb & Berthel. 42.11 31.58 5.26 5.26 - 19.2 Fagaceae Quercus* 10.53 10.53 - - - 1.1 Lamiaceae Thymus 26.32 26.32 - - - 1.7 Liliaceae Asphodelus microcarpus Parl. 10.53 10.53 - - - 1.2 Malvaceae Malva 15.79 15.79 - - - 2.7 Myrtaceae Eucalyptus 10.53 10.53 - - - 1.7 Oleaceae Olea* 21.05 21.05 - - - 1.4 Papaveraceae Papaver rhoeas L.* 10.53 10.53 - - - 2.2 Pinaceae Pinaceae* 5.26 5.26 - - - 1.1 Plantaginaceae Plantaginaceae* 15.79 15.79 - - - 5.2 Poaceae Poaceae* 31.58 31.58 - - - 6.3 Continued on the next page Table1: Frequency of distribution of taxa and their frequency classes in honey samples Acta agriculturae Slovenica, 118/3 – 2022 5 Pollen quality and sensory attributes of Algerian jujube (Ziziphus lotus (L.) Lam.) honeys in relation to the widespread distribution in the region (Battesti, 1990). Qualitative pollen analysis highlighted the dominance of Z. lotus (Figure 1) in honey samples as predominant with a maximum frequency of 97.1 %. The determination of botanical origin was based on the relative frequencies of the pollen types of nectarifer- ous species. In general, a honey is considered unifloral when the relative pollen frequency of onetaxon exceeds 45 % (Von Der Ohe et al., 2004). Z. lotus constitutes an abundant source of nectar frequently collected by honey bees justifying the unifloral designation “Jujube honey” attributed by beekeepers. Jujube is a species also visited for the pollen harvest. Moreover, we noted the second- ary presence of pollens from Thapsia garganica, Euphor- bia bupleuroides, Retama retam and Peganum harmala, important sources of nectar and pollen, with respective maximum levels of 17.3  %, 23.7  %, 19.2  % and 27.6  % (Table 1). 3.2 SENSORY ANALYSIS Regarding visual analysis, slight variations in color, ranging from 61 to 99 mm Pfund, were observed, which corresponds to a color range from amber yellow to light brown. Studies reported that the color interval of Al- gerian honeys ranged from 18 to 119  mm Pfund cor- responding to a color range varying from very light to dark brown. The very light honey samples are dominated by Citrus and Hedysarum while dark brown honeys are characterized by predominance ofEucalyptus, Apiaceae as Daucus and Rubus (Ouchemoukhet al., 2007; Bena- ziza et al., 2010). The profile of the aromas and flavors perceived in the 19 honey samples analyzed were shown in Table 2. The odor classes determined by the tasters were warm, floral and woody, with medium intensity. In terms of the perception of aromas, the general intensity is medium, and the aromatic classes perceived were warm caramel- ized, floral fruity and woody. The perception of chemical aroma was found only in two samples with low intensity. The sweet flavor was perceived with medium to high in- tensity. Z. lotus honey samples are characterized by higher fructose contents than glucose, and pH values between 5.17-5.8 for jujube honey from North Africa (Mekious et al., 2015; Chakir et al., 2016; Zerrouket al., 2017), and an average value of pH equal to 6.71 for jujube honey from China (Zhou et al., 2013). In all samples, the acidic flavor was weak, and the astringent sensation was medium. The piquant note was perceived in all the samples, but with a lower rate in 84.21 % of honey samples and *: nectarless species. F: Frequency of distribution of taxa in honey samples, M: Minor pollen (< 3 %), I: important minor pollen (3-15 %), A: Second- ary pollen (16-45 %), P: Predominant (> 45 %), Max: maximum recorded of pollen frequency Ranunculaceae Ranunculaceae 26.32 26.32 - - - 1.7 Resedaceae Reseda 10.53 10.53 - - - 1.2 Rhamnaceae Ziziphus lotus (L.) Lam. 100.00 - - - 100 97.1 Rosaceae Rosaceae 15.79 15.79 - - - 1.2 Tamaricaceae Tamarix 36.84 36.84 - - - 7.2 Nitrariaceae Peganum harmala L. 68.42 31.58 26.32 10.53 - 27.6 Sensory characteristics Olfactory assessment Intensity: medium Description: warm, floral, woody Intensity: low Description: chemical Tasting assessment Intensity Medium Sweetness Medium to high Acidity Low Aroma Intensity: medium Description: warm caramelised, floral-fruity, woody Persistence Medium Other sensations Intensity: low to medium Description: astringent, piquant Crystallization Absent Table 2: Sensory characteristics of Ziziphus lotus honey samples Acta agriculturae Slovenica, 118/3 – 20226 S. MEKIOUS et al. with medium rate in the rest of the samples. Pollen analy- sis of these samples showed the presence of Euphorbia bupleuroides pollen, either as an accompaniment with a maximum pollen frequency of 23.7 % or in rare, isolated cases at <3 %(Table 1). The nectar of this species gives a piquant note to honey; this is confirmed by the reference system for unifloral honeys established by the laboratory specifying that the honey samples were obtained from nectars of the Euphorbiaceae family species. Some notes are linked to the flora foraged by bees without exogenous contamination (Guyot-Declerck, 2001). The unifloral honey samples derived mainly from a single plant species (at least 45  % of pollen grain), may considerably differ in their sensory properties with highly prominent flavor and aroma (Lippolis, 2020).In all honey samples crystallization was absent. 4 CONCLUSION This study describes the melissopalynological and sensory characteristics of honey from the same floral ori- gin “Ziziphus lotus” produced in arid and semi-arid areas in Algeria. Overall, all the honey samples were character- ized by the predominance of Z. lotus pollen. This type of honey has slight variations in color, ranging from amber yellow to light brown. The perceived odor and aromatic classes were warm caramelized, floral fruity and spicy woody, with medium intensity. Sweet flavor, astringent sensation, acid flavor and spicy notes were also perceived in the honey samples. This work proves to be important for improving the knowledge in typical Algerian honeys and in particular in Jujube honey. In this context, the protection and the promotion of regional honeys will be ensured effectively when the various physicochemical, pollen and aromatic components can be described quite precisely. The definition of floral and regional appella- tions should be supported, and they are necessary for any quality approach to honey. It is therefore necessary to de- fine a set of pollen and sensory physicochemical stand- ards characteristic of unifloral honey. The acquisition of new data regarding melissopalynological and sensory analysis of honey allows the protection and the promo- tion of specific regional honeys. In this context, more studies based on the characterization of Z. lotus L. are needed to provide a normative framework and to deter- mine future specific standards composition for this type of honey. 5 ACKNOWLEDGMENTS We would like to thank the collaboration of the CARI laboratory, in particular the tasters Beatrice Per- at, Laurence Leclercq and Christine Delcourt. 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Delo je prispelo 12. novembra 2021, sprejeto 13. julija 2022 1 Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani, Ljubljana, Slovenija 2 Korespondenčni avtor, e-naslov: irena.macek@bf.uni-lj.si Development of research methods to characterise arbuscular mycorrhizal fungal communities and potential effects of fun- gal endophyte biodiversity on vegetation Abstract: Characterization and quantification of the functional and taxonomic diversity of microbial communities is essential for understanding all aspects of microbial ecology and is closely related to ecosystem function. Arbuscular mycor- rhiza is the most widespread symbiosis on Earth, with arbus- cular mycorrhizal (AM) fungi present in more than 2/3 of all plant species. Just over a decade after the publication of the first review article on molecular approaches to study the ecology of AM fungi in Acta Agriculturae Slovenica (Maček, 2009), the rapid development of molecular tools, especially next genera- tion sequencing (NGS) technology, has accelerated the study of the research field of plant root endophytes. In this paper, the current approach to study the ecology and taxonomy of AM fungi is presented, which also provides some insights into the study of other plant root endophytes. In addition, a widely used system for classifying AM fungi with so-called virtual taxa (VT) is presented, which is used for ecological studies and comparison between different studies. Finally, a brief overview of the importance of climate and soil properties for AM fungal community composition and taxa distribution in global ecosys- tems is presented. Key words: arbuscular mycorrhiza; biodiversity; ecology; endophytes; rhizosphere; sequencing; soil Razvoj raziskovalnih metod za karakterizacijo združb arbu- skularnih mikoriznih gliv in potencialni vpliv biodiverzitete glivnih endofitov na vegetacijo Izvleček: Karakterizacija in kvantifikacija funkcionalne in taksonomske raznolikosti mikrobnih združb je ključnega pomena za razumevanje vseh vidikov mikrobne ekologije in je povezana tudi širše z razumevanjem delovanja ekosistemov. Arbuskularna mikoriza predstavlja najbolj razširjeno in staro- davno simbiozo na Zemlji, saj so arbuskularne mikorizne (AM) glive prisotne v koreninah več kot dveh tretjin vseh rastlinskih vrst. V dobrem desetletju od objave preglednega članka o upo- rabi molekulskih pristopov pri raziskavah arbuskularne miko- rize v reviji Acta Agriculturae Slovenica (Maček, 2009) je razvoj metodologije, predvsem tehnologije določanja nukleotidnega zaporedja (sekvenciranja) naslednjih generacij (NGS), močno pospešil raziskave raznolikosti in ekologije združb AM gliv in drugih koreninskih endofitov. V tem članku so predstavljene novosti na področju raziskav endofitskih gliv v koreninah rast- lin, s poudarkom na aktualnem pristopu k raziskavam v eko- logiji in taksonomiji AM gliv, ter sistem njihove klasifikacije s tako imenovanimi virtualnimi taksoni (VT). Slednji je zelo uporaben za namen ekoloških raziskav in širše primerjave raz- ličnih študij med sabo. Na kratko je predstavljen tudi vpliv kli- matskih in talnih lastnosti okolja na sestavo združb in pojavl- janje posameznih taksonov AM gliv v različnih ekosistemih. Ključne besede: arbuskularna mikoriza; biodiverziteta; ekologija; endofiti; rizosfera; sekvenciranje; tla Acta agriculturae Slovenica, 118/3 – 20222 I. MAČEK 1 UVOD V RAZISKAVE DIVERZITETE AM IN DRUGIH SKUPIN ENDOFITSKIH GLIV V KORENINAH RASTLIN Arbuskularne mikorizne (AM) glive (skupina Glo- meromycotina ali tudi Glomeromycota) (Spatafora in sod., 2016, Tedersoo in sod., 2018) predstavljajo ključno skupino talnih mikroorganizmov v številnih kopenskih ekosistemih in najbolj široko razširjeno simbiozo na planetu (Brachmann in sod., 2006). Povezava med AM glivami in rastlinami je starodavna, saj so bile AM gli- ve prisotne že ob prehodu rastlin iz morja na kopno v paleozoiku pred več kot 450 milijoni let. Poleg AM gliv (Glomeromycotina) pa so bile ob prehodu rastlin na kop- no prisotne tudi endofitske glive iz starodavne in delno saprotrofne skupine Endogonales (Mucoromycotina, Mucoromycota), katerih predstavnike so dolgo uvršča- li med AM glive zaradi podobnih morfoloških struktur (drobne hife in njihov razrastki podobni abuskulom), ki jih te glive tvorijo v koreninah rastlin. Na podlagi mo- lekulskih označevalcev (markerjev) je bilo ugotovljeno, da predstavljajo t.i. drobni koreninski endofiti iz skupine Mucoromycotina (ang. ‚fine root endophytes‘ ali MFRE) filogenetsko ločeno skupino globalno razširjenih rastlin- skih endosimbiontov (Orchard in sod. 2017). Slednji tudi danes še vedno tvorijo endosimbiozo z večino skupin ko- penskih rastlin, pogosto tudi istočasno in funkcionalno komplementarno z AM glivami (Field on sod., 2015; Or- chard in sod., 2017, Hoysted in sod., 2019, Besiana et al., 2021; Sinanaj in sod., 2021). Ker so ugotovitve o pomenu simbioze rastlin z glivami iz skupine Mucoromycotina še relativno nove, je podatkov o njihovi povezavi z rastlina- mi manj kot za arbuskularno mikorizo, tako da obstaja še kar nekaj odprtih vprašanj za boljše razumevanje te skupine gliv in njihove funkcije v ekosistemih (Sinanaj in sod., 2021). V tem preglednem članku se osredotočam predvsem na arbuskularno mikorizo, torej AM glive iz skupine Glomeromycotina, vsekakor pa bo v prihodnosti potrebno spremljati tudi razvoj raziskav drugih skupin rastlinskih endofitov, tako že dlje časa poznanih temnih septiranih endofitov (DSE) (Rodriguez in sod., 2009, Knapp in sod., 2018, Tonjer in sod., 2021), kot tudi drob- nih koreninskih endofitov iz skupine Mucoromycotina (MFRE). Razvoj novih molekulskih metod v zadnjem desetletju vsekakor omogoča lažje odkrivanje in razume- vanje celotnega spektra raznolikosti organizmov, ki živijo v območju rastlinskih korenin oz. rizosferi. V bližnji pri- hodnosti zato lahko pričakujemo veliko novih informacij o starodavnih simbiozah med glivami in rastlinami, nji- hovi biodiverziteti in pomenu za ekosisteme, vključno z agroekosistemi. Že dolgo je znano, da arbuskularna mikoriza rast- linam prinaša številne koristi, od izboljšane mineralne prehrane in preskrbe z vodo ter obrambe pred patogeni, do posrednih koristi npr. izboljšane strukture tal in man- jše erozije tal. AM glive so obvezni biotrofi, od rastlin dobivajo fotoasimilate, ki predstavljajo njihov edini vir organskega ogljika (Smith & Read, 2008). Simbioza vpli- va tudi na sestavo združb kopenskih rastlin, s tem pa tudi na funkcioniranje ekosistemov in njihovo produktivnost (Fitter, 2005, Schnitzer & Klironomos, 2011, Wurzburger in sod., 2017). Taksonomsko lahko AM glive klasificira- mo na več načinov, od tradicionalnih klasifikacij, ki te- meljijo na morfologiji celične stene njihovih spor (npr. Oehl in sod., 2011, Blszkowski, 2012), kar je naslovljeno v prvem delu članka, do molekulskih pristopov, pri kate- rih uporabljamo različne molekulske označevalce, kar je obravnavano v drugem delu članka. Definicija vrste je pri mikroorganizmih, vključno z mikroglivami, na splošno zelo težavna in podvržena konsenzu na podlagi trenut- nega poznavanja posamezne skupine organizmov. Prav zato lahko na tem področju v prihodnosti pričakujemo tudi nadaljnje spremembe klasifikacije, ki bodo temeljile na novo pridobljenih podatkih o AM glivah. V zadnjem delu članka so na kratko povzete tudi glavne omejitve različnih pristopov k raziskovanju raznolikosti AM gliv, s poudarki, kje je na mestu previdnost, z namenom čim bolj kvalitetne interpretacije rezultatov in pridobivanja novega znanja o tej zanimivi in pomembni skupini or- ganizmov. 1.1 GOJENJE AM GLIV V ČISTIH KULTURAH IN KLASIČNA IDENTIFIKACIJA VRST AM GLIV Veliko težavo pri morfološkem določanju vrst AM gliv predstavlja njihovo gojenje v čistih kulturah, saj AM glive brez primerne gostiteljske rastline ne uspevajo. V sklopu raziskav AM gliv čisto, enovrstno (monospecifič- no) kulturo predstavlja kultura, kjer je prisotna samo ena vrsta glive, ki je bila vzgojena iz ene same spore. Postopek vzpostavitve take kulture je zapleten in dolgotrajen, zah- teva veliko ekspertnega znanja ter rastne razmere, ki pre- prečujejo navzkrižno kontaminacijo lončnih kultur rast- lin z drugimi glivami iz okolja (npr. ob zalivanju rastlin, zaradi prenosa z živalskimi vektorji, kot so insekti itd.). Spore nekaterih vrst AM gliv lahko izoliramo iz njihovega okolja (običajno iz talnih vzorcev ali pri ne- katerih vrstah tudi iz korenin) s postopkom redčenja in koncentriranja v vodi in/ali raztopini saharoze ter is- kanjem in prepoznavanjem spor s stereo mikroskopom. Novo, enovrstno lončno kulturo AM gliv vzpostavimo s prenosom posamezne spore v bližino korenin izbranih vrst rastlin. Rastline so običajno za namen tega postop- ka vzgojene iz semen, ki so bila predhodno površinsko sterilizirana. Da povečamo možnost neposrednega stika Acta agriculturae Slovenica, 118/3 – 2022 3 Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo med korenino rastline in viabilno sporo AM glive, vzpo- stavimo začetno interakcijo med obema organizmoma v omejenem prostoru. Za ta namen se je izkazala primer- na uporaba nastavkov za pipete, napolnjenih z vlažnim, sterilnim substratom (Slika 1). Kasneje rastline skupaj z nastavkom prenesemo v večje lonce, napolnjene s steril- nim substratom (Slika 1). Da zagotovimo bolj raznolik življenjski prostor za glive (koreninska biomasa) lahko v kasnejši fazi rasti dosejemo še več gostiteljskih rastlin. Ko- ristno je, če uporabimo več rastlinskih vrst, kar omogoči večji nabor potencialnih gostiteljev za AM glive, večjo raznolikost in biomaso korenin in s tem večjo možnost, da se simbioza (mikoriza) v rizosferi dejansko vzpostavi. Pomembno je, da tudi kasneje, tekom rasti rastlin, mak- simalno zmanjšamo možnost navzkrižne kontaminaci- je s sporami ali delci infektivnih hif med posameznimi lončnimi kulturami. Taki preventivni postopki zajemajo gojenje rastlin v zaprtih prostorih, kontrolo škodljivcev, kontrolirano odtekanje vode iz loncev z mikorizo, ustrez- no zalivanje, ki je lahko urejeno z avtomatiziranim kapl- jičnim sistemom, in omejeno ventilacijo zraka znotraj prostora. Rastline lahko gojimo tudi v posebnih vrečkah z vgrajenim filtrom, kjer se ustvari mikroklima in so izo- lirane od ostalih rastlin, obenem pa je poseganje v tak sistem (npr. zalivanje) omejeno na minimum. Vzpostavitev enovrstne kulture AM gliv je dol- gotrajni postopek, ki je lahko odvisen od številnih de- javnikov, med drugimi vrste oz. genotipa glive, rastne sezone, gostiteljskih rastlin in rastnih razmer. Tudi spo- rulacija (tvorba spor) je pri AM glivah zelo nepredvidlji- va in odvisna od vrste glive. Namen vseh teh postopkov je zagotovitev zadostne količine materiala (nepoškodo- vanih, živih spor) za njihovo izolacijo in identifikacijo, saj za opis morfološke vrste AM gliv ne zadostuje ena sama spora, ampak jih potrebujemo vsaj nekaj deset za pripravo preparatov in še več za kasnejše shranjevanje glivnega materiala v banko gliv (Oehl in sod., 2011), kar je pogoj za opis nove vrste (Slika 2). Običajno se shranju- je posušen substrat s sporami v suhem, temnem in hlad- nem prostoru oz. se vzdržuje aktivno kulturo AM gliv, skupaj z živimi rastlinskimi simbionti v rastlinjaku, kar pa je časovno in finančno zelo zahtevno. Taksonomske in ekološke raziskave AM gliv na pod- lagi morfološh znakov so torej zelo težavne iz več razlogov. Če povzamemo, so glavne omejitve: (1) veliko taksonov AM gliv ne moremo gojiti v čistih (lončnih) kulturah, obenem je glive nemogoče identificirati na podlagi kolo- nizacije v koreninah (Slika 3), (2) zahteve za rast in spor- ulacijo različnih taksonov AM gliv so zelo raznolike in kompleksne, (3) pri številnih taksonih (še) nismo uspeli izolirati njihovih spor in tako morfološko določene vrste povezati s podatki, ki izvirajo iz molekulskih raziskav na osnovi DNA, (4) taksonomsko določanje AM gliv je zelo zapleteno in zahteva kompleksno ekspertno znanje, ki je Slika 1: Vzpostavitev enovrstnih (monospecifičnih) kultur AM gliv, vzgojenih iz ene same spore, v nastavkih za pipete (levo) in kasneje v lončnih kulturah (sredina, desno). Namen tega postopka je, da omogočimo čim boljši stik korenine rastline s posamezno izolirano sporo AM gliv in kasneje pomnoževanje AM gliv v kulturi. Rastline so vzgojene iz semena, ki je bilo predhodno površin- sko sterilizirano (foto: I. Maček) Figure 1: Monospecific cultures of AM fungi grown from a single AM fungal spore in pipette tips (left) and later in pot cultures (centre, right). The method allows good spatial contact between plant roots grown from a single surface-sterilised seed and a single AM fungal spore and further fungal development (photo: I. Maček) Acta agriculturae Slovenica, 118/3 – 20224 I. MAČEK kljub temu, da na to temo obstaja kar nekaj strokovne lit- erature (Oehl in sod., 2011, Blaszkowski, 2012), omejeno na samo nekaj strokovnjakov (Slika 2), (5) taksonomski znaki različnih skupin AM gliv se lahko tudi prekrivajo, oz. ni znano, če lahko npr. ista gliva v različnih okoljih tvori tudi več različnih morfoloških tipov spor. Prav zato je razvoj molekulskih metod zelo pomem- ben za razumevanje ekologije in diverzitete AM in dru- gih endofitskih gliv. Ena izmed prednosti tega pristopa je tudi ta, da lahko DNA izoliramo direktno iz korenin (Slika 3), s tem pa dobimo podatke o združbi gliv, ki je fiziološko aktivna in v času vzorčenja v dejanski interak- ciji z rastlinami. Molekulske metode in njihov hitri razvoj tako predstavljajo pravo revolucijo v razumevanju teh or- ganizmov in so še vedno med najbolj obetavnimi orodji za raziskave združb endofitskih gliv (Clapp in sod., 2003, Dickie & FitzJohn, 2007, Dumbrell in sod., 2017, Sinanaj in sod., 2021). 2 MOLEKULSKA IDENTIFIKACIJA AM GLIV V OKOLJSKIH VZORCIH IN GEN- SKI MARKERJI Dejstvo je, da obstaja relativno malo vrst oz. tak- sonov AM gliv, ki jih lahko gojimo v lončnih kulturah. Pri številnih genotipih AM gliv, ki jih poznamo samo na osnovi molekulskih označevalcev izolirane DNA iz okoljskih študij, raziskovalci še nismo uspeli izolirati spor ali celo ugotoviti, če ti taksoni sploh sporulirajo v njihovem naravnem okolju. Taki genotipi so poznani samo na osnovi nukleotidnih zaporedij oz. sekvenc določenih genskih označevalcev. V okoljskih študijah se kot ustrezni označevalec za raziskave raznolikosti in ekologije združb AM gliv največkrat uporablja gen 18S rRNA za malo podenoto ribosoma (SSU) (Simon in sod., 1992, Helgason in sod., 1998; Maček in sod., 2019), v zadnjem času pa v manjšem obsegu tudi regija notran- jega prepisanega vmesnika – ITS (nuclear ribosomal in- ternal transcribed spacer region) (npr. Alzarhani in sod., 2019) (Slika 4). Slednja se največkrat uporablja v prim- eru, da so v raziskavo vključene tudi druge skupine gliv (ne samo AM glive), za katere je regija ITS bolj primeren označevalec kot 18S rRNA, obenem pa uporaba ene same regije za vse zmanjša stroške raziskave (uporaba enega označevalca, namesto dveh, čeprav regija ITS ni optimal- no za identifikacijo AM gliv) (Alzarhani in sod., 2019). Molekulsko določanje omogoča številčno ovrednotenje (kvantifikacijo) taksonov AM gliv v tleh ali v koreninah rastlin. Geni, ki kodirajo to 18S rRNA (ali 18S SSU) genomsko regijo, so dostopni v velikem številu kopij in vsebujejo veliko ohranjenih kot tudi variabilnih regij, kar omogoča ločevanje taksonov na različnih rav- neh. Sekvence male podenote ribosoma (18S SSU) se v ekologiji AM gliv uporabljajo nekje od začetka devet- desetih let prejšnjega stoletja (Simon in sod., 1992). Do Slika 2: Spore dveh različnih vrst AM gliv iz rodu Rhizophagus (levo, sredina). Spora AM glive s strto celično steno iz rodu Acau- lospora (desno). Vidna je večplastna, strukturirana celična stena. Morfološke značilnosti celične stene, njenih plasti ter pritrjenih hif služijo kot taksonomski znaki v taksonomiji AM gliv (foto: I. Maček) Figure 2: Spores of two different AM fungal species from the genus Rhizophagus (left, centre). Crushed AM fungal spore of the genus Acaulospora with visible multi-layered structured cell wall (right). The morphological structures of the cell wall, its layers and attached hyphae serve as taxonomic features in the taxonomy of AM fungi (photo: I. Maček) Acta agriculturae Slovenica, 118/3 – 2022 5 Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo danes je bilo objavljenih kar nekaj različnih začetnih oli- gonukleotidov za pomnoževanje odsekov sekvenc, spe- cifičnih za različne skupine AM gliv, v postopku verižne reakcije s polimerazo (PCR). Najpogosteje uporabljen par začetnih oligonukleotidov male podenote ribosoma v okoljskih raziskavah je AM1 (Helgason in sod., 1998) in NS31 (Simon in sod., 1992), kjer je dolžina pomnože- nega fragmenta DNA približno 550 baznih parov. Za ta par začetnih oligonukleotidov je značilno tudi za AM gli- ve nespecifično pomnoževanje drugih fragmentov, sploh, kadar je glivne DNA v okolju oz. ekstraktu malo. To je bil tudi povod za izdelavo novih začetnih oligonukleoti- dov, kot je par AML1-AML2 (Lee in sod., 2008; dolžina pomnoženega fragmenta DNA je okrog 800 baznih pa- rov) (Slika 4). Regija vsebuje tudi začetno regijo pomno- ževanja oligonukleotidov AM1-NS31, in pomnožuje širok nabor taksonomskih skupin AM gliv, ne pa vseh – izključujeta na primer skupino Paraglomeraceae (Lee in sod., 2008). Ribosomska DNA je v posamezni spori AM gliv zelo polimorfna, kar pomeni, da so sekvence rRNA gena med posameznimi taksoni AM gliv močno variabilne (Tisserant in sod., 2013). Dokazano je tudi, da hife in spore AM gliv vsebujejo na stotine jeder (Tisserant in Slika 3: Kolonizacija korenin koruze (Zea mays L.) z AM glivami (levo) in mikroskopski preparati za oceno stopnje kolonizacije korenin z AM glivami (desno). Na sliki levo so vidne znotraj-koreninske hife AM gliv in arbuskuli (drevescom-podobne struktu- re), ki so pomembne za izmenjavo hranil med rastlinami in glivami (foto: I. Maček) Figure 3:. Arbuscular mycorrhizal fungi colonising maize (Zea mays L.) roots (left), with visible intraradical hyphae and arbuscu- les (tree-like structures) important for nutrient exchange between plants and fungi. Slides for microscopy and estimation of AM fungal colonisation in plant roots (right) (photo: I. Maček) Slika 4: Shematski pregled najpogosteje uporabljenih parov začetnih oligonukleotidov, ki se uporabljajo v ekoloških raziskavah AM gliv (NS31-AM1 in AML1-AML2). Na vrhu slike so prikazani molekulski označevalci jedrne ribosomske RNA – mala po- denota ribosoma (18S SSU), velika podenota ribosoma (28S LSU), 5.8S medgenski vmesnik (IGS) in notranji prepisani vmesnik (ITS). Trikotne puščice prikazujejo smer in mesto za prijemanje začetnih oligonukleotidov Figure 4: Schematic of the most common primer pairs and DNA regions used in the community ecology of AM fungi (NS31- -AM1 and AML1-AML2). At the top of the figure are shown molecular markers of nuclear ribosomal RNA (rRNA) – small ribosomal subunit (18S SSU), large ribosomal subunit (28S LSU), 5.8S intergenic spacer (IGS) and the internal transcribed spacer (ITS). The arrows indicate the direction and alignment range of the primers Acta agriculturae Slovenica, 118/3 – 20226 I. MAČEK sod., 2013). Zaradi polimorfizma trenutno razpoložljivih genskih označevalcev je težko določiti vrsto AM gliv z molekulskimi metodami. V večini študij združb AM gliv različne taksone identificiramo kot skupine sorodnih sekvenc (npr. operativne taksonomske enote – OTU), ki pa verjetno bolj kot nivoju posamezne vrste AM gliv ustrezajo posameznim rodovom (npr. Krüger in sod., 2009). Največ okoljskih raziskav raznolikosti AM gliv je bilo izvedenih s pomnoževanjem regije 18S rRNA (npr. Schwarzott & Schuβler, 2001; Helgason in sod., 2002; Vandenkoornhuyse in sod., 2002; Öpik in sod., 2006), najprej z uporabo pirosekvenciranja s platformo Roche 454 GS-FLX (npr. Öpik in sod., 2009, Dumbrell in sod., 2011), kasneje pa tudi s z določanjem nukleotidnega zaporedja (sekvenciranjem) s platformo Illumina (npr. Alzarhani in sod. 2019; Maček in sod., 2019, Davison in sod., 2021). Trenutno poznamo več kot 300 morfotipov AM gliv, določenih na podlagi morfologije spor (http:// www.amf-phylogeny.com/). Na podlagi molekulskih analiz lahko ocenimo, da je število različnih taksonov AM gliv v okolju bistveno večje, kot kažejo taksonom- ske študije na podlagi morfoloških znakov. Virtualna taksonomija (virtualni taksoni – VT) AM gliv, ki je bila vzpostavljena v specializirani bazi podatkov s področja raziskav AM gliv, ki se imenuje MaarjAM (Öpik in sod., 2010), tudi bazira na uporabi genskega markerja 18S rRNA. Enote, definirane znotraj tega sistema, so t.i. vir- tualni taksoni (VT), ki po navedbah avtorjev baze Ma- arjAM verjetno predstavljajo monofiletske skupine AM gliv, pri katerih podobnost sekvenc znotraj skupine pre- sega dogovorno določeno mejo 97 %. Resolucija VT naj bi približno ustrezala tisti, ki definira vrste AM gliv, do- ločene na podlagi morfoloških znakov (Öpik & Davison, 2016). Baza MaarjAM (http://maarjam.botany.ut.ee) vse- buje več kot 450 virtualnih taksonov, identificiranih na podlagi sekvenc za malo podenoto ribosoma (18S rRNA) (Öpik in sod., 2014), vendar se številke spreminjajo oz. so večje, odvisno, katere genske označevalce uporabimo za identifikacijo taksonov. Klasifikacija sekvenc genov 18S rRNA z uporabo virtualnih taksonov (VT), ki so določe- ni na podlagi baze MaarjAM (Öpik in sod., 2009, 2010), nam omogoča poenoten sistem poimenovanja genotipov, ki jih lahko v ekoloških študijah uporabimo za najboljši približek identifikaciji do nivoja vrst oz. rodov (Öpik in sod., 2014). Pogosta kritika uporabe molekulskih metod za številčno ovrednotenje mikrobnih združb so tudi napake posamezne metode, ki lahko vplivajo na končni rezultat analize. Ena izmed najpogosteje omenjenih napak je po- vezana z metodo PCR in neenakomernim pomnoževan- jem DNA različnih taksonov znotraj združbe (t.i. PCR bias ali PCR pristranskost). PCR je ključni del praktič- no vseh molekulskih pristopov za analizo mikrobnih združb, vključno z združbami AM gliv. Znotraj izolata DNA iz vzorca korenin namreč glivna DNA predstavl- ja le majhen del celokupne ekstrahirane DNA, zato je potrebno specifično pomnoževanje (amplifikacija) regij DNA, ki so informativne za ločevanje različnih taksonov AM gliv. Odvisno od ciljev in namena raziskave se za to lahko uporablja različne genske označevalce, najpogos- teje pa je v ekoloških raziskavah AM gliv uporabljena že omenjena regija 18S rRNA. Potencialne napake oz. pristranskost metode PCR za pomnoževanje specifičnih taksonov (genotipov) AM gliv ter primernost uporabe presejalne metode polimorfizma dolžine terminalnih restrikcijskih fragmentov (TRFLP) za kvantitativne ra- ziskave je bila za regijo 18S rRNA AM gliv testirana v študiji Cotton in sod. (2014). V raziskavi so potrdili, da pri uporabi metod PCR za gene 18S rRNA ne prihaja do bistvenih razlik v pomnoževanju DNA med različnimi genotipi AM gliv in se zato te metode lahko uporablja tudi za kvantitativne analize združb AM gliv, zavedati pa se moramo določenih omejitev. Napake v pomnoževanju regij DNA z metodo PCR se lahko zgodijo, če se različni genotipi pomnožujejo različno hitro zaradi razlik v nji- hovih nukleotidnih zaporedjih ali zaradi same kinetike reakcije pomnoževanja (Kanagawa, 2003). Argument, da se to ne pojavlja pri AM glivah ob uporabi najpogosteje uporabljenih začetnih oligonukleotidov AM1 in NS31 je ta, da so regije DNA, na katere se ti oligonukleotidi vežejo zelo konzervativne (ohranjene med sorodstveno oddalje- nimi taksoni) in imajo obenem zelo majhno variabilnost v vsebnosti baz gvanina (G) in citozina (C) v ampliko- nih (Dumbrell in sod., 2010). Odsotnost tovrstnih napak (pristranskosti pri pomnoževanju med različnimi genoti- pi) pri reakciji PCR, je eden izmed osnovnih pogojev, da je neka analiza lahko kvantitativna in obenem omogoča oceno različnih indeksov pestrosti oz. diverzitetnih in- deksov (Cotton in sod., 2014). V nadaljnjih testih so uporabili tudi analizo TRFLP za karakterizacijo umetno ustvarjene združbe z znanimi razmerji količin vhodne DNA različnih genotipov AM gliv. Podatki študije kažejo, da je protokol TRFLP v kom- binaciji s PCR močno in konsistentno orodje za analizo združb AM gliv, medtem, ko na nivoju analiz populacij posameznih taksonov (genotipov) avtorji študije pripo- ročajo več previdnosti (Cotton in sod., 2014). Ta ekspe- riment je torej potrdil hipotezo, da je možno z uporabo PCR in metodo TRFLP ustrezno ugotoviti relativne raz- like med različnimi združbami AM gliv, v smislu njiho- ve diverzitete in sestave. Študija je tudi pokazala, da pri pomnoževanju različnih genotipov AM gliv za gene 18S rRNA metoda PCR posameznih genotipov ne pomno- žuje bolj, kot drugih, kar pomeni, da se ta metoda lah- ko uporablja tudi v sklopu drugih kvantitativnih analiz združb AM gliv, kjer predstavlja metoda PCR pomemb- Acta agriculturae Slovenica, 118/3 – 2022 7 Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo no komponento. Mednje vsekakor sodijo tudi vse meto- de sekvenciranja naslednjih generacij (NGS), ki so danes za raziskave ekologije združb AM gliv največ v uporabi in katerih sestavni del je tudi pomnoževanje sekvenc s PCR (npr. platforma Illumina). 2.1 UPORABA KLONIRANJA IN DOLOČANJA NUKLEOTIDNEGA ZAPOREDJA PO SANGER- JU TER PRESEJALNIH METOD V času nastanka prvega preglednega članka o upo- rabi molekulskih pristopov pri raziskavi AM gliv (Ma- ček, 2009) se je za raziskave sestave mikrobnih združb, vključno z združbami rizosfernih AM gliv iz okoljskih vzorcev (tal ali korenin), v veliki meri uporabljalo mo- lekulske pristope, ki so temeljili na postopkih kloniranja in določanja nukleotidnega zaporedja po Sangerju. Če povzamemo na kratko, tak pristop običajno vsebuje na- slednje korake: (1) pomnoževanju glivne DNA z reakci- jo PCR, (2) ‚in vivo‘ ločevanje pomnoženih fragmentov DNA s kloniranjem pomnožkov (amplikonov) v plaz- mide in uporabo kolonij bakterij vrste Escherichia coli za ločevanje sekvenc, (3) ponovna/sekundarna reakcija PCR za namen ločevanja sekvenc, potrebnega za upo- rabo (4) sekventorjev prve generacije (sekvenciranje po Sangerju) (glej opis postopka v Maček, 2009). Vsi na- šteti koraki, predvsem pa kloniranje in uporaba celičnih kultur, so predstavljali velik časovni in finančni zalogaj, zato je bilo število vzorcev (biološke ponovitve), kot tudi število sekvenc, ki so bile sekvencirane za posamezni vzorec (globina sekvenciranja), zelo omejeno. Za posa- mezno okoljsko študijo sestave združbe AM gliv smo na tak način tipično lahko pregledali nekje med 500 in 1000 sekvenc za posamezni genski marker (npr. fragment 18S rRNA). Posledica tega je bila, da so bile združbe opisane zelo pomanjkljivo. Lahko se namreč zgodi, da na ta na- čin nevede izpustimo tiste taksone, ki so znotraj združbe manj pogosti (redki), združbo pa zaradi metodoloških omejitev opisujemo zgolj na podlagi bolj dominantnih in bolj številčnih predstavnikov skupine. Vmesna faza med zgoraj opisanim postopkom in novimi postopki sekvenciranja naslednjih generacij (NGS) je bila uporaba različnih presejalnih metod, ki temeljijo na primerjavah t.i. prstnih odtisov združbe oz. ločevanju produktov PCR z dvojno vijačnico podobne dolžine, vendar z različno sekvenco. Te metode so omo- gočile zajem nekoliko večjega števila vzorcev in tudi večjo globino vzorčenja (število obravnavanih sekvenc). Mednje sodijo metode, kot so denaturacijska gradientna gelska elektroforeza (DGGE), temperaturna gradientna gelska elektroforeza (TGGE) in že omenjeni polimorfi- zem dolžine terminalnih restrikcijskih fragmentov (TR- FLP). Med naštetimi metodami ima TRFLP največjo zmogljivost v smislu možnosti obdelave večjega števila bioloških vzorcev, medtem, ko so tehnike gelske elektro- foreze (DGGE, TGGE) zamudne in omogočajo obdela- vo le manjšega števila vzorcev. To pomeni, da so manj uporabne za ekološke študije, kjer je med vzorci običajno velika variabilnost in je zato potrebno v študijo vključiti veliko število vzorcev. Slaba stran vseh teh metod je tudi ta, da so primarno presejalne narave, zato v končni fazi običajno nimamo vpogleda v sekvence posameznega markerskega gena raziskovanih organizmov. Tako meto- do lahko zato uporabljamo samo za relativne primerjave sestave združb in biodiverzitete med primerjanimi vzorci znotraj ene študije, ne omogočajo pa primerjave različ- nih študij med sabo. Tako je bila pred razvojem in širšo uporabo metod NGS relativno pogosto uporabljana ana- liza za relativne primerjave sestave združb organizmov iz okoljskih vzorcev preučevanje polimorfizma dolžine ter- minalnih restrikcijskih fragmentov (TRFLP) tarčnih ge- nov (prokariontskih 16S ali evkariontskih 18S rRNA), ki v nekaterih primerih omogoča tudi kvantitativne študi- je. Metoda je bila pogosto uporabljana tudi na področju mikrobne ekologije in okoljske mikrobiologije, predvsem zaradi cenovne dostopnosti, filogenetske ločljivosti in enostavne analize večjega števila vzorcev. Analiza TRFLP ima torej dovolj veliko zmogljivost (omogoča obdelavo zadostnega števila vzorcev), da lahko z njo preučimo tudi vplive različnih okoljskih dejavnik- ov na strukturo in dinamiko mikrobnih združb. Na tak način dobimo t.i. ‚prstni odtis‘ (finger-print) združbe raziskovane skupine organizmov za analizirane vzorce, zato tehniko TRFLP imenujemo tudi tehnika prstnih odtisov. Take podatke lahko uporabimo za analizo diver- zitete (izračun različnih indeksov raznolikosti) in sestave združbe, pri čemer lahko izvajamo primerjave med vzor- ci, zajetimi znotraj ene študije, ne pa tudi med različnimi študijami, kar je pomanjkljivost tehnik, ki uporabljajo t. i. tehniko prstnih odtisov. Poznati moramo tudi specifike posamezne preučevane skupine organizmov in omejitve uporabe posamezne metode za to skupino. Primernost metode TRFLP so testirali tudi za kvantitativne analize združb AM gliv, pri tem pa avtorji študije opozarjajo, da tehnika TRFLP v vseh testiranih vzorcih precenjuje vrstno pestrost AM gliv znotraj raziskovane združbe, zato je pri kvantitativnem vrednotenju indeksov razno- likosti potrebna pozornost pri interpretaciji s to tehniko pridobljenih rezultatov (Cotton in sod., 2014). Sama me- toda TRFLP ne producira celotnih sekvenc posameznih organizmov znotraj združbe, se pa lahko s TRFLP pri- dobljeni podatki primerjajo z obstoječo bazo sekvenc, če želimo pridobiti podatke o identiteti posmeznega vzorca (Dickie & FitzJohn, 2007, Roberts in sod., 2012). Danes so tehniko TRFLP v raziskavah ekologije združb mik- Acta agriculturae Slovenica, 118/3 – 20228 I. MAČEK roorganizmov v veliki meri nadomestile tehnike NGS, katerih velika prednost je, da končne izhodne podatke predstavljajo celotne sekvence posameznih amplikonov, kar omogoča lažjo in neposredno identifikacijo organ- izmov, ki pa je odvisna od informativnosti regije, ki jo sekvenciramo za posamezno skupino organizmov. Kot že rečeno, naj bi regija 18S rRNA pri AM glivah približno us- trezala nivoju morfološke vrste, se pa to področje še zelo intenzivno raziskuje in lahko v prihodnosti pričakujemo novosti. 2.2 DOLOČANJE NUKLEOTIDNEGA ZAPOREDJA (SEKVENCIRANJE NASLEDNJIH GENERACIJ – NGS) Metode NGS so najprej uporabljali v medicini in pri poskusih sekvenciranja človeka in drugih primatov (Wheeler in sod., 2008). Relativno hitro so se te metode razširile tudi na področje ekoloških raziskav in raziskav mikrobnih združb (mikrobiomov) v različnih okoljih, saj so omogočale vključevanje bistveno večjega števila vzorcev v raziskavo in obenem bistveno večjo globino sekvenciranja znotraj posameznega vzorca (najprej od več sto tisoč pa vse do več sto milijonov sekvenc pri danes uporabljanih pristopih). Ti novi principi so močno vplivali na številna področja v mikrobni ekologiji, okoljs- ki mikrobiologiji, ekologiji tal in raziskavah rizosfere, kot tudi pri raziskavah različnih interakcij med organizmi, vključno z interakcijami rastlin z drugimi organizmi (npr. mikoriza). Danes je tudi v okoljskih raziskavah pogosta praksa pomnoževanja tarčnih filogenetskih in/ali funkcional- nih genskih označevalcev in obenem uporaba pristop- ov NGS za karakterizacijo njihove raznolikosti. Nova tehnologija omogoča vse številčnejše sete vzorcev, kar je bistvenega pomena za ekološke raziskave, ki se lahko iz- vajajo v različnih dimenzijah, tako prostora, kot tudi časa (vzorčenja v več prostorskih in časovnih točkah, raziskave dinamike procesov in sukcesije). Zavedati pa se moramo, da so prav vsi pristopi NGS podvrženi metodološkim napakam oz. so lahko pristranski, pri čemer gre na eni strani za produkcijo visoko kvalitetnih podatkov, ki jih zahtevajo raziskave, in na drugi napačnih sekvenc ter metodološkega šuma. Vsi pristopi z uporabo NGS zato zahtevajo natančno bioinformacijsko analizo in proce- siranje podatkov, kar omogoča kvalitativno filtriranje in procesiranje sekvenc z namenom izogibanja zavajajočih interferenc iz metodoloških napak. Podobna previdnost je potrebna tudi pri vseh nadaljnjih statističnih anali- zah pridobljenih podatkov, saj lahko nepravilen izbor statističnega pristopa rezultira v napačnih zaključkih raziskave. Prav s tem namenom je nastalo tudi kar nekaj preglednih objav oz. pregleda metodoloških pristopov, ki sistematično podajajo navodila za lažje spopadanje z izzivom obdelave podatkov, ki izhajajo iz NGS (npr. za osnovno analizo podatkov, ki izhajajo iz sekven- ciranja amplikonov z namenom raziskav raznolikosti in ekologije združb AM gliv (Dumbrell in sod., 2017). Tipično bioinformacijski pristopi vključujejo metode za preverjanje kakovosti sekvenc in odstranjevanja šuma, formiranje operacijskih taksonomskih enot (Operational Taxonomic Unit – OTU), taksonomsko določanje posa- meznih sklopov sekvenc ter osnovne statistične analize za testiranje hipotez. Prikaz teh metod za dva pogosto uporabljena pristopa (QIIME in mothur), skupaj s sa- mostojnimi orodji (vključno z odprtokodnim program- skim okoljem ter jezikom R), so predstavljene v objavi Dumbrell in sod. (2017), kjer so predstavljeni pristopi za obdelavo podatkov sekvenciranja amplikonov, ki izhajajo iz dveh pogosto uporabljenih tehnologij NGS, v pretek- losti več uporabljane tehnologije 454-pirosekvenciranja ter danes široko uporabljane platforme Illumina. Zaradi hitrega napredka tehnologije in z njo povezane bioinformatike pa je nujno redno spreml- janje tekočih objav in drugih virov na to temo. Zaradi obsežnosti in kompleksnosti podatkov, ki izhajajo iz tehnik sekvenciranja amplikonov z NGS je pogost izziv tudi njihova ustrezna predstavitev. Izziv, ki se skriva v novih tehnologijah je predvsem ta, da nas lahko za- slepi analitična moč novih metod, obenem pa je naše zavedanje o tem, da zaradi hitrega razvoja teh pris- topov lahko pridemo tudi do napačnih zaključkov, po- manjkljivo. Napačni zaključki so lahko rezultat napak v sami tehnologiji, pomanjkljivega testiranja metod in/ ali pomanjkanja izkušenj. Nujno je, da nek problem oz. študijo že v začetni fazi načrtovanja naslovimo z jasni- mi vprašanji, posledica katerih so tudi jasno zastavljene raziskovalne hipoteze in ciljni metodološki pristopi, ki izhajajo iz teh hipotez. Vsekakor pa predstavljajo me- tode NGS ob pravilni uporabi močno analitično orodje, ki odpira povsem nove možnosti v raziskavah ekologije tako bolj vidnih in karizmatičnih nadzemnih organ- izmov, kot tudi bolj skritih, a pomembnih akterjev v podzemnem delu kopenskih ekosistemov. 3 VPLIV OKOLJSKIH IN GEOGRAFSKIH DEJAVNIKOV NA SESTAVO ZDRUŽB AM GLIV IN NJIHOVO RAZŠIRJENOST Znano je, da sestavo združb AM gliv določajo raz- lični dejavniki okolja, vključno s klimatskimi in talnimi specifikami, ki delujejo tako na lokalni, kot tudi na glo- balni ravni (npr. Dumbrell in sod., 2010, Kivlin in sod., Acta agriculturae Slovenica, 118/3 – 2022 9 Razvoj raziskovalnih metod za karakterizacijo združb arbuskularnih mikoriznih gliv in potencialni vpliv ... na vegetacijo 2011, Lekberg in sod., 2011, Maček in sod., 2011, Ha- zard in sod., 2013, Davison in sod., 2015, Maček in sod., 2019, Vetrovsky in sod., 2019, Davison in sod., 2021). Na splošno je pri pojavljanju različnih taksonov AM gliv še vedno relativno malo dostopnih informacij na ravni odnosa posameznega organizma in okolja (Davison in sod., 2020). Po podatkih iz številnih študij, ki so zbrane v bazi MaarjAM (Öpik in sod., 2010) lahko vidimo, da so mnogi virtualni taksoni (VT) AM gliv široko geograf- sko razširjeni in se pojavljajo v številnih habitatnih tipih (Davison in sod., 2015, Savary in sod., 2018). Taka opa- žanja pa so nastala večinoma na podatkih o prisotnosti taksonov v določenem habitatu, ni pa podatkov v ko- likšni meri številčnost (abundanca) posameznih VT va- riira vzdolž gradienta določenega abiotskega dejavnika. Taksone AM gliv so v preteklosti klasificirali v različne ekotipe (Alzarhani in sod., 2019) ter v ekološke skupine, kot so generalisti in specialisti na osnovi različnih ran- gov, ki vključujejo geografske dejavnike (Moora in sod., 2011, Bouffaud in sod., 2016), habitat (Sykorova in sod., 2007, Oehl in sod., 2010, Vályi in sod., 2015) ali rastlinske gostiteljske vrste (Helgason in sod., 2007). Poudariti pa je treba, da je tovrstno klasificiranje taksonov v posamezne ekološke niše omejeno na obseg okoljskih dejavnikov, ki jih pokriva posamezna študija in posplošenje tega poja- va ni mogoče. V nekaterih novejših objavah poročajo, da so si določene funkcionalne lastnosti med sorodnimi morfološkimi vrstami AM gliv podobne (Powell in sod., 2009, Hoeksema in sod., 2018). Dejstvo pa je, da pred- stavljajo morfološko opisane vrste AM gliv, identificira- ne na podlagi morfologije celične stene njihovih spor, le majhen del raznolikosti AM gliv, ki so bile določene na osnovi molekulskih označevalcev (markerskih genov) (Öpik in sod., 2014). V zelo obsežni nedavni študiji, ki je vključevala več kot 300 talnih vzorcev iz različnih naravnih ekosistemov iz celega sveta so ugotovili, da so porazdelitev VT v različnih ekosistemih skupaj pojasnile tako okoljske, kot tudi prostorske (geografske) variable. Med njimi sta bili temperatura okolja in vrednost pH tal najpomembnejša okoljska določevalnika porazdelitve in lokalne relativne abundance (številčnosti) različnih taksonov AM gliv (Davison in sod., 2021). V študiji so ugotovili tudi različne vzorce ekoloških niš, ki so bili naj- bolj opazni na ravni družin AM gliv, kar kaže, da sorod- ni taksoni oz. VT zavzemajo podobne ekološke niše. Za predstavnike družine AM gliv Acaulosporaceae je tako značilen optimum pri manjši vrednosti pH tal, in nižji temperaturi, medtem, ko so predstavniki družine Gi- gaporaceae bolj številčni v bolj vlažnih območjih z več dežja (Davison in sod., 2021). Na to temo pa bo v prihod- njem desetletju sigurno še veliko novih podatkov, tudi v luči vpliva klimatskih sprememb na AM glive in njihovo simbiozo z rastlinami (Maček in sod., 2019). 4 ZAKLJUČEK Razumevanje vpliva globalnih sprememb na terest- rične ekosisteme zahteva povezovalen pristop med raz- ličnimi disciplinami, ki raziskuje odzive skozi vse ravni biološke organizacije in skozi različne prostorsko-ča- sovne skale. Obenem mora ta pristop vključevati tako nadzemno, kot tudi podzemno raznolikost znotraj eko- sistemov, saj sta obe neločljivo povezani in prepleteni. V večini primerov še vedno ni celostnega razumevanja odziva interakcij komponent nadzemne in podzemne raznolikosti na akutne kratkoročne (npr. suša, toplotni valovi) ter kronične dolgoročne globalne in klimatske spremembe (npr. segrevanje, povečana koncentracija CO2, onesnaženje). Prav zato so nujno potrebni ekspe- rimenti, ki naslavljajo te vrzeli v razumevanju delovanja ekosistemov. Nove metode sekvenciranja omogočajo ve- liko ponovljivost in s tem dokaj robusten pristop k ra- ziskavam v ekologiji, predvsem v smislu velikega števila bioloških ponovitev (zadosti ponovitev za ustrezno ana- lizo podatkov), obravnavo vseh ravni ekoloških združb (nadzemnega in podzemnega – rizosfernega), in v smislu primernega trajanja eksperimenta (zaželene so dolgoroč- ne študije, ki zajemajo vzorčenje preko več sezon ali celo let). Slednje je nujno za razumevanje in ločevanje dol- goročnih in kratkoročnih odzivov na vseh ravneh biod- iverzitete. Najnovejša molekulska orodja za raziskave v eko- logiji združb, kot so metode NGS, ki omogočajo prido- bivanje podatkov o sekvencah organizmov iz različnih okolij, postavljajo vse cenejše in široko dostopne. Pri tem pa je nujno zavedanje, da kljub široki dostopnosti, kva- litetna biološka interpretacija molekulske karakterizacije združb posameznih skupin organizmov zahteva veliko specialističnega znanja o specifični skupini organizmov. Zavedati se moramo na primer, da tako tehnika TRFLP, kot tudi NGS, predstavljata analize relativne številčnosti (abundance) taksonov znotraj vzorcev, zato ju ne smemo uporabljati za ugotavljanje razlik v absolutni številčnosti med posameznimi vzorci. Na koncu se moramo zaveda- ti tudi dejstva, da lahko podajajo ocene sestave združb, ki temeljijo na ekstraktih DNA, le podatke o genski raz- nolikosti in genski sestavi združb. Nemogoča je namreč ocena biomase posameznih vrst teh organizmov v nekem okolju na osnovi takih podatkov zaradi medvrstne in ča- sovne variabilnosti števila kopij posameznih genov na enoto rasti, kar posebej velja za nitaste organizme, kot so AM glive (Corradi in sod., 2007, Jansa in sod., 2008). Bistveno je torej, da se obenem zavedamo tako moči te- hnologije, da v kratkem času producira ogromno količi- no podatkov, kot tudi vseh omejitev in novosti, vključno s hitro spreminjajočimi se postopki bioinformatike, ki je neločljivo povezana z vsakim postopkom NGS. Tak pris- Acta agriculturae Slovenica, 118/3 – 202210 I. 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Delo je prispelo 30. decembra 2021, sprejeto 04. julija 2022 1 Poltava University of Economics and Trade, Faculty of Commodity science, Trade and Marketing, Department of Commodity research, Biotechnology, Expertise and Customs, Poltava, Ukraine 2 Corresponding author, e-mail: asemen2015@gmail.com 3 Poltava University of Economics and Trade, BA in International Economics, Master`s degree student Ultraviolet disinfection of water in recirculating aquaculture system: a case study at sturgeon caviar fish farm Abstract: In this report, we present a practical example of ultraviolet (UV) water disinfection in an aquaculture facility for sturgeon caviar production. Among the methods of water disinfection in recirculating aquaculture systems, the technical approaches using ozonation or ultraviolet radiation in com- bination with other methods are the most effective. However, improper use of ozonation can result in excessive ozone con- centrations that can cause serious harm to fish and be harm- ful to the environment and personnel. Therefore, we describe an example of a reagent-free ultraviolet water disinfection system. Preliminary results show that filtration followed by ultraviolet irradiation inactivates microorganisms in fish tank water. Total microbial count, total coliform bacteria, and E. coli (CFU/m3) did not exceed the permissible values. The described UV system provides an irradiance of 180 W/m2. For a pool with a water volume of 300 m3, bacteriological purity of the water was achieved with 480 W of UV-light. Key words: aquaculture; fish; sturgeon; recirculation sys- tem; UV water disinfection Razkuževanje vode z ultravijolično svetlobo v recirkulacij- skem akvakulturnem sistemu reje: primer ribogojnice jese- trov za prirejo iker, namenjenih za proizvodnjo kaviarja Izvleček: V članku predstavljamo primer razkuževanja vode z ultravijolično (UV) svetlobo v recirkulacijskem akva- kulturnem sistemu reje jesetrov za prirejo iker, namenjenih za kaviar. Med metodami razkuževanja vode pri gojenju rib v zaprtih akvakulturnih sistemih reje so najučinkovitejši pristopi z uporabo ozoniranja ali UV sevanja v kombinaciji z drugimi metodami. Nepravilna uporaba ozoniranja za razkuževanje vode lahko povzroči nastanek prevelikih koncentracij ozona, ki lahko negativno vplivajo na zdravje rib in škodujejo okolju in osebju v ribogojnici. Predstavljamo primer razkuževanja vode, ki temelji na uporabi UV svetlobe. Preliminarni rezultati so pokazali, da razkuževanje vode z metodo filtracije in UV ste- rilizacije zagotavlja učinkovito inaktivacijo mikroorganizmov v bazenu za gojenje rib, saj skupno število mikrobov, skupno število koliformnih bakterij in E. coli (CFU/m3) ni preseglo pri- poročenih vrednosti. Opisani sistem proizvaja jakost UV seva- nja 180 W/m2, kar ob uporabi 480 W kvarčnih UV žarnic zago- tavlja bakteriološko čistost vode za bazen prostornine 300 m3. Ključne besede: akvakultura; ribogojstvo; ribe; jeseter; recirkulacijski sistem; razkuževanje vode; UV svetloba Acta agriculturae Slovenica, 118/3 – 20222 A. SEMENOV and K. SEMENOVA 1 INTRODUCTION In recent years, in Ukraine, as in other countries, industrial farming methods in aquaculture facilities have become increasingly important. These include fish farming in recirculating aquaculture systems (RAS) (Martins et al., 2010; Bulc et al., 2011). This approach can achieve high growth rates with minimal energy costs (Zainal et al., 2021) and is economically advanta- geous because of the reuse of water resources and the possibility of optimizing the hydrochemical regime without depending on environmental conditions. The development of aquaculture farms and the as- sociated increase in production volume has led to prob- lems with effective disinfection of water in RAS. The choice of the method and means of purification of recy- cled water is of crucial importance for the technological cycle of fish farming. New technologies offer alterna- tives to classical water treatment methods (e.g., parti- cle filtration, biofiltration, and gas exchange) (Huyben et al., 2018). Gundula et al. (2019) consider recircula- tion systems as systems that incorporate a number of water purification stages, which consists of: 1) devices for removing solid particles from water, 2) biofilters for ammonia reduction, and 3) gas exchange devices for carbon dioxide removal and oxygen addition. Rearing fish in RAS may be beneficial for increased fish survival rates, when compared to standard cage systems, mostly due to stable microbial environment that prevents op- portunistic microbe multiplication (Dahle et al., 2020). UV irradiation and ozonation are the most com- mon methods of water disinfection in aquaculture. Studies have shown that it is possible to achieve opti- mal conditions for the microbiological composition of water also with the combined effects of filtration, UV radiation, and ozonation (Gregersen et al., 2020; Mid- dlemiss et al., 2015). The effectiveness of these methods, as well as their combination depends on the presence of dissolved and suspended organic compounds in the water (Semenov et al., 2021a). Overexposure to ozone can cause serious damage to fish and can be harmful to the environment (Sharrer et al., 2005). During ozone treatment, microparticles are broken down into molec- ular structures and then removed at different stages of filtration. This method of water purification is suitable for fish incubators that are sensitive to microparticles and bacteria in the water. However, there are arguments against the use of ozonation in RAS (Attramadal et al., 2012) as such systems require a large amount of ozone, which is mostly consumed in the reactions with organic substances, however residual ozone and reaction by- products can be toxic for fish and live feed. On the other hand, UV irradiation of water is considered a safe alter- native to ozonation. When using ultraviolet radiation, the number of microorganisms is significantly reduced (Moriarty et al., 2018) as it inactivates microorgan- isms through photochemical reactions of nucleic acids, which occurs in a special ultraviolet chamber (Semenov et al., 2018) with no harmful effects on fish, environ- ment, and personnel. According to Runia (1995), different irradiances are required for different types of microorganisms: for inactivation of bacteria and fungi from 100 mJ/cm2 and for viruses from 250 mJ/cm2. These relatively high doses compensate for the possible change in turbidity of the water and the change in transmittance of UV ra- diation energy. For example, Sharrer et al. (2005) used UV doses ranging from 75 to 1800 mW/cm2 to achieve inactivation of coliform bacteria in rearing salmonids. However, the inactivation process is not guaranteed if suspended solids are present in the water stream. In practice, radiation intensity of at least 400 mJ/m2 is re- quired for the operation of fish incubators and RAS. In Ukraine, ozonation is the predominant method of water disinfection in aquaculture (Semenov et al., 2021a; Se- menov et al., 2021b). In this report we present an exam- ple of an alternative solution – UV disinfection of water in aquaculture facility for caviar production. 2 MATERIALS AND METHODS Experimental work was carried out in an aquacul- ture farm (Zhashkov, Cherkasy region) when growing sturgeon (Acipenser gueldenstaedtii) for caviar produc- tion. Fish farming was carried out in an insulated hang- ar. Bioload of the system was 53–55 kg of live fish per 1 m2 of pool area. Fish were fed four times a day using commercial diet. All experimental work was carried out in a closed water supply system with a volume of 300 m3, water temperature of 21–22 °С, pH level of 7.3–7.7, and dissolved oxygen content of 5.6–5.8 mg/l. Water purifi- cation was carried out continuously through the water recirculation channel with width of 200 mm and water flow height of 840-860 mm. The recirculation channel provides a water flow of 75 m3/h. For UV disinfection of water, quartz lamps (type ZW80D19W) were used with the power of 80 W, lamp current of 800–1200 mА, and UV (254 nm) irradiance (d = 1 m) of 240–270 μW/cm2. Presence of bacteria was determined with bacteriologi- cal cultures on dense nutrient media, followed by iden- tification of phenotypic or serological properties of the studied strains. Acta agriculturae Slovenica, 118/3 – 2022 3 Ultraviolet disinfection of water in recirculating aquaculture system: a case study at sturgeon caviar fish farm 3 RESULTS AND DISCUSSION Among the types of UV disinfection units consid- ered, there are two types – surface and submersible. A surface sterilizer consists of a battery of UV lamps set-up above the water. Submersible sterilizers, in which water disinfection takes place in the irradiation chamber are more efficient and reliable (Semenov et al., 2018). In or- der to obtain satisfactory results in terms of water quality it is necessary to continuously treat the water. For this, filtration and bacterial disinfection are used together. For The obtained results of bacteriological studies of water when growing fish are presented in Table 1. Bacteriological studies of the water in the pool showed that ultraviolet disinfection combined with fil- tration provides the necessary bacteriological purity of water in pools with a volume of up to 300 m3. With the proposed system, bacteria causing fish diseases such as Flexibacter Cytophaga, Aeromonas and mycoses (Sapro- legniales) were not detected within first three months and after six months. 4 CONCLUSIONS A reagent-free system for disinfecting water in fish breeding pools based on UV irradiation was assembled and tested. In the case of RAS with a water volume of 300 m3, the proper bacteriological quality of the water was ensured for six monitored months by installing UV quartz lamps with a power of 480 W and a UV irradia- tion intensity of 180 W/m2. 5 REFERENCES Attramadal, K. J. K., Øie G., Størseth, T. R., Alver, M.O., Vad- stein, O., Olsen, Y. (2012). The effects of moderate ozonation or high intensity UV-irradiation on the microbial environ- ment in RAS for marine larvae. Aquaculture, 330, 121–129. https://doi.org/10.1016/j.aquaculture.2011.11.042 Bulc, T. G., Istenic, D., Klemenci, A. K. (2011). The efficiency of a closed-loop chemical-free water treatment system for cyprinid fish farms. Ecological Engineering, 37(6), 873–882. https://doi.org/10.1016/j.ecoleng.2011.01.004 Dahle, S. W., Bakke, I., Birkeland, M., Nordøy, K., Dalum, A. S., Attramadal, K. J. K. (2020). Production of lumpfish (Cy- clopterus lumpus L.) in RAS with distinct water treatments: Effects on fish survival, growth, gill health and microbial communities in rearing water and biofilm. Aquaculture, 522, 735097. https://doi.org/10.1016/j.aquaculture.2020.735097 Gregersen, K. J., Pedersen, P. B., Pedersen, L.-F., Liu, D., Dals- gaard, J. (2020). UV irradiation and micro filtration effects on micro particle development and microbial water qual- ity in recirculation aquaculture systems. Aquaculture, 518, 734785. https://doi.org/10.1016/j.aquaculture.2019.734785 Figure 1: Scheme of the water purification and disinfection installation the purification and disinfection of water for sturgeon fish farming in RAS we assembled a UV irradiation sys- tem schematically presented in Figure 1. The system includes the following: 1) installation for removing coarse dirt, 2) installation for removing highly dispersed impurities (fine cleaning), 3) installation for ultraviolet water disinfection, and 4) equipment for pH correction, water saturation with oxygen, water heating and electronic control system. For the inactivation of mi- croorganisms, a 480 W UV installation has been devel- oped, which consists of six low-pressure ultraviolet lamps with the previously described characteristics. Lamps are placed after the filtration units and fitted vertically within the water flow. They are inserted in quartz glass covers to maximize the irradiation area. The total bactericidal flow is 180 W/m2. 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