ACTA s AGRiCULTURAE SLOVENICA Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica • ISSN 1581-9175 • 115 - 1 • Ljubljana, marec 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 1 26. 03. 2020 12:05:37 AAS_vsebina_2020_115_1_koncna 180320.indd 2 26. 03. 2020 12:05:37 Acta agriculturae Slovenica Volume / Letnik 115 • Number / Številka 1 • 2020 VSEBINA / CONTENTS Abdelkader AMEUR AMEUR, Onur YILMAZ, Nezih ATA, Ibrahim CEMAL, Semir Bechir Suheil GAOUAR 5 Assessment of genetic diversity of Turkish and Algerian native sheep breeds Pregled genetske pestrosti turških in alžirskih avtohotnih populacij ovac Oksana DMITRIEVA, Semen IVANOV 15 Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation Primerjalna raziskava vrst ščira (Amaranthus spp.) v zmernem kontinentalnem podnebju Ruske Federacije Josiah E. IFIE, Sandra IFIE-ETUMAH, Beckley IKHAJIAGBE 25 Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity Fiziološki in biokemični odziv akcesij kitajske vinje (Vigna unguiculata (L.) Walp.) na toksičnost železa Mohammad MALMIR, Rahim MOHAMMADIAN, Ali SOROOSHZADEH, Ali MOKHTASSI-BIDGOLI, Somayeh EHSANFAR 39 The response of the sugar beet (Beta vulgaris L.ssp. vulgaris var. altissima Doll) genotypes to heat stress in initial growth stage Odziv genotipov sladkorne pese (Beta vulgaris L. ssp. vulgaris var. altissima Doll) na vročinski stres v začetnih fazah rasti Leila AMRAEE, Fatemeh RAHMANI 53 Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.) CTAB protokol za ekstraskcijo RNK iz melise (Melissa officinalis L.) Alexandr V. AMELIN, Aleksey N. FESENKO, Evgeniy I. CHEKALIN, Ivan N. FESENKO, Valeriy V. ZAIKIN 59 Higher yielding varieties of common buckwheat (Fagopyrum esculentum Moench) with determinate growth habit (single mutation det) manifest higher photosynthesis rate at stage of grain filling Bolj donosne sorte navadne ajde (Fagopyrum esculentum Moench) z determinantno rastjo (enojna det mutacija) imajo večjo fotosintezo v fazi polnjenja zrnja Valeriia BONDAR, Nataliia MAKARENKO 67 Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Pridelava ozimne pšenice v Ukrajini: ekološka ocena tehnologij glede na vplive na rodovitnost tal Abhijit Kishanrao KADAM, Agbesi Kwadzo KETEKU, Suchada DANA , Precious Kwaku BLEGE 79 Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility Vpliv priprave zemljišča in gnojilnih tehnik na pridelek soje (Glycine max (L.) Merrill.), na vlažnost in rodovitnost tal Sandra PESTAR BIZJAK, Hristo HRISTOV, Aleš KUHAR 89 Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers Dimenzije zaznane vrednosti vina s perspektive slovenskih kupcev vina Asgar EBADOLLAHI, Reza SADEGHI 97 Comparison of the toxicity and repellency of two conventional neonicotinoids and a coconut-derived insecticide soap toward the parasitoid wasp Aphelinus mali Haldeman, 1851 Primerjava strupenosti in odvračalnega delovanja dveh konvencionalnih neonikotinoidov in insekticidnega mila iz kokosa na krvavkinega najezdnika (Aphelinus mali Haldeman, 1851) AAS_vsebina_2020_115_1_koncna 180320.indd 3 26. 03. 2020 12:05:37 Sara KHOSRAVI, Reza AZIZINEZHAD, Amin BAGHIZADEH, Mahmood MALEKI 105 Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid and hexaploid cultivars of wheat using stress tolerance indices Ovrednotenje in primerjava tolerance na sušo nekaterih divjih diploidnih populacij, tetraploidnih in heksaploidnih sort pšenice z uporabo indeksov tolerance na stres Seyyed Shahaboddin HOSEINI, Asad ROKHZADI, Ezzat KARAMI 113 Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions Medsebojni vpliv sejalnih metod in uporabe zeolita na komponente pridelka čičerke (Cicer arietinum L.) v sušnih razmerah Mahmoud o. HASSAN 123 Allelopathic interference of Sonchus oleraceus L. with wheat and the associated weeds: a field study Alelopatični učinek navadne škrbinke (Sonchus oleraceus L.) na pšenico in njene plevele: poljski poskus Emmanuel T. NNEJI, Robert O. UDDIN II, Abdulrasak K. MUSA 133 Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. King & H. Rob. extracts on some field insect pests of okra (Aabelmoschus esculentus (L.) Moench) Učinki izvlečkov iz rastlin Boerhavia diffusa L. nom. cons. in Chromolaena odorata (L.) R.M. King & H. Rob. na nekatere škodljive žuželke na jedilnem oslezu (Aabelmoschus esculentus (L.) Moench) Jure ČOP, Klemen ELER 141 Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): primerjava in agronomska vrednost Agro-biological diversity of Slovene ecotypes and standard varieties of cocksfoot (Dactylis glomerata L.): comparison and agronomic value Hajar SABET, Mahmood MALEKI, Maryam ABDOLI NASAB, Saeid MIRZAEI 151 Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Somatska embriogeneza hipokotilnih kalusov izbrane sorte jajčevca Yimam ALI ABTEW, Alemu ABATE 161 Association of traits in Ethiopian fenugreek (Trigonella foenum-graecum L.) genotypes regarding to seed yield by using phenotypic data Povezava med fenološkimi značilnostmi pri različnih genotipih sabljastega triplata (Trigonella foenum-graecum L.) Faraj MOAYEDI, Sajad KORDI, Ali Ashraf MEHRABI 171 Evaluation of yield, chemical composition and yield of essential oil of four cultivars of sweet basil (Ocimum basilicum L.) affected by different levels of nitrogen Ovrednotenje pridelka, kemijske sestave in pridelka eteričnih olj štirih sort navadne bazilike (Ocimum basilicum L.) pri različnem gnojenju z dušikom Paolo CASINI, Gherardo BIANCOFIORE 183 Optimizing sowing time for boosting productivity and nutritional quality of amaranth (Amaranthus cruentus L.) genotypes under Mediterranean climate Optimizacija časa setve za povečanje produktivnosti in prehranske kakovosti genotipov zrnatega ščira (Amaranthus cruentus L.) v mediteranskih klimatskih razmerah Andrej SEČNIK, Sebastjan RADIŠEK, Nataša ŠTAJNER, Jernej JAKŠE 193 Študij polarnosti verig različnih viroidov in njihovih kombinacij pri okuženih rastlinah hmelja Studying strands polarity of different viroids and their combinations in infected hop plants Paulina G. FLORES 203 Cows Save the Planet: And Other Improbable Ways of Restoring Soil to Heal the Earth", by Judith Schwartz. Chelsea Green Publishing. ISBN: 9781603584326. Pp 240; USD 17.95 "Krave rešite planet: in drugi neverjetni načini za ohranitev tal pri zdravljenju Zemlje", Judith Schwartz. Chelsea Green Publishing. ISBN: 9781603584326. Pp 240; USD 17.95 207 Navodila avtorjem Author guidelines AAS_vsebina_2020_115_1_koncna 180320.indd 4 26. 03. 2020 12:05:37 doi:10.14720/aas.2020.115.1.1229 Original research article / izvirni znanstveni članek Assessment of genetic diversity of Turkish and Algerian native sheep breeds Abdelkader AMEUR AMEUR 1 2, Onur YILMAZ 3, Nezih ATA 3, Ibrahim CEMAL 3, Semir Bechir Suheil GAOUAR 1 Received July 18, 2019; accepted December 27, 2019. Delo je prispelo 18. julija 2019, sprejeto 27. decembra 2019. Assessment of genetic diversity of Turkish and Algerian native sheep breeds Abstract: In Algeria and Turkey, the sheep production systems are based on the under extensive rural conditions and their genetic management has led to increased homozygosity and hence productivity loss. The identification of inter-breed and intra-breed genetic diversity plays a key role in the shaping of conservation and breeding programs. The present study was conducted to investigate the genetic diversity of native sheep breeds reared in Turkey and Algeria. A total of 240 animals from four Algerian (Hamra, Ouled Djellal, Sidaou, and Tazegzawt) and four Turkish (White Karaman, South Karaman, Karacabey Merino, and Kivircik) native sheep breeds were gen-otyped with fourteen microsatellite markers recommended by FAO. A total of 340 alleles were detected from fourteen markers studied. All the eight breeds exhibited moderate to high levels of genetic diversity, with a slight superiority of the Algerian sheep breeds. Overall FIS value was low, but highly significant (p < 0.001). It may have been due to the high inbreeding within the population. The mean global coefficient of gene differentiation (GST) showed that approximately 94.0 % of the genetic variation was within-population. The highest number of private alleles with a frequency above 5 % was observed in Ouled Djellal sheep. Structure analysis of populations studied revealed the most appropriate K with four genetic clusters. As the result, the dendrogram showed that the Algerian sheep breeds were completely separated from the Turkish sheep breeds furthermore the Bayesian clustering revealed a high level of admixture, especially in Algerian sheep populations. Key words: small ruminants; native sheep breeds; genetic diversity; microsatellite; genetic distances Pregled genetske pestrosti turških in alžirskih avtohotnih populacij ovac Izvleček: Sistemi reje ovac v Alžiriji in Turčiji temeljijo na ekstenzivni kmečki reji, upravljanje s temi populacijami pa je privedlo do povečanja homozigotnosti in s tem do poslabšanja proizvodnih lastnosti. Ocena medpasemske in znotraj-pasemske genske pestrosti igra ključno vlogo pri oblikovanju programov za ohranjanje teh pasem. Pričujoča študija je bila izvedena z namenom raziskovanja genske pestrosti avtohtonih pasem ovac v Turčiji in v Alžiriji. Skupno 240 živali štirih alžirskih (hamra, ouled djellal, sidaou in tazegzawt) in štirih turških (white karaman, south karaman, karacabey merino in kivircik) pasem je bilo genotipiziranih s štirinajstimi mi-krosatelitnimi markerji, ki jih priporoča FAO. Na štirinajstih analiziranih markerskih lokusih je bilo zabeleženih 340 alelov. Vseh osem pasem je imelo zmerno do visoko stopnjo genske pestrosti z rahlo prevlado alžirskih pasem ovac. Skupna vrednost FIS je bila nizka, vendar statistično značilna (p < 0,001). To je verjetno posledica visokega inbridinga v populaciji. Povprečni globalni koeficient diferenciacije genov (GST) je pokazal, da je približno 94,0 % genske pestrosti znotraj populacije. Največ privatnih alelov s pogostostjo nad 5 % je bilo ugotovljenih pri ovcah pasme ouled djellal. Analiza strukture populacij je pokazala, da je K, ki predvideva štiri genetske klastre najprimernejši. Dendrogram, ki je rezultat študije, je pokazal, da so alžirske pasme ovac popolnoma ločene od turških, poleg tega pa je Bayesovo klastriranje pokazalo visoko stopnjo križanja, zlasti v alžirskih populacijah ovac. Ključne besede: drobnica; avtohtone pasme ovac; genska pestrost; mikrosateliti; genetske distance 1 University of Tlemcen, Department of Biology, Laboratory of Physiopathology and Biochemistry of Nutrition, Algeria 2 Corresponding author, e-mail: ameurabdelkader@gmail.com 3 Adnan Menderes University, Faculty of Agriculture, Department of Animal Science, Aydin, Turkey Acta agriculturae Slovenica, 115/1, 5-14, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 5 26. 03. 2020 12:05:37 A. AMEUR AMEUR et al. 1 INTRODUCTION Domestic sheep (Ovis aries) has been a very important farm animal species for the people economically and culturally since its domestication time around the world (Ryder, 1983). Algeria and Turkey are endowed with diverse farm animal genetic resources including the sheep breeds they are favored by the different climates existed and vegetation, which has since generated a very specific sheep breeding practice. The majority of the sheep population of Turkey and Algeria is composed of multipurpose native breed producing meat, milk, and wool. According to Turkish Statistics Agency, there are 31.2 million sheep heads distributed among twenty sheep breeds have been officially registered while Algeria has a large local sheep population with around 28 million head (Faostat, 2016), the latter contains twelve local ovine breeds with very different phenotypic characteristics (Ameur Ameur et al., 2017). Turkey has a great genetic diversity that can be characterized by numerous sheep breeds are categorized into four main groups: Fat-tailed, thin-tailed, crossbreeds and extinct breeds (Yilmaz et al., 2013). South Karaman sheep breed, which is one of these breeds and especially raised in the Taurus Mountains located in the Mediterranean region, is a fat tail native sheep breed. It was reported that the hides of this breed, which is very similar to the Karagul breed, could be used in making inner fur because of having a curly pattern of fleece (Ertugrul et al., 2009; Kiraz et al., 2014). White Karaman sheep breed is the most commonly used breed in central Anatolia in Turkey for lamb production, with a population of around 16.000,000 it makes up more than 50 % of the national sheep herd (TUIK, 2018). Kivircik and Karacabey Merino sheep breeds are especially raised in western part of Turkey are known for their meat quality, wool, and meat production (Oner et al., 2014; Yilmaz et al., 2011, Karaca et al., 2009). Karacabey Merino was developed by crossbreeding of Kivircik and German Black Head Mutton (Yalcin, 1986). Algerian domestic sheep breeds were classified according to many different methods such as phenotypic data, morphological and molecular descriptions throughout history (Chelig, 1992; Djaout et al., 2017). The Ouled Djellal breed (white Arabian breed), which constitutes more than half of the Algerian sheep population and is widely raised in Algeria. This breed has a good body conformation, has a high level of adaptability to different climatic conditions (Chelig, 1992; Djaout et al., 2017). The Hamra sheep breed known as Deghma is a very important sheep breed for meat tenderness in Algeria (Chelig, 1992; Djaout et al., 2017). The Sidaou breed, known as Targuia, is raised in the Sahara between Libya-Niger and southern part of Algeria with several more than one million head (Chelig, 1992; Djaout et al., 2017). Tazegzawt sheep breed which is constituted 0.02 % of the total population raised in Kabyle and Ham in the region of Mechria (Chelig, 1992; Moulla, 2015; Djaout et al., 2017). In recent years, non-systematic cross-breeding practices and changes in consumer habits have triggered a quantitative reduction of this breed and quickly faced the risk of extinction threat (Moulla, 2015). Autosomal microsatellites are a well-known effective and powerful tool to investigate genetic structure and diversity have been widely used in sheep breeds, in all over the world, for Turkish sheep breeds (Koban, 2004; Gutiérrez-Gil et al., 2007; Yilmaz and Karaca, 2012; Yilmaz et al.; 2013; Cemal et al., 2013; Yilmaz et al., 2014) and for the Algerian sheep breeds (Gaouar et al., 2014; Gaouar et al.,2015; Gaouar et al.,2016a; Ghernouti et al., 2017; Ameur Ameur et al., 2018). Figure 1: Geographical location of Algeria and Turkish sheep breeds WK: White Karaman, SK: South Karaman, KM: Karacabey Merino, KIV: Kivircik, HR: Hamra, OD: Ouled Djellal, SD: Sidaou, TG: Tazegzawt Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 6 26. 03. 2020 12:05:36 6 Assessment of genetic diversity of Turkish and Algerian native sheep breeds The first step for a well-structured and sustainable animal breeding and conservation program is to reveal detailed information on intra and inter-breed genetic diversity. This situation indicates how important it is to reveal the genetic structure of breeds. The objective of the present study was to determine genetic diversity and population structure of different native sheep breeds raised in two different countries. 2 MATERIALS AND METHODS 2.1 ANIMAL MATERIAL AND DNA ISOLATION Blood samples were obtained from 240 sheep, which consist of Hamra (30), Ouled Djellal (30), Sidaou (30), Tazegzawt (30) raised in Algeria and White Karaman (30), South Karaman (30), Karacabey Merino (30) and Kivircik sheep (30) breeds raised in Turkey (Figure 1). 2.2 SAMPLING METHOD AND DNA ISOLATION Blood samples were obtained from 240 head sheep. Blood samples were collected from Vena jugularis into tubes containing K3-EDTA as anticoagulant and stored at -20 °C until DNA extraction. DNA was extracted by using the salting-out technique reported by Miller et al. (1988) and Montgomery and Sise (1990). NanoDrop 2000 (Thermo Scientific, Waltham, MA) spectropho- tometer device was used to determinate the quality and quantity of DNA samples. 2.3 PCR AND FRAGMENT ANALYSIS Fourteen microsatellite markers labeled with a fluorescent dye (D2, D3, and D4) were used according to the recommendation of FAO (2011). Two multiplex groups were created according to the fragment length of microsatellites. Touchdown PCR protocols reported by Hecker and Roux, (1996) were used for the amplification of specific genomic regions (Table 1). The total volume of the amplification mixture amounted to 25 pL. Amplification mixture contained 0.1 pM/each primer, 0.2 mM dNTPs (Applied Biological Materials Inc., Canada), 2.0 mM MgCl2, 1X PCR buffer, 1U of Taq DNA polymerase (Applied Biological Materials Inc.. Canada) and ~50 ng genomic DNA. Capillary electrophoresis was used for the separation of the PCR fragments labeled with fluorescent dye in the Beckman Coulter GeXP genetic analyzer (Beckman Coulter. Inc. USA).GenomeLab™ DNA Size Standard Kit 400 was used for the determination of fragment size. 2.4 STATISTICAL ANALYSIS The polymorphism statistics such as number of al-leles per locus (Na), mean number of alleles (MNa), effective number of alleles (Ne), observed heterozygosity Table 1: Thermal cycling conditions according to Touchdown PCR Loci (Dye) Multiplex First group denaturation Denaturation Annealing Extension Cycle Final extension OarFCB193 (D3) 1 95 °C 95 °C 63-54°C 72 °C 40 72 °C 0arFCB304 (D3) (5 min) (40 sec) (40 sec) (60 sec) (10 min) INRA0023 (D3) oarCP34 (D4) D5s2 (D4) BM1818 (D4) BM8125 (D3) McM0527 (D3) CsRD0247 (D3) OarFCB128 (D2) BM1329 (D2) 2 95 °C (5 min) 95 °C (40 sec) 60-50 °C (40 sec) 72 °C (60 sec) 34 72 °C (10 min) HSC (D2) OarJMP29 (D4) MAF214 (D4) Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 7 26. 03. 2020 12:05:37 7 A. AMEUR AMEUR et al. (Ho), expected heterozygosity (He), Wright's F-statistics (FIT> FIs, FsT), Hardy-Weinberg equilibrium and Principal component analysis (PCoA) was performed using the software GENALEX (Peakall & Smouse, 2006) were calculated using GenAlEx (Peakall and Smouse, 2012). Polymorphic information content (PIC) and null allele frequencies were calculated using CERVUS 3.0.3 (Marshall, 1998). Populations 1.2.32 (Langella, 1999) and FigTree 1.4.2. (Rambout, 2006) software was used to generate neighbor-joining (NJ) tree phylogenetic tree between breeds according to Nei's Da distance matrix (Nei et al., 1983). Robustness of the dendrogram topology was tested by bootstrap resampling (n = 1000). FSTAT version 2.9.3 software (Goudet, 2001) was used to obtain values belong to genetic diversity statistics such as Nei's gene diversity (HT), diversity between breeds (DST), and coefficient of gene differentiation (GST). Analysis of molecular variance (AMOVA) was performed using the ARLEQUIN v3.5.2.2 (Excoffier and Lischer, 2010). The STRUCTURE software was used to analyze population structure using independent allele frequencies and an admixture model (burn of 20.000, followed by 100.000 MCMC iterations with 20 replicate runs for each K) (Pritchard et al., 2000). The appropriate number of clusters was identified using AK values (K = 2 to 8) that expressing the proportion of alteration in the logarithmic probability Pr(X|K) (Evanno et al., 2005). The most suitable K value was determined according to AK value calculated by the STRUCTURE harvester program (Earl and Vonholdt, 2012). The CLUMPAK program reported by Kopelman et al. (2015) was used to find the best alignment from the obtained STRUCTURE results. 3 RESULTS In this study, a total of 340 alleles were identified from fourteen microsatellites used in the present study. Molecular genetic statistical parameters obtained from the fourteen microsatellites used was given in Table 2. The number of alleles ranged from 17 (OarCP34, D5S2) to 32 (CSRD0247), while the average number of effective alleles was 10.99. observed heterozygosity (Ho) values varied from 0.67 (0arFCB304) to 0.85 (OarCP34). PIC values were found to be between 0.87 and 0.93. F values that use the local gene pool as a reference point were obtained as positive in thirteen of fourteen microsatellites. The FIT value expressing general heterozygosity loss was higher in BM1329 (0.254) locus than the other. FST described as an indicator of genetic variation among individuals within the population was observed varied from OarJMP29 (0.041) to BM1329 (0.107) with a mean of 0.068. Mean value of DST indicating genetic diversity between breeds, GST, which is an important indicator of Table 2: Genetic polymorphism parameters of the fourteen investigated loci in sheep breeds studied Locus N Na Ne PIC Ho He F * is F* it F* st dst gst ht HWE F(Null) 0arFCB304 237 25 8.22 0.87 0.67 0.88 0.172*** 0.252*** 0.096*** 0.074 0.084 0.88 *** 0.134 OarFCB193 239 23 9.17 0.89 0.80 0.89 0.066** 0.105*** 0.042*** 0.033 0.036 0.89 *** 0.053 BM1818 234 28 14.17 0.93 0.83 0.93 0.047* 0.118*** 0.075*** 0.061 0.065 0.93 *** 0.059 INRA0132 240 21 11.92 0.91 0.82 0.92 0.052* 0.113*** 0.064*** 0.052 0.056 0.92 *** 0.057 OarCP34 239 17 7.68 0.86 0.85 0.87 -0.029ns 0.027ns 0.055*** 0.042 0.049 0.87 *** 0.008 D5S2 207 17 8.19 0.87 0.73 0.88 0.109** 0.181*** 0.080*** 0.061 0.069 0.88 *** 0.092 CSRD0247 230 32 13.21 0.92 0.76 0.92 0.120*** 0.186*** 0.075*** 0.062 0.067 0.93 *** 0.103 MCM0527 232 20 9.59 0.89 0.68 0.90 0.207*** 0.252*** 0.056*** 0.044 0.049 0.90 *** 0.140 BM8125 240 20 12.70 0.92 0.78 0.92 0.094*** 0.169*** 0.083*** 0.068 0.074 0.92 *** 0.083 HSC 231 24 14.48 0.93 0.72 0.93 0.191*** 0.230*** 0.048*** 0.039 0.042 0.93 *** 0.122 BM1329 237 31 14.13 0.93 0.70 0.93 0.164*** 0.254*** 0.107*** 0.088 0.094 0.93 *** 0.137 OarFCB128 240 22 10.22 0.90 0.76 0.90 0.116*** 0.162*** 0.052*** 0.041 0.046 0.90 *** 0.086 OarJMP29 240 31 11.20 0.91 0.81 0.91 0.081*** 0.119*** 0.041*** 0.033 0.036 0.91 *** 0.058 MAF214 240 29 8.92 0.88 0.77 0.89 0.071* 0.142*** 0.076*** 0.060 0.068 0.89 *** 0.077 Mean 24.29 10.99 0.90 0.76 0.90 0.104 0.165 0.068 0.054 0.060 0.91 N: Number of genotyped individuals, Na: number of alleles, Ne: effective number of alleles, PIC: polymorphic information content, Fjp F , FST: Wright's F-statistics, Ho: observed heterozygosity, He: expected heterozygosity, HWE: Hardy-Weinberg Equilibrium, F(Null): null allele frequency, Ht: Nei's gene diversity, DST: the diversity between breeds, GST: coefficient of gene differentiation, *: Wright's statistics according to Weir and Cocker-ham (1984), *: p < 0.05, **: p < 0.01, ***: p < 0.001 8 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 8 26. 03. 2020 12:05:37 Assessment of genetic diversity of Turkish and Algerian native sheep breeds Table 3: Genetic polymorphism parameters according to studied Turkish and Algerian sheep breeds across 14 loci Mean Heterozigosity NPA Freq. Freq. Breeds MNA Ho (SE) He (SE) fis HWE > 5% < 5% Total WK 9.93 0.78 (0.040) 0.80 (0.016) 0.047ns 5 5 3 8 KIV 9.57 0.58 (0.056) 0.81 (0.012) 0.326*** 9 3 1 4 KM 10.21 0.67 (0.058) 0.76 (0.030) 0.114*** 11 1 5 6 SK 9.57 0.72 (0.063) 0.74 (0.059) 0.055** 2 2 3 5 HR 14.71 0.84 (0.023) 0.89 (0.006) 0.055*** 8 1 5 6 OD 17.00 0.83 (0.022) 0.90 (0.005) 0.075*** 5 - 17 17 SD 13.86 0.82 (0.028) 0.87 (0.010) 0.025ns 4 1 7 8 TG 15.07 0.83 (0.028) 0.86 (0.014) 0.038* 7 1 11 12 WK: White Karaman, SK: South Karaman, KM: Karacabey Merino, KIV: Kivircik, HR: Hamra, OD: Ouled djellal, SD: Sidaou, TG: Tazegzawt, MNA: number of alleles, Ho: observed heterozygosity. He: expected heterozygosity, HWE: number of loci not in the Hardy-Weinberg equilibrium (p < 0.05), NPA: private alleles, within-breed, FIS: heterozygote deficiency, *: p < 0.05, **: p < 0.01, ***: p < 0.001 the relative magnitude of genetic differentiation, and HT described as total genetic diversity, values were found as 0.054, 0.060 and 0.91, respectively. Microsatellite loci genotyped in the present study were tested using the x2 test in terms of compliance with HWE. All fourteen loci and SK) to 17.00 (OD). The highest expected heterozygosity was observed in OD (0.90) sheep breed reared in Algeria. All the studied breeds showed positive F values. Although a total of 66 private alleles have been identified in all breeds studied, only fourteen of them have a fre- Table 4: AMOVA of the eight sheep breeds genotyped with fourteen microsatellite markers Variation Sources DF SS VC PV (%) FI Among population 7 204.88 0.3893 Va 6.71 fis : = 0.092 Among individuals within populations 232 1370.53 0.4964 Vb 8.56 F st = 0.067 Within individuals 240 1179.50 4.9145 Vc 84.73 fit = 0.153 Total 479 2754.91 5.800 DF: degree of freedom, SS: sum of square, VC: variance components, PV: percentage of variance, FI: fixation index deviated from the HWE (p < 0.001). Null allele frequency belonging to microsatellite loci used were found to be below 20 %. The results of genetic diversity statistics for each breed are summarized in Table 3. The mean number of alleles varied from 9.57 (KIV White Karaman Klvlrclk South Karaman ^ ♦ ♦ Karacabey Merino ♦ □uled djelal Sidaou ♦ Hamra * Tazegzawt 35.37% Figure 2: Principal component analysis. Plot of the first (PC1: X axis) second (PC2: Y axis) principal components for 8 sheep populations. quency greater than 5 %. Analysis of Molecular Variance (AMOVA), which is a method to detect population differentiation utilizing molecular markers, was performed to detect genetic variation between individuals and populations (Table 4). It was revealed 84.73 % of the total variance was found within individuals while 8.56 % among individuals within populations and 6.71 % among the population. In the PCA analysis (Figure 2) of the Nei's genetic distance, the first two axes represent 35.38 % and 25.83 % of the total genetic variability, respectively. The phylogenetic network of eight sheep breeds (Figure 3) confirmed and complemented the PCA analysis results. Four cluster belonging to eight breeds studied was revealed in dendrogram based on Nei's Da distance matrix. The first cluster consisted of WK, Acta agriculturae Slovenica, 115/1 - 2020 and AAS_vsebina_2020_115_1_koncna 180320.indd 9 26. 03. 2020 12:05:37 9 A. AMEUR AMEUR et al. 0.7 Figure 3: Dendrogram based on Nei's Da distance matrix in studied sheep breeds (bootstrap resampling methodology (1000 replicates)) (WK: White Karaman, SK: South Karaman, KM: Karacabey Merino, KIV: Kivircik, HR: Hamra, OD: Ouled djel-lal, SD: Sidaou, TG: Tazegzawt) Table 5: Estimated posterior probabilities [Ln Pr(X|K)] for different numbers of inferred clusters (K) and AK statistic K Mean LnP(K) AK 2 -16.760,605 — 3 -16.232,850 3.7415 4 -16.025,320 6.1730 5 -16.209,625 0.5631 6 -15.647,060 3.9293 7 -15.638,650 2.5657 8 -16.353,680 — To present the suitable cluster number (K) in structure analysis results were given in Table 5. The results obtained from the STRUCTURE analysis were similar to the dendrogram drawn according to Nei's Da distance matrix (Nei et al., 1983) as expected. It is seen that the optimal number of groups was 4 considering the value of AK obtained by the method reported by Evanno et al. (2005). 4 DISCUSSION SK and KIV, the second cluster was formed by KM sheep breed raised in Turkey, the third cluster was formed by OD sheep breed and fourth cluster was formed by HR, TG and SD sheep breeds sampled from Algeria. The results of the Population structure analysis containing different numbers of clustering (K = 2-8) and performed to determine the population structure of the studied breeds are given in Figure 4. K=2 K=3 K=4 K=5 K=6 K=7 K=8 WK SK KIV KM TG OD HR SD Figure 4: Estimation of the population structure with different K values (WK: White Karaman, SK: South Karaman, KM: Karacabey Merino, KIV: Kivircik, HR: Hamra, OD: Ouled Djellal, SD: Sidaou, TG: Tazegzawt) Today, conservation of farm animal species and the determination of the genetic diversity is currently on the agenda of animal breeders. In this context, conservation activities and characterization of genetic diversity for animal genetic resources have become very important phenomena in all over the world. Genetic variation needed for genetic improvement of domestic animals is a basic requirement for animal breeding (Askari et al., 2011). Mean number of alleles and polymorphic information content values observed in the present study were higher than values obtained from the other sheep breeds (Yilmaz et al., 2015; Guang-Xin et al., 2016; Kirikçi et al., 2018). This situation can be regarded as an important indicator of high genetic diversity in sheep populations studied. F values, which is a measure of the deviation of genotypic frequencies from panmixia in populations in terms of heterozygous deficiency or excess, showed that loss of heterozygosity at just one microsatellite locus (OarCP34). The value of overall F was low, but highly significant (p < 0.001). It may have been due to the high inbreeding within the population. The local inbreeding coefficient (F ) values, which is used the local gene pool as the point of reference, was low but highly significant (p < 0.01) except OarCP34. Similar findings have been found in previous experiments, conducted in different sheep breeds (Yilmaz et al., 2015). Overall FST value indicated that a moderate 10 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 10 26. 03. 2020 12:05:37 Assessment of genetic diversity of Turkish and Algerian native sheep breeds genetic drift occurred in populations. It can be said that this situation is an expected finding given that populations are reared under extensive conditions and are mated freely. The global GsT value showed that 94.00 % of the total genetic variation can be explained by genetic differences among individuals. It can be accepted that the overall genetic diversity value (DST) obtained from the present study was an indication that the inter-population variability is not high. This finding supported the previously mentioned FST and GST results. Overall HT described as Nei's gene diversity value was 0.91 which was higher than the values obtained from Algerian native sheep breeds (Ameur Ameur et al., 2018), Turkish native sheep breeds (Yilmaz et al., 2015) and Albanian native sheep breeds (Hoda and Marsan, 2012) This finding supported Na and PIC values is an indication that goats have a high genetic diversity of populations studied. The Hardy-Weinberg equilibrium (HWE), which is stated that allele and genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary mechanisms, was analyzed using x2 test. Test results showed that allele distributions of all studied loci deviated from Hardy-Weinberg equilibrium. This is an expected finding due to the intensive selection studies conducted in the studied populations for long years. Null allele, which was first introduced by Paetkau and Strobeck (1995), causes to misreading of the microsatellite peaks. It has been reported by Dakin and Avise (2004) that null allele frequencies below 0.20 have no significant effect on molecular genetic studies performed with microsatellites. When the null allele frequencies obtained are examined, it is seen that the null allele frequency values of fourteen microsatellites to be studied are below 0.20. Taking this value into consideration, it has been demonstrated that working locus can be used safely in genetic diversity. In the present study, the calculated MNa values belonging to Algerian sheep populations studied were found to be higher than the values stated in some studies on domestic and foreign breeds (Gaouar et al., 2016b; Loukovitis et al., 2016; Naqvi et al., 2017) but MNa values observed in Turkish sheep breeds were lower than those of native sheep breeds raised in Tunisia (Sassi-Zaidy et al., 2016). This is thought to be due to the difference in the number of microsatellites and sampling methodology used in this study. FIS value, defined as the inbreeding coefficient, indicated that there is no loss of heterozygosi-ty in populations. Deviations from the Hardy-Weinberg equilibrium should be regarded as a natural consequence of the intensive animal breeding activities that have been practiced in the populations for many years. Although the number of private alleles defined as the source of genetic diversity which has a frequency above 5 % are limited, it can be said that they have sufficient efficiency to identify populations studied. It is seen that the essential genetic diversity is realized within individuals when the results of the analysis of molecular variance (AMOVA) are examined. Fixation index values give an idea in terms of the inbreeding coefficient and population differences. Analysis of molecular variance (AMOVA) results pointed out that these eight native sheep breeds can be differentiated weakly. The FST value obtained from the AMOVA analysis was parallel to the GST value. This finding proves that most of the genetic diversity is caused by the difference between individuals in the present study. It was noticed that there were four clusters when the dendrogram was examined. Dendrogram, which is showed the position of the Turkish sheep breeds in the present study was different from the findings obtained from the study conducted on the same breeds by Yilmaz et al. (2015). It is known that the Karacabey Merino breed was obtained by crossbreeding the sheep breeds of Kivircik reared in the Marmara region and German Black Head Mutton (Sezenler and Ozder, 2009). It has a large number of local Kivircik form that is adapted to the different regions in Turkey (Oner et al., 2014). Kivircik sheep breed which is used as animal material in the study material is raised in the Aegean region. In the context of this information, it is expected result that these two breeds will take place in different clusters in the obtained dendrogram. STRUCTURE results showed a low level of differentiation and a high level of admixture, especially in Algerian sheep populations. The value of AK obtained by the method reported by Evanno et al. (2005) shows that the optimal number of groups is four as in the dendrogram. This revealed that STRUCTURE analysis and dendrogram results supported each other. It can be said that there was a high gene flow between Algerian sheep populations when examined the results obtaoined by CLUMPACK software. 5 CONCLUSION Domestic sheep reared all over the world, are raised for meat, milk, or fiber production, or conservation purposes. Non-systematic cross-breeding practices applied to increase the production capabilities of domestic animals carried out by breeders are among the main problems of animal husbandry in North African countries as in Turkey (Karaca et al., 2009). This type of practice leads Acta agriculturae Slovenica, 115/1 - 2020 11 AAS_vsebina_2020_115_1_koncna 180320.indd 11 26. 03. 2020 12:05:37 A. AMEUR AMEUR et al. to one of the major threats such as the disappearance of local genetic diversity (Herold et al., 2012). The present study, which was carried out for the first time, was not described any genetic similarities between Algeria and Turkey sheep breeds. However, the result shows that populations studied have a low level of differentiation and a high level of admixture. Results obtained from the present study revealed that Algerian and Turkish sheep breeds have a high genetic variability. While the knowledge of genetic diversity between breeds is important, the benefit of understanding the genetic variation within a population is considerable. There is no research focused on within and between breed genetic variations in Algerian and Turkish sheep breeds. Microsatellites used in the study have a highly accurate identification potency for the genetic diversity of the studied breeds. In conclusion, the present study has revealed an important knowledge about genetic diversity and the relationship between some sheep breeds raised in Algeria and Turkey. 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Turkish Journal of Veterinary and Animal Sciences, 1411(46), 576-582. https://doi.org/10.3906/vet-1411-46 14 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 14 26. 03. 2020 12:05:37 doi:10.14720/aas.2020.115.1.1281 Original research article / izvirni znanstveni članek Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation Oksana DMITRIEVA 1 2 Semen IVANOV 3 Received September 26, 2019; accepted December 2, 2019. Delo je prispelo 26. septembra 2019, sprejeto 2. decembra 2019. Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation Abstract: Field experiments were carried out in the Chuvash Republic, which is located in the center of the European part of Russia on the banks of the Volga River and has a moderately continental climate with warm summers, cold winters, well-defined transitional seasons and average annual rainfall of 500 mm. There are many cultural and wild species of amaranth in nature, differing in morphological and biological features. The objects of research were four common species of amaranth (Amaranthus spp.): A. cruentus L., A. caudatus L., A. hybridus L., A. spinosus L.. Particular attention in the experiment was paid to the morphological and biological characteristics of plants according to the growth stages during growing season and the formation of the yield of green mass and grain during 3 years of research. As a result of the experiment, the possibility of cultivating a heat-loving amaranth culture in the climatic conditions of the Chuvash Republic was proved and adaptive species for growing green mass (A.cruentus and A.caudatus) and for grain (A.cruentus) were identified. Key words: A. cruentus; A. caudatus; A. hybridus; A. spinosus; climatic conditions; growing season; growth stages Primerjalna raziskava vrst ščira (Amaranthus spp.) v zmernem kontinentalnem podnebju Ruske Federacije Izvleček: Poljski poskus je bil izveden v Čuvaški republiki, v osrednem delu evropskega dela Rusije, na bregovih Volge, ki ima zmerno kontinentalno podnebje s toplimi poletji, mrzlimi zimami, z dobro izraženimi prehodnimi obdobji in s povprečno letno količino padavin 500 mm. Na območju uspeva več gojenih in podivjanih vrst ščira z različnimi morfološkimi in biološkimi lastnostmi. Predmet raziskave so bile štiri pogoste vrste ščira: A. cruentus L., A. caudatus L., A. hybridus L., A. spinosus L.. Posebna pozornost je bila posvečena morfološkim in biološkim lastnostim rastlin glede na fazo v rastni sezoni, tvorbi biomase in zrnja v 3 letih raziskav. Kot rezultat poskusa je bila dokazana možnost gojitve toploljubnih ščirov v klimatski razmerah Čuvaške republike, za zeleno biomaso vrsti A.cruentus and A.caudatus in za zrnje vrsto A.cruentus. Ključne besede: A. cruentus; A. caudatus; A. hybridus; A. spinosus; rastna sezona; faze rasti; prilagodljiva vrsta 1 I. N. Ulyanov Chuvash state University, Medical faculty, Department of medical biology with Microbiology and Virology course, Cheboksary, Chuvash Republic, Russian Federation 2 Corresponding author, e-mail: dofa1967@mail.ru 3 Ministry of natural resources and ecology of the Chuvash Republic, Cheboksary, Chuvash Republic, Russian Federation Acta agriculturae Slovenica, 115/1, 15-24, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 15 26. 03. 2020 12:05:37 O. DMITRIEVA and S. IVANOV 1 INTRODUCTION Agriculture faces a great pressure to produce greater quantities of food, feed and biofuel on declining land resources for the projected nine billion people on the planet by 2050 (Godfray et al., 2010). It is predicted that agricultural production has to increase by 70 % by 2050 to cope with an estimated 40 % increase in world population. So, utilization of underutilized food crop performs a massive function for providing food, feed and vitamins to such increasing population (Bruinsma, 2009). In recent years producers and consumers have been interested in new plant species, which are referred as alternative plants or new crops. Whether they are actually new or just recently rediscovered, these species not only create valuable crude material for a number of industrial branches but they also constitute an important source of renewable energy. In addition, they add to the human menu, making it more diverse (Rastogi & Shukla, 2013). Amaranth is one of the few multipurpose crops who provide grain, leafy vegetable, fodder, and greater diet then the predominant staple crops. Nutrition value and use of grain amaranth is a potential future application in bread making (Mlakar et al., 2009). It is a tremendously short-lived annual, which develop vigorously, drought resistant and adapt effortlessly to new environments. It originates from tropical America, archeological excavations proved it also in India but from tropical America is has spread all over the tropical World, as China, Nepal, Italy, Greece, Africa, and Australia (Ozsoy, 2009). Amaranth is not a "true cereal" such as wheat, corn or barley, but it is considered as "pseudo-cereal" like buckwheat (Fagopirum esculentum Moench) and quinoa (Che-nopodium quinoa Willd.). Amaranth belongs to the order of Caryophyllales and family of Amaranthaceae and to the genus of Amaranthus. Amaranth leaves have excellentl chemical composition with mild spinach-like taste so it comes beneath an accurate leafy vegetable (Amicarelli & Camaggio, 2012). Amaranth species (A. blitum L., A. caudatus L., A. cruentus L., A. tricolor L.) are collectively known as amaranths or pigweed. They have a common name such as African spinach, India spinach and Chinese spinach; approximately 60 species are recognized with inflorescence and foliage ranging from purple and red to gold. Members of this genus share many characteristics and uses with members of the closely related genus Celosia (Juan, 2007). Amaranth originates from south America, from where it was widely distributed in most tropical regions and has been used as a grain, leafy vegetable and forage crop (Ebert, 2010). Amaranths could be divided into two groups, based on their consumption, grain and vegetable amaranths. species grown for vegetables are represented mainly by A. tricolor, A. dubius, A. lividus L., A. creuntus, A. palmeri S. Wats. and A. hybridus. Three principal species considered for grain include, A. hypochondriacus L., A. cruentus and A. caudatus (Topwal, 2019). Amaranth is a dicotyledonous, herbaceous plant with an erect stem and large inflorescence. Amaranth is C4 plant and belongs into group of NAD-malic enzyme-type of C4 metabolism. Some anatomical characteristics of amaranth and its C4-photosynthesis pathway result in increased efficiency of the usage of C02 below an extensive vary of temperature (from 25 °C to 40 °C), below greater mild intensity, and moisture stress environments. All this contribute to the crop's huge geographic adaptability to numerous environmental conditions (Kaufman, 1992). Amaranth leaves can be used as greens in salads, boiled or fried in oil and mixed with meat or fish. This can be used as side dish in soups or as an ingredient in sauce and baby food (Mlakar et al., 2010). The leaves are high in fibers and contain high concentration of vitamin A, B6 and C, riboflavin and foliate. Minerals include calcium, iron, magnesium, phosphorus, potassium, zinc, copper and manganese. An amaranth grain can be ground for use in bread, noodle, pancakes, cereals, granola cookies or other flour baked products. More than 40 products containing amaranths are currently on the market in the USA (Putnam, 2007). Leafy vegetables of amaranth supply protein, minerals and vitamins in diet. Their lush, green, succulent crisp are eaten raw or cooked as vegetables in soup, they are best when the plant is young and tender. Amaranth grain is high in protein and contains two essential amino acids: lysine and methionine, which are frequently found in other cereal grains. It is higher in fiber and iron than wheat, and higher in calcium (Uusikua, 2010). Amaranth is a widely adapted genus, and can be grown in different climate conditions. But the more limiting factor is the temperature and less limiting is the rainfall amount during the growing season (Dmitrieva, 2018 b). Amaranth can be grown together with corn and sunflower to obtain high-nutrient silage. To do this, the seeds of amaranth, corn and sunflower are sown in separate rows in one field and cut together for silage without mixing in the future (Dmitrieva, 1993). The aim of the research was to compare amaranth species (Amaranthus spp.) by morphological and biological characteristics and to determine adaptive species among them for cultivation for fodder and grain purposes in the soil and climatic conditions of the Chuvash Republic. 2 MATERIALS AND METHODS 2.1 FIELD EXPERIMENT Field experiment was conducted in period from 16 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 16 26. 03. 2020 12:05:37 Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation 2015 to 2017 at the experimental field of the University located on the right Bank of the Volga river in Cheboksary (53°58'N; 47°15'E). The soil type of the experimental field is light gray forest loam, medium podzolic according to the Russian classification that corresponds to soils with average nutritional values (Shishova et al., 2004). The pH of the salt extract (pHkcl) is 5.4, the humus content according to Tyurin is 2.2 %. The arable layer was characterized by moderate concentrations of phosphorus (12.0 mg 100 g-1 of soil), potassium (12.0 mg 100 g-1 of soil) and magnesium (4.5 mg 100 g-1 of soil). The thickness of the arable horizon is 21-23 cm, relief the fields is flat. Judging by the agrochemical characteristics, the soils of the experimental plot do not have the properties to obtain high yields of amaranth without the application of mineral fertilizers. There was one background of mineral nutrition in the experiment. Before sowing, N, P, K fertilizers were applied to all plots at the following rates: 60 kg N ha-1, 80 kg P205 ha-1 and 90 kg K20 ha-1 according to soil analysis. The area of the experimental plot was 40 m2 respectively 10 m2 for each species of amaranth. The experiment had a randomized block design with four replications. The analyzed species were four of the most common species of amaranth (Amaranthus spp.): A. cruentus, A. caudatus, A. hybridus, A. spinosus. from different groups of plants according to plant height: long-stemmed, medium-stemmed and short-stemmed (Table 1). Every year, amaranth was sown after potatoes. Since the seeds are very small (mass of 1000 seeds is 0.6 g) amaranth requires thorough soil preparation in the spring (double loosening of the soil to a depth of 6 and 8 cm). For the same reason seeds were mixed with sand at a ratio of 1 g seed to 100 g sand to facilitate the sowing process and to obtain a uniform stand (Dmitrieva, 2018a, Fadeeva & Dmitrieva, 2017). Seeds were sown in late May at soil temperatures above 16-18 0C to a depth of 1 cm. Method of planting was broadcasting and seeds were spaced at 70 cm between rows. Seeding rate was 1.0 kg ha-1. Weeding was done using hoe. First weeding was carried out at two weeks after sowing and second weeding at 4 weeks after sowing. Harvesting for green mass (silage) and grain was done by cutting the whole plant on different dates depending on the Amaranthus spp. and weather conditions (Table 2). Observations of plant growth, plant height, number of leaves per plant, leaf area were carried out in accordance with the growth stages: vegetative state, inflorescence formation, flowering, seed maturation. To measure plant height a meter ruler was used to take the height of four tagged plants of amaranths in each experimental unit, and the mean was calculated and recorded. The height was measured from the ground level to the tip of the plant. Leaves of four tagged plants were counted in each experimental, and the mean was determined and recorded. Only fully opened leaves were counted. To calculate the leaf area, the length and width were multiplied using the constant (6.6) for four tagged plants of each amaranth species. The fresh mass of the plants was determined after cutting of whole plant in the net plots, and then extrapolated to per hectare. The harvested amaranth (dry mass of the plant) was dried on 60-70 0C in laboratory and weighed to determine the dry mass of the net plot and then extrapolated to per hectare. Seeds were harvested once when inflorescence change color to yellow or pink. Plants were cut, threshed and seed cleaned. Seed harvesting was carried out by cutting inflorescences and threshing seeds only in dry and warm weather for two weeks (Table 2). The content of nutrients in the green mass and grain (protein, lipids, carotene, sugar) was determined in the agrochemical laboratory of the Universityby near infrared spectroscopy (Russian Organization for Standardization, 2012). 2.2 WEATHER CONDITIONS Chuvash Republic has moderately continental climate with warm summers, cold winters, well-defined transitional seasons and average annual rainfall of 500 mm. Weather conditions during 2015-2017 are presented in Table 2. The information is given together with the dates of sowing and harvesting and respectively the duration of the growing season of A. cruentus and with such indicators as the rainfall amount and the amount of active temperatures above 10 0C accumulated during the growing season (ZT > 10 0C). Table 2 shows that growing season for the formation Tablel: Description of amaranth species (Amaranthus spp.) used in the experiment Amaranthus spp. Height group Origin Stem colour Inflorescence form Inflorescence colour Seed colour A. cruentus long-stemmed USA Pink Erect Pink Black A. caudatus long-stemmed USA Pink Drooping Pink Pink A. hybridus medium-stemmed China Green Erect Green Cream A. spinosus short-stemmed India Green Erect Green Cream Acta agriculturae Slovenica, 115/1 - 2020 17 17 AAS_vsebina_2020_115_1_koncna 180320.indd 21 26. 03. 2020 12:05:37 O. DMITRIEVA and S. IVANOV Table 2: Weather conditions for the growing season of A. cruentus in the years 2015-2017 Years Seeding date Harvest date Growing season Rainfalls (mm) ET > 10 0C Green fresh mass (biomass) 2015 23 May 20 August 91 165 1496 2016 25 May 17 August 85 178 1654 2017 23 May 18 August 87 269 1636 Average 88 204 1595 Grain (seeds) 2015 23 May 19 September 120 215 1850 2016 25 May 10 September 109 222 2080 2017 23 May 9 September 111 298 2112 Average 113 245 2014 of green mass for silage in 2016 and 2017 was shorter due to warmer weather (IT > 10 0C - 1654 0C and 1636 0C) and was not particularly dependent on rainfall. The same situation was observed in the cultivation of grain amaranth when ZT > 10 0C accumulated up to 2080-2112 0C. In general, it can be noted that the weather conditions of Chuvash Republic were quite optimal for formation of green fresh mass (biomass) and seeds of A. cruentus during the years of the experiment. 2.3 STATISTICAL ANALYSIS The results were processed statistically by analysis of variance (One-Way ANOVA) in the Statistica 12.0 program. The significance of differences was determined with Tukey's test at p < 0.05. 3 RESULTS AND DISCUSSIONS 3.1 GROWTH ANALYSIS OF AMARANTH SPECIES (Amaranthus SPP.) Amaranth growing season includes the following growth stages from sowing to mature seeds: seedlings, vegetative state, inflorescence formation, flowering, seed maturation. Each growth stage was determined at the time when it was observed in 75 % of the control plants (Dmitrieva, 2018c). The results of growth stages duration of amaranth species in the years of the experiment are presented in Table 3. Table 3 shows that all amaranth species (Amaran-thus spp.) have two longest stages of growth: vegetative state reaching 35-45 days and flowering reaching 28-45 days. Comparing species in the vegetative state when plants form the root system and stems with leaves it is determined that A. cruentus passes this period faster by 8-16 days which is a good indicator for temperate climatic conditions of the experiment. These results are compatible with those reported by investigators (Thapa & Blair, 2018; Saratovsky et al., 2018). The same pattern was observed during flowering when the same species A. cruentus bloomed 6-17 days earlier compared to other species that made it possible to grow A. cruentus for grain in these conditions for three years of experiment. As a result, during the growing season A. cruentus formed biomass for silage for 88 days and after 24-29 days of seed maturation formed grain suitable for use for fodder and food purposes. A. caudatus having a growing season of about 100 days is also suitable for growing fresh biomass for silage in experimental conditions. But analysis of the growth stages showed their longer duration due to lack of heat that contributed only to the partial maturation of seeds. A. hybridus was distinguished by the longest duration of the growing season and did not have time to grow to full flowering due to higher requirements for temperature (Costea et al., 2001). In general, the climatic conditions of the Chuvash Republic allow growing all amaranth species for fresh green biomass but considering the early onset of frost it is preferable to grow species with a shorter growing season (A. cruentus, A. caudatus). The main indicator of the physiological state is the growth of plants. Therefore, the experiment involved the analysis of changes in the linear growth (height) and stem diameter of amaranth species. Amaranth species differed significantly in height and were conventionally divided into three groups: long-stemmed (A. cruentus, A. caudatus), medium-stemmed (A. hybridus), short-stemmed (A. spinosus). The change in plant height during the growing season is presented in Table 4. Table 4 shows that early stages of all amaranth species growth were characterized by very slow growth of the stem and leaves due to the small supply of nutrients 18 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 18 26. 03. 2020 12:05:37 Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation Table 3: Duration of amaranth species (Amaranthus spp.) growth stages (days) Growth stages A. cruentus A. caudatus A. hybridus A. spinosus Seedlings 15 15 15 15 Vegetative state 34 42 45 40 Inflorescence formation 14 14 15 15 Flowering 30 35 45 30 Growing season (biomass) 91 106 120 100 Seed maturation 29 - - - Growing season (grain) 120 - - - Seedlings 12 12 12 12 Vegetative state 35 42 45 40 Inflorescence formation 12 12 14 14 Flowering 26 32 45 30 Growing season (biomass) 85 98 116 96 Seed maturation 24 - - - Growing season (grain) 109 - - - Seedlings 12 12 12 12 Vegetative state 36 44 45 40 Inflorescence formation 10 10 12 11 Flowering 29 33 45 31 Growing season (biomass) 87 99 114 94 Seed maturation 24 - - - Growing season (grain) 111 - - - 2015-2017 Seedlings 13 13 13 13 Vegetative state 35 43 45 40 Inflorescence formation 12 12 14 13 Flowering 28 33 45 31 Growing season (biomass) 88 101 117 97 Seed maturation 25 - - - Growing season (grain) 113 - - - in small seeds. During this period, the root system developed very actively from seedlings to inflorescences. After 4-5 weeks, intensive plant growth began in the middle of the vegetative stage and reached 40 cm in long-stemmed, 30 cm in medium-stemmed and 16 cm in short-stemmed of amaranth species (Amaranthus spp.). Since the inflorescence formation, the growth of the stem and the increase in its diameter were very active and by the end of flowering when harvesting green biomass for silage reached maximum height of 173-175 cm and a diameter of more than 2.0 cm (A. cruentus, A. caudatus), 150 cm and 1.7 cm (A. hybridus), 71 cm and 0.7 cm (A. spino-suss). Plant growth during this period was due to the growth of a long inflorescence and in the previous stages due to the elongation of the internodes of the stem. The same results were reported by other investigators. They noted the maximum growth of plants in the period between formation of inflorescences and flowering (Abbas et al., 2017; Carlquist, 2003; Archipova & Breus, 2004). It was determined that the daily increase in plant height Acta agriculturae Slovenica, 115/1 - 2020 19 19 AAS_vsebina_2020_115_1_koncna 180320.indd 21 26. 03. 2020 12:05:37 O. DMITRIEVA and S. IVANOV Table 4: Dynamics of changes in plant height and stem diameter of amaranth species (Amaranthus spp.) during the growing season in the year 2016 (cm) Growth stages A. cruentus A. caudatus A. hybridus A. spinosus Plant height Seedlings 5.5 a 5.5 a 5.2 a 5.4 a Vegetative state 37.9 a 39.0 a 28.8 b 16.4 c Inflorescence formation 91.0 a 93.6 a 66.6 b 40.8 c Flowering 173.3 a 175.2 a 150.0 b 71.4 c Seed maturation 186.9 - - - Stem diameter Seedlings 0.3 a 0.3 a 0. 3 a 0.2 a Vegetative state 0.7 a 0.7 a 0.6 a 0.4 b Inflorescence formation 1.6 a 1.5 a 1.2 b 0.7 c Flowering 2.2 a 2.1 a 1.7 b 1.0 c Seed maturation 2.2 - - - Values within a lines marked with different letter are significantly different atp < 0.05. Table 5: Dynamics of changes in in the year 2016 number of leaves and leaf area of amaranth species (Amaranthus spp.) during the growing season Growth stages A. cruentus A. caudatus A. hybridus A. spinosus Number of leaves Vegetative state 10.5 a 10.0 a 9.5 a 7.0 b Inflorescence formation 30.9 a 28.7 a 27.2 a 21.4 b Flowering 59.2 a 57.4 a 54.7 a 39.3 b Seed maturation 64.9 - - - Leaf area (cm2) Vegetative state 446.9 a 450.2 a 429.4 a 289.6. b Inflorescence formation 2007.5 a 1988.8 a 1907.6 a 1686.1 b Flowering 4796.7 a 4773.3 a 4689.8 a 3044.5 b Seed maturation 5710.4 - - - Values within a lines marked with different letter are significantly different atp < 0.05. was 5 cm in the flowering stage and 2 cm throughout the growing season. A. cruentus and A. caudatus had the best and significantly different indicators of growth processes and are preferred for growing in climatic conditions of the experiment. Plant growth depends on metabolic processes and, above all, on the photosynthetic activity of the plant organism. Therefore, it is necessary to select such forms of plants that have a high capacity of photosynthesis and a high rate of growth processes. Number of plant leaves plays an important role because they manufacture and Supply food material synthesized during photosynthesis. Favorable conditions for harvest formation of the plants are created when the leaf area exceeds 3 times the area of 20 Acta agriculturae Slovenica, 115/1 - 2020 the plant and the leaves do not shade each other (Taipova & Kuluev, 2015; Dmitrieva, 2014). The change in number of leaves and leaf area of one plant during the growing season is presented in Table 5. Analysis of the increase in the number of leaves showed the same pattern as with the height of plants according to the growth periods. The maximum increase in the number of leaves and the leaf area per plant was established after vegetative state and inflorescence formation until the end of flowering at the time of harvesting green biomass for silage (Betschart et al., 2001; Saratovsky et al., 2018). The number of leaves and leaf area in these stages increased by 2-4 times and reached 55-60 leaves and 4600-4800 cm2 in long-stemmed and AAS_vsebina_2020_115_1_koncna 180320.indd 20 26. 03. 2020 12:05:37 Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation medium- stemmed species, 40 leaves and 3000 cm2 in short-stemmed of amaranth species (Amaranthus spp.). The area of one plant in the experiment was 700 cm2, the leaf area of A. cruentus and A. caudatus was 7 times larger and reached more than 5000 cm2, which indicates the optimal ratio of these parameters for photosynthesis and as a result for better growth and development of plants in the climatic conditions of the experiment. Magome-dov (2008) made similar experiments in the Republic of Tatarstan and observed the same pattern in the development of the leaf surface during the amaranth growth. 3.2 YIELD OF AMARANTH SPECIES (Amaranthus spp.) Yield is a comprehensive generalizing indicator of the degree of favorable growth conditions for the cultivation of agricultural plants. To determine the yield it is necessary to analyze the increase in fresh and dry mass of plants during growth periods, which are presented in Table 6. Table 6 shows that since the growth processes were not so active in the first 50 days after sowing, the increase in fresh and dry biomass of all amaranth species (Ama-ranthus spp.) was not intensive during this period. From the stage of inflorescence formation to the end of flowering within 40-50 days, fresh and dry biomass increased almost 5 times (from 12.4 to 57.2 t ha-1 fresh mass and from 2.0 to 10.6 t ha-1 dry mass in A. cruentus) due to active growth in length, thickening of the stem, increasing the number of leaves and elongation of inflorescences (Pospisil et al., 2009; Ulbricht et al. 2009). The same pattern was observed with other species of amaranth. Comparing amaranth species found a significant increase in biomass in the group of long-stemmed species (A. cruentus and A. caudatus) due to more active growth in length, thickening of the stem, increasing the number of leaves and elongation of inflorescences. The dry matter content increased from 16 % in the vegetative state to 18.5 % at the end of flowering when harvesting fresh green biomass (Norman & Shongwe, 1993). Comparing Amaranthus spp. found that the dry matter content did not differ significantly depending on the species of amaranth. By the time of harvesting mature A. cruentus seeds, the dry matter content increased by another 1 %. The results of the yield of fresh green biomass at the end of the flowering stage and mature seeds are presented in Table 7. Yield analysis of fresh green biomass showed that all amaranth species except A. spinosus had high biological productivity. Soil and climatic conditions during the experiment allowed the plants to form well-developed stems, leaves, inflorescences that contributed to intensive photosynthesis and significantly high yield especially among long-stemmed species (A. cruentus - 53.7 t ha-1 and A. caudatus - 49.0 t ha-1). Analysis of seed productivity showed that only one species of amaranth (A. cruentus - 1.32 t ha-1) had time to form seeds in full maturity in all years of the experiment. In other species, seed maturation in climatic conditions of the experiment was not observed due to higher temperature requirements. Experiments with heat-loving amaranth in other climatic conditions prove the possibility of seed maturation of Table 6: Dynamics of increase of fresh and dry mass of amaranth species (Amaranthus spp.) during the growing season in the year 2016 (t ha-1) Growth stages A. cruentus A. caudatus A. hybridus A. spinosus Fresh mass (t ha 1) Vegetative state 12.4 a 12.9 a 9.3 b 6.3 c Inflorescence formation 31.2 a 29.7 a 19.4 b 15.0 c Flowering 57.2 a 53.8 a 39.5 b 28.6 c Dry mass (t ha 1) Vegetative state 2.0 a 2.1 a 1.5 b 1.0 c Inflorescence formation 5.5 a 5.2 a 3.4 b 2.8 c Flowering 10.6 a 10.0 a 6.3 b 4.6 c Dry matter content (%) Vegetative state 16.1 a 16.2 a 16.0 a 16.2 a Inflorescence formation 17.6 a 17.5 a 17.5 a 17.6 a Flowering 18.5 a 18.2 a 18.2 a 18.3 a Values within a lines marked with different letter are significantly different atp < 0.05.. Acta agriculturae Slovenica, 115/1 - 2020 21 AAS_vsebina_2020_115_1_koncna 180320.indd 21 26. 03. 2020 12:05:37 O. DMITRIEVA and S. IVANOV Table 7: Yield of amaranth species (t ha 1) Years A. cruentus A. caudatus A. hybridus A. spinosus Green fresh mass (biomass) 2015 48.0 a 42.6 a 37.2 b 25.4 c 2016 57.2 a 53.8 a 39.5 b 28.6 c 2017 55.9 a 50.6 a 40.2 b 28.0 c Average 53.7 a 49.0 a 39.0 b 27.3 c Grain (seeds) 2015 2016 2017 Average 1.11 1.46 1.40 1.32 Values within a lines marked with different letter are significantly different atp < 0.05.. Table 8: Chemical composition and nutritional value of biomass (Amaranthus spp.) Indicators A. cruentus A. caudatus A. hybridus A. spinosus Chemical composition (% in dry mass) Protein 20.87 21.56 20.75 21.12 Lipids 1.38 1.50 1.33 1.28 Cellulose 17.54 16.12 18.06 15.90 Ash elements 18.73 18.35 17.98 18.07 Sugar 2.65 2.54 2.76 3.05 Nutritional value (g kg-1 dry mass) Protein 161.70 162.43 160.74 162.78 Calcium 26.39 26.14 25.98 27.09 Phosphorus 4.19 4.49 3.57 4.54 Carotene (mg) 99 93 79 88 Vitamin C (mg) 23 24 31 36 all amaranth species (Caselato-Sousa & Amaya-Farfan, 2012). 3.3 CHEMICAL COMPOSITION AND NUTRITIONAL VALUE OF AMARANTH SPECIES (Amaranthus spp.) The value of forage crops in agriculture is determined by the nutrient content of green biomass for silage and seeds. Main nutritional information of amaranth species is presented in Table 8. The chemical composition of biomass indicates a quite good feed value of amaranth species (Amaranthus spp.). Protein content did not differ significantly among amaranth species and ranged from 20.75 % to 21.56 % in dry mass. The same pattern is established with other 22 Acta agriculturae Slovenica, 115/1 - 2020 indicators of nutritional value comparing species of amaranth (Andini et al., 2013)It can be stated that the soil and climatic conditions of the experiment were quite favorable for the accumulation of nutrients in the fresh green biomass of all amaranth species (Amaranthus spp.). 4 CONCLUSIONS The results of comparative study of amaranth species (Amaranthus spp.) indicated that soil and climatic conditions of the Chuvash Republic were quite favorable for growing all species to produce green biomass for silage. But given that amaranth is more demanding to heat than to moisture it is preferable to grow more rapidly developing species with a growing season of not more than 100 days (A. cruentus, A. caudatus) in order AAS_vsebina_2020_115_1_koncna 180320.indd 22 26. 03. 2020 12:05:37 Comparative study of amaranth species (Amaranthus spp.) in the temperate continental climate of Russian Federation to avoid being damaged by early frosts. Comparing the dynamics of changes in growth parameters in 2016 year (height and diameter of stem, number and area of leaves, mass of plants), it was found that long-stemmed species of amaranth (A. cruentus, A. caudatus) are more suitable for growing under experimental conditions. 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Realizing the potential of grain Amaranth. Food Reviews International, 8, 5-21. https://doi. org/10.1080/87559129209540927 Magomedov, I. M. (2008). Physiological bases of amaranth competitiveness. Successes of modern natural science, 5, 57-59 [in Russian]. Mlakar, S. G., Turinek, M., Jakop, M., Bavec, M. Bavec, F. (2010). Grain Amaranth as an alternative and perspective crop in temperate climate. Journal for Geography, 5(1), 135-145. Mlakar, S. G, Turine, M., Jakop, M., Bavec, M. Bavec, F. (2009). Nutrition value and use of grain amaranth: potential future application in bread making. Agricultura, 6, 43-53. Norman, J. C. & Shongwe, V. D. (1993). Influence of some cultural practices on the yield and quality of amaranth (Ama-ranthus hybridus L.). Advances in horticultural science, 7, 169-172. Acta agriculturae Slovenica, 115/1 - 2020 23 23 AAS_vsebina_2020_115_1_koncna 180320.indd 21 26. 03. 2020 12:05:37 O. DMITRIEVA and S. IVANOV Ozsoy, N., Yilmaz, T., Kurt. O., Can, A., Yanardag, R. (2009). In vitro antioxidant activity of Amaranthus lividus L. Food Chemistry, 116(4), 867-872. https://doi.org/10.1016/j.food-chem.2009.03.036 Pospisil, A., Pospisil, M., Macesic, D., Svecnjak, Z. (2009). Yield and quality of forage sorghum and different amaranth species (Amaranthus spp.) biomass. Agriculture conspectus scientific, 74(2), 85-89. Putnam, D. H. (2007). Center for alternative plant and animal products Minnesota extension services. University of Minnesota. St Paul Mn. 1-5. Rastogi, A. & Shukla, S. (2013). Amaranth: a new millennium crop of nutraceutical values. Food science and nutrition 53, 109-125. https://doi.org/10.1080/10408398.2010.517876 Russian Organization for Standardization (2012). Near infrared spectroscopy (State standard No. 32041-2012. Retrieved from https://www.shimadzu.ru/. Saratovsky, L. I., Vashchenko, E. G, Fedotov, V. A., Kazazyan, V. V. (2018). Elements of varietal technology of amaranth cultivation in the steppe zone of the Central black earth region. Bulletin of the Voronezh state agrarian University, 1(56), 22-31. [in Russian]. https://doi.org/10.17238/ issn2071-2243.2018.1.22 Shishova, L. L., Tonkonogov, V. D., Lebedeva, I. I., Gerasimova, M. I. (2004). Classification and diagnostics of soils in Russia. Smolensk: Oikumen [in Russian]. Taipova, R. M. & Kuluev, B. R. (2015). Amaranth: features of culture, application, and prospects of cultivation in Russia. Biomics, 4(2), 84-90. [in Russian]. Thapa, R. & Blair, M. (2018). Morphological assessment of cultivated and wild amaranth species diversity. Agronomy, 8, 272-280. https://doi.org/10.3390/agronomy8110272 Topwal M. (2019). A review on Amaranth: nutraceutical and virtual plant for providing food security and nutrients. Acta Scientific Agriculture, 3(1), 9-15. Ulbricht, C., Abrams, T., Conquer, J., Costa, D., Serrano, J. M. G., Taylor, S., Varghese, M. (2009). An evidence-based systematic review of amaranth (Amaranthus spp.) by the natural standard research collaboration. Journal of dietary supplements, 6(4), 390-417. https://doi. org/10.3109/19390210903280348 Uusikua, N. P., Oelofsea, K. G., Duodub, M. J., Besterc, M. Megan, J. (2010). Nutritional value of leafy vegetables of sub-Saharan Africa and their potential contribution to human health. Food composition and analysis, 23, 499-509. https://doi.org/10.1016/j.jfca.2010.05.002 24 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 24 26. 03. 2020 12:05:37 doi:10.14720/aas.2020.115.1.969 Original research article / izvirni znanstveni članek Physiological and biochemical responses of selected cowpea (Vigna un-guiculata (L.) Walp.) accessions to iron toxicity Josiah E. IFIE 1 2 3, Sandra IFIE-ETUMAH 2, Beckley IKHAJIAGBE 2 Received November 26, 2018; accepted December 31, 2019. Delo je prispelo 26. novembra 2018, sprejeto 31. decembra 2019. Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity Abstract: This study aimed to investigate the effect of iron toxicity in cowpea using physiological and biochemical responses of selected accessions. Fifteen accessions of cowpea were exposed to two treatments of iron using FeS04 solution (100 mg l-1 and 400 mg l-1) and distilled water at pH 6.2 as control. The results showed that there was a general reduction in germination morphology; germination percentage among the 400 mg l-1 Fe-treated accessions. Seed mortality rates were significantly higher among the 400 mg l-1Fe-treated accessions (> 35 %). Water imbibition capacity and relative mass gained were higher for Fe-treated accessions. Furthermore, significant increase in the total sugar and percentage utilization of sugars was accompanied by an insignificant decrease in chlorophyll a, a significant decrease in chlorophyll b contents and the persistence of foliar chlorosis, among the 400 mg l-1 Fe-treated accessions. MDA levels were significantly increased while proline remained unchanged, mean SOD activity was insignificantly increased, whereas Cat decreased among the 400 mg l-1 Fe-treated accessions. Documentation of these observable changes in physiological and biochemical parameters will be useful in understanding the impact of elevated iron concentrations on the cultivation of cowpea accessions in soils associated with ferruginous ultisols. Key words: ferruginous ultisol; Vigna unguiculata; cow-pea accessions; iron toxicity; plant antioxidants; cowpea tolerance; physiological response; biochemical response Fiziološki in biokemični odziv akcesij kitajske vinje (Vigna unguiculata (L.) Walp.) na toksičnost železa Izvleček: Raziskava je bila izvedena z namenom preučevanja učinkov toksičnosti železa na kitajsko vinjo na osnovi fiziološkega in biokemičnega odziva izbranih akcesij. 15 akce-sij kitajske vinje je bilo izpostavljeno dvema obravnavanjema z raztopino FeS04 (100 mg l-1 in 400 mg l-1) in destilirano vodo pri pH 6,2 kot kontrolo. Rezultati so pokazali, da je bilo splošno zmanjšanje v morfoloških parametrih kalitve in v odstotku ka-litve pri akcesijah, ki so bile tretirane s 400 mg l-1 Fe. Tudi smrtnost semen je bila pri akcesijah, tretiranih s 400 mg l-1Fe značilno večja (> 35 %). Sposobnost nabrekanja z vodo in relativno povečanje mase sta bila večja pri z železom tretiranih akcesijah. Nadalje je bilo opaženo pri s 400 mg l-1 Fe tretiranih akcesijah značilno povečanje vsebnosti celokupnih sladkorjev in njihove porabe, kar je bilo spremljano z neznačilnim upadom vsebnosti klorofila a, z značilnim upadom klorofila b in pojavom listnih kloroz. Pri obravnavanjih s 400 mg l-1 Fe se je vsebnost MDA značilno povečala, medtem, ko so vsebnosti prolina ostale nespremenjene, poprečna aktivnost soD se je neznačilno povečala, aktivnost Cat pa zmanjšala. Dokumentiranje teh sprememb v fizioloških in biokemičnih parametrih bo koristno za razumevanje vpliva povečanih koncentracij železa pri gojenju akcesij kitajske vinje v tleh povezanih z železovimi ultisoli. Ključne besede: železov ultisol; Vigna unguiculata; akce-sije kitajske vinje ; toksičnost železa; rastlinski antioksidanti; strpnost kitajske vinje; fiziološki odziv; biokemični odziv 1 Kampala International University, Western Campus, Medical Biochemistry Department, Ishaka, Uganda 2 University of Benin, Department of Plant Biology and Biotechnology, Environmental Biotechnology and Sustainability Research Group, Benin City, Nigeria 3 Corresponding author, e-mail: josiahifie@gmail.com Acta agriculturae Slovenica, 115/1, 25-38, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 25 26. 03. 2020 12:05:37 J. E. IFIE et al. 1 INTRODUCTION The importance of proteins in humans cannot be over-emphasized especially in its use for the development of body structures as well as regulation of human metabolism. Dietary proteins can be obtained from either the plant or animal sources according to Ahenkora et al. (1998). Many of African countries are currently struggling economically and as such it has become difficult for the citizens to purchase animal products which are major sources of proteins. Notwithstanding the economic situation especially in Nigeria, there is also the issue of health disorders associated with over-dependence on animal proteins (Spence et al., 2010) and these challenges make it imperative for the drive for cheaper and healthier alternative sources which the plant sources provide (Clinfton, 2011). During austere times such as is prevalence in most African countries, legumes are among the safer and cheaper plant sources of proteins (Nielsen et al., 1993). Averagely, about 70 million people consume legumes globally and attempts aimed at achieving food security through these sources have prompted the need to set up farmlands and improve the technological inputs for better yields while at same time ensuring that adequate and efficient storage systems for preservation of harvested crops are in place (Diouf & Hilu, 2005). Though the above efforts in the achievement of nutritional sufficiency are a welcome development, environmental factors have been shown to impact significantly on crop productivity. However, human activities such as mining and other industrial activities may be a major source of heavy metal pollution to soil and these impacts negatively on crop productivity (Ikhajiagbe, 2016). The presence of heavy metal in soils usually above the limit of tolerance leads to metal toxicity and a good example is the presence of high concentrations of iron in ferruginous soils in which the available iron (Fe2+) is greater than 300 mg kg-1 (Yamauchi and Peng, 1995; Ratering & Schnell, 2000). At such high concentrations, iron becomes toxic to plants' growth and development as seen in lowland rice which has been reported in various countries including Nigeria, Colombia, Malaysia (Suresh 2005; Mitra et al., 2009). Reports have shown that most cowpea; about 66 % of global production, are grown in the African continent particularly in Nigeria and Niger. In Nigeria, most of these productions are domiciled in the Northern region (Blade et al., 1997). The conditions needed for the cultivation of cowpea such as above 80 % sandy content are also present in some other states such as Edo State, Nigeria, Edo State but have recorded very low production (IITA, 2003). One of the reasons for this shortfall 26 Acta agriculturae Slovenica, 115/1 - 2020 may be due to the presence of high iron in most of these soils (ferruginous ultisols) as shown by the geological evidence of Ikhile (2016) and collaborated by Imasuen & Onyeobi (2013). There is a dearth of information on the physiological and biochemical response of cowpea to elevated iron levels as most existing literature focus on rice. Therefore, the study aims to investigate iron toxicity in cowpea using physiological and biochemical responses of selected accessions which will serve as a background for the understanding of the basis of tolerance of cowpea accessions planted in a ferruginous ultisol. 2 MATERIAL AND METHODS 2.1 EXPERIMENTAL DESIGN, SEED COLLECTION AND PLANTING Fifteen accessions (TVu-3742, TVu-3769, TVu-5348, TVu-5760, TVu-5768, TVu-5782, TVu-5883, TVu-6102, TVu-6193, TVu-6219, TVu-6290, TVu-10600, TVu-10881, TVu-11114, and TVu-11214) of cowpea were used in this study. The study was undertaken in the greenhouse of the Department of Plant Biology and Biotechnology, University of Benin, Nigeria. The accessions were provided by the Genetic Recourses Centre of the International Institute for Tropical Agriculture (GRC, IITA), Ibadan, Nigeria. The accessions were exposed to two treatments (100 mg l-1 and 400 mg l-1) of iron sulphate (FeSO4) solution based on toxicity reference for soil iron (300 mg l-1) and distilled water at pH 6.2 as control. The No. 1 Whatman's Filter papers were placed in Petri dishes moistened with the treatment solutions and thereafter ten seeds (mean mass: 0.18 ± 0.09 g) each of the fifteen accessions were planted in three replicates. 2.2 DATA COLLECTION 2.2.1 Germination Germination parameters were assessed by methods described by Sadeghi et al. (2011). These include germination percentage, seminal root length, shoot length, leaf area of germinant, seed mortality, mean water imbibitions capacity, water imbibitions rate and relative mass gain. 2.2.2 Total sugars and pigment analyses The productive capacity and pigmentation of the AAS_vsebina_2020_115_1_koncna 180320.indd 26 26. 03. 2020 12:05:38 Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity seeds and cotyledons such as total sugars, chlorophyll a and b were assessed. The total sugar was estimated according to the method described by Nelson (1944) and modified by Sankar & Selvaraju (2015). Chlorophyll a and b contents were investigated according to methods described by Arnon et al (1949) and Maxwell & Johnson (2000). 2.2.3 Characterization of iron toxicity symptoms For characterization of FE-induced chlorosis, morphological observations of the plant in response to the experimental conditions were recorded on an intermittent basis. These observations include the colour, shape, form or the appearance of the leaves and the stem of the plant as well as the positioning of flowers and nodes. Care was also taken to make sure that the succession of chlorosis was recorded. 2.2.4 Antioxidant activities Enzymatic (catalase, superoxide dismutase) and non-enzymatic (proline and malondialdehyde) anti-oxidants parameters were evaluated in the seeds and cotyledons of successfully germinated accessions. SOD activity was determined by the methods of Beauchamp & Fridovich (1971) and described by Ranganayakulu et al. (2013). Catalase activity was measured by the method of Luck (1971) and modified by Esma & Gulnur, (2016). MDA was determined using the thiobarbituric acid assay method described by Health & Packer (1968) and modified by Erja et al (2001). The extraction and estimation of proline were done according to the methods of Bates et al (1973) and Marin et al (2006). 2.3 STATISTICAL ANALYSIS. Data obtained from this study were subjected to a two-way analysis of variance using the SPSS statistical analysis software (SPSS-20"). Students' T-test and the least significant difference; LSD was used for equality of means while the Levene's test was used for the analysis of equality of variances. All statistical analysis was performed at a confidence limit of 95 % (p = 0.05) and values are presented as mean ± standard deviation. 3 RESULTS AND DISCUSSION 3.1 RESULTS The results obtained from this study showed the germination (%) of TVu-3769, 5760 and 6219 were significantly higher for control as well as the mean germ (%) when compared to the 100 mg l-1 and the 400 mg l-1 Fe-treated accessions (Fig 1). The seminal root length of TVu-5348, 6219, 11114 and mean value of control accessions were significantly higher compared to the 100 mg l-1 and the 400 mg l-1 Fe-treated accessions (Fig 2). For shoot length, TVu-3769 and 11214 showed significantly higher values for control accessions whereas TVu-5348, 5760, 5782, 6290 and 10600 were significantly higher for 100 mg l-1 Fe-treated accessions. Comparison of the mean values of control, 100 mg l-1 and the 400 mg l-1, showed a significantly higher mean for 100 mg l-1 Fe-treated accessions (Fig 3). Figure 1: Final germination percentage of accessions after 1 week following germination initiation. A: Seeds treated with 100 mg l-1 FeS04 Soln., B: Seeds treated with 400 mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. Acta agriculturae Slovenica, 115/1 - 2020 27 27 AAS_vsebina_2020_115_1_koncna 180320.indd 35 26. 03. 2020 12:05:39 J. E. IFIE et al. The leaf area of TVu-5348, 5883, 6193 and 10600 was significantly higher in control accessions. Also, the mean value of control accessions was higher when compared to 100 mg l-1 and 400 mg l-1 Fe-treated accessions, the mean differences were statistically insignificant (Fig 4). The mean seed mortality (%) showed a significantly higher value for 400 mg l-1 accessions compared to the control and insignificant when compared to 100 mg l-1 Fe-treated accessions. The least mean value was observed in the control accessions (Fig 5). The water imbibition capacity, water imbibition rate and relative mass gain of TVu-10881 were significantly higher in control (Fig 6, 7 and 8) but, comparison of the mean values of the control, 100 mg l-1 and 400 mg l-1 Fe-treated accessions show no significance (Fig 6). Tvu-3769 showed a significantly higher value for water imbibition rate and relative mass gain for the 100 mg l-1 and 400 mg l-1 Fe-treated accessions while the comparison of the mean values was statistically insignificant (Fig 7 and 8). For the plants' productive capacity, the total sugar of germinated seeds was compared to the seeds obtained for studies from the IITA. The results showed that the seeds from IITA had significantly higher total sugar (Fig 9). However, the mean total sugar of 100 mg l-1 was higher than 400 mg l-1 Fe-treated and control accessions. These mean differences among 100 mg l-1, 400 mg l-1 Fe-treated and control accessions were insignificant (p > 0.05). Accessing the total sugar contents of the cotyledons showed a significantly higher value for TVu-6219 in control accessions meanwhile comparison the mean values among Figure 2: Seminal root length of accessions after 5 days following germination initiation. A: Seeds treated with 100 mg l-1 FeSO4 Soln., B: Seeds treated with 400 mg l-1 FeSO4 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. Figure 3: Shoot length of accessions after 5 days following germination initiation. A: Seeds treated with 100 mg l-1 FeSO4 Soln., B: Seeds treated with 400 mg l-1 FeSO4 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. 28 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 28 26. 03. 2020 12:05:38 Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity Figure 4: Leaf area of early developing accessions after 1 week following germination initiation. A: Seeds treated with 100 mg l-1 FeS04 Soln., B: Seeds treated with 400 mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance.. Figure 5: Percentage seed mortality at 21 days after germination initiation. A: Seeds treated with 100 mg l-1 FeS04 Soln., B: Seeds treated with 400 mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. 100 mg l-1, 400 mg l-1 Fe-treated and control accessions were insignificant (p > 0.05) as seen in Figure 10. The percentage utilization of sugar showed a significantly higher mean value for 400 mg l-1 compared to 100 mg l-1 Fe-treated and control accessions (p < 0.05) as seen in Table 1.0. Table 2.0 showed the Chlorophyll-a (Chl a) and b (Chl b) contents of cotyledon after 1 week following germination initiation. For Chl a, the highest value was seen in Tvu-10881 (0.139 mg g-1) among 100mg l-1 Fe-treated accessions and the least in Tvu-10600 (0.032 mg g-1) among control accessions. Statistically, insignificant differential mean values were observed when the control accessions were compared with the Fe-treated accessions. For Chl b, the highest value was observed in Tvu-10881 (0.251 mg g-1) among 100 mg l-1 Fe-treated accessions and the least in Tvu-10600 (0.057 mg g-1) among control. The mean chlorophyll b value of 100 mg l-1 Fe-treated accessions was higher while the 400 mg l-1 Fe-treated was lower compared to the control. These mean differences were statistically significant. The mean total chlorophyll value for the 100 mg l-1 Fe-treated accessions was higher while the 400 mg l-1 Fe-treated was lower when compared to the control. However, these mean differences are statistically insignificant. The ratio of Chl a to Chl b showed insignificant equal mean values among the 100 mg l-1, 400 mg l-1 and the control accessions. Table 3.0 shows the persistence of chlorosis on germinants at 5, 6 and 7 days after germination with the mean persistence of chlorosis being more evident (++ and +++) among the Fe-treated Acta agriculturae Slovenica, 115/1 - 2020 29 29 AAS_vsebina_2020_115_1_koncna 180320.indd 35 26. 03. 2020 12:05:39 J. E. IFIE et al. Figure 6: Water imbibition capacity at 4 days after germination initiation. A: Seeds treated with 100 mg l-1 FeS04 Soln., B: Seeds treated with 400 mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. Figure 7: Water imbibition rate 4 days after germination initiation A: Seeds treated with 100 mg l-1 FeS04 Soln., B: Seeds treated with 400 mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance accessions (100 mg l-1 and 400 mg l-1) at early days of germination compared to the controlled accessions (+). Antioxidant capacities of the accessions showed that the MDA levels of TVu-3769, 6193, 10881 and 11114 were significantly higher among the control group (Fig 11). Comparison of the mean MDA values of 100 mg l-1, 400 mg l-1 and control showed that the mean value of control was significantly higher but those of proline were statistically insignificant (Fig 12). Catalase activities of TVu-6193 and 11214 were significantly higher in the control group compared to Fe-treated accessions (Fig 13). Comparison of the mean CAT activities among 100 mg l-1, 400 mg l-1 and control accessions showed a significant least value for 400mg l-1 Fe-treated accessions while a significantly higher mean value for the 400 mg l-1 30 Acta agriculturae Slovenica, 115/1 - 2020 Fe-treated accessions was observed for SOD activities when compared to the 100 mg l-1 and control accessions (Fig 14). 3.2 DISCUSSIONS The findings of this study showed that iron toxicity significantly reduced germination parameters as evident in the lower germination (%), seminal root and shoot lengths and leaf area of germinants whereas seed mortality is significantly increased. Also, the productive capacities of the accessions were significantly reduced among 400 mg l-1 Fe-treated accessions while insignificant mean differences were observed for chlorophyll a. However, Fe- AAS_vsebina_2020_115_1_koncna 180320.indd 30 26. 03. 2020 12:05:38 Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity Accessions of Cowpea Figure 8: Relative seed mass of imbibed seeds 4 days after germination initiation. A: Seeds treated with 100 mg l-1 FeSO4 Soln., B: Seeds treated with 400 mg l-1 FeSO4 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. Figure 9: Total sugars of germinated seeds at 4 days following germination initiation. A: Seeds treated with 100mgl-1 FeSO4 Soln., B: Seeds treated with 400mg l-1 FeSO4 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. toxicity significantly reduced the chlorophyll b content as well as increased persistence of chlorosis among the 400 mg l-1 Fe-treated accessions. The plants' antioxidants defence capacity showed significant lower catalase and higher SOD activities among the 400 mg l-1 Fe-treated accessions which corresponded with significant higher MDA values while the proline content is unchanged. Plant germination is usually affected by the difference in genetic make-ups of the different plants as well as environmental factors such as the presence of heavy metals (Bhagyashree et al., 2016). Seed germination is a significant stage in the seedling establishment; it decides successful crop and yield production (Bhattacharjee, 2008). The reduced values for germination parameters obtained from this study is consistent with the results of Ahmad et al. (2012) and Abdel-Haleem (2015). Though arguably, the reduced germination parameters may not be due to iron overload but rather due to general nutrient deficiencies, the presence of iron oxide deposits on the roots of wetland plants could act as a filter for nutrients such as phosphate, thereby causing a deficiency in the aerial parts of the plant. Furthermore, the physic-chemical effects of iron on osmotic balance through the increase of osmotic pressure with the tendency to disrupt seed hydration may be a contributory factor for the reduced seed germination (Nabil & Coudret, 1995; On-yango et al., 2019). The significance of water in seed germination is critical; thus the capacity for water imbibition by plant seeds is necessary for successful germination initiation Acta agriculturae Slovenica, 115/1 - 2020 31 31 AAS_vsebina_2020_115_1_koncna 180320.indd 35 26. 03. 2020 12:05:39 J. E. IFIE et al. 0.025 J» 0.02 uj E Accessions of Cowpea Figure 10: Total sugars of cotyledons at 4 days following germination initiation. A: Seeds treated with 100mg l-1 FeS04 Soln., B: Seeds treated with 400mg l-1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. Table 1: Percentage utilization of sugars during germination (taken at 4 days after germination initiation) Accessions Utilisation (%) Group A Group B Group C p-value TVu-3742 30.76f 25.06« 40.26e 0.192 TVu-3769 23.83h 13.33h 11.06h 0.114 TVu-5348 76.47b 67.71c 25.88 « 0.011 TVu-5760 34.01f 30.54f 15.57 f 0.089 TVu-5768 20.75« 73.66b 24.74 « 0.012 TVu-5782 14.21h 66.88c 13.69 h 0.033 TVu-5883 21.81« 35.53e 45.40e 0.051 TVu-6102 25.26« 40.23e 35.39 e 0.216 TVu-6193 35.62f 29.33«-f 64.81c 0.037 TVu-6219 17.72h 28.31«-f 40.61 e 0.033 TVu-6290 25.37« 28.44«f 40.19e 0.052 TVu-10600 36.20f 53.55d 55.05d 0.051 TVu-10881 28.63« 82.85a 6.50 i 0.001 TVu-11114 18.09h 45.39e 13.32 h 0.050 TVu-11214 30.76« 25.06« 40.26 e 0.060 p-value 0.023 0.032 0.026 A: Seeds treated with 100mg l 1 FeS04 Soln., B: Seeds treated with 400mg l 1 FeS04 Soln. and C: Control. *Mean values of accessions with different letters show statistical significance. and this has been shown to be affected by the difference in the permeability of the seed testa, seed composition and the availability of water in the environment (Wada and Abubbakar, 2013). Water imbibition capacity responses differed across the accessions. The low osmotic potential has been shown to extend the time needed for imbibition as well as delaying the onset of germination. Results obtained were similar to those of Olasoji et al. (2013) and Araujo et al. (2016) who also observed differential response for the rate of water of imbibition for 32 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 32 26. 03. 2020 12:05:38 Physiological and biochemical responses of selected cowpea (Vigna unguiculata (L.) Walp.) accessions to iron toxicity U m d LA LA LA U") LA LA LA LA LT> Ln Ln Ln Ln U") O O O o O LA LA LA LA U-) LA LA LA LA U-) Ln Ln Ln Ln U-> O o O C3 o LA LA LA LA LA LA U-) LA LA LA u^ U-) O O C3 C3 o m m m m m m m m m m m m m m m m o o o U o o ^ in 00 > H H m m m m a a > > H H o o\ md m m a a > > H H a a > > H H O ^ m on c^ o o O O O O O O a a H H o Oi o JS co fcts Ph v "ii ' g 6 ¡2 o S # ttf H3 ^ iu O -a > ^ G CO O Table 3: SD: stomatal density (SD), stomatal pore length (SPL), epidermal cell density (ECD), stomatal pore area (SPA), stomatal pore area index (SPAI), net photosynthesis rate (An), stomatal conductance (gs), Water use efficiency (WUE), leaf temperature depression (LTD) and vapor pressure deficit (VPD) of different sugar beet genotype at 30. Genotype SD SPL ECD SPA SPAI An & WUE LTD VPD (stomata mm"2) (|Jm) fepiderm mm"2) (|Jm-) (%) (mmol m s j (mol m s j (%) CC) (kPa) 419 192.67 ahc 14.99 h 1274.1 a 33.46 fgh 0.647 a-d 11.44 c-h 0.057 b-f 0.0085 c-g -0.161 d-j 3.77 h-k 7112 192.67 ahc 16.22 gh 764.4 c-f 34.87 d-h 0.667 ahc 11.95 h-g 0.044 c-i 0.0076 c-g -0.144.0c-j 4.07 a-e 31714 99.44 e-j 16.38 fgh 714.7 c-h 33.59 fgh 0.334 fgh 10.87 e-i 0.042 d-i 0.0064 d-h -0.035 h-i 3.92 d-i 110-7-8 142.95 c-g 18.11 c-h 696.1 c-h 38.41 h-g 0.548 a-g 11.50 c-h 0.054 b-g 0.0089 b-f -0.236 e-k 4.11 a-d 111-52-25 149.16 h-g 18.84 h-g 783.1 cde 39.43 a-g 0.588 a-f 9.88 i 0.028 hi 0.0038 h -0.130 b-j 3.9 e-j Sl-92685 111.87 e-j 21.72 ah 677.4 c-i 43.66 ah 0.492 a-h 15.24 a 0.039 d-i 0.0089 b-f -0.237 e-k 4.08 a-e Sl-92747 124.30 d-j 19.23 a-g 565.6 e-j 38.80 h-g 0.482 a-h 11.01 d-i 0.036 e-i 0.0058 e-h -0.126 b-j 4.14 ahc SI-92748 111.87 e-j 17.45 c-h 689.9 c-i 34.71 d-h 0.400 a-h 14.57 a 0.054 h-g 0.011 a-c -0.068 h-i 4.03 a-e SI-92749 223.74 a 16.33 fgh 888.7 hcd 32.91 gh 0.742 a 13.16 h 0.041 d-i 0.0079 c-g -0.367 h-k 4.05 a-e Sl-92750 74.58 ij 17.51 c-h 540.7 e-j 34.79 d-h 0.260 h 12.19 b-f 0.043 c-i 0.0068 d-h 0.038 h-h 3.71 jkl Sl-92751 99.44 e-j 20.49 ahc 658.8 c-i 41.20 a-d 0.413 c-h 11.92 h-g 0.055 b-g 0.0088 b-f -0.172 d-j 3.78 g-k SB27-H-1 111.87 e-j 21.50 ah 515.8 f-j 43.56 ah 0.487 a-h 12.22 b-e 0.050 h-h 0.008 c-g 0.102 c-f 3.77 h-k SB27-H-2 186.45 a-d 21.07 a-e 745.5 c-g 38.52 h-g 0.704 ah 11.86 h-g 0.038 d-i 0.0069 d-h -0.504 jk 4.22 a SB27-H-3 211.31 ah 17.01 e-h 895.0 hcd 34.15 e-h 0.730 a-h 11.94 h-g 0.039 d-i 0.0066 d-h -0.045 h-i 3.98 h-g SB33-H-1 111.87 e-j 16.89 e-h 491.0 hij 34.37 e-h 0.389 d-h 11.94 h-g 0.039 d-i 0.0068 d-h -0.192 d-j 4.12 a-d SB33-H-2 149.16 h-g 19.37 a-g 907.4 h-c 40.67 a-e 0.601 a-e 11.51 c-h 0.041 d-i 0.0069 d-h -0.081 b-j 4.18 ab SB33-H-3 136.73 c-i 17.15 c-h 783.1 cde 35.37 c-h 0.487 a-h 11.10 d-i 0.036 f-i 0.0053 fgh 0.016 h-h 3.82 f-k SI-92521 111.87 e-j 16.42 fgh 553.1 e-j 33.92 fgh 0.381 e-h 10.44 hi 0.024 i 0.0036 h -0.639 k 4.12 a-d SI-92615 155.38 b-f 18.96 a-g 646.4 d-i 38.38 h-g 0.593 a-f 12.75 he 0.069 ah 0.0118 ah 0.072 h-g 3.89 e-j SI-92006 87.01 g-i 15.27 h 733.4 c-h 31.45 h 0.276 h 11.96b-g 0.086 a 0.0127 a 214 a-d 3.57 1 Sl-92039 118.09 e-j 19.54 a-g 602.9 e-j 39.83 a-f 0.472 h-h 10.67 ghi 0.060 bed 0.0096 a-e 0.300 ab 4.05 a-e Sl-92128 118.09 e-j 17.77 c-h 559.3 e-j 37.22 h-h 0.440 c-h 12.19 b-f 0.039 d-i 0.0068 d-h -0.119 b-j 4.04 a-e 7233-P.29 93.23 f-j 22.28 a 441.3 ij 45.68 a 0.426 c-h 11.15 d-i 0.059 b-e 0.0091 b-f -0.030 h-i 3.9 e-j DR1-HSF14-P.35 93.23 f-j 20.44 a-d 609.1 e-j 41.80 ahc 0.399 d-h 10.79 ghi 0.053 h-g 0.0082 c-g 0.212 a-d 4.05 a-e Fodder beet 118.09 e-j 20.21 a-e 596.6 e-j 41.12 a-d 0.484 a-h 11.24 d-h 0.038 d-i 0.0059 e-h -0.334 g-k 3.9 e-j O.T.607 68.37 j 19.64 a-f 366.7 j 39.73 a-f 0.270 h 10.96 d-i 0.036 f-i 0.0056 e-h -0.320 f-k 4.01 b-f SI-24 74.58 ij 20.04 a-e 441.3 ij 38.30 h-g 0.286 g-h 11.33 d-h 0.036 f-i 0.0054 fgh 0.070 b-g 3.76 h-1 SB26 192.67 ahc 14.83 h 1106.3 ah 31.08 h 0.600 a-e 10.83 f-i 0.034 ghi 0.0049 gh 0.281 ahc 3.71 jkl SB36 80.80 hij 17.08 d-h 497.2 g-j 34.28 e-h 0.277 h 12.23 hcd 0.065 ahc 0.0098 a-d 0.144 b-e 3.75 i-1 SHR01-P.12 161.59 a-e 16.60 fgh 733.4 c-h 35.62 c-d 0.576 a-f 13.01 b 0.053 b-g 0.0096 a-d -0.092 h-ij 3.95 c-h SHR02-P.4 155.38 d-f 19.53 a-g 652.6 d-i 38.69 h-g 0.600 a-e 11.30 d-h 0.044 c-i 0.0064 d-h 0.635 a 3.66 kl Different letters indicate significant differences using the LSD Test (p < 0.05). £ 2 > — 2 53 a p: The response of the sugar beet (Beta vulgaris L.ssp. vulgaris var. altissima Doll) genotypes to heat stress in initial growth stage different from other genotypes at 30 regarding this trait (Table 3). The lowest amount of An was obtained for '111-52-25' (29.65 mmol m-2 s-1). Stomatal conductance (g) (p < 0.002) of 'S1-92006' and 'S1-92615' (0.09 and 0.07 mol m-2 s-1, respectively) indicated the highest values and the minimum level of this trait was related to 'S1-92521' (0.02 mol m-2 s-1), with no significant difference from 18 of other genotypes (Table 3). The best WUE was observed in 'S1-92006' (0.0130 %) which had no significant difference from 'S1-92615', 'S1-92748', 'SB36, 'SHR01-P.12', and 'S1-92039'. On the other hand, the minimum WUE value belonged to 'S1-92521' (0.0036 %) and '111-52-25' (0.0038 %) (Table 3). 'SHR02-P.4' (0.635) produced the highest value of LTD. The least value for LTD was obtained from 'S1-92521' (-0.639) (Table 3). Also in case of VPD, 'S192006' (3.57 kPa) and 'SB27-H-2' (4.22 kPa) had the highest and the lowest values, respectively (Table 3). In order to have a better assessment of sugar beet genotypes based on important measured traits, the PCA was performed. The PCA revealed that two first components together accounted for 56.82 % (38.23 % and 18.59 %, respectively) (Figure 2). Genotypes placed in the upper quarter on the right (S1-92039, DR1-HSF14-P.35, 7233-P.29, 110-7-8, S1-92749, S1-92685, 7112, SB27-H2 and SB33-H-2) had a high leaf area, total fresh mass, total dry mass, Fv/Fm and greenness index. On the other hand, genotypes placed in the upper quarter on the left (S1-92006, S1-92615, S1-92748, SHR01-P.12, SB36, S1-92751 and 419) had high values of LTD, WUE, gs and An. Also it was revealed that leaf area, total fresh mass and total dry mass were not related to WUE, gs and An. However, Fv/Fm was positively related to all of these six traits. 4 DISCUSSION This study was conducted to determine the tolerant genotypes of sugar beet in high temperature, as well as investigating the suitable criteria for screening tolerant genotypes. According to the results, in laboratory experiment, 'S1-92521', '111-52-25', 'SHR01-P.12', 'O.T. 607', fodder beet, '7233-P.29' and 'SB26' demonstrated a better performance, compared to the other genotypes, almost in all traits in high temperature (Figure 1). Records of 'S1-92521', '111-52-25' and 'SHR01-P.12', in 30 were higher than those in 20, or did not change significantly (Figure 1). Specifically, all measured traits of 's1-92521' showed higher records in 30 except seminal root length which did not change significantly. similarly, except germination percentage, which did not change significantly, all traits of 'SHR01-P.12' improved in 30 compared with 20. There was not a significant change in any of the measured traits of '111-52-25' either. Genotypes O.T. 607, fodder beet, 7233-P.29 and sB26 performed well in all of the traits, too, except in the case of seminal root length which decreased in 30. In 'SB26', a decline in germination percentage was observed as well which can be compensated through planting more seeds. Genotypes with poor performance in laboratory experiment included 110-7-8, SB33-H2 and SHR02-P.4 (Figure 1). In most of the genotypes, seminal root length was negatively affected by heat stress, but concerning the whole seedling, this loss was, to some ex- Figure 2: Biplot of the first and second principal component (PC) axes for greenness index (GI), leaf area (LA), leaf temperature depression (LTD), net photosynthesis (An), photochemical efficiency of PSII (Fv/Fm), stomatal conductance (g5), total dry mass (TDM), total fresh mass (TFM) and water use efficiency (WUE) traits on sugar beet genotypes at 30. Acta agriculturae Slovenica, 115/1 - 2020 47 AAS_vsebina_2020_115_1_koncna 180320.indd 41 26. 03. 2020 12:05:39 M. MALMIR et al. tent, compensated by increasing shoot length under high temperature (Figures 1C, 1D, 1E). According to the results, two of the traits could be good criteria for screening tolerant genotypes to high temperature, seed vigor index and seminal root length. Genetically difference among the genotypes is a criteria to select a trait as a screening tool for stress tolerance (El-Hendawy et al., 2007). In the present study, by increasing the temperature, high differences among the genotypes were observed regarding seed vigor index and seminal root length (Figures 1B, 1C). In other words, almost all of the genotypes showed decreased records under heat stress, and just a few of them could show an increase or maintain without a significant change (Figures 1B, 1C). At this point, by having a more precise look, it can be seen that seed vigor index could be a better screening criteria rather seminal root length. Because genotypes which showed a better or unchanged seed vigor, generally had a good record in other measured traits in the laboratory experiment, too (like '7233-P.29' and 'O.T.607'), but this was not observed in the case of genotypes with a better or unchanged seminal root length (like 'S1-92750' and 'S1-92128') (Figure 1). Seed vigor index has been considered as a screening criteria in previous studies, too (ISTA, 2014). Seed germination factors and seedling properties have been considered as evaluation criteria in breeding programs that worked on environmental stresses such as drought stress (Sadeghian & Yavari, 2004). In greenhouse experiment, high amounts of leaf area, total fresh mass and total dry mass which were accounted for plant yield, were observed in 'S1-92521', 'S1-92039, 'SB33-H-2' and 'SB27-H2' (Table 2). Two other genotypes, S1-92747 and S1-92749, also showed high records for total dry mass and greenness index as well as quantum efficiency of PSII (Table 2, Figure 2). The potential of multivariate analysis technique such as PCA for the identification of tolerant genotypes to environmental stress has been shown in different crops such as rice (Cha-um et al., 2009), sugarcane (Cha-um et al., 2012), tomato (Juan et al., 2005), peanut (Liu et al., 2012) and soybean (Shelke et al., 2017). PCA identifies the probable grouping and establishment of relationships among variables (Martínez-Calvo et al., 2008; Sarabi et al., 2016). Regards lack of significant relationship between stomatal indices and gas exchange parameters maybe due to small number of samples (McElwain et al., 2016), we did not use stomatal characteristics in PCA. According to the PCA analysis, WUE and g produced the most variance among the genotypes, and the least was related to greenness index and Fv/Fm (Figure 2). Furthermore, there was a positive relationship among total dry mass, total fresh mass, leaf area, greenness index and Fv/Fm (Figure 2). This implies that with more quantum efficiency of PsII, indicating less stress inside the plants, photosynthesis got more efficient, and consequently more assimilate were produced. Fv/ Fm had a positive relationship with WUE, gs and An, as well, indicating that more tolerance caused the net photosynthesis to rise (Figure 2). There is a positive relationship between g and An, showing that increasing stomatal conductance leads the An to rise (Urban et al., 2017). Stomatal conductance and An, did not show much relation with the yield parts of the plants, total dry mass and leaf area which could be because of the fact that these traits were measured instantaneously. In Figure 3: Relationship between WUE and An (a) and gs (b) in sugar beet genotypes (n = 31). ** indicate significant correlation at p < 0.05 and 0.01, respectively 48 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 48 26. 03. 2020 12:05:39 The response of the sugar beet (Beta vulgaris L.ssp. vulgaris var. altissima Doll) genotypes to heat stress in initial growth stage addition, it might be due to the increasing of transpiration under high temperature. High VPD values of 'S1-92749', 'S1-92039', 'S1-92521' and 'SB27-H2' resulted higher total dry mass and leaf area; while, the least values of mentioned traits observed from 'S1-92006' and 'SB36' (Figure 2). Difference between the leaf and air temperatures (LTD) showed a negative relationship with yield parameters and also with VPD and a positive relationship with WUE, gs and An (Figure 3). This indicates that under high temperature, tolerant plants could maintain their stomata open resulting in cooling the leaves as a consequence of transpiration. Stomatal conductance to C02 is strictly proportional to stomatal conductance to water (Gilbert et al., 2011). Gas exchange reaction to temperature was related to the interaction effect of some parameters like internal plant water status and VPD (Chaves, 1991). In addition, it has been mentioned that high temperature causes rising in gs (Killi et al., 2017). By increasing VPD, we witness an increase in stomatal conductance and leaf transpiration and consequently the mobility of water and nutrient in the plant (Caird et al., 2007). It has been well established that plants regulate rates of transpiration and photosynthesis in parallel, maintaining a balance between gs and An (Lawson et al., 2011). High positive correlation between canopy temperature depression and stomatal conductance has been observed in sugar beet (Mohammadian et al., 2001; Fukuoka, 2005). Under stress-free conditions the water transpired by the plants evaporates and cools the leaves (González-Dugo et al., 2006; Schauberger et al., 2017). In the present study, plants could maintain their transpiration because of the VPD and because the plants were not in shortage of water, as sufficient water was supplied for them. However, LTD of sugar beet genotypes was varied (Table 3). It was clear that sugar beet genotypes had different ability for cooling their leaves under high temperature. In order to choose a screening criteria for heat tolerance in the sugar beet genotypes, the level of reaction to heat stress should be taken into consideration, as it was done in the laboratory experiment. High reaction intensities among the genotypes were observed in leaf area, total dry mass, gs, An and LTD among which the first two traits need destructive harvest most of the time, so it is better not to choose them as criteria. Stomatal conductance (g) and net photosynthetic rate (An) are not suitable either, because according to the PCA, they were not related to the yield parameters like total dry mass (Figure 2). Leaf temperature depression (LTD), however, could be considered as a screening criteria for heat tolerance, because it does not show the two weakness point mentioned above. Temperature difference between leaves and air has been considered as a screening criteria in previous studies as well (Reynolds et al., 2009). Canopy temperature depression was selected as a suitable screening tool for selecting drought-tolerance cultivars of wheat, because there was a genotypic variation for it, and also it was directly correlated with grain yield (Thapa et al., 2018). Quantum efficiency of PSII (Fv/Fm) could be considered as a good screening criterion for heat tolerance, because it was positively related to gs, An, WUE and yield parameters (Figure 2). Quantum efficiency of PSII (Fv/Fm) has been introduced as a tool for early detection of heat (Zhou et al., 2015) and drought stress (Mohammadian et al., 2003). It was observed that Fv/Fm, shoot fresh mass, shoot dry mass and root dry mass of tomato genotypes decreased under heat stress conditions (Zhou et al., 2015). Also, Fv/ Fm had decreased in sugar beet by drought stress (Mo-hammadian et al., 2003). In addition, possibility of using biomass, canopy temperature depression, greenness index and Fv/Fm for precise screening in heat-tolerant wheat genotypes has been proved (Joshi et al., 2007; Nagar et al., 2015). A similarity was observed between the performance of some genotypes under high temperature in laboratory and that in greenhouse. Genotype S1-92521 showed a good performance in high temperature in both experiments in all traits (Figure 2 and Table 2 and 3). On the other hand, 'SB-27-H-1', 'S1-92006' and '11152-25' performed poorly in both laboratory and greenhouse experiments, especially in yield traits, leaf area and total dry mass. However, other genotypes did not show any noticeable similarity in their reaction to heat stress between laboratory and greenhouse experiments. 5 CONCLUSION The present study investigated the sugar beet genotypes behavior under high temperature condition as well as identifying good screening tools at germination and early growth stages (4-6 leave stage). A high variation was observed among the genotypes in the present study, which is a valuable feature for breeding programs. Based on important traits, 'S1-92039, 'S1-92521, 'SB33-H-2', 'S1-92747, 'S1-92749' and 'SB27-H-2' were tolerant genotypes to high temperature in greenhouse. The only genotype which showed a good tolerance in both laboratory and greenhouse experiments, was S1-92521. Three genotypes, 110-7-8, SB33-H3, and SHR02-P.4, did not perform well in neither of the two experiments. So, in general, except for 'S1-92521, we could not say the genotypes which performed well in laboratory could also tolerate high temperature in greenhouse. In Acta agriculturae Slovenica, 115/1 - 2020 49 49 AAS_vsebina_2020_115_1_koncna 180320.indd 41 26. 03. 2020 12:05:39 M. MALMIR et al. laboratory experiment, seed vigor index was chosen as a good screening tool for selecting heat-tolerant genotypes. In greenhouse, LTD and Fv/Fm were considered as beneficial non-destructive screening tools to find tolerant sugar beet genotypes to high temperature at early growth stages. 6 ACKNOWLEDGEMENTS This research was supported by Tarbiat Modares University and Sugar Beet Seed Institute of Iran. 7 REFERENCES Agrawal, R. L. (2003). Seed technology. Pub. Co. Pvt. Ltd. New Delhi. India. 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Environmental and Experimental Botany, 118, 1-11. https://doi.org/10.1016/j. envexpbot.2015.05.006 52 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 52 26. 03. 2020 12:05:39 doi:10.14720/aas.2020.115.1.692 Original research article / izvirni znanstveni članek Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.) Leila AMRAEE \ Fatemeh RAHMANI 1, 2 Received February 23, 2018, accepted January 1, 2020. Delo je prispelo 23. februarja 2018, sprejeto 1. januarja 2020. Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.) Abstract: Ribonucleic acid (RNA) quality and integrity are crucial for many studies in plant molecular biology. High-quality RNA extraction from plants with high levels of compounds such as polysaccharides, polyphenols, and other secondary metabolites are problematic. RNA extraction from Lemon balm tissues can be difficult due to the presence of polyphenolic and polysaccharide compounds or can be done by expensive protocols. This study shows improvement of a CTAB-based protocol which allows rapid and easy isolation of high-quality RNA from Lemon balm plant. The RNA obtained is suitable for cDNA synthesis and RT-PCR experiments. Key words: Melissa officinalis; CTAB; RNA; RT-PCR CTAB protokol za ekstraskcijo RNK iz melise (Melissa officinalis L.) Izvleček: Kakovost in ohranjenost ribonukleinske kisline (RNK) sta bistveni za mnoge raziskave v molekularni biologiji rastlin. Visoko kakovostni izvlečki RNK iz rastlin z veliko vsebnostjo spojin kot so polisaharidi, polifenoli in drugi sekundarni metaboliti so problematični. Ekstrakcija RNK iz tkiv melise je lahko težavna zaradi prisotnosti polifenolov in polisaharidov ali pa je lahko narejena le z dragimi protokoli. Raziskava predstavlja izboljšanje protokola na osnovi CTAB, ki omogoča hitro in enostavno izolacijo kvalitetne RNK iz melise. Pridobljena RNK je primerna za cDNK sintezo in RT-PCR poskuse. Ključne besede: Melissa officinalis; CTAB; RNK; RT-PCR 1 Urmia University, Faculty of Sciences, Department of Biology, Urmia, Iran 2 Corresponding author, e-mail: f.rahmani@urmia.ac.ir Acta agriculturae Slovenica, 115/1, 53-57, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 53 26. 03. 2020 12:05:37 L. AMRAEE and F. RAHMANI 1 INTRODUCTION The extraction of high quality ribonucleic acid (RNA) is an important step for many studies in plant molecular biology, such as northern blotting, microar-ray hybridization, both targeted real-time PCR (RT-qPCR) analysis and next generation sequencing (NGS) (Gambino et al., 2008; Guerriero et al., 2016). Extraction of RNA in sufficient quantity and quality from the tissues of aromatic, woody, and aquatic plants is particularly challenging, because of high levels of compounds such as polysaccharides, polyphenols, and other secondary metabolites (Gambino et al., 2008; Jordon-Thaden et al., 2015). Phenolic compounds form high molecular weight complexes tend to co-precipitate with RNA by binding to nucleic acids and polysaccharides in the presence of alcohols. Hence, they contaminate the final extract and interfere with subsequent applications (Gambino et al., 2008). Conventional protocols for RNA isolation usually involve the use of detergents, such as cetyl trimethyl ammonium bromide (CTAB) or sodium dodecyl sulphate (SDS), denaturing organic solvents (phenol and chloroform), reducing agents (ß-mercaptoethanol and dithiothreitol), or denaturing agents (guanidinium isothiocyanate salts) (Gambino et al., 2008). Methods involving CTAB, initially were developed for pine tree tissues (Chang et al., 1993) and subsequently used to extract RNA from a wide range of polysaccharide- and polyphenol-rich plant tissues (Iandolino et al., 2004; Meisel et al., 2005; Gambino et al., 2008). Lemon balm is one of the oldest and widely used medicinal plant in the mint family (Lamiaceae), native to southern Europe, Mediterranean region and northern Iran (Döring et al., 2014; Jalal et al., 2015). It is a perennial herb and a rich source of natural antioxidants (Saraydin et al., 2012). Reports indicated that lemon balm had many beneficial effects such as anti-oxidant, anti-bacterial, anti-viral, anti-inflammatory, sedative, mnemonic improvement, reducing excitability, anxiety, stress, gastrointestinal disorders and sleep disturbance. However, the mechanisms underlying these medicine effects remain largely unknown (Jalal et al., 2015). Nutritional efficacy testing, based on selected bio-marker approaches, do not provide global information while application of microarrays and high-throughput qPCR allows quantification of a large number of bio-markers, and consequently enables evaluation of the global effects of nutrients on cells (Jun et al., 2012). Hence, a clean and intact RNA is important for functional genomic studies (Rubio-Piña & Zapata-Pérez, 2011). The aim of this study was to develop an optimized 54 Acta agriculturae Slovenica, 115/1 - 2020 CTAB-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA from polyphenolics and polysaccharide-rich tissues of old lemon balm. 2 MATERIALS AND METHODS 2.1 PLANT SPECIES USED Leaf tissues was collected from 3 months old lemon balm plant (Agricultural and Natural Resources and Education Center, West Azerbaijan, Iran) and frozen in liquid nitrogen. Then, leaf samples were transferred to -80 °C until analyses. Total RNA was extracted with TRIzol (Sigma Alderich), RNX- plus (Sina Clone) and (CTAB)-based protocol. 2.2 NUCLEIC ACID EXTRACTION 2.2.1 (CTAB)-based protocol 2.2.2 Reagents Extraction buffer: 2 % (w/v) CTAB (hexadecyltri-methylammonium bromide), 0.1 M Tris-Hcl (pH: 8), 1.4 M NaCl, 20 mM EDTA (pH: 8), 2 % PVP (polyvinylpyrrolidone), BME (^-mercaptoethanol) (to a final concentration of 10 % (v/v), added right before use). Sodium acetate 3 M (pH: 5) Chloroform/ IAA (isoamyl alcohol) (24/1) Phenol/ chloroform/ isoamyl alcohol (25/24/1) 70 % ethanol RNA/RNase-free water DNase inactivation reagent Liquid nitrogen in a Dewar flask Equipment list Water bath at 65 °C Vortexer 24-place centrifuge cooled to 4 °C Incubator at 37 °C with orbital shaker Procedure 1- About 80-100 mg of leaf tissue was ground to a fine powder using liquid nitrogen and transferred into a 2 ml centrifuge tube. Then, 900 pl of extraction buffer and 100 pl of ^-mercaptoethanol were added. The mixture was shaken for 30 s and then incubated at 65 °C for 10 min, inverting the tube 3-4 times every now and then during incubation. 2- Then, 800 pl of chloroform was added. The mixture was shaken for 30 s and centrifuged at 12000 rpm AAS_vsebina_2020_115_1_koncna 180320.indd 54 26. 03. 2020 12:05:41 Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.) for 10 min at 4 °C. The supernatant was transferred to a new tube. 3- 800 pl of phenol/chloroform/ isoamyl alcohol (25/24/1) was added and shaken for 1 min. Then, the mixture was centrifuged at 12000 rpm for 15 min at 4 °C (this step was repeated twice). 4- The supernatant was transferred to a new tube and an equal volume of chloroform/isoamyl alcohol (24/1) was added. Samples were shaken for 30 s and centrifuged at 12000 rpm for 10 min at 4 °C. 5- The final supernatant was transferred to a new tube. 1/10 volume of sodium acetate (3 M, pH: 5) and equal volume of cool isopropanol were added to the tube, then kept at -20 °C for at least 1 h. Next, the samples were centrifuged at 13000 rpm for 15 min at 4 °C. 6- The pellets were successively washed with 70 % ethanol for one or two times. After a short drying time at room temperature, the pellets were dissolved in 2050 pl of DEPC water. 7- Finally, 1 pl of DNase enzyme was added and incubated at 37 °C for 30 min. 2.3 ESTIMATION OF RNA QUALITY The purified RNA was measured by spectrophotometry analysis (Nanodrop 2000c; Thermo Fisher Scientific, Waltham, USA). Contamination due to proteins and phenol/carbohydrates was determined by recording the OD ratios; A260/280 and A260/230, respectively. In order to verify RNA integrity, extracts were fractioned by electrophoresis in a 1.5 % agarose gel, stained with ethidium bromide and visualized in a gel documentation system (InGenius3, Syngene, UK). 2.4 RT-PCR Single-stranded cDNA was synthesized from 500 ng total RNA using reverse transcriptase and oligo (dT), following the manufacturer's protocol (Thermo Scientific). The synthesized cDNA was used in a PCR reaction in order to estimate the expression level of the tubulin gene using following primers: Tubulin-FWD: 5 '-GCTTTCAACACCTTCTTCAGTG-3 'and Tubulin REV: 5'-CTTTCTCAGCTGAGATCACTG G-3'. 3 RESULTS AND DISCUSSION 3.1 ESTIMATION OF RNA QUALITY The isolation of high-quality RNA from enrich polysaccharides and polyphenols plant tissues is quite challenging (Liao et al., 2014). Lemon balm leaves contain secondary metabolites such as, polyphenolic acid (rosmaric acid, trimeric compounds and some flavonoids), terpenoids, carboxylic acid, essential oils, various sugars, golden and pectic materials (Jafarpour & Fard, 2016) which affect the quality of the isolated RNA. The biosynthesis of secondary metabolites in plants is influenced by season harvesting, growing region, agronomic conditions and type of processing (Wahby, 2016). Therefore, RNA extraction from lemon balm tissues can be difficult due to the presence of these compounds. Polyphenolic compounds (particularly tannins) are readily oxidized to form quinones, which bind irreversibly to nucleic acids and proteins, and may decrease RNA yield, as well as inhibit PCR amplification (Shu et al., 2014). In contrast, polysaccharides can co-precipitate and degrade RNA, constitute the major obstacle of RNA isolation in low ionic strength buffers, and cause the browning effect and make RNA difficult to be dissolved (Shu et al., 2014). In addition, these interfering chemicals severely interfere with RNA-de-pendent RT and DNA polymerases and cause the RT-PCR to fail. Thus, these contaminating substances must be eliminated during RNA isolation (Hou et al., 2011; Ouyang et al., 2014; Sabzevari & Hosseini, 2014). In this case, successful isolation of intact RNA from tissues rich in polysaccharides and polyphenolic compounds, is a basic requirement for many molecular studies Figure 1: (A): Ethidium bromide agarose gel (1.5 % (w/v)) was used to separate total RNA and (B): pellet of RNA extracted from leaf tissues of three months old lemon balm plant by (a) CTAB; (b) TRIzol and (c) RNX-Plus. Acta agriculturae Slovenica, 115/1 - 2020 55 55 AAS_vsebina_2020_115_1_koncna 180320.indd 57 26. 03. 2020 12:05:41 L. AMRAEE and F. RAHMANI Table 1: Quality of total RNA isolated from three months old leaves of lemon balm based on three different protocols Method a260^280 ValUe a260^ 230 ValUe CTAB 1.96 1.80 TRIzol 1.52 0.54 RNX-Plus 1.67 0.38 (Hunter & Reid, 1999; Martínez-Fuentes et al., 2015). The currently commercial kits such as RNeasy, BioZOL and BIOLINE are simple, rapid, non-toxic with good yields of high-quality RNA from plant tissues, but too expensive. Moreover, successful RNA extraction by RNX- Plus and TRIzol from lemon balm plant has only been reported in seedling stage and explants grown in solid MS medium, respectively (Kim et al., 2011; Nasiri-Bezenjani et al., 2014). Upon this status, we tested three different methods (modified CTAB, TRIzol and RNX- Plus) to evaluate the best and effective RNA extraction method from 3 months old lemon balm plant. In current study, evaluation of quality and quantity of total RNA isolated from three different protocols was done by agarose denatured gel (Figure 1a) and nanodrop (Table 1), respectively. The CTAB method already showed a much better RNA quality than TRIzol and RNX-Plus methods by only visualizing the pellet. The RNA pellet appeared in white color in CTAB method while TRIzol and RNX-Plus methods produced brown colored pellet (Figure 1b). The brown color indicates the presence of carbohydrates, proteins and phenolic compounds mixed with the extracted RNA (Rubio-Piña & Zapata-Pérez, 2011). In the CTAB method, brown color and contamination (phenolic compounds) were reduced by addition of PVP and high concentrations of ^-mercaptoetanol in the extraction buffer. The PVP strongly binds to the polyphenol compounds through hydrogen bonds, and removes phenolic compounds and secondary metabolites from nucleic acid, also preventing browning effect of polyphenols (Rubio-Piña & Zapata-Pérez, 2011; Shu et al., 2014). The strong reductant ^-mercaptoetanol is conventionally used at 10 % (v/v) to inhibit RNase activity and prevent any possible oxidation reactions (Ouyang et al., 2014). On the other hand, chloroform: isoamyl alcohol is used to eliminate remaining phenolic compounds and PVP from solution (Shu et al., 2014; Martínez-Fuentes et al., 2015). Total RNA isolated by modified CTAB and RNX- Plus method showed two bright bands (28S rRNA and 18S rRNA). However, the gel electrophoresis result of TRIzol method revealed no production of clear and bright RNA band, indicating low quantity of RNA. The A ratio detected below 0.6 in both TRIzol 26U 230 and RNX-Plus methods (Table 1), representing high con-56 Acta agriculturae Slovenica, 115/1 - 2020 Figure 2: RT- PCR product with tubulin specific primers using total RNA isolated from leaves of three months old lemon balm plant based an CTAB method. Lane 1: 50 bp DNA ladder (Thermo Fisher Scientific), Lane 2: 750 bp amplified Tubulin gene. tamination with phenolic compounds or polysaccharides and very low molecular grade of RNA (Martínez-Fuentes et al., 2015). It is important to note that RNA solution with A260/280 and A260/230 ratios of 1.8~2.0 corresponds to a high quality (Hou et al., 2011; Martínez-Fuentes et al., 2015). However, CTAB based method produced highly purified RNA with AJ™ and A, ratios of 1.96 and 1.80, re- 260 280 260 230 spectively (Table 1). 3.2 RT-PCR RNA sample extracted by the improved CTAB method, was further characterized with RT-PCR using tubulin primers. The results showed amplification of an expected fragment of approximately 750 bp (Figure 2), suggesting high quality of extracted RNA at the molecular level. Due to RNA contamination, reverse transcriptase enzyme will fail to produce cDNA, which consequently, RT-PCR could result in failure to amplify the target sequence (Sabze-vari & Hosseini, 2014). Thus, the successful synthesis of the first-strand cDNA and PCR amplification (Figure 2) clearly shows that the RNA is free of contaminants. 4 CONCLUSION Our results demonstrated that, modified CTAB AAS_vsebina_2020_115_1_koncna 180320.indd 56 26. 03. 2020 12:05:41 Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.) protocol extracted highly qualified RNA from lemon balm old leaves compared to other tested methods. The method is cost effective and may be simply used as an alternative method for RNA isolation from recalcitrant plant tissues. 5 REFERENCES Chang, S., Puryear J., Cairney J. (1993). 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Melissa officinalis essential oil reduces plasma triglycerides in human apolipoprotein E2 transgenic mice by inhibiting sterol regulatory element-binding protein-1c-dependent fatty acid synthesis. The Journal of Nutrition, 142(3), 432-440. https://doi.org/10.3945/ jn.111.152538 Kim, J. H., Hossain, A. M., Kim, N. H., Lee, D. H., Lee, H. J. (2011). Identification and functional characterization of the GALACTINOL SYNTHASE (MoGolSl) gene in Melissa officinalis plants. Journal of Applied Biological Chemistry, 54(4), 244-251. https://doi.org/10.3839/jabc.2011.040 Liao, C. M., Li, J., Liu, X. H., Zhang, Y. S. (2014). An effective method for extracting total RNA from Dioscorea oppos-ita Thunb. Genetics and Molecular Research, 13, 462-468. https://doi.org/10.4238/2014.January.21.15 Martínez-Fuentes, A., Mesejo, C., Agustí, M., Reig, C. (2015). Toward a more efficient isolation of total RNA from loquat (Eriobotrya japonica Lindl.) tissues. Fruits, 70(1), 47-51. https://doi.org/10.1051/fruits/2014042 Meisel, L., Fonseca, B., González, S., Baeza-Yates, R., Cambiazo, V, Campos, R., Gonzalez, M., Orellana, A., Retamales, J., Silva, H. (2005). A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biological Research, 38(1), 83-88. https://doi.org/10.4067/S0716-97602005000100010 Nasiri-Bezenjani, M. A., Riahi-Madvar, A., Baghizadeh, A., Ah-madi, A. R. (2014). Rosmarinic acid production and expression of tyrosine aminotransferase gene in Melissa officinalis seedlings in response to yeast extract. Journal of Agricultural Science and Technology, 16(4), 921-930. Ouyang, K., Li, J., Huang, H., Que, Q., Li, P., Chen, X. (2014). A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba. Biotechnology & Biotechno-logical Equipment, 28(6), 1008-1013. https://doi.org/10.1080 /13102818.2014.981086 Rubio-Piña, J. A., & Zapata-Pérez, O. (2011). Isolation of total RNA from tissues rich in polyphenols and polysaccha-rides of mangrove plants. Electronic Journal of Biotechnology, 14(5), 11-11. https://doi.org/10.2225/vol14-issue5-fulltext-10 Sabzevari, A. G., & Hosseini, R. (2014). A quick, efficient, and cost-effective method for isolating high-quality total rna from tomato fruits, suitable for molecular biology studies. Preparative Biochemistry and Biotechnology, 44(4), 418431. https://doi.org/10.1080/10826068.2013.833109 Saraydin, S. U., Tuncer, E., Tepe, B., Karadayi, S., Ozer, H., Sen, M., Karadayi, K., Inan, D., Elagoz, S., Polat, Z., Duman, M. (2012). Antitumoral effects of Melissa officinalis on breast cancer in vitro and in vivo. Asian Pacific Journal of Cancer Prevention, 13(6), 2765-2770. https://doi.org/10.7314/ APJCP.2012.13.6.2765 Shu, C., Sun, S., Chen, J., Chen, J., Zhou, E. (2014). Comparison of different methods for total RNA extraction from scle-rotia of Rhizoctonia solani. Electronic Journal of Biotechnology, 17(1), 50-54. https://doi.org/10.1016Zj.ejbt.2013.12.009 Wahby, M. (2016). Effect of drying period and harvesting times on herb, essential oil content and its constituent's from different parts of Melissa officinalis. Journal of Chemical and Pharmaceutical Research, 8(5), 919-933. Acta agriculturae Slovenica, 115/1 - 2020 57 AAS_vsebina_2020_115_1_koncna 180320.indd 57 26. 03. 2020 12:05:41 AAS_vsebina_2020_115_1_koncna 180320.indd 58 26. 03. 2020 12:05:37 doi:10.14720/aas.2020.115.1.1316 Original research article / izvirni znanstveni članek Higher yielding varieties of common buckwheat (Fagopyrum escu-lentum Moench) with determinate growth habit (single mutation det) manifest higher photosynthesis rate at stage of grain filling Alexandr V. AMELIN \ Aleksey N. FESENKO 2, Evgeniy I. CHEKALIN \ Ivan N. FESENKO 2, 3, Valeriy V. ZAIKIN 1 Received October 28, 2019; accepted January 4, 2020. Delo je prispelo 28. oktobra 2019, sprejeto 4. januarja 2020. Higher yielding varieties of common buckwheat (Fagopyrum esculentum Moench) with determinate growth habit (single mutation det) manifest higher photosynthesis rate at stage of grain filling Abstract: Comparison of common buckwheat varieties with determinate vs. indeterminate growth habit reveals no differences in leaf photosynthesis rate at stage before flowering. However, at stage of seed filling the difference was significant. Maximal difference was 20 days after early flowering, i.e in period of most intensive seed formation. These results show that determinate varieties have higher sink strength providing by developing seeds. It is correlated with higher yield ability of such varieties. Probably, growth limitation resulting from det-mutation leads to some shifts in system of sink priorities of buckwheat plant and allows initiate the development of additional seeds. 0ne more possible cause of alteration of the physiological parameters in determinate varieties is some optimization of plant structure: in terms of physiology the determinate buckwheat is a plant which is more similar to cereals than indeterminate buckwheat. However, underlying physiological changes accompanying the transition from indeterminate toward determinate growth in buckwheat remain almost unknown. Assumption about strong effect of det-mutation per se on photosynthesis rate was not supported in our work. Alternative assumption about accumulation of additional genes enhancing the sink ability suggests opportunities for additional progress in the selection work using tools evaluating photosynthesis intensity at stage of grain filling. Key words: Fagopyrum esculentum; buckwheat; photosynthesis rate; sink strength; growth habit Bolj donosne sorte navadne ajde (Fagopyrum esculentum Moench) z determinantno rastjo (enojna det mutacija) imajo večjo fotosintezo v fazi polnjenja zrnja Izvleček: Primerjava sort navadne ajde z determinantno in nedeterminatno rastjo ne kaže razlik v fotosintezi listov v fazi pred cvetenjem, vendar je razlika v fazi polnjenja zrnja značilna. Največja razlika je bila20 dni po začetku cvetenja, to je v fazi najbolj intezivnega oblikovanja semen. Ti izsledki kažejo, da imajo determinantne sorte večjo moč ponora, ki jo dajejo razvijajoča se semena. To je povezano tudi s sposobnostjo večjega pridelka teh sort. Verjetno je omejitev rasti posledica det-mutacije, kar vodi v nekatere premike v sistemu prioritet ponora v rastlinah ajde in, kar vzpodbudi razvoj dodatnih semen. Nadaljni možni vzrok v spremembi fizioloških parametrov determinatnih sort je v optimizaciji zgradbe rastline, determinatna ajda je v fiziološkem pogledu bolj podobna žitom kot pa nedeterminatni ajdi. Kljub vsemu, pa ostajajo fiziološke spremembe, ki spremljajo prehod od nede-terminantne k determinatni rasti skoraj popolnoma neznane. Domneva o močnemu činku det-mutacije per se na fotosintezo v našem delu ni bila potrjena. Alternativna domneva o kopičenju dodatnih genov, ki pospešujejo sposobnost ponora daje priložnosti za nadaljni napredek pri selekcijskem delu z uporabo ovrednotenja jakosti fotosinteze kot orodja v fazi polnjenja zrnja. Ključne besede: Fagopyrum esculentum; navadna ajda; velikost fotosinteze; moč ponora; rastna oblika 1 Orel State Agrarian University, 302019, Generala Rodina 69, Orel, Russia 2 Federal Scientific Center of Legumes and Groats Crops, 302502, p/o Streletskoe, Orel, Russia 3 Corresponding author, e-mail: ivanfesenko@rambler.ru Acta agriculturae Slovenica, 115/1, 59-65, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 59 26. 03. 2020 12:05:37 A. V. AMELIN et al. 1 INTRODUCTION Fagopyrum esculentum Moench (common buckwheat) is a species cultivated as groats or grain crop in many countries, mainly in Russia and China (Wang & Campbell, 2004; Fesenko et al., 2016). During a last half of century, crop evolution of common buckwheat have resulted the increasing of the species grain productivity. Physiological basis of the result is mainly unknown. Probably, it was associated with correction of source-sink relationships. In Russia, breeding of this crop on scientific basis was started in 1900s on Shatilov's Experimental Station (Orel region, Russia). First Russian commercial variety, Bogatyr, was bred by selection of heavier (i.e. larger and better filled) fraction of grain from cultivated local buckwheat. Beginning from 1960s buckwheat breeding in Russia is based on application of several morphological mutations (Fesenko, 1983; Fesenko et al., 2006). Agricultural practice has chosen mainly determinate varieties based on a mutation d (det) (Fesenko, 1968; ohnishi, 1990) that limits the generative development of shoots by 3-5 inflorescences without possibility for development of any additional ones (Fesenko, 1983; Fesenko et al., 2009)(Fig. 1). First variety of this type was registered in 1985. Since the beginning of 21st century the share of the determinate varieties in the buckwheat sowing area in Russia was increased from 8.2 % to 56.2 %, which led to an increase in average buckwheat yield by 1.5 times (FAO, 2014). Breeding and research work with determinate type buckwheat was conducted also in Slovenia (Bohanec & Kreft, 1981; Luthar et al., 1986; Kreft, 1989), Serbia (Neskovic et al., 1990) and Japan (Kasajima et al., 2016). The steady increase in productivity of determinate varieties compared to traditional ones implies, among others, changes in their physiology, which probably include correcting some processes associated with regulation of photosynthesis. Although the results of experiments evaluating correlation between photosynthesis rate and plant productivity are not always unambiguous (Peng et al., 1991; Long et al., 2006; Driever et al., 2014), the intensification of the assimilate synthesis looks as one of major factors in plant productivity growth. Since changes in the intensity of photosynthesis are one of the supposed reasons for a higher grain productivity of buckwheat with determinate growth, we compared the common buckwheat varieties of indeterminate, i.e. traditional, and determinate types on seasonal dynamics of the photosynthesis rate. Also, we evaluated the influence of det-mutation itself on photosynthesis rate in buckwheat. The aim of this article was to describe results of this work and to discuss it. 2 MATERIAL AND METHODS 2.1 PLANT MATERIAL Two local cultivars from Orel region represented by accessions k-406 and k-1709 from collection of Vavilov's Institute of Plant Industry, St.-Petersburg; three varieties of traditional type with indeterminate growth habit (genotype DET/DET) Bogatyr (registered in 1938), Kalininskaya (1954) and Shatilovskaya 5 (1967); four varieties with determinate growth (genotype det/det) Demetra (1995), Dozhdik (1998), Dikul (1999) and Devyatka (2004). All the varieties are similar in characteristics of vegetation period and manifest similar time of flowering beginning. F2 hybrids 'Dikul Figure 1: Shoots of a) determinate and b) indeterminate buckwheat shoots 60 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 60 26. 03. 2020 12:05:42 Higher yielding varieties of common buckwheat ... habit (single mutation det) manifest higher photosynthesis rate at stage of grain filling Table 1: Some essential dates of the experiment Year Sowing date Date of seedlings appearance Varieties groups *Dates of early flowering 2013 May 23 May 29 Indeterminate June 19-20 Determinate June 19-22 2014 May 21 May 27 Indeterminate June 15-19 Determinate June 18-20 2015 May 29 June 4 Indeterminate June 28 Determinate June 28-29 * 10-15 % of plants have any opened flowers Table 2: Weather conditions in days when photosynthesis rate was measured Air temperature, °C Air humidity, % Year Date average max average min 2013 June 13 19.5 24.4 61 41 June 26 21.8 30.9 81 49 July 6 23.9 31.1 64 34 July 16 17.1 21.6 90 73 2014 July 3 21.8 26.5 39 22 July 14 13.6 18.0 81 61 July 24 13.6 19.2 74 49 2015 June 20 15.6 18.0 93 90 July 6 21.2 27.1 60 42 July 16 17.4 24.2 76 43 July 26 26.3 33.8 57 37 2018 July 13 22.1 28.8 69 39 x Bogatyr' were used to evaluate the influence of det-mutation itself on photosynthesis intensity. 2.2 EXPERIMENTAL DESIGN The experiment was conducted in 2013-2015 in crop rotation of buckwheat breeding laboratory of the All-Russia Research Institute of Grain Legumes and Groats Crops, Orel, Russia. A plot area was 10m2. The plots locations were random, with fourfold replication. Sowing rate was 300 seeds per square meter. Dates of sowing and early flowering are presented in Table 1. The photosynthesis intensity was evaluated on intact plants in real-time regime with a portable gas analyzer Li-COR - 6400 using the original methodology of the company Li-COR. The evaluations were conducted three times in 2014 and four times in both 2013 and 2015 at different developmental stages (see Results). Fifteen plants of every determinate variety and twelve plants of every indeterminate one were analyzed every time of the experiment (60 plants of every type in sum). The measurements were made in order "indeterminate - determinate - indeterminate - etc" with alteration every five plants. Yield data were obtained by weighting of the grain yield from each plot. All these parameters were used to compare the two groups of varieties, i.e. indeterminate (traditional) vs. determinate. Significance of the differences between the groups was evaluated using t-statistics. 2.3 WEATHER CONDITIONS Weather conditions deviate during experiments, but in permissible range (Table 2). In addition, gas exchange was evaluated in morning time, 9AM - 11AM, when conditions were maximally suitable. They did not notably influence the results of gas exchange evaluation. For example, photosynthesis rate was not decreased in 2014 July 14, the coldest day of the work, in comparison Acta agriculturae Slovenica, 115/1 - 2020 61 AAS_vsebina_2020_115_1_koncna 180320.indd 61 26. 03. 2020 12:05:42 A. V. AMELIN et al. with results at this developmental stage in two other years. 3 RESULTS 3.1 GRAIN PRODUCTIVITY OF VARIETIES IN THE EXPERIMENT In this experiment the determinate varieties manifest higher grain productivity than the indeterminate varieties, on average; the difference was significant in all years of the study (Table 3). 3.2 PHOTOSYNTHESIS RATE AT DIFFERENT DEVELOPMENTAL STAGES At stage before flowering the photosynthesis rate was measured only in 2013 and 2015. In a season scale, in 2013 this stage values of photosynthesis rate were maximal, but in 2015 - minimal. Comparison between 2013 and 2015 reveals at least twice difference (Table 4), but comparison between indeterminate and determinate varieties in every year reveals no any differences. During period of flowering, i.e. 10, 20 and 30 days followed to early flowering, the measuring of photosynthesis rate was conducted in 2013, 2014 and 2015. Determinate varieties manifest significantly higher mean values of CO2 exchange in all cases with an exception of 10 days after early flowering in 2014 (Table 4). In all years of the experiment the maximal differences between determinate and indeterminate varieties were at stage of 20 days after early flowering. Maximal values of photosynthesis rate among determinate and indeterminate varieties sometimes were almost identical, and sometimes were even higher for indeterminants. Therefore, really, the possible maximal CO2 exchange at level of individual plant of determinate varieties is not always higher, but CO2 exchange at population level is always sufficiently more consistent Table 3: Grain yield (t ha 1) of varieties with indeterminate and determinate growth habits Year Varieties group X±m t P 2013 Indeterminate 1.02±0.12 Determinate 1.38±0.11 2.21 0.05 2014 Indeterminate 1.49±0.06 Determinate 1.97±0.07 5.21 0.001 2015 Indeterminate 1.25±0.09 Determinate 1.52±0.08 2.24 0.05 at stage of seed filling. It correlates with the sufficiently higher and more consistent grain productivity of determinate varieties in Russia. 3.3 THE DET-MUTATION PER SE DOES NOT AFFECT THE PHOTOSYNTHESIS RATE The differences between varieties with indeterminate and determinate growth habits in the photosynthesis rate at stage of grain filling may be due to either the effect of the det-allele per se or the accumulation of additional genes affecting the intensity of gas exchange. We analyzed F2 hybrids between indeterminate (Boga-tyr) and determinate (Dikul) varieties. As expected, all F1 hybrids were indeterminate; F2 segregation was Men-delian, 182 indeterminate : 67 determinate (x2 = 0.48; p = 0.49). For the test 55 plants of each type were selected and labeled. Measurements were made alternately: one determinate plant, one indeterminate plant etc. The experiment shown no differences in photosynthesis rate between the indeterminate and determinate groups of F2 hybrids: photosynthesis rate was 10.89 ± 0.51 with range 1.32 - 18.22 for indeterminate sample and 10.07 ± 0.57 with range 1.00 - 20.12 for determinate sample from F2 population (t = 1.07; p > 0.1). Therefore, the advantages of determinant varieties in photosynthesis rate are not directly conditioned by det-allele. Obviously, some other genes were accumulated which increase the photosynthesis rate at stage of grain filling. This indicates the possibility of selection for the intensity of photosynthesis. 4 DISCUSSION Photosynthesis rate is regulated by sink strength (assimilate demand) and source strength (assimilate supply) (King et al., 1967; Marcelis et al., 2004; Wubs et al., 2009; Borrill et al., 2015; Zhang et al., 2015; White et al., 2016). Obviously, there are limitations for the photosynthesis intensity, which are various between crops. So, maximal values of CO2 assimilation was 42.5 pmol m-2 s-1 for sorghum (Salas-Fernandes et al., 2015) and 30-33 pmol m-2 s-1 for a high-yielding indica cultivar of rice (Adachi et al., 2014). Sometimes, photosynthesis rate is restricted by CO2 concentration in air: several studies on rice revealed polymorphism for reaction on increasing of CO2 concentration in air that was interpreted as differences in sink ability of filling grain between different varieties (Chen et al., 2014; Zhu et al., 2014). However, photosynthetic apparatus usually does not work at full capacity. So, reported maximum indi- 62 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 62 26. 03. 2020 12:05:42 Higher yielding varieties of common buckwheat ... habit (single mutation det) manifest higher photosynthesis rate at stage of grain filling Table 4: Leaf photosynthesis rate (|imol m-2 s 1) of buckwheat plants at different stage of their life cycle in field conditions Indeterminate varieties Determinate varieties t P Year Developmental stage Date X ± m (range) X ±m (range) 2013 Before flowering June 13 14.04 ± 0.75 (8.54 - 21.85) 14.78 ± 1.05 (8.45 - 21.97) 0.57 - 10 days after early flowering June 26 9.59 ± 0.62 (4.14 - 17.30) 11.84 ± 0.64 (5.77 - 16.25) 2.53 0.02 20 days after early flowering July 6 9.03 ± 0.80 (4.25 - 15.69) 14.68 ± 0.51 (10.75 - 18.60) 5.96 0.001 30 days after early flowering July 16 9.54 ± 0.47 (5.32 - 12.98) 12.08 ± 0.43 (8.43 - 15.47) 3.96 0.001 2014 10 days after early flowering July 3 10.24 ± 0.60 (6.23 - 17.30) 11.04 ± 0.51 (7.98 - 14.67) 1.02 - 20 days after efflorescence July 14 11.76 ± 0.62 (5.77 - 17.62) 13.56 ± 0.57 (8.81 - 17.54) 2.14 0.05 30 days after early flowering July 24 8.55 ± 0.27 (6.05 - 11.99) 9.63 ± 0.46 (6.44 - 14.00) 2.02 0.05 2015 Before flowering June 20 6.50 ± 0.09 (5.87 - 6.96) 6.24 ± 0.13 (5.58 - 6.78) 1.64 - 10 days after early flowering July 6 11.44 ± 0.19 (10.48 - 12.96) 12.57 ± 0.27 (11.21 - 14.28) 3.42 0.001 20 days after early flowering July 16 12.19 ± 0.18 (11.14 - 13.10) 14.07 ± 0.17 (13.33 - 14.70) 7.59 0.001 30 days after early flowering July 26 8.27 ± 0.19 (6.97 - 9.30) 9.38 ± 0.15 (8.32 - 10.17) 4.59 0.001 vidual leaf net C02 assimilation rates for V. vinifera L. and other Vitis species approach 20 |mol m-2 s-1(Roper & Williams, 1989; Gamon & Pearcy, 1990). But more commonly reported maximum rates fall in the range of 8 to 13 |mol C02 m-2 s-1 (Downton et al., 1987; Cor-reia et al., 1990). 0n Eucalyptus globulus Labill. excision of several leaves causes increased photosynthesis in the remained leaves (Eyles et al., 2013). In addition, the increased assimilate demand also enhances photosynthesis (Aranjuelo et al., 2013). Finally, total sink strength can be increased as result of interactions with other organisms: for example, soybean plants inoculated with two different strains of Bradyrhizobium japonicum (Kirchner,1896) Jordan, 1982 had 14-31 % higher rates of photosynthesis than N-fertilized plants (Kaschuk et al., 2012). Different groups of buckwheat varieties were not different in both sink and source strength at stage of vegetative development. However, at stage of seed filling the significant differences in photosynthesis rate were revealed between varieties with determinate vs. indeterminate growth habits. At present time the varieties with determinate growth habit cover more than a half of sowing area under buckwheat in Russia. Earlier, the higher productivity of such varieties was interpret- ed only in terms of shift in balance between competitive sinks, i.e. vegetative growth and seed development. 0ur work elucidates that grain filling in buckwheat is limited by itself sink capacity rather than source capacity of leaves and competitive interactions with other growing organs. All buckwheat varieties produce redundant number of flowers. 0bviously, not all of the flowers produce seeds. It was discussed that there is often a hierarchy among sinks (Wardlaw, 1990), i.e. some organs have priority and suffer less from a reduction in assimilate supply than other organs. Such hierarchy usually is resulted from evolution of certain strategy of a species adaptation (Wardlaw, 1990). Since main adaptive property of common buckwheat is ability to continuous intensive growth, primary sinks in buckwheat plant are shoot meristems; seed production is only secondary sink (Fesenko, 1983). It explains the very little increasing of buckwheat seed productivity due to selection of most vigorous and productive plants. Progeny of such plants also had vigorous growth (maybe more vigorous than parental population), but competition between plants in the canopy was also very strong, and seed production was poor: only few plants produce sufficient num- Acta agriculturae Slovenica, 115/1 - 2020 63 AAS_vsebina_2020_115_1_koncna 180320.indd 63 26. 03. 2020 12:05:42 A. V. AMELIN et al. ber of seeds, and productivity of whole canopy remains low (Fesenko et al., 2006). Determinate varieties manifest higher and more consistent yield obviously due to set several additional seeds per plant in comparison to indeterminate varieties. Setting the additional seeds on determinate plants could be explained by essentially reduced competition from vegetative growth at time of seed formation in comparison to indeterminate ones. However, this hypothesis does not explain why seed filling together with indeterminate growth in varieties of traditional type drive less sink strength than seed filling together with reduced vegetative growth in varieties of determinate type. Besides, it does not answer a question, why indeterminate varieties do not set additionally seeds with possible following growth of photosynthesis rate? Probably, growth limitation resulting from det-mutation leads to some shifts in the priorities and allows initiate the development of additional seeds. One more possible base of the alteration of physiological and grain yield parameters in determinate varieties is some optimization of plant structure: determinate buckwheat is a plant, which is more similar with cereals than indeterminate buckwheat (det-mutation is a first step of buckwheat to became "cereal" in terms of physiology). However, underlying physiological changes accompanying the transition from indeterminate toward determinate growth in buckwheat remain almost unknown. Attempting to determine any genes influencing photosynthesis rate led to discovering QTLs affecting, for example, chlorophyll content, stomatal resistance, transpiration rate (Teng et al., 2004; Wang et al., 2015), mesophyll conductance, and root surface area determining hydraulic conductance (Adachi et al., 2014). A mutation of rice erect panicle 3 (ep3) decreases photosynthesis due to reducing stomatal conductance (Yu et al., 2015). Assumption about strong effect on photosynthesis rate of det-mutation per se was not supported in our work. Possible alternative explanation for higher photosynthesis rate together with higher seed productivity of the determinate varieties is accumulation of some additional genes enhancing the sink ability of filling seeds. The mechanisms of functioning of these genes are currently unknown. However, such assumption suggests opportunities for additional progress in the selection work using tools evaluating photosynthesis intensity at stage of grain filling. 5 CONCLUSION The present study revealed the buckwheat varieties 64 Acta agriculturae Slovenica, 115/1 - 2020 with determinate growth habit (a mutation det) manifested higher photosynthesis rate at stage of grain filling compared to varieties with indeterminate growth habit. The mutation det itself is not determining the difference. 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Genetic differences in fruit-set patterns are determined by differences in fruit sink strength and source: sink threshold for fruit set. Annals of Botany, 104, 957-964. https:// doi:10.1093/aob/mcp181 Yu, H., Murchie, E.H., Gonzalez-Carranza, Z.H., Pyke, K.A., Roberts, J.A. (2015). Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development. Journal of Experimental Botany, 66, 1543-1552. htt-ps://doi:10.1093/jxb/eru525 Zhang, C., Tanabe, K., Tamura, F., Matsumoto, K., Yoshida, A. (2015).13C-photosynthate accumulation in Japanese pear fruit during the period of rapid fruit growth is limited by the sink strength of fruit rather than by the transport capacity of the pedicel. Journal of Experimental Botany, 56, 2713-2719. https://doi:10.1093/jxb/eri264 Zhu, C., Zhu, J., Cao, J., Jiang, Q., Liu, G., Ziska, L.H. (2014). Biochemical and molecular characteristics of leaf photosynthesis and relative seed yield of two contrasting rice culti-vars in response to elevated [CO2]. Journal of Experimental Botany, 65, 6049-6056. https://doi:10.1093/jxb/eru344 Acta agriculturae Slovenica, 115/1 - 2020 65 AAS_vsebina_2020_115_1_koncna 180320.indd 65 26. 03. 2020 12:05:42 AAS_vsebina_2020_115_1_koncna 180320.indd 66 26. 03. 2020 12:05:46 doi:10.14720/aas.2020.115.1.982 Original research article / izvirni znanstveni članek Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Valeriia BONDAR 1 2, Nataliia MAKARENKO 1 Received December 12, 2018; accepted December 27, 2019. Delo je prispelo 12. decembra 2018, sprejeto 27. decembra 2019. Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Abstract: Modern technologies of winter wheat growing need to be improved taking into account the results of ecological evaluation of their impact on soil fertility indices. We aimed to assess the technologies of winter wheat growing in different soil and climatic conditions of Ukraine by their influence on soil fertility. It is known that in order to estimate ecological safety of crop growing technologies, it is advisable to use a method based on identifying negative impacts on soil fertility. We propose the group of deviation values from the optimum as follows: (i) strong, which leads to an unsatisfactory ecological condition (> 50 %), (ii) average that provides a satisfactory state (> 25 %, but < 50 %), (iii) moderate, which provides a normal state (< 10 %, but < 25 %), (iv) absent, an optimal condition is provided (< 10 %). It is revealed that technologies of winter wheat growing in the conditions of Polissya, Forest-steppe and Steppe Zones of Ukraine can have a negative influence on potassium regime in soils, the influence by intensity can vary from moderate to strong. In Polissya and Forest-steppe, winter wheat growing can lead to deterioration of soil pH status. In Steppe, along with the potassium regime, the technologies can negatively influence soil nitrogen status and the effect may be characterized as strong. Key words: ecological assessment; growing technology; limiting factors; soil fertility Pridelava ozimne pšenice v Ukrajini: ekološka ocena tehnologij glede na vplive na rodovitnost tal Izvleček: Moderne tehnologije pridelave ozimne pšenice je potrebno izboljšati glede na ekološko ovrednotenje njihovega vpliva na indekse rodovitnosti tal. Namen raziskave je bil oceniti tehnologije pridelave ozimne pšenice na različnih tleh in klimatskih razmerah Ukrajine po njihovem vplivu na rodovitnost tal. Ugotovljeno je bilo, da je za oceno ekološke varnosti pridelave poljščin priporočljivo uporabiti metode, ki temeljijo na prepoznavanju negativnih učinkov na rodovitnost tal. Predlagamo naslednje skupine glede na odstopanja od optimalnega stanja: (i) močno odstopanje, ki vodi v nezadovoljive ekološke razmere (> 50 %), (ii) povprečno odstopanje, ki daje zadovoljivo stanje (> 25 %, a manj kot 50 %), (iii) zmerno odstopanje, ki daje normalno stanje (< 10 %, a manj kot 25 %), (iv) odstopanja ni, optimalno stanje (< 10 %). Ugotovljeno je bilo, da lahko imajo tehnologije pridelave ozimne pšenice v razmerah con polesja (Polissya), lesostepe (Forest-steppe) in stepe (Steppe) v Ukrajini negativni vpliv na režim kalija v tleh, jakost vpliva se spreminja od zmerne do močne. V polesju in lesostepi lahko pridelava ozimne pšenice vodi do poslapšanja pH tal. V stepi lahko tehnologije ob vplivu na režim kalija še močno negativno vplivajo na status dušika v tleh. Ključne besede: ekološko ovrednotenje; tehnologija pridelave; omejujoči dejavniki; rodovitnost tal 1 National University of Life and Environmental Sciences of Ukraine, Faculty of Plant Protection, Biotechnology and Ecology, General Ecology and Safety Department, Kyiv, Ukraine 2 Corresponding author, e-mail: lera_bond@email.ua Acta agriculturae Slovenica, 115/1, 67-78, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 67 26. 03. 2020 12:05:42 V. BONDAR and N. MAKARENKO 1 INTRODUCTION At the International Congress on Environmental Issues in Agriculture (Lefebvre et al., 2005) where the most important issues of modern agrotechnologies were discussed, it was stated that their ecological safety is one of the main requirements nowadays. It has been shown by researches (Davidson, 2000; Arshad and Martin, 2002; lenu et al., 2009; Killebrew and Wolff, 2010; Makarenko and Bondar, 2012) that it is impossible to identify environmental risks and to develop technologies that would meet the modern environmental standards without studying the mechanisms of negative influence of agrotechnical methods of crops growing on ecosystem components (Karlen et al., 2003; Killebrew and Wolff, 2010; Makarenko and Bondar, 2013). Winter wheat - is one of the leading crops for Ukraine. This crop is grown on an area of 6.4 million hectares and occupies about 37-40 % in the structure of crops (according to the Ministry of Agrarian Policy of Ukraine, 2018). In spite of its role as a major food in Eastern Europe (Petrenko et al., 2017) wheat is growing by technologies that causes environmentally negative impact. Thus, soil tillage, application of pesticides, mineral fertilizers, growth regulators, which can lead to violation of natural processes in the soil system and to soil degradation (Puskas and Farsang, 2009; Killebrew and Wolff, 2010; Mueller et al., 2012). Baliuk and Medvediev pointed out that soil degradation is a result of maintaining old technologies in Ukrainian agriculture (Baliuk et al., 2012; Ukraine: Soil fertility to strengthen climate resilience. Preliminary assessment of the potential benefits of conservation agri- culture. 2014). Researches stated that pesticides application leads to soil and terrestrial ecosystems missfunction (Wasim et al., 2009). Application of agrochemicals violating the optimal doses, with expired dates, inappropriate proportions of nutrient elements will - decrease the effective and potential soil fertility, pollute natural water resources by toxicants and reduce the quality of agricultural products (Shang et al., 2019). Taking into account that the vast majority of agrochemicals are recycled products of industrial wastes. For instance, low enriched agro-ores have a high probability of impact on living organisms and ecosystems due to the presence of impurities of heavy metals, radionuclides, organic and inorganic substances (Hazrat et al., 2019). Therefore, the study of the influence of wheat growing technologies on the soil conditions of agroecosystems is of high importance from scientific and practical points of view. The main purpose of the study was to identify (1) the negative processes in the soils of agroecosystems of different climatic zones of Ukraine for winter wheat cultivation; (2) the implementation of environmental regulation of the effects of winter wheat cultivation technology on the soils in agroecosystems; (3) application of the obtained results for the development of recommendations for winter wheat cultivation in the conditions of Ukraine. 2 MATERIALS AND METHODS 2.1 SITE DESCRIPTION The research was carried out within the framework of scientific and technical program of National Acade- Figure 1: Mapping scheme of long-term field experiments location in various soil-climatic zones of Ukraine: 1 - Rivne SARS, 2 - Khmelnytskyi SARS, 3 - Poltava Institute AIP named after M. Vavilov, 4 - SS NULES of Ukraine "Agronomic Research Station", 5 - Zaporizhzhya RS IOS NAAS. 68 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 68 26. 03. 2020 12:05:42 Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Table 1: Characteristic of technologies of winter wheat growing in different soil-climatic zones of Ukraine Parameter Characteristic of the parameter Poltava Institute AIP named after M. Vavilov Soil type Soil tillage Variety / biological yield Planting rate / method Preceding crop Fertilization system Protection system Khmelnytskyi SARS Soil type Soil tillage Variety / biological yield Planting rate / method Preceding crop Fertilization system Protection system SS NULES of Ukraine "Agronomic Research Station Soil type Soil tillage Variety / biological yield Planting rate / method Preceding crop Fertilization system Protection system Rivne SARS Soil type Soil tillage Variety / biological yield Planting rate / method Preceding crop Fertilization system Protection system Zaporizhzhya RS IOS NAAS Soil type Soil tillage Variety / biological yield Planting rate / method Preceding crop Fertilization system Protection system chernozem typical low-humus heavy loamy surface tillage, plowing, pre-sowing cultivation Kosoch / 5.93 t ha-1 200 kg ha-1/ conventional sowing pea organic fertilizers (manure 10 t ha-1) and mineral fertilizers N52P52K5 recommended for this soil-climatic zone chernozem podzolized slightly eroded medium loamy with deep groundwater occurrence plowing, disking Astet / 6.85 t ha-1 135 kg ha-1 /conventional sowing pea organic fertilizers (manure 16 t ha-1 and 8 t ha-1) and mineral fertilizers (n116p60k120 and n55p30kj recommended for this soil-climatic zone chernozem typical low-humus medium loamy surface tillage, plowing, pre-sowing cultivation Natsionalna / 6 t ha-1 150 kg ha-1 /conventional sowing pea mineral fertilizers N P K 60 60 60 recommended for this soil-climatic zone dark grey podzolized surface tillage, plowing, pre-sowing cultivation Poliska 90 / 6.5 t ha-1 145 kg ha-1 / conventional sowing pea mineral fertilizers N90P60K60, organic (manure 10 t ha-1), organic-mineral (straw + green manure + N90P60K60) recommended for this soil-climatic zone chernozem ordinary low-humus basic cultivation, plowing, pre-sowing cultivation Dalnytska / 7 t ha-1 200 kg ha-1 / conventional sowing black fallow mineral fertilizers in the following doses: N75P50K25, organic fertilizers: semi-decomposed manure 5.7 t ha-1 recommended for this soil-climatic zone n50p50k50, n15p10k5, Acta agriculturae Slovenica, 115/1 - 2020 69 AAS_vsebina_2020_115_1_koncna 180320.indd 69 26. 03. 2020 12:05:43 V. BONDAR and N. MAKARENKO my of Agrarian Sciences of Ukraine (NAAS) "Scientific and practical substantiation of sustainable development of agro ecosystems in Ukraine", which envisaged integrated research of crops growing technologies in various soil and climatic zones of Ukraine. The following institutions were involved in this study: Institute of Agroecology and Environmental management of National Academy of Agrarian Sciences (NAAS, 48°18'N, 25°56'E); Rivne State Agricultural Research Station (Rivne SARS, 50°37'N, 26°15'E); Khmelnytskyi State Agricultural Research Station (Khmelnytskyi SARS, 49°25'N, 27°0'E); Poltava Institute of agro-industrial production (Poltava Institute AIP, 49°35'N, 34°34'E) named after M. Vavilov; Zaporizhzhya Agricultural Research Station of the Institute of Oilseeds (Zaporizhzhya RS IOS NAAS, 47°50'N, 35°10'E); Separated subdivision of National University of Life and Environmental Sciences of Ukraine (SS NULES of Ukraine, 50°10'N, 30°19'E) "Agronomic Research Station". The research was conducted during 2007 - 2018 years in the conditions of long-term field experiments (established more than 20 years ago) located in Polissya, Forest-Steppe and Steppe regions of Ukraine (Figure 1). 2.2 METHODOLOGY OF TECHNOLOGIES ASSESSMENT The basic technology (BT) of winter wheat growing which envisaged soil tillage depending on its type, selection of regional variety, seed rates and method of sowing, system of plant protection. In addition, technologies with application of mineral and organic fertilizers in various combinations were studied, taking into account soil-climatic conditions of the of the experiments location (Table 1). The basic agrochemical parameters of the soil were determined according to standardized methods: pHKCl - by potentiometric method (displacement of exchange ions H+ and Al3+ 1N KCl (pH = 5.56) ratio of soil to solution was 1 : 2.5), nitrogen content that is easily hydro-lysed - by Cornflind (the method is based on alkaline hydrolysis of organic nitrogen-containing compounds of soil under the action of 1N NaOH for two days at 260 °C. The released ammonia is absorbed by boric acid and is determined by titration with sulfuric acid), content of exchangeable potassium and labile phosphorus - according to the modified Machigin method (the method is based on the removal of mobile compounds of phosphorus and potassium from the soil with a 1 % ((NH4)2 CO3), (pH 9.0) at the soil:solution ratio 1 : 20 and a temperature of 25 ± 2 ° C. The soil suspension was shaken for - 5 minutes and than, insulating the soil 70 Acta agriculturae Slovenica, 115/1 - 2020 in solution - 20 hours. Before determining the phosphorus, the colored soil extraction was discolored with activated carbon. Phosphorus was determined calori-metrically after ammonium molibdate addition. Potassium was determined by flame photometry. Soil organic matter (SOM) was determined - by Tyurin method (the method is based on the oxidation of organic carbon to CO2 by a solution of potassium bichromate and sulfuric acid, the excess of which is titrated with Mora salt). 2.3 STATISTICAL ANALYSIS Data are presented as mean values of four replicates and standard deviations. The significance of the experimental data was estimated by the analysis of variance (two-factor ANOVA) following calculation of the least significant difference LSD05. 3 RESULTS AND DISCUSSION Following the concept of limiting factors, a method for evaluating the technology was proposed the intensity of its negative influence on soil fertility indices: strong influence, which leads to an unsatisfactory ecological state of components of agroecosystems or individual processes that occur in them (the deviation from the optimum in the direction of deterioration is more than 50 %); medium influence that provides a satisfactory state of components of agroecosystems or individual processes that occur in them (the deviation from the optimum in the direction of deterioration is greater than 25 % but does not exceed 50 %); moderate influence, which ensures a normal state of components of agroecosystems or individual processes that occur in them (the deviation from optimum in the direction of deterioration more than 10 % but not exceeding 25 %); no influence, which ensures an optimal state of components of agroecosystems or individual processes that occur in them (the deviation from the optimum in the direction of deterioration does not exceed 10 %) is provided. The optimal soil parameters by fertility indices were determined according to DSTU 4362: 2004 "Soil quality. Soil fertility indices". The optimal parameters of fertility indexes are presented in Table 2. According to the results of our research, the technologies of winter wheat growing in different soil and climatic conditions of Ukraine influenced the formation of edaphic soil parameters of the agroecosystem, AAS_vsebina_2020_115_1_koncna 180320.indd 70 26. 03. 2020 12:05:42 Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Table 2: The range of soil parameters and respective ecological state Ecological state Nitrogen that is easily hydrolyzed Labile phi osphorus Exchangeable potassium PH KCl SOM, % mg kg-1 chernozem typical low-humus heavy loamy Standard 35-45 45-60 300-400 6.3-7.0 4.5-5.7 Optimal 32-34 41-44 270-299 5.7-6.2 4.1-4.4 Normal 27-33 34-43 228-269 4.8-5.6 3.4-4.0 Satisfactory 18-26 23-33 150-227 3.2-4.7 2.3-3.3 Unsatisfactory < 18 < 23 < 150 < 3.2 < 2.3 chernozem podzolized medium loamy slightly ero ded Standard 35-45 150-200 120-170 5.7-6.4 2.8-4.2 optimal 34-32 135-149 108-119 5.1-5.6 2.5-2.7 Normal 33-27 113-134 90-107 4.3-5.5 2.1-2.4 Satisfactory 18-26 75-112 59-89 2.9-4.4 1.3-2.0 Unsatisfactory < 18 < 75 < 60 < 2.9 < 1.4 chernozem typical low-humus medium loamy Standard 35-45 45-60 300-400 6.0-6.8 3.5-5.0 Optimal 34-32 41-44 270-299 5.4-5.9 3.2-3.4 Normal 33-27 34-40 228-269 4.5-5.3 2.6-3.1 satisfactory 18-26 22-33 150-227 3.0-4.4 1.8-2.5 Unsatisfactory < 18 < 23 < 150 < 3.0 < 1.8 dark grey podzolized standard 35-45 150-200 170-220 5.3-6.0 1.6-2.6 optimal 34-32 135-149 153-169 4.8-5.2 1.4-1.5 Normal 33-27 113-134 128-152 4.0-4.7 1.2-1.3 satisfactory 18-26 75-112 85-127 2.7-3.9 0.8-1.1 Unsatisfactory < 18 < 75 < 85 < 2.7 < 0.8 chernozem ordinary low -humus standard 35-45 45-60 300-400 6.8-7.6 3.2-5.3 optimal 34-32 41-44 270-299 6.1-6.7 2.9-3.1 Normal 33-27 34-43 228-269 5.1-6.0 2.4-2.8 satisfactory 18-26 23-33 150-227 3.4-5.0 1.6-2.3 Unsatisfactory < 18 < 23 < 150 < 3.4 < 1.6 and, consequently, the conditions of plant nutrition. The soil fertility indicators by long-term influence of the winter wheat growing technologies are presented in Table 3. A comparison of the soil actual parameters with the optimum level allowed to establish general patterns of changes occurring in the soil component of agro-ecosystems, and to identify the main negative effects of technologies (Table 4): nitrogen regime: there was no negative influence of the technologies on the content of nitrogen forms in typical, podzolized and dark gray podzolized soils. The exception was chernozem, in which the technology led to the decrease of nitrogen content. The effect varied from medium to strong. Negative phenomenon was not eliminated even with nitrogen application at the rates N15 - N75 kg ha-1; - phosphorus regime: the technologies provided optimal soil parameters by the content of phosphorus form in all soils. The exception was observed in chernozem podzolized, where the basic technology implementation led to the depletion of mobile phosphorus Acta agriculturae Slovenica, 115/1 - 2020 71 AAS_vsebina_2020_115_1_koncna 180320.indd 71 26. 03. 2020 12:05:43 V. BONDAR and N. MAKARENKO Table 3: Indicators of soil fertility for different technologies of winter wheat growing Indicators of soil fertility Technology Nitrogen that is eas ily hydrolyzed Labile phosphorus Exchangeable potassium PH KCl SOM, % mg kg-1 chernozem typical low-humus heavy loamy BT 102 ± 1.70 78 ± 3.40 65 ± 1.63 4.8 ± 0.09 5.4 ± 0.16 BT + manure 10 t ha-1 109 ± 2.49 92 ± 0.94 103 ± 1.41 5.0 ± 0.09 5.5 ± 0.22 BT + N52P52K52 + straw +N10 112 ± 1.25 132 ± 2.83 112 ± 0.82 5.5 ± 0.21 5.3 ± 0.05 BT + manure 10 t ha-1 + N52P52K52 119 ± 0.94 170 ± 2.62 153 ± 3.09 5.2 ± 0.09 5.5 ± 0.05 LSD0.05 12.11 3.37 6.67 0.18 0.21 chernozem podzolized medium loamy slightly eroded BT 101.5 ± 0.26 78 ± 0.47 72 ± 0.94 5.0 ± 0.05 3.1 ± 0.05 BT + N116P60K120 119.0 ± 1.89 146 ± 0.94 106 ± 0.29 3.5 ± 0.03 5.0 ± 0.22 BT + manure 16 t ha-1 105.6 ± 0.19 246 ± 2.83 235 ± 0.31 3.8 ± 0.09 6.3 ± 0.16 BT + manure 8 t ha-1 + N55P30K60 103.3 ± 0.14 490 ± 0.47 160 ± 0.12 3.5 ± 0.05 6.4 ± 0.09 LSD0.05 22.12 14.05 17.23 0.86 1.16 chernozem typical low-humus medium loamy BT 84.2 ± 0.08 67 ± 0.38 230 ± 0.45 7.7 ± 0.02 2.9 ± 0.05 BT + N60P60K60 94.5 ± 0.37 62 ± 0.05 240 ± 0.37 7.7 ± 0.03 3.2 ± 0.09 LSD0.05 4.67 1.16 dark grey podzolized 8.04 0.14 0.57 BT 75.4 ± 0.05 165 ± 0.12 53 ± 0.16 4.9 ± 0.09 1.3 ± 0.02 BT + N90P60K60 87.5 ± 0.16 245 ± 0.14 65 ± 0.21 5.2 ± 0.05 1.5 ± 0.04 BT + manure 10 t ha-1 91.0 ± 0.17 295 ± 0.08 85 ± 0.19 5.1 ± 0.16 1.5 ± 0.05 BT + N90P60K60 + straw + green 91.0 ± 0.16 340 ± 0.09 91±0.26 5.6 ± 0.16 1.4 ± 0.05 manures LSD0.05 2.96 15.11 6.12 0.92 0.58 chernozem ordinary low-humus BT 9.8 ± 0.09 100 ± 0.41 150 ± 0.16 7.15 ± 0.16 3.9 ± 0.02 BT + N75P50K25 15.6 ± 0.34 243 ± 0.17 183 ± 0.17 7.00 ± 0.05 4.1 ± 0.05 BT + N50P50K50 22.3 ± 0.17 280 ± 0.19 205 ± 0.21 6.72 ± 0.09 3.8 ± 0.02 BT + N15P10K5 14.7 ± 0.08 199 ± 0.47 159 ± 0.37 6.98 ± 0.16 4.1 ± 0.09 BT+ manure 5.7 t ha-1 11.4 ± 0.19 144 ± 0.19 219 ± 0.19 7.07 ± 0.08 3.8 ± 0.05 LSD0.05 3.82 22.14 20.10 3.57 2.63 level and the influence of technology was characterized as strong; potassium regime: there was a negative influence of the technologies on the content of exchangeable forms of potassium in all soil types, it varied from moderate to strong. The most dangerous impact of the technologies was recorded in dark gray podzolized soils (strong); soil pH: the influence of technologies within the limits of the moderate and average in chernozem typical low-humus heavy-loamy and chernozem pod-zolized; in dark gray podzolized soil, the negative influence was observed only in the basic technology; SOM content: technologies did not lead to negative changes in the SOM. The obtained results indicate the inappropriateness of using SOM status indicator as a diagnostic tool for assessing the influence of technologies on soil fertility. Thus, it was established that in Polissya, Forest-steppe and Steppe of Ukraine the technologies of win- 72 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 72 26. 03. 2020 12:05:42 Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility Table 4: Influence of the winter wheat growing technologies on soil fertility indicators Negative influence of technology on indicators of soil condition Nitrogen that is Labile Exchangeable Technology easily hydrolyzed phosphorus potassium PH KCl SOM, % chernozem typical low-humus heavy loamy BT absent absent strong moderate absent BT + manure 10 t ha-1 absent absent strong moderate absent BT + N52P52K52 + straw + N10 absent absent strong moderate absent BT + manure 10 t ha 1 + N52P52K52 absent absent strong moderate absent chernozem podzolized medium loamy slightly eroded BT absent strong strong moderate absent BT + N116P60K120 absent absent moderate medium absent BT + manure 16 t ha-1 absent absent absent medium absent BT + manure 8 t ha-1+ N55P30K60 absent absent absent medium absent chernozem typical low-humus medium loamy BT absent absent moderate absent moderate BT + N60P60K60 absent absent moderate absent absent dark grey podzolized BT absent absent strong moderate moderate BT + N90P60K60 absent absent strong absent absent BT + manure 10 t ha-1 absent absent strong absent absent BT + N P „K „ + straw + green manures absent 90 60 60 absent strong absent absent chernozem ordinary low-humus BT strong absent medium absent moderate BT + N75P50K25 strong absent medium absent absent BT + N50P50K50 medium absent medium absent moderate BT + N15P10K5 strong absent medium absent absent BT + manure 5.7 t ha- 1 strong absent medium absent moderate ter wheat growing negatively affected potassium regime of soils: - there was a depletion of mobile potassium forms; in chernozem typical low-humus and chernozem podzolized medium-loamy winter wheat growing led to deterioration of the acid-base conditions of the soil - there were processes of acidification of the soil solution. In Steppe, along with potassium regime, the technology negatively affected nitrogen regime of the soil - there were observed processes of depletion of stocks of nitrogen compounds. It is possible to predict that the indicated negative phenomena in the soil led to a violation of the interconnections in the soil-plant system. To confirm this, the productivity of winter wheat cultivation under various technologies was investigated. It is known that productivity, that is the ability of ecosystem to form biomass over a certain period of time on a certain area, is an integral indicator of its state. In agroecosystems, it was decided to determine productiv- ity of a main agricultural crop (a determinant). In our case, the determinant was winter wheat and, according to the level of its productivity, an assessment of the impact of technology on the agroecosystem was carried out. Potential productivity of a certain wheat variety was taken as a standard (etalon) since this indicator indicates the maximum possible biological potential the crop can use for the formation of biomass. At the same time, in case of reduced wheat productivity relative to the standard, the limiting edaphic soil factors, that could negatively affect plant growth and development, were determined. In Forest-steppe zone based on Poltava Institute AIP named after M. Vavilov winter wheat of the Kosoch variety was studied. The potential yield of this variety is 5.93 t ha-1. Climate of Poltava region is continental. The mean annual temperature is +8.2 oC. The mean annual precipitation is 580-480 mm. The productivity of wheat at the po- Acta agriculturae Slovenica, 115/1 - 2020 73 AAS_vsebina_2020_115_1_koncna 180320.indd 73 26. 03. 2020 12:05:43 V. BONDAR and N. MAKARENKO Figure 2: Influence of the technologies on productivity of winter wheat variety Kosoch (soil - chernozem typical low-humus heavy-loamy; 1. BT; 2. BT + manure 10 t ha-1; 3. BT + N52P52K52 + straw + N10; 4. BT + manure 10 t ha-1 + N52P52K52; 5. Standard) Figure 3: Influence of the technologies on productivity of winter wheat variety Astet (soil - chernozem podzolized medium loamy slightly eroded; 1. BT; 2. BT + N116P60K120; 3. BT + manure 16 t ha-1; 4. BT + manure 8 t ha-1 + N55P30K60; 5. Standard) tential level was ensured by the technology with integrated mineral and organic fertilizer application (BT + N52P52K52 + manure of 10 t ha-1). The basic technology, the technology with applied organic fertilizers did not allow achieving the standard level of crop productivity. The deviation from the standard was 26.4-35.5 %, that is, the negative impact of technology is estimated as medium (Figure 2). It can be assumed that one of the reasons for unsatisfactory growth and development of plants using these technologies was insufficient level of mobile potassium supply to plants. This effect was amplified by the discrepancy of the reaction of the soil environment with requirements of the crop. In the conditions of Khmelnytskyi SARS (Forest-Steppe zone), winter wheat of the Astet variety was studied, whose potential yield is 6.85 t ha-1. Climate of Khmelnytsky region is continental. The mean annual temperature is + 7.3°C. The mean annual precipitation is 530-670 mm. The basic technology, as well as the technologies of mineral fertilizers BT + N116P60K120, BT + manure of 16 t ha-1 application did not provide the optimal conditions for plant growth and development. The productivity level of wheat was 27.3-52.8 % lower comparing the standard. The influence of the technologies on the state of agroecosystem by the level of wheat productivity can be estimated as strong and average (Figure 3). One of the reasons for this phenomenon could be the depletion of the mobile forms of soil phosphorus, potassium and the discrepancy of soil pH to requirements of the crop. In the conditions of SS NULES of Ukraine "Agronomic Research Station" (Forest-Steppe zone) winter wheat of the Natsionalna variety was studied, the potential yield of which is 6 t ha-1. Climate of Kyiv re- 74 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 74 26. 03. 2020 12:05:42 Winter wheat growing in Ukraine: ecological assessment of technologies by the influence on soil fertility 01234567 productivity of Natsionalna variety, t ha1 Figure 4: Influence of the technologies on productivity of winter wheat variety Natsionalna (soil - chernozem typical low-humus medium loamy; 1. BT; 2. BT + N60P60K60; 3. Standard) (Note: Averages in groups are significantly different from each other, ANOVA with Bonferroni correction, except of those, marked by * p < 0.05) Figure 5: Influence of the technologies on productivity of winter wheat variety Poliska 90 (soil - dark grey podzolized; 1. BT; 2. BT + N90P60K60; 3*. BT + manure 10 t ha-1; 4*. BT + N90P60K60+ straw + green manure; 5. Standard) (Note: Averages in groups are significantly different from each other, AN0VA with Bonferroni correction, except of those, marked by * p < 0.05) gion is continental. The mean annual temperature is + 10.3 °C. The mean annual precipitation is 500-600 mm. The basic technology and the technology that envisaged mineral fertilizers application at the rate N60P60K60 did not ensure the implementation of biopotential of the variety. The reduction of wheat productivity in relation to the standard was 62.3-65.2 %. Cosequently, the influence of the technology on the state of agroecosystem by wheat productivity can be estimated as strong (Figure 4). One of the reasons could be an insufficient level of plant's supply with mobile potassium forms. In Polissya zone, based on Rivne SARS, winter wheat of the Poliska 90 variety was studied. According to the characteristics of the variety, the potential yield of this variety is 6.5 t ha-1. Climate of Rivne region is continental. The mean annual temperature is + 7.8 °C. The mean annual precipitation is 600-700 mm. The actual yield level was close to the potential and achieved with the technology included mineral fertilizers application in combination with straw and green manure (N90P60K60 + straw + green manure). The basic technology, as well as technologies that envisaged application of mineral and organic fertilizers separately, did not ensure the implementation of the variety biopotential. The deviation from the standard was 10.6-37.2 %, the impact of technology on the agroecosystem state was moderate and average (Figure 5). It can be assumed that one of the reasons was insufficient level of plant provision with mobile potassium forms. In Steppe zone, in the conditions of Zaporizhzhya Acta agriculturae Slovenica, 115/1 - 2020 75 AAS_vsebina_2020_115_1_koncna 180320.indd 75 26. 03. 2020 12:05:43 V. BONDAR and N. MAKARENKO Figure 6: influence of the technologies on productivity of winter wheat variety Dalnytska (soil - chernozem ordinary low-humus; 1. BT; 2. BT + N75P50K25; 3. BT + N50P50K50; 4. BT + N15P10K5; 5. BT + manure 5.7 t ha-1; 6. Standard) RS IOS NAAS winter wheat of the Dalnytska variety was studied. According to characteristics of the variety, the potential yield is 7 t ha-1. Climate of Zaporizhzhia region is continental. The mean annual temperature is + 12.8 °C. The mean annual precipitation is 430-475 mm. The technology with mineral fertilizers application provided optimum conditions for the growth and development of plants - wheat productivity exceeded the standard and amounted to 7.56 t ha-1. Despite the fact that the average negative impact of this technology on nitrogen and potassium regimes of chernozem was noted, plant nutrition was balanced. At the same time, other combinations of fertilizers led to strong negative influence of the technologies on nitrogen regime of the soil and average influence on potassium regime. Consequently, the influence of these technologies can be estimated as strong and moderate according to wheat productivity (Figure 6). Thus, it has been established that modern technologies of winter wheat growing, in most cases, do not provide plants with balanced mineral nutrition, which may be reason for the decreasing crop productivity if compared to its biological potential. provides a normal state (<10 %, but < 25 %), absent, an optimal condition is provided (< 10 %). It is revealed that technologies of winter wheat growing in Polissya, Forest-steppe and Steppe Zones of Ukraine can have a negative influence on potassium regime of soils; the influence by intensity can vary from moderate to strong. In Polissya and Forest-steppe, technologies of winter wheat growing can lead to deterioration of soil pH status. In the conditions of Steppe, along with potassium regime, the technologies can negatively influence nitrogen status and the effect may be characterized as strong. It is shown that modern technologies of winter wheat growing, in most cases, do not provide plants with balanced nutrition, which may be one of the reasons for the low level of productivity of the crop relative to the biological potential of the variety. The decrease in productivity can fluctuate within 10.6-73.6 %. Modern technologies of winter wheat growing need to be improved and revised taking into account the results of ecological evaluation of their impact on soil fertility indices. 4 CONCLUSIONS We recommend using a method based on identifying negative impacts of crop growing technologies on soil fertility in order to estimate ecological safety. Negative impacts of deviations from the optimum to the direction of deterioration are proposed to be grouped as follows: strong, which leads to an unsatisfactory ecological condition (> 50 %), average that provides a satisfactory state (> 25 %, but < 50 %), moderate, which 76 Acta agriculturae Slovenica, 115/1 - 2020 5 REFERENCES Arshad, M., Martin, S. (2002). Identifying critical limits for soil quality indicators in agroecosystems. Agriculture Ecosystems & Environment, 88(2), 153-160. https://doi. org/10.1016/S0167-8809(01)00252-3 Andrist-Rangel, Y., Edwards, A., Hillier, S., Öborn, I. (2007). Long-term K dynamics in organic and conventional mixed cropping systems as related to management and soil properties. 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Merrill.) productivity, soil moisture and fertility Abhijit Kishanrao KADAM 1, Agbesi Kwadzo KETEKU 1 2 3, Suchada DANA 4, Precious Kwaku BLEGE 5 Received July 11, 2019 accepted January 2, 2020. Delo je prispelo 11. julija 2019, sprejeto 2. januarja 2020. Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility Abstract: An experiment was conducted to investigate the impact of flatbed (FB), ridges and furrows (RF) and broad bed furrows (BBF) combined with recommended fertilizer dose N30P60K30 kg ha-1 (F1), 75 % NPK (F2), 125 % NPK (F3), 75 % NPK + 25 % N through farm yard manure (FYM)-F4, 75 % NPK + 2 sprays of micro nutrient mixture (Fe, Zn, Cu, Mn, B and Mo) - 0.5 % at 35 and 60 days after sowing (DAS)-F5 and 75 % NPK + 2 sprays of KNO3 - 0.5 % at 35 and 1.0 % at 60 DAS (F6) on the productivity of soybean in a split plot design. BBF stored 14.15 % more soil water and produced 1058.97 kg ha-1 more yield than FB. A significant 3.76 kg ha-1-mm rain water use efficiency was notice in BBF compared to FB. The yield increment recorded under F6 was 15.6 % higher than F1. Grain nitrogen and oil contents were highest in F3. The residual soil fertility was much improve by F3 and F5. Our result demonstrated that the combination of BBF and F6 were the best technique to increase soybean yield in the Vertisol soil. Key words: foliar fertilization; land configuration; soil moisture; soybean nutrition; yield Vpliv priprave zemljišča in gnojilnih tehnik na pridelek soje (Glycine max (L.) Merrill.), na vlažnost in rodovitnost tal Izvleček: Poskus z deljenkami je bil narejen za preučitev načinov priprave zemljišča kot so ravno zemljišče (FB), grebeni in brazde (RF) in široki grebeni (BBF) v kombinaciji s priporočenimi odmerki gnojenja: N30P60K30 kg ha-1 (F^, 75 % NPK (F2), 125 % NPK (F3), 75 % NPK + 25 % N kot hlevski gnoj (FYM) (F4), 75 % NPK + 2 kratno pršenje z mešanico mikrohranil (Fe, Zn, Cu, Mn, B in Mo), 0,5 % 35 in 60 dni po setvi (DAS)(F5) in 75 % NPK + 2 pršenja s KN03 - 0,5 % 35 in 1,0 % 60 dni po setvi DAS (F6) na pridelek soje. Pri BBF se je ohranilo 14,15 % več talne vode in dalo za 1058,97 kg ha-1 več pridelka kot FB. Pri BBF je bila ugotovljena tudi značilno večja učinkovitost (3,76 kg ha-1-mm) izrabe deževnice kot pri FB. Povečanje pridelka je bilo pri F6 za 15,6 % večje kot pri F1.Vsebnost dušika in olja v zrnih sta bili največji pri F3. Rodovitnost tal se je znatno povečala pri F3 in F5. Rezultati so pokazali, da je bila kombinacija BBF in F6 najboljša tehnika za povečanje pridelka soje v tleh na vertisolu. Ključne besede: foliarno gnojenje; oblikovanost zemljišča; vlažnost tal; gnojenje soje; pridelek 1 Vasantrao Naik Marathwada Agric. University, Dept. of Agronomy and AICRP on Dryland Agriculture, India 2 Naresuan University, Faculty of Agriculture, Natural Resource and Environment, Thailand 3 Corresponding author, e-mail: keteku.agbesi@yahoo.com 4 Udon Thani Rice Research Center, Nikhom Song Khro, Thailand 5 University for Development Studies, Faculty of Agriculture, Dept. of Agricultural Mechanization and Irrigation Technology, Ghana This paper is a part of the MS.c thesis of A.K.K. under supervision of A.K.K. Acta agriculturae Slovenica, 115/1, 79-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 A. K. KADAM et al. 1 INTRODUCTION Soybean (Glycine max (L.) Merrill.) is the most important oil seed crop in India, owing to its several domestic and industrial uses, besides its use in numerous food preparations and animal feed formulations. Soybean accounts for about 53 % of the world production share among the oilseed crops, and has therefore, occupied an important place in most farming systems in the Marathwada region of Maharashtra State, India (Talukdar & Shivakumar, 2016). However, under rain fed systems, soil moisture stress of 15-21 days at any growth phase of the soybean crop results in a significant yield loss in the Maharashtra State (Patil, 1992). These yield losses are especially severe in the early determinate genotypes (Chaturvedi et al., 2012). The drying and cracking nature of the vertisol soils in the region, coupled with their low fertility aggravates the situation (Keteku et al., 2016). To effectively manage the problem amidst irrigation difficulties, technologies targeted at soil moisture and nutrient conservation such as land layout are very crucial. Thus soil, water and nutrient conservation technologies are the key adaptation strategies to mitigate the rapid loss of moisture (Kurukulas-uriya & Rosenthal, 2003); and help plants withstand the occurrence of short dry spells under rain fed farming. An earlier studies on soil management strategies, aimed to increase crop productivity revealed that, modification of land such as broad bed furrow, and ridges and furrows in vertisol soils were superior to flatbed under watershed development (Raut & Taware, 1997). It is noteworthy to also mention that, soybean is a high protein and energy crop, as such it has a high nutrient requirement. Unfortunately, the inadequate and imbalance fertilization practiced by farmers, also adds to the problem of decreasing yield (Chaturvedi et al., 2012). Usually, the farmer's fertilizer programs focus solely on soil applied NPK, without plans for foliar application, however recent studies had shown the foliar fertilization enhance soybean yield (Gowthami & Rama, 2014; Chaturvedi et al., 2012). Others had also reported that micronutrients are essential for the optimum utilization of major nutrients, and also for the production of organic compounds (Gowthami & Rama, 2014; In-tanon, 2013). Numerous previous studies had equally reported the impact of combine major and micro nu- trients on crop yield (Keteku et al., 2018; Intanon, 2013; Salem & El-Gizawy 2012). Soybean is a focus crop for the realization of the Sustainable Development Goals (Shinde et al., 2009). Climate change threatens rainfall pattern and therefore, the achievement of the Sustainable Development Goals aimed at poverty and hunger reduction. Therefore our work is designed to investigate (i) the influence of land modification on soil moisture and soybean yield, and (ii) the effect of different fertilization techniques on soybean yield and soil properties. This is necessary to find the appropriate land configuration and techno-economic nutrient package for soybean production under such vertisol soil. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE The experiment was conducted at the experimental farms of the All India Coordinated Research Project on Dryland Agriculture (AICRP) station at Vasantrao Naik Marathwada Agriculture University (VNMKV), Marathwada, India, during rainy season, 2017. VNMKV is situated on a latitude and longitude of 19° 15' 28.0440'' N and 76° 46' 25.4748'' E respectively, and at 409 m above mean sea level. The average annual precipitation of the region is 963 mm, distributed in 48 rainy days, mostly during June - October. The mean maximum and minimum temperatures are 32.2 °C and 19.0 °C respectively. The total rainfall received during the trail was 308.4 mm, distributed in 35 rainy days. A total effective rainfall of 281.7 mm was recorded. Relative humidity was in the ranges of 81.8 % - 48.1 %, while that of mean bright sunshine was 6.7 hr. The mean evapotranspiration was 5.4 mm as well. The research soil was vertisol in classification (WRB), medium deep black, well drained, low in fertility, except K and levelled in topography. The soil nutrients and moisture contents at a depth of (0-20 cm) before the trial are shown in (Table 1). The pH of the soil was alkaline. 2.2 EXPERIMENTAL PLAN The experiment was 2 factorial, conducted in split plot design with 3 replications. The treatments were Table 1: Soil properties before the trial (sample size (n) = 3) N P K Fe Zn Cu Mn B pH (1:1.5) Moisture % % mg/kg 0.514 0.313 1.27 5.24 1.33 0.32 2.24 0.37 7.9 17.85 80 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 80 26. 03. 2020 12:05:44 Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility composed from 3 land configurations as main plots treatments and 6 fertilization strategies as subplots treatments, making a total of 18 treatment combinations. The 3 land configurations were; flatbed (FB), ridges & furrow (RF) and broad bed furrow (BBF), while the 6 fertilizers were a combination of urea, single super phosphate and murate of potash; recommended fertilizer dose N30P60K30 kg ha-1 F1 = 100 % by mass, 75 % NPK (F2), 125 % NPK (F3), 75 % NPK + 25 % N through 5 tons FYM ha-1 (F4), 75 % NPK + 2 sprays of 0.5 % micro nutrient mixture (Fe, Zn, Cu, Mn, B and Mo)- 50 ml per 10 L water at 35 and 60 DAS (F5) and 75 % NPK + 2 sprays of 0.5 % KN03 - 50 ml per 10 L water at 35 and 1.0 % - 100 ml per 10 L water at 60 DAS (F6). Thus, 2500 ml for 0.5 % and 5000 ml for 1 % to 500 L water was used for ha.The KN03 contained 13 % and 45 % nitrogen and potassium, respectively. The gross and net plot sizes used were 5.4 x 6.0 m and 4.5 x 5.0 m, in length and width respectively. The land was ploughed with a tractor drawn plough to a depth of 20 cm and harrowed twice before the preparation of the ridges and furrows, and broad bed furrows. The ridges measured 45 cm wide and 15 cm high while that of the broad bed furrows were 120 cm x 30 cm x 15 cm in width, length and height, respectively. The seeds of determinant soybean variety ('MAUS-162') were treated with Rhizobium culture (Bradyrhizobium japonicum (Kirchner 1896) Jordan 1982) and phosphate solubilizing bacteria (PSB), and sown at the recommended spacing of 45 x 5 cm2. But on the broad bed furrows a planting distance of 37.5 x 5 cm2 was used so as to obtain uniform plant population in all plots. A rate of 65 kg ha-1 was used, two seeds were dibbled per hill and thinned out after 14 DAS to maintain one seedling per hill. The solid fertilizers were applied by side placement method, 30 % was applied at sowing and the remaining 70 % applied at 30 DAS. The FYM was broadcasted and raked into the soil on the flatbed and broad bed furrows, but in the ridges and furrows, it was applied uniformly in the lines opened for sowing. The 'MAUS-162' seeds were sourced from the seed processing plant, VNMKV while the fertilizers, Rhizobium culture and PSB were obtained from AICRP, VNMKV. One spraying of Chloropyriphos 20 EC was performed to control leaf eating caterpillar. Two hand weeding and one hoeing were performed to control weeds and also loosen the soil for good aeration. 2.3 DATA COLLECTION Before the trial, soil cores were collected from 12 spots on the research site at a depth of (0-20 cm) with the hand auger for the assessment of soil fertility and pH. The routine methods of Lu (1999) were followed for the determination of soil nutrients. Total nitrogen, phosphorus and potassium were determine by the Kjeldahl method, Bray's no. II method and Neutral N ammonium method, respectively. The wet digestion (nitric-perchloric digestion) method was adopted for the analysis of iron, zinc, copper, manganese and boron. Soil pH was measured at 1:1.5 solution ratio, using the electrode (H19017 Microprocessor) pH meter. Another soil cores were also taken from a depth of (0-15 cm and 15-30 cm) for the determination of soil moisture content. Using the gravimetric method, the percentage soil moisture content were calculated for each depth and the mean worked out using the formula in (Equation 1). 50 g of the sample soil was oven dried at 105 °C ± 5 °C for 12 h. Moisture %= (m2-mi)/(m2) x 100 (Eqn 1) Where; m = mass of wet soil sample, m2 = mass of oven dried soil sample Fifteen representative sampled plants were randomly selected in each plot and tagged for the measurement of vegetative growth. Plant height, number of leaves, number of branches, leaf area and total dry matter mass per plant were measured after 30 DAs at 15 days interval. At each periodic data collection, two representative plant were uprooted, processed and oven dried at 72 °C ± 2 °C for 12 h for total dry matter measurement. However on the harvest day, total dry matter weight was again measured from the 15 sampled plants in each plot. Leaf area was measured from the sampled plants uprooted for dry matter studies. The leaves were aerated into leaflets and grouped into three class viz., small, medium and big. The maximum length and diameter of five leaflets from each group were measured using the hand held laser leaf area meter (CID Bio-science, Inc.), and the method of Pawar (1978) was used to calculate the leaf area/plant (Equation 2). Leaf area/plant (dm2) = £P=T03(X X D)JC (Eqn 2) Where; L, D, n and K are leaf length, leaf diameter, number of leaves and leaf area constant for soybean (0.689), respectively. Only the final values were reported here. Also, yield components namely; number of pod plant-1, pod mass plant-1, grain mass plant-1 and 1000 seeds mass were measured from the fifteen sampled plants. After harvesting (120 DAS), grain mass plot-1 and straw mass plot-1 were measured, all the plants in the net plots were consider. The values were later converted to grain yield ha-1. The biological yield produce Acta agriculturae Slovenica, 115/1 - 2020 81 AAS_vsebina_2020_115_1_koncna 180320.indd 81 26. 03. 2020 12:05:44 A. K. KADAM et al. was determined as the summation of grain mass plot-1 and straw mass plot-1 and again converted into ha. Harvest index (HI) was calculated as indicated in (Equation 3). HI % = (Biological yield (kg))/(Grain yield (kg)) x 100 (Eqn 3) The protein and oil content of the seeds were determined for quality assessment, grain nitrogen content was estimated by the micro Kjeldahal method (A.O.A.C., 1975), and was converted into crude protein percentage by multiplying the percent nitrogen with 6.25. Soxhlet ether extraction method was used to estimate the oil content. After the trial, soil samples were again sampled and the properties estimated by the same methods above. Rain water use efficiency (RWUE) was computed by the formula (Equation 4) and expressed in kg/ha-mm. RWUE (kg ha-1-mm) = (Yield kg ha"')/(Moisture use(effective rainfall) mm) (Eqn 4) 2.4 STATISTICAL ANALYSIS The data recorded were subjected to Analysis of Variance (ANOVA) using SPSS 21 statistical package. The variation between treatments means were quantified at a probability of 5 %. Duncan's Multiple Range Test (DMRT) analysis was performed and presented in tables, in alphabets with 'a' depicting highest value. Interactions between factors were not significant, hence not presented. Regression analysis was used to show the relationship between some variables. 3 RESULTS AND DISCUSSION 3.1 INFLUENCE OF LAND CONFIGURATION ON SOIL MOISTURE CONTENT The impact of land configuration on soil moisture content (0-30 cm) was significant (p < 0.05) as shown in Figure 1. Soil moisture content increased gradually from 30 to 90 DAS in the BBF and RF, compared to FB which recorded a decrease at 90 DAS. At 30, 60, 75 and 90 DAS, BBF conserved the highest significant soil moisture of 19.86, 27.30, 23.55 and 20.43 %, respectively when compared to RF and FB. Similarly, RF stored a significant amount of moisture on 60 and 90 DAS (27.29 % and 18.30 %, respectively) than FB. The furrows between the BBF and RF prevented the runoff of rain water and enhanced the infiltration of water into the soil. Probably the size of BBF also enhanced water conservation, as it has less surface area for evapora- 82 Acta agriculturae Slovenica, 115/1 - 2020 tion when compare to the RF. In-situ land management strategies that reduces water lost caused by runoff and evaporation, and improves water infiltration and storage would lead to increase the amount of water retained in the soil for crops (Singh et al., 2014). BBF conserved 14.15 % more water than FB. Our results agrees with the previous findings of Shinde et al. (2009), Bhar-ambe et al.(1999) and Patil et al.(1992) that BBF and RF conserved more water than FB. According to Kumar et al. (2010), furrow irrigated bed planting systems, on an average retained 40 % more water, compared to FB planting systems. Consistently, Selvarajua et al. (1999) also reported 17 % more soil moisture in BBF compare to FB. In contrasts to our findings, Singh et al. (2018) reported 28.54 % soil moisture in RF and 27.58 % in BBF, nevertheless they similarly reported the least soil moisture in FB. The principal aim of land configuration are; the preparation of a conducive seedbed for seed germination and seedling growth, conservation of soil moisture that influences the infiltration characteristics of the soil, and also, provides adequate soil depth for optimum root growth and proper fertilizers placement. The land configuration that stores enough moisture will reduce soil moisture tension, while improving nutrient flow and their availability for crop uptake (Singh & Kumar, 2009). our findings has demonstrated that BBF and RF can conserve more moisture than FB. 3.2 INFLUENCE OF TREATMENTS ON SOYBEAN GROWTH The results in Table 2 showed that soybean growth variables were significantly (p < 0.05) influenced by the various land configurations and fertilizers strategies. Interaction between factors were not significant and were therefore not discussed. Soybean height and number of branches (55.98 cm and 4.95) respectively, were superior in BBF compared to RF and FB. However, leaf area plant-1 was equal between BBF and RF, while that of total dry matter produced did not significantly differ among the land layouts. But the greatest dry matter mass of 24.20 g was produced by BBF. The high growth observed in BBF and RF could be related to the availability of optimum soil moisture at the key vegetative phase of the crop. When soil moisture tension is low, the ability of crops to absorb nutrients and that of the soil to supply nutrients are optimal, and so, nutrients availability are improved (Singh & Kumar 2009). In addition, the BBF and RF could also provide adequate aeration and a good soil depth for root expansion and nutrient exploration (Singh et al., 2014). From our re- AAS_vsebina_2020_115_1_koncna 180320.indd 82 26. 03. 2020 12:05:44 Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility DAYS AFTER SOWING Figure 1: Effect of land configuration on soil moisture content (0-30 cm) during the trial sults, it is evident that land layout had significant effect on growth. Also, the application of F6 (75 % NPK + 2 sprays of KNO3- 0.5 % at 35 DAS and 1.0 % at 60 DAS) recorded the highest soybean height plant-1, number of branches plant-1, leaf area plant-1 and total dry matter mass plant-1 of 53.12 cm, 5.17, 22.10 dm2 and 25.80 g respectively, but it was not significant when compared to F3 and F5 (dry matter). Increasing the NPK rate from F1 to F3 also increased soybean growth variables but did not give significant results. According to our result, F1, F2 and F4 treatments had a similar effect on soybean growth. The performance of F6 might probably be due to the rapid and efficient nutrients absorption resulting from the foliar spray of KNO3 which contained 13 % N and 46 % K, while that of F3 can be attributed to its higher NPK nutrients. Nitrogen particularly, is a principal constituent of protein, chlorophyll and the hormones which are essential for cell expansion and an increase in the vegetative apparatus of crops (Keteku et al., 2016; Nsoanya & Nweke, 2013). Besides having nitrogen which is an integral component of cell division in the fertilizers, the micronutrients do also influence cell division, chlorophyll construction and photosynthesis (Intanon 2013). This may probably explains why F5 produced a similarly high total dry matter mass of 24.83 g, despite its low NPK content when compared to F6 (25.80 g) and F3 (24.50 g). It is noteworthy to indicate that the application of F5 produced greater dry matter mass of 24.83 g when compared to F3 (24.50); and a higher leaf area and dry matter mass when compare to F1 (19.20 dm2 and 23.30 g) respectively. Previous studies by Khaliq et al. (2006) reported that, the sink capacity of a plant is mainly dependent on its vigorous vegetative growth; as such in our study, the treatments that recorded a large leaf area plant-1, had more green areas available for the interception of active radiation during photosynthesis, for greater dry matter production (Azarpour et al., 2014). The regression analysis showed the impact of leaf area on dry matter produced by the fertilizers (R2 = 0.8361) as shown in Figure 2.Dry matter production responded positively to an increase in leaf area plant-1. Our results are also in agreement with Raj & Mallick (2017), in their studies the application of 80 kg N ha-1 + mixed spray of 0.203 % Ca (NO3)2 + 0.25 % KNO3 produced the maximum leaf area index values (1.748 and 1.592), dry matter accumulation (1404.3 and 1288.8 g m-2) and crop growth rate (27.87 and 25.68 g m-2 day-1). 3.3 INFLUENCE OF TREATMENTS ON SOYBEAN YIELD COMPONENTS, YIELD, QUALITY AND WATER USE EFFICIENCY The data in Table 3 showed a significant (p < 0.05) impact of land configuration and fertilizer on soybean yield components. Interactions between the factors were not significant. Pod mass plant-1, grain mass plant-1 and 1000 seeds mass were significantly influenced by the different land configurations. The highest (12.32 g, 5.09 g and 85.56 g) respectively, were produced by BBF. This resulted to its greatest grain yield of 1058.97 kg ha-1 as well, but it was comparable to RF (1026.77 kg ha-1) as shown in the Table 4. The soybean yield were in accordance with the vegetative growth record by the land configurations. BBF significantly increased grain yield by 8.8 %, when compared to FB. Straw yield, biological yield and harvest index did not vary significantly among the land configurations, nevertheless the greatest values were observed in BBF, and was followed by RF. A similar results had been previously reported in Acta agriculturae Slovenica, 115/1 - 2020 81 AAS_vsebina_2020_115_1_koncna 180320.indd 83 26. 03. 2020 12:05:44 A. K. KADAM et al. Table 2: Influence of treatments on soybean growth Treatments Plant height plant-1 (cm) Number of leaves plant-1 Number of branches plant-1 Leaf area plant-1 (dm2) Total dry matter mass plant-1 (g) Land Configurations FB 46.83b 16.68 3.82c 17.98b 23.20 RF 49.60b 19.52 4.05b 18.90ab 23.72 BBF 55.98a 19.53 4.95a 21.47a 24.20 CD @ 5 % 3.51 NS 0.19 2.63 NS Fertilizers f1 50.90* 18.43 4.10cd 19.20bc 23.30ab f2 46.57b 16.97 3.70e 17.40c 22.23b f3 53.67a 19.20 4.47b 20.47ab 24.50ab f4 49.44* 17.60 3.93de 18.03c 21.57b f5 51.13* 19.10 4.27bc 19.50bc 24.83ab f6 53.12a 20.17 5.17a 22.10a 25.80a CD @ 5 % 5.42 NS 0.32 2.22 2.72 Note: mean values with different superscript letter within each column denotes significance (p < 0.05) between different groups. CD = critical difference between means; NS = non-significant (n = 15) other crops. In a study, Pramanik et al. (2009) reported a significant 16.8 % and 15.9 % rise in chickpea grain yield under raised bed planting over flatbed planting, in two seasons. According to Selvarajua et al. (1999) also, planting on BBF increased sorghum and pearl millet yields by 34 % and 33 % respectively, compared to FB. This they ascribed to the optimum water storage and safe disposal of excess rain water by BBF. Among all the in situ soil moisture conservation techniques, rain water use efficiency was the highest (3.76 kg ha-1-mm) in BBF as well (Table 4). It is evident from the Table 4 that, an increase in water use efficiency corresponded to a greater soybean yield. When water utilization increased, nutrient uptake was enhanced by the mass flow process. Previous work of Lomte et al. (2006) also showed that, opening of furrows in every row recorded the highest water use efficiency of 3.15 kg ha-1-mm than flat bed. Among the fertilizers also, the application of F6 recorded the greatest yield components; pod number plant-1 (30.65), pod mass plant-1 (13.40 g), grain mass plant-1 (5.36 g) and 1000 seeds mass (95.56 g). However, grain yield ha-1 was on a par between F6 (1160.33 kg) and F5 (1086.50 kg). A similar trend was noticed for straw yield and biological yield between the two treatments, and were followed by F3. In addition, an increase in NPK rate from F1 to F3, also significantly increased grain yield by 2.3 % (Table 4). Additionally, the yield increment realized for F6 and F5 were 15.6 % and 9.9 % respectively, higher compared to the recommended fertilizer rate (F1). The higher yield of F6 was mainly due to its greater grain mass plant-1 and 1000 seed mass. The KN03 sprayed during the seed filling stages (60 DAS) might had increased the availability of N and K to the plants. N is central in organic compounds formation in plants (Intanon, 2013). Besides the beneficial functions of nitrate nitrogen, the prevalence of K+ in KN03, may had also improved grain filling and phytomass production, and the translocation of assimilates to reproductive apparatus (Ravikiran et al., 2012; Waraich et al., 2011). The relationship between leaf area plant-1 and grain mass plant-1 (R2 = 0.8276) showed that, the high vegetative growth produced affected grain yield positively (Figure 2). Our result are in line with those of other previous experiments as well. Soil application of 80 kg N ha-1 + foliar spray of 0.25 % KNO3 + 0.203 % Ca (NO3)2, led to an increased soybean yield of 1.68 t ha-1, about 10.7 % increase over the 1.5 t ha-1 produced by 80 kg N ha-1 + water spray (Raj & Mallick, 2017). Again, Vekaria et al. (2013) reported that foliar application of 0.4 % KNo3 ha-1 significantly increased soybean yield by 18.4 % when compared to water spray only. Intanon (2013) did mentioned that, the micronutrient (Fe, Zn and Cu) are important for carbohydrate formation. The presence of this element in F5 could partly account for its high yield output. 84 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 84 26. 03. 2020 12:05:44 Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility Table 3: Influence of treatments on soybean yield components Treatments Pods plant 1 Pod mass plant-1 (g) Grain mass plant 1 (g) 1000 seeds mass (g) Land Configurations FB 27.71 11.45b 3.76b 81.77b RF 28.19 11.87* 4.55a 82.72ab BBF 29.68 12.32a 5.09a 85.56a CD @ 5% NS 0.48 0.58 3.24 Fertilizers F, 28.28 11.41bc 4.23bcd 79.84b f2 26.85 10.26c 3.75d 76.52b f3 28.51 12.44* 4.50bc 82.86b f4 27.65 10.95c 4.01cd 77.35b f5 29.20 12.83* 4.92b 84.95b f6 30.65 13.40a 5.36a 96.56a CD @ 5 % NS 1.75 0.74 9.04 Note: mean values with different superscript letter within each column denotes significance (p <0.05) between different groups. CD = critical difference between means; NS = non-significant (n = 15). TO O) c 3 tc T' t/3 O) C CD TO E a i- (/> (1) (T> a CD ¡E Q 30 x 25 20 '-15 -10 5 f 0 Dry matter mass plant"1 (g) Grain mass plant"1 (g) Linearna (Dry matter mass plant"1 (g)) Linearna (Grain mass plant"1 (g)) ~i—I—.——.—I—i— y = 0,8765x + 6,6562 R2 = 0,8361 y = 0,3207x- 1,7757 R2 = 0,8276 0 15 20 25 5 10 Leaf area plant"1 (dm2) Figure 2: Regression analysis of leaf area plant-1 (dm2) to total dry matter plant-1 and grain mass plant-1 (g) The impact of boron (1 kg ha-1) and molybdenum (0.5 kg ha-1) on soybean yield had been demonstrated (Adkine et al., 2011); while the combination of NPK with 400 g Fe ha-1 and 20 g Mo ha-1 had also been reported by (Zahoor et al., 2013). In our work, land configuration had no significant effect on seed protein and oil content; likewise was the fertilizers on seed oil content, but numerically, the highest oil content of 19.65 % was obtained in F5 and the lowest in F2 (Figure 3). Seed protein content was significantly (p < 0.05) influenced by the fertilizers, with the highest (39.64 %, 39.38 %, 39.33 % and 39.18 %) realized in F , F, R, ' 6 5 3 and F4, respectively. The seed qualities observed in our study concur with those of (Kiran et al., 2008). 3.4 SOIL FERTILITY AFTER THE TRAIL The different in situ soil moisture conservation techniques had no significant influence on soil fertility after the trial, though the best recordings were noticed in BBF (Table 5). The more soil water conserved in BBF, probably increased the crop residues added to the soil due to high vegetative growth (Sel-varajua et al., 1999; Lal, 1995). The residual soil nitrogen (11.14 %), phosphorus (0.73 %) and potassium (1.85 %) contents were greatest in F3 plots, probably due to the high NPK levels of this fertilizer formula. Phosphorous and potassium contents in particular, were significantly (p < 0.05) improved by F3, when Acta agriculturae Slovenica, 115/1 - 2020 85 AAS_vsebina_2020_115_1_koncna 180320.indd 85 26. 03. 2020 12:05:44 A. K. KADAM et al. Table 4: Influence of treatments on soybean yield, harvest index and rain water use efficiency. Grain yield Straw yield Biological yield Harvest index RWUE Treatments (kg ha-1) (kg ha-1) (kg ha-1) (HI) (kg ha-1 mm-1) Land Configurations FB 966.10b 1913.00 2879.10 0.34 3.43b RF 1026.77ab 1985.83 3012.60 0.34 3.64* BBF 1058.97a 2085.67 3144.63 0.34 3.76a CD @ 5 % 63.13 NS NS NS 0.22 Fertilizers F, 979.03c 1992.00abc 2971.03bc 0.33 3.48c f2 916.37c 1820.33c 2736.70d 0.34 3.25c f3 1001.70b 2019.33ab 3021.03bc 0.33 3.56bc f4 959.73c 1905.00bc 2864.73cd 0.34 3.41c f5 1086.50ab 2062.33ab 3148.83* 0.34 3.86ab f6 1160.33a 2170.00a 3330.33a 0.35 4.12a CD @ 5 % 99.91 197.79 216.12 NS 0.35 Note: mean values with different superscript letter within each column denotes significance (p < 0.05) between different groups. CD = critical difference between means; NS = non-significant. Figure 3: Influence of fertilizers on grain quality (n = 3) compared to the other fertilizers. Generally, soil nitrogen and phosphorus content improve in all the plots, when compared to the levels before the trial. 0ur findings could be due to the Rhizobium culture (Bradyrhizobium japonicum) and phosphate solubi-lizing bacteria (PSB) treatment given to the seeds, as root nodules formations were generally observed on most roots. Also, the soil micronutrients; Fe, Zn, Cu, Mn and B contents were found the highest in F5 with 8.75, 2.10, 0.60, 3.31 and 1.40 mg kg-1, respectively. This could be due to the micronutrient spray given under this treatment. The lowest performance were observed in the F2 plots. 86 Acta agriculturae Slovenica, 115/1 - 2020 4 CONCLUSION Our result supports our hypothesis that (i) proper land configuration can promote soybean yield than flatbed; (ii) proper fertilization technique (foliar spray) can improve soybean yield and soil fertility. Our work has shown that BBF and RF can conserve more soil moisture for greater soybean growth and yield in ver-tisol soil. The application of F6 also produced the highest soybean growth and yield. Therefore, for general soil improvement, the application of F5 is recommended as it improved both the macro and micro soil nutrients elements, however for maximum yield, farmers in the AAS_vsebina_2020_115_1_koncna 180320.indd 86 26. 03. 2020 12:05:44 Influence of land configuration and fertilization techniques on soybean (Glycine max (L.) Merrill.) productivity, soil moisture and fertility Table 5: Soil fertility after the trail Treatments N P K Fe Zn Cu Mn B % mg/kg Land Configurations mg/kg FB 0.99 0.67 1.71 6.11 1.50 0.40 2.65 0.62 RF 0.98 0.66 1.71 6.23 1.53 0.39 2.66 0.58 BBF 0.99 0.69 1.73 6.43 1.57 0.42 2.68 0.62 CD @ 5 % NS NS NS NS NS NS NS NS Fertilizers F1 1.06b 0.71* 1.79b 5.68c 1.50b 0.34c 2.50bc 0.40d F2 0.84c 0.60d 1.60d 5.59c 1.44b 0.35c 2.39c 0.41d F3 1.14a 0.73a 8 a 5.77c 1.36b 0.37b 2.36c 0.43cd F4 0.89c 0.67c 1.71c 6.16b 1.43b 0.39b 2.88b 0.48bc F5 0.85c 0.64cd 1.61d 8.75a 2.10a 0.60a 3.31a 1.40a F6 1.13ab 0.68bc 1.74bc 5.58c 1.39b 0.36b 2.46c 0.54b CD @ 5 % 0.07 0.04 0.05 0.29 0.22 0.03 0.38 0.06 Note: mean values with different superscript letter within each column denotes difference between means; NS = non-significant (n = 3). significance (p < 0.05) between different groups. 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(2013). Soybean (Glycine max L.) response to micronutrients. Turkish Journal of Field Crops, 18, 134-138. 88 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 88 26. 03. 2020 12:05:44 doi:10.14720/aas.2020.115.1.1267 Original research article / izvirni znanstveni članek Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers Sandra PESTAR BIZJAK 1 2, Hristo HRISTOV \ Aleš KUHAR 1 Received September 11, 2019; accepted January 3, 2020. Delo je prispelo 11. septembra 2019, sprejeto 3. januarja 2020. Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers Abstract: Perceived value is a subjective impression of the consumer about the value of a product or a service. There is a lack of research and understanding of Slovenian wine consumers preferences, while at the same time the importance of the Slovenian wine sector is increasing. 221 adults from the two biggest Slovenian winegrowing regions who at least occasionally consume and purchase wine were interviewed with a structured questionnaire. The Perval scale was used to measure and identify the factors (dimensions) of the perceived value for wine. Further, the relation between Slovenian winegrowing regions and the perceived value dimensions was investigated. It was shown that respondents from the two largest Slovenian winegrowing regions perceive three different dimensions of value for wine: quality-price, emotional-social, and a dimension where indicators for humane, environmental and region of origin factors highly correlated, and were therefore named terroir. We also found that respondents value Primorska winegrowing region the most, followed by Podravje and other wine regions. Both winegrowing regions correlate to terroir and quality-price value dimensions. It was also found which value dimensions are more important to different socio-demographic groups, which can give wine producers and wine-sellers some ideas on consumer segmentation and marketing strategy. Key words: perceived value; wine; consumer; Perval scale; Slovenia; factor analysis Dimenzije zaznane vrednosti vina s perspektive slovenskih kupcev vina Izvleček: Zaznana vrednost je subjektivna ocena potrošnika o vrednosti izdelka oziroma storitve. Ker v Sloveniji primanjkuje raziskav, ki bi pripomogle k boljšemu razumevanju izbire potrošnikov vina, hkrati pa vinarski sektor pridobiva na pomembnosti, je bila med 221 potrošniki iz dveh največjih vinorodnih dežel v Sloveniji, ki vsaj občasno pijejo in kupijo vino, opravljena raziskava. Za ugotavljanje števila dimenzij zaznane vrednosti vina je bila uporabljena lestvica Perval, nato pa nas je zanimala povezava med dimenzijami in različnimi vinorodnimi deželami. Ugotovili smo, da vprašani potrošniki ločijo tri dimenzije zaznane vrednosti vina in sicer kakovostno-cenov-no, emocionalno-socialno in tretjo dimenzijo, kjer so močno korelirali indikatorji okoljskih in človeških faktorjev, ter indikatorji, ki opisujejo regijo porekla vina; in smo jo poimenovali terroir. Anketiranci najbolj cenijo vinorodno deželo Primorska, sledi Podravje in nato ostale vinorodne dežele. Obe omenjeni vinorodni deželi korelirata s kakovostno-cenovno in terroir dimenzijo zaznane vrednosti vina. Ugotovili smo tudi, katere dimenzije zaznane vrednosti so pomembnejše respondentom z določenimi socio-demografskimi značilnostmi, kar bo vinskim pridelovalcem in prodajalcem lahko dobro izhodišče za se-gmentacijo potrošnikov in oblikovanje marketinške strategije. Ključne besede: zaznana vrednost; vino; potrošnik; lestvica Perval; Slovenija; faktorska analiza 1 University of Ljubljana, Biotechnical faculty, Department for animal science, Ljubljana, Slovenia 2 Corresponding author, e-mail: sandra@santei.si Comment: This paper is part of first author's Ph.D. thesis, done in 2018 by the mentorship of assoc. prof. Aleš Kuhar, Ph.D., and co-mentorship of prof. Tatjana Košmerl, Ph.D. Acta agriculturae Slovenica, 115/1, 89-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 S. PESTAR BIZJAK et al. 1 INTRODUCTION Perceived value is the perception about the product (or service) by the consumers, rather than something objectively determined by the seller and these perceptions involve a trade-off between what the consumer receives and what she or he gives up in order to acquire and/or use a product (Woodruff, 1997). It is a subjective value from the perspective of the consumer and is not equal to objective value of the product (Zeithaml, 1988). According to Woodruff (1997), consumer value is the source for competitive advantage and, as a concept, it is moving beyond the focus just on the quality. A promising direction for research toward an enhanced understanding of value is the dual perspective of value measurement, which includes assessing (intra-variable approach) and linking (inter-variable approach) value dimensions to other related measures, where the main flow of effects moves from perceived quality and perceived price to perceived value, satisfaction and to loyalty (Gallarza et al., 2011). Olson and Jacobi (1972) defined consumer value as a sum of intrinsic and extrinsic information about a product. Intrinsic characteristics are physical part of the product (colour, taste, smell, sweetness, etc.), while extrinsic characteristics are connected with the product, but are not a physical part of it (label, cork, brand name, region of origin etc.). They showed, that for determining the quality of the product, intrinsic features are more important than extrinsic. Intrinsic and extrinsic features are important for the perception and willingness to buy in the case of wine. Agnoli et al. (2016) found, that intrinsic features are more important for more regular consumers who are more familiar with wines. The concept of consumer value differs also with regard to circumstances within which a consumer thinks about value; when making a purchasing decision (when extrinsic features are more important) or when experiencing product performance during or after use, when intrinsic features come first (Woodruff, 1997). Price can be an indicator of the products quality when there is no other information to imply quality of the product, when there are many similar products on the market and when the consumer does not know the price (Zeithaml, 1988). Perceived value is closely connected to value proposition. A brand's value proposition is a statement of the functional, emotional and self-expressive benefits delivered by the brand that provide value to the consumer (Aaker, 1996). Aaker (1996) considers price separately, as the price could reduce the value proposition of the product, however it can at the same time imply its higher quality. There are different classifications of value found in the literature. Generally, value is defined as a two- or multidimensional construct, however Lin et al. (2005) claim that both theories are inadequate and contradictory at some points. They explain perceived value as a secondary indirect construct, where components of value and cost, both of which are manifested by numerous indicators, work as indirect indicators of perceived value. Most authors consider value as a multidimensional construct, but the agreement on the number of dimensions has not yet been reached (Gallarza et al., 2011). Number of dimensions most likely depends on the consumer and on the type of product in question. Sheth-Newman-Gross model, also known as the theory of consumption values, is one of the analytical cognitive models of consumer behaviour. According to this model the consumer choice is a function of multiple consumption values, which make differential contributions in any given choice situation. Consumption values are independent, and are defined as: functional value, which is the perceived utility acquired from an alternative's capacity for functional, utilitarian or physical performance and is presumed to be the primary driver of consumer choice; social value, which is the perceived utility acquired from an alternative's association with one or more specific social groups; emotional value is the perceived utility acquired from an alternative's capacity to arouse feelings or affective states; epistemic value is the perceived utility acquired from an alternative's capacity to arouse curiosity, provide novelty, and/or satisfy a desire for knowledge; and conditional value, which is defined as the perceived utility acquired by an alternative as the result of the specific situation or set of circumstances facing the choice maker (Sheth et al., 1991). Even products, generally thought to be of high functional value are frequently selected on the basis of their social or emotional value (for example cars, kitchen appliances etc.). on the basis of the Sheth-Newman-Gross model of consumer behaviour (Sheth et al., 1991), Sweeney & Soutar (2001) developed PERVAL, a multiple-item scale to assess consumers perception of value for different product types. The PERVAL scale consists of four dimensions, which are: functional/quality, emotional, social and price/value for money, and has been assessed for validity and reliability in assessing perceived value for different types of products (i.e. orth et al., 2005; Sigala, 2006; Gill et al., 2007; Brown & Mazzarol, 2009; Walsh et al., 2014...). The PERVAL scale was used to assess the perceived value for wine in U.S. in 2005 and has been adapted due to the nature of the product. Adjustments included the addition of an environmental and a humane dimension and re-phrasing of few items 90 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 90 26. 03. 2020 12:05:45 Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers (Orth et al., 2005). The outcome was that the drivers of preference for wine in U.S. were: functional benefit/ quality, price/value for money, social benefit, emotional benefit, environmental benefit and humane benefit. The PERVAL scale had been previously used also in Slovenian environment, where the author found that region of origin has a significant influence on the perceived value of chicken salami, which found to be at least as important as the four elements of the marketing mix (Vukasovic, 2010). our study aimed to identify the dimensions of perceived value for wine from the perspective of Slovenian wine consumers and understand how they influence wine purchasing decisions. We also wanted to understand how the preferences of Slovenian wine regions differ depending on the dimensions of the perceived value for wine. Finally, we aimed to understand if the dimensions of the perceived value for wine differ depending on the socio-demographics of the respondents; and in the conclusion part of the paper also to give wine producers some guidance based on our findings, which could improve their marketing impact. 2 MATERIALS AND METHODS 2.1 QUALITATIVE RESEARCH Prior to conducting the quantitative market research, a series of focus group meetings were conducted in order to understand all aspects of the perceived value of wine from the perspective of Slovenian wine consumers. A test focus group, represented by seven participants was held in June 2011 to prepare questions for other focus groups in a language understandable to average wine drinkers. In July 2011, three focus groups were conducted with a total of 20 Slovenian wine consumers (seven, five and eight participants). Each focus group consisted of participants from different socioeconomic statuses and adult age groups. The main purpose was to obtain a set of indicators which would best describe the perceived value of wine from the perspective of Slovenian wine consumers so that, if required, we could upgrade the PERVAL scale for wine, which was previously used to study the perceived value of wine in a U.S. environment (orth et al., 2005). Accordingly, the scale was updated by adding two indicators which were frequently mentioned during focus group discussions: »produced in a wine region that has the potential to produce high quality wines« and »produced in wine region with long wine-making tradition«. Focus group meetings were used as a basis to develop the questionnaire for the quantitative part of the study. 2.2 QUANTITATIVE RESEARCH The data for the study were gathered in July and August 2016 from 221 Slovenian wine consumers, who were residents of two major Slovenian wine regions: 109 from the Primorska and 112 from the Podravje wine regions. The majority of high quality Slovenian wine is produced in the two biggest Slovenian wine regions, and while both have a long wine-making history and tradition, they belong to different wine zones ('Council regulation (EC) No 479/2008 on the common organization of the market in wine'), and they consequently produce different varieties and styles of wines. The online questionnaire used in this study was pretested online on a sample of 20 respondents from each studied wine regions to ensure that no semantic and measurement problems existed. Data were gathered through an online questionnaire using 1KA online software (University of Ljubljana, Faculty of Social Sciences), by means of convenience sampling with respect to balance in age, gender and place of residence. A specialized market research service provider was contracted to recruit their panel of respondents by email according to the sampling requirements. The screening section of the questionnaire asked four inclusion questions: the respondents had to (a) be born and reside in one of the two studied wine regions, (b) be above 18 years of age, (c) drink wine at least once per month and d) buy wine at least twice per year. Wine professionals, winemakers and sommeliers were excluded with a subsequent exclusion question. Those who were included were administered the remainder of the questionnaire, which was divided into three sections relevant for this paper: (1) wine purchasing and consumption habits; (2) PERVAL measures and wine region preferences; and (3) socio-demographic questions. The response rate of the contacted respondents was 48 %. 2.3 CONSTRUCTS MEASUREMENT To assess wine purchasing, consumption habits and socio-demographic characteristics of the sample, closed questions with one possible answer were used. Standard socio-demographic questions were used. To assess preferences for wine regions, 7-point Likert scale was used. The main focus of the study was to identify the dimensions of the perceived value for wine. Consumer perceived value was measured using the PERVAL scale, which was developed by Sweeney & Soutar (2001) and adapted by Orth et al. (2005) to investigate the perceived value for wine, where it was validated in a U.S. environment as a six-factor scale. We adjusted Acta agriculturae Slovenica, 115/1 - 2020 91 AAS_vsebina_2020_115_1_koncna 180320.indd 91 26. 03. 2020 12:05:45 S. PESTAR BIZJAK et al. the PERVAL scale by including two region of origin specific indicators, which were frequently mentioned during focus group discussions. We also re-phrased some of the claims due to semantic differences that were realized during focus group discussions to ensure the respondents would have a clear understanding of the claims. Our PERVAL scale consisted of 21 items, and a seven-point Likert scale ranging from 1 (strongly disagree) to 7 (strongly agree) was used to measure the intensity of all indicators. value for wine and socio-demographic data, Pearson's and Spearman's correlation coefficients were used. To calculate how the preferences towards different wine regions differ according to the dimensions of the perceived value for wine, multiple regression analysis was used. Preferences towards different winegrowing regions (measured on a Likert scale from 1 to 7) were independent variables, dependent variable was each dimension of the perceived value for wine. 2.4 DATA ANALYSIS Analysis of wine purchasing and consumption habits were based on the type of variables and analyzed with t-test or chi-square test to understand the differences between the two studied wine regions. Exploratory factor analysis was used for the identification of number of factors in the PERVAL scale and confirmatory factor analysis was used to demonstrate the reliability and validity of the scale. To assess the connection between the dimensions of the perceived 3 RESULTS AND DISCUSSION 3.1 SAMPLE DESCRIPTION Total sample consisted of 53 % male and 47 % female respondents. Average age of the respondent was close to 41 years of age, with most of them having at least secondary school education. 79 % of respondents were living in a small town or village. 21 % had below, 17 % above, and the rest average monthly income relative to the national average. More than half of respondents from both wine regions (53 %) Table 1: Socio-demographic characteristics of the sample (n = 221) Primorska winegrowing Podravje winegrowing region region Total sample Socio-demographic characteristics Count Column N % Count Column N % Count Column N % Gender Male 62 57 56 50 118 53 Female 47 43 56 50 103 47 Employment status Student 8 7 13 12 21 10 Unemployed 8 7 11 10 19 9 Employed 81 74 78 70 159 72 Retired 12 11 10 9 22 10 Size of place of residence Town (above 100.000 inhabitants) 9 8 37 33 46 21 Small town (10.000100.000 inhabitants) 34 32 20 18 54 25 Village (below 10.000 inhabitants) 64 60 54 49 118 54 Education status High school and below 41 38 48 43 89 40 Graduate degree 30 28 26 23 56 25 Post-graduate degree 38 35 38 34 76 34 Monthly income Under average 14 13 32 29 46 21 Average 73 68 62 56 135 62 Above average 20 19 17 15 37 17 Values in the same row and subtable marked with bolded fonts are significantly different at p < .05 in the two-sided test of equality for column proportions. Tests assume equal variances. Tests are adjusted for all pairwise comparisons within a row of each innermost subtable using the Bonferroni correction. 92 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 92 26. 03. 2020 12:05:45 Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers usually consumed white wines, 44 % reds and less than 3 % sparkling or rose wine. 48 % of respondents most commonly purchased wine at the supermarket stores and 35 % at winemakers. Average price of purchased wine was 8 EUR or below, highest number of respondents (35 %) in the last year usually bought wine for the price between 3 and 5 EUR. Looking at the preferences toward Slovenian winegrowing regions, we found that Primorska is the most preferred wine region (M = 6.15; SD = 1.28), followed by wine region Podravje (M = 5.48; SD = 1.45). Posavje and foreign wine regions were less preferred (M = 4.59; SD = 1.61; and M = 4.35; SD = 1.61, respectively). A comparison of socio-demographic characteristics of the respondents from the two winegrowing region samples indicates that samples were similar in relation to gender distribution, employment status and level of education. Some socio-demographic differences occurred due to demographic differences between the compared wine regions, namely size of place of residence and monthly income; significantly more respondents from winegrowing region Podravje had their monthly income below national average and more of them came from bigger town (table 1). 3.2 VALIDITY AND RELIABILITY OF THE PER- VAL SCALE We conducted exploratory factor analysis using SPSS software version 21 to verify the reliability and uni-dimensionality of the constructs. Statistically significant Bartlett test of sphericity and the Kaiser-Meyer-Olkin (KMO) above the 0.5 threshold confirmed suitability of data for factor analysis (Sharifpour et al., 2014). By use of the scree diagram, three factors for perceived value of wine were extracted: quality-price (quality and price indicators were highly correlated), emotional-social (high correlation of social and emotional indicators) and a factor we named terroir, where indicators for region of origin, humane and environmental indicators were highly correlated. The mean importance of quality-price was 5.3 (SD = 1.4), indicating that respondents paid most attention to the product's quality and price, followed by terroir (M = 5.1; SD = 1.6) and the factor explaining the emotional-social value of wine (M = 3.1; SD = 1.7). The factor loadings on all items were significant and exceeded the desired 0.7 threshold (Hulland, 1999) except for four items with loadings above 0.6; which were expected to reflect important information and were thus kept in for further Table 2: Quality of the PERVAL scale (n = 221) Constructs and items of the PERVAL scale (Crombach alpha) Standardized loadings % of Variance CR AVE Factor Terroir (0.9) 24.1 0.95 0.74 „.is produced in a wine region that has the potential... 0.8 .. .is produced in an environmentlly friendly manner 0.8 „is produced in wine region with long wine-making tradition 0.8 .is crafted by dedicated individuals 0.8 „is made from grapes under strictly controlled environment 0.8 „is crafted by very special and unique experts 0.8 „is made without polluting the environment 0.6 Factor Emotional-social (0.8) 14.5 0.9 0.7 ...improves the way I am perceived by others 0.7 „gives its owner social approval 0.9 „makes me feel good 0.9 „would give me pleasure 0.6 Factor Quality-price (0.8) 12.7 0.87 0.62 „has consistent quality 0.6 „offers value for money 0.8 „is reasonably priced 0.7 ... is a good product for the price 0.6 Inexes of fit: x2(84) = 178.62; p < 0.001; RMSEA = 0.07; NFI = 0.94; NNFI = 0.96; CFI = 0.97; IFI = 0.97; SRMR = 0.07 CR = composite reliability coefficient, AVE = average variance extracted. Acta agriculturae Slovenica, 115/1 - 2020 93 AAS_vsebina_2020_115_1_koncna 180320.indd 93 26. 03. 2020 12:05:45 S. PESTAR BIZJAK et al. analysis. The loadings for two indicators did not exceed 0.4 threshold and were excluded from further analysis. Cronbach's alpha for all factors was well above the desired 0.7 threshold. The variance explained by the three dimensions was satisfactory at 53 %. In addition to the reliability analyses, we used indexes to demonstrate the fit of model to the data, as demonstrated in Table 2. Confirmatory factor analysis was done with AM0S IBM 21 program with robust maximum likelihood method. 3.3 DIMENSIONS OF THE PERCEIVED VALUE FOR WINE WITH REGARD TO WINE REGIONS Slovenian wine consumers perceive three different dimensions of value in wine: quality-price, emotional-social and a terroir dimension. In American paper from 2005 they found, that American consumers perceive six different dimensions of value for wine and those were: quality, price, emotional, social, environmental and humane dimension. In this research it was also shown that the consumers for which quality dimension is more important, prefer French wines, and those for which price is more important, prefer Californian and Australian wines (Orth et al., 2005). We were interested to find, which dimensions of the perceived value for wine Slovenian consumers link to which wine regions. The relationships between the dimensions of the perceived value for wine and preferences for different wine regions are depicted in Table 3. We found that both wine regions, Primorska and Podravje, are associated with the terroir dimension of the perceived value for wine (p < 0.001 for both wine regions), and quality-price dimension (p < 0.001 for both regions), meaning that consumers, who value these two dimensions of the perceived value more, will rather choose wine from either of those two wine regions. Regression coefficient tells us, how much each of the perceived value dimension changes, if the preference for a wine region changes by one point. This means that wine region Podravje is more tightly connected with the terroir dimension of the per- ceived value for wine, than Primorska. Both wine regions are connected to the same extent also with quality-price dimension of the perceived value for wine. There is no connection between the preference for any wine region and emotional-social dimension and also, there is no connection between the wine region Posavje or foreign wine regions and any of the dimensions of the perceived value for wine. Despite the fact, that the overall preference is higher for Primorska wine region, the total sample believes that the same two value dimensions are important when purchasing wine from either Primorska or Podravje wine regions, namely terroir and quality-price. The respondents in our sample, who appreciate the terroir of wine more, would more likely choose wines from Podravje winegrowing region. It is possible that the respondents from our sample were not familiar enough with wines from region Posavje and foreign countries, and this could be the reason why these wines are not percieved as being connected with any of the studied value dimensions. Emotional-social was rated much lower than the other two value dimensions and it seems that to Slovenian consumers from our sample this dimension is so unimportant, that they do not consider it when choosing wine from any of the winegrowing regions. Another question is why American consumers consider five, and Slovenian consumers only three value dimensions while choosing wine. It is likely that because American consumers come from a more mature capitalist market, they are therefor more susceptible to a variety of marketing messages. On the other hand Slovenian wine consumers are more traditional, considering mainly the connection between the quality and price and the terroir of wine, meaning the region of origin and the tradition of the winegrowing region. 3.4 DIMENSI0NS 0F THE PERCEIVED VALUE F0R WINE WITH REGARD T0 S0CI0-DEM0-GRAPHIC CHARACTERISTICS Lastly, we aimed to understand if the dimensions of the perceived value for wine differ depending on the Table 3: Connection between wine region preferences and dimensions of the perceived value for wine Perceived value dimension Terroir Emotional-social Quality-price B P-value B P-value B P-value Constant 2.58 < 0.001 3.22 < 0.001 2.98 < 0.001 Primorska 0.25 < 0.001 - 0.094 0.172 0.2 < 0.001 Podravje 0.28 < 0.001 0.105 0.133 0.2 < 0.001 Posavje - 0.11 0.061 - 0.014 0.819 - 0.04 0.446 Foreign wine regions - 0.03 0.637 0.027 0.639 0.02 0.615 B = regression coefficient. 94 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 94 26. 03. 2020 12:05:45 Dimensions of the perceived value for wine from the perspective of Slovenian wine consumers Table 4: Relation between socio-demographics and perceived value for wine (n = 221) Perceived value dimension Terroir Emotional-social Quality-price Age Pearsons r 0.15 0.07 0.08 P-value 0.029 0.276 0.245 Number of persons in the hausehold Pearsons r - 0.03 0.07 - 0.05 P-value 0.646 0.335 0.448 Size of place of residence Spearmans r - 0.01 - 0.04 - 0.02 P-value 0.914 0.543 0.819 Education Spearmans r - 0.04 -0.10 - 0.17 P-value 0.589 0.124 0.013 Monthly income Spearmans r 0.05 0.01 - 0.14 P-value 0.468 0.908 0.039 socio-demographic characteristics of the respondents. We found that there is weak, but statistically significant connection between respondents age and terroir dimension of the perceived value for wine, meaning that older respondents value this dimension more than younger. We also found weak negative statistically significant link between education and monthly income and quality-price dimension of the perceived value for wine, meaning that for these consumers wine price or the ratio between quality and price is less important when purchasing wine. There were no differences in terms of consumers employment status and family status and the perceived value for wine (Table 4). These results are relevant when creating consumer segmentation, having in mind which are the most important consumer segments that are the basis for the creation of marketing strategy. 4 CONCLUSIONS Our research demonstrated, that in the sample of Slovenian wine consumers from the two largest Slovenian winegrowing regions, who at least occasionally buy and drink wine, perceive three different dimensions of the perceived value for wine: quality-price, emotional-social and a dimension which we named "terroir", where the indicators for region of wine origin, humane and environmental claims highly correlated. Quality-price and terroir dimensions were rated above five on a seven-point Likert scale, indicating that both dimensions are very important to Slovenian wine consumers. Emotional-social dimension was rated just over three, meaning that marketing strategy from wine-makers and wine-sellers should focus on supporting superior terroir and quality-price ratio messages. Both, quality-price and terroir dimensions corre- lated with Primorska and Podravje wine regions, but not with others. When making wine purchasing decisions, older respondents give greater value to the terroir dimension, while respondents with higher education and income are less concerned with the quality-price ratio. We propose that these information are taken into account in order to improve consumer segmentation and targeting. According to the methodological requirements of this research, while measuring the constructs of consumer regiocentrism and regional identity, only consumers from wine regions Primorska and Podravje were included. It would be interesting to check if the perception of other (non-winegrowing regions and the third Slovenian winegrowing region-Posavje) consumers with regard to the perceived value would differ or confirm our findings. 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Comparison of the toxicity and repellency of two conventional neonicotinoids and a coconut-derived insecticide soap toward the parasitoid wasp Aphelinus mali Haldeman, 1851 Abstract: The parasitoid wasp Aphelinus mali Haldeman, 1851 (Hymenoptera: Aphelinidae) is the most important biological control agent against the woolly apple aphid, Eriosoma lanigerum (Hausemann, 1802) (Hemiptera: Aphididae), which is an important apple orchards pest throughout the world. Based on the importance of using low-risk compounds to protect beneficial agents, the present study was carried out to evaluate the toxic and repellent effects of two conventional chemicals (imi-dacloprid and thiacloprid) and coconut-derived biopesticide soap (Palizin*) toward A. mali. The results of residual bioassays on apple leaf discs indicated that imidacloprid after 24 h and insecticide soap after 72 h exposure time categorized at the highest and no/little toxicity rates, respectively. Ingestion bioassays on filter papers revealed that imidacloprid and thiacloprid had moderate toxicity rate, while insecticide soap had a low-level toxicity rate. Repellency test at Y-tube olfactometer showed that the repellent effects of both chemicals were more than that of insecticide soap. It is concluded that coconut-derived soap was compatible with the parasitoid activity, and the caution should be paid when including the two neonicotinoid insecticides imi-dacloprid and thiacloprid for E. lanigerum management. Key words: Eriosoma lanigerum; Aphelinus mali; insecticides; toxicity; repellency Primerjava strupenosti in odvračalnega delovanja dveh konvencionalnih neonikotinoidov in insekticidnega mila iz kokosa na krvavkinega najezdnika (Aphelinus mali Haldeman, 1851) Izvleček: Parazitoidna osica krvavkin najezdnik (Aphelinus mali Haldeman, 1851, Hymenoptera: Aphelinidae) je najpomembnejši naravni sovražnik krvave uši (Eriosoma lanigerum [Hausemann, 1802], Hemiptera: Aphididae), ki je pomemben škodljivec jablan širom po svetu. Zaradi vse večjega pomena okoljsko sprejemljivejših snovi pri zatiranju rastlinskih škodljivcev, saj na ta način varujemo tudi koristne žuželke, smo v raziskavi ovrednotili toksične in repelentne učinke dveh konvencionalnih kemičnih sredstev za varstvo rastlin (imidakloprid in tiakloprid) in bioinsekticidnega mila (Palizin*) iz kokosa na vrsto A. mali. Rezultati poskusa na di-skastih izsečkih jabolčnih listov so pokazali, da sta imela imi-dakloprid 24 h po nanosu in insekticidno milo 72 h po nanosu največji učinek na uši in nič ali le malo strupenega učinka na parazitoida. Prehranjevalni poskus na filtrirnem papirju je pokazal, da sta imela imidakloprid in tiakloprid zmeren toksični učinek, medtem, ko je imelo insekticidno milo zelo majhnega. Preizkusi odvračalnega delovanja snovi v Y-ceva-stem olfaktometru so pokazali, da so bili odvračalni učinki obeh kemikalij večji kot pri insekticidnem milu. Zaključimo lahko, da je kokosovo milo kompatibilno s parazitoidom, več pozornosti pa je potrebno nameniti, kadar za zatiranje krvave uši uporabimo oba neonikotinoidna insekticida, imidaklo-prid in tiakloprid. Ključne besede: Eriosoma lanigerum; Aphelinus mali; in-sekticidi; toksičnost; repelenca 1 University of Mohaghegh Ardabili, Moghan College of Agriculture and Natural Resources, Ardabil, Iran 2 Corresponding author, e-mail: ebadollahi@uma.ac.ir 3 University of Tehran, College of Abouraihan, Department of Entomology and Plant Pathology, Tehran, Iran Acta agriculturae Slovenica, 115/1, 97-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 A. EBADOLLAHI, R. SADEGHI 1 INTRODUCTION The woolly apple aphid (Eriosoma lanigerum (Hausemann, 1802), Homoptera: Aphididae) is important pest in apple orchards. It can causes significant damages to the apple trees by sucking the sap from new shoots and branches (Rogers et al., 2011; Lordan et al., 2015). Its feeding activity leads to the formation of galls on woody tissue and young shoots. Buds can be destroyed and apple calyces may also be contaminated with the aphid honeydew and sooty molds (Beers et al., 2007). The woolly aphid parasitoid [Aphelinus mali Hal-deman, 1851, (Hymenoptera: Aphelinidae)], is a specific and very efficient endoparasitoid of E. lanigerum that had been introduced from North America to other apple-growing regions of the world, such as in Iran (Heu-nis & Pringle, 2003, Su et al., 2017). Although the application of synthetic insecticides is the conventional method for the management of E. lanigerum, such strategy can have various negative side effects, such as impact on non-target organisms, pest resistance and threats to human health (Desneux et al., 2007; Damalas & Eleftherohorinos, 2011). For example, organic phosphorus insecticides, which are widely applied to reduce populations of E. lanigerum, cause severe damage in its parasitoid wasp populations (Beers et al., 2007). Recent researches have shown that the chemical agents have lethal and sublethal effects on the hymenopterous parasitoids. For example, Desneux et al. (2004) indicated that lambda-cyhalothrin had a high toxicity on Aphidius ervi Haliday, 1834 (Aphidii-nae). Further, low doses of this chemical weakened the orientation behavior of A. ervi. Toxic effects of the del-tamethrin and its negative effects on recolonization capacities of A. ervi was also documented (Desneux et al., 2006). The adults of egg parasitoid wasp Trichogramma chilonis Ishii, 1941 (Trichogrammatidae) were very susceptible to chlorfenapyr, fipronil, spinosad, avermectins, 6-cypermethrin, and cartap (Wang et al., 2012). In other study, acute toxicity of mineral oil and pyriproxyfen (a juvenile hormone mimic) was tested against the parasitoid wasp Aphytis melinus DeBach, 1959 (Aphelinidae). Mineral oil had high mortality on the adults of A. melinus, but pyriproxyfen had neither lethal nor sublethal effects. However, parasitoid larvae were very susceptible to pyriproxyfen (Biondi et al., 2015). Accordingly, the application of suitable chemical control tools that can reduce the side effects of pesticides and prevent damage to a pest's natural enemies is necessary. The aim of the present study was, therefore, to assess the non-target toxicity of two conventional neonicotinoids imidacloprid and thiacloprid and a novel coconut-derived bioinsecticide soap (Palizin) on the parasitoid wasp A. mali adults in the experimental conditions. 2 MATERIALS AND METHODS 2.1 TEST INSECT All bioassays were conducted using a partheno-genetic (all females) strain of A. mali. Apple branches ('Red Delicious') infested with E. lanigerum were collected from apple orchards at Miami County (Semnan province, Iran) (36°N 55°E), in June 2016, and placed in one-liter flasks until emergence of the parasitoid wasps. Prior to the experiment, the A. mali colony was reared for 3 to 4 generations in an incubator at 27 ± 1 °C, 55 ± 5 % Relative Humidity (RH) and 14 L: 10 D photoperiod, using apple leaves infested with E. lani-gerum. Adult parasitoids were kept in glass tubes and fed on 10 % honey-water solution smeared inside the test tubes' wall. 2.2 INSECTICIDES Three commercial formulations of insecticides were used in the present study: imidacloprid concentrate suspension (Confidor*), provided as a commercial product by Paksame Iranian Company with 35 % active ingredient, thiacloprid (Calypso*) concentrate suspension, provided as a commercial product formulized by Bayer Company with 48 % active ingredient, and the coconut soap (Palizin*) as concentrate suspension was purchased from Iranian Kimia Sabzavar Company with 65 % active ingredient. For bioassays, maximum recommended concentrations were used: 0.5, 0.2 and 1.5 ml l-1 insecticide/ distilled water imidacloprid, thiacloprid and the insecticide soap, respectively. 2.3. RESIDUAL CONTACT BIOASSAY ON APPLE LEAVES Fresh apple leaves (Malus pumila Miller) were sampled from apple orchards, which had no history of chemical control in the past three years. Fresh apple leaves were sprayed by recommended concentrations of insecticides until runoff by using a hand sprayer (Williams et al., 2003). The leaves were put into a chemical hood for an hour and then transferred to Petri dishes, 9 cm of diameter. Twenty-five, one-day-old female parasitoids were placed in each Petri dish, and then the dishes were kept at room conditions at 27 ± 1 °C and 55 98 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2C2C_115_1_koncna 18C32C.indd 1CC 26. C3. 2C2C 12:C5:46 Comparison of the toxicity and repellency of two conventional ... toward the parasitoid wasp Aphelinus mali Haldeman, 1851 ± 5 RH. In the untreated control groups, distilled water was used instead of insecticide concentrations. Each experiment was repeated four times and mortality was recorded 24, 48 and 72 h after the exposure beginning. 2.4 INGESTION BIOASSAY ON FILTER PAPER To improve the feeding of the parasitoid adults and to mimic the field situation where they custom external nutrients, the same mentioned concentrations were prepared by adding 10 % of sugar. The filter papers (5 cm of diameter) was soaked in one ml of the solutions and were placed in the center of the 9cm Petri dishes (Rogers et al., 2011). Twenty-five parasitoid adults were transferred to each Petri dish under the laboratory conditions as described above. In the control groups, distilled water with 10 % sugar was also used instead of insecticides. The experiment was repeated 4 times and mortality rates was recorded 72 h post exposure. 2.5 REPELLENT EFFECT The Y-tube olfactometer was assembled by 3 linked glass tubes (each 10 cm long and 1cm in diameter). The flow of air was established by an adjustable fan. To evaluate the repellency of the insecticides, infested apple leaves with 15 adult females of aphids were sprayed at the recommended concentrations and allowed to dry for one hour under test conditions. The insecticide-treated leaves were inserted in one of the openings of the Y-tube olfactometer and the other opening was considered as a control. The control groups consisted of leaves treated only with distilled water. Thirty adult parasitoids were released inside a one-liter glass container covered by a black cloth for stimulating the wasps to move from darkness to light, thus allowing the selection of one of the arms of the device. Number of insects were recorded on each side of the tube every 3 h for 18-hour duration. Y-tube olfactometer was kept in experimental conditions with 27 ± 1 °C and 55 ± 5 RH and tests were repeated 3 times (Sadeghi et al., 2014). uted and thus the one-way analysis of variance (ANOVA) was run for each dataset. Mean separation was assessed using the Tukey's post hoc test at p < 0.05. Insecticide toxicity was rated according to IOBC toxicity categories (Sterk et al., 1999): (1) harmless, (2) slightly harmful, (3) moderately harmful, and (4) harmful (corresponding to reductions below 30 %, between 31-79 %, between 80-99 % and higher than 99 %, respectively). Percentage repellency (PR) was calculated as follows: PR = [(C - T) / (C + T)] x 100, where C = number of wasps on the treated area with distilled water, and T = number of wasps on the area treated with insecticide (Nerio et al., 2009). Classification of PR values was done according to Juliana and Su (1983): class 0 (PR = 0.1 %), class I (PR = 0.1-20 %), class II (PR = 20.1-40 %), class III (PR = 40.1-60 %), class IV (PR = 60.1-80 %) and class V (PR = 80.1-100 %). 3 RESULTS 3.1 RESIDUAL CONTACT BIOASSAY In the residual contact bioassay on apple, although all the insecticides caused significant mortality (F = 75.044, df = 2, 11, p < 0.001) after 24 h, imidacloprid caused the highest rate of parasitoid mortality (78.850 ± 4.703%); while insecticide soap caused the lowest mortality (28.351 ± 2.413%) at this duration. Mortality was also statistically significant after 48 (F = 274.485, df = 2, 11, p < 0.001) and 72 (F = 209.515, df = 2, 11, p < 0.001) h. In general, the mortality rates decreased as time pass due to reduction in residual concentrations. For example, mortality significantly decreased from 32.609 ± 2.413 % at the 24 hour exposure time to 16.304 ± 2.030 % after 72 h for thiacloprid (Figure 1 and Table 1). Observed mean mortality in the control groups were 5.00 ± 0.25 % after 24, 48 and 72 h. Toxicity ratings for insecticidal activity of imidacloprid, thiacloprid and insecticide soap in bioassays with apple leaves are also shown in Table 1. According to toxicity rating of IOBC, imidacloprid had moderately harmful effect after 24-hour and insecticide soap had lowest toxicity (harmless) after 72 hour exposure period (Table 1). 2.6 DATA ANALYSIS 3.2 INGESTION BIOASSAY Data of parasitoid mortality in the bioassays were corrected using Abbott's formula (Abbott, 1925) when mortality was observed in the control groups. Data analysis was carried out by SPSS software (Version 24). Data obtained after 24, 48 and 72 h of residual exposure and those from the ingestion bioassay were checked for normality using the Kolmogorov-Smirnov test. All data were normally distrib- All tested insecticides had significant effects on the parasitoid mortality (F = 174.915, df = 2, 11, p < 0.001) in the ingestion bioassays. After 72 h of exposure, imidacloprid caused the highest rate of parasitoid mortality (69.042 ± 1.011 %) and insecticide soap showed the lowest rate of mortality (29.875 ± 1.628%). However, insignificant differences were found between imidacloprid and thiaclo- Acta agriculturae Slovenica, 115/1 - 2020 99 AAS_vsebina_2020_115_1_koncna 180320.indd 99 26. 03. 2020 12:05:46 A. EBADOLLAHI, R. SADEGHI Figure 1: Mean percentage mortality (± SE) of Aphelinus mali females after exposure to dry residues of imidacloprid, thiaclo-prid and a coconut-derived insecticide soap on apple leaves. Mortalities (%) compared with Tukey's test at p < 0.05 in which same letters are not significantly different. Table 1: International Organization for Biological Control (IOBC) toxicity classes for Aphelinus mali females exposed to dry residues in apple leaves or that have fed on sugary solutions contaminated with imidacloprid, thiacloprid and insecticide soap Bioassay Insecticide Exposure Time (h) Toxicity Rating Residual Contact imidacloprid 24 moderately harmful 48 slightly harmful 72 slightly harmful thiacloprid 24 slightly harmful 48 harmless 72 harmless insecticide soap 24 harmless 48 harmless 72 harmless Ingestion imidacloprid 72 slightly harmful thiacloprid 72 slightly harmless insecticide soap 72 harmless Classification of PR values followed that cited in Juliana and Su (1983): Harmless, slightly harmful, moderately harmful, and harmful corresponding to mortality below 30 %, between 31 and 79 %, between 80 and 99 % and higher than 99 %, respectively. prid (Table 1and Figure 2). Observed mean mortality in the control groups was 3.0 % after 72 h. Rating of insecticide toxicity indicated that imidacloprid and thiacloprid were slightly harmful while insecticide soap was harmless against A. mali (Table 1). In general, coconut-derived insecticide soap had lower toxicity against A. mali than the chemical insecticides: imidacloprid and thiacloprid in both bioassays with apple leaves and filter papers. 3.3 REPELLENT EFFECT The repellent activity of the two neonicotinoids was 100 Acta agriculturae Slovenica, 115/1 - 2020 more evident than that of the insecticide soap. Classification of the percentage of repellency (PR) values according to Juliana and Su (1983), indicated that insecticide soap ranked as class I (PR = 0.1-20 %) within an exposure time of 3 to 18 h. In contrast, the PR values of imidacloprid were classified as class III for all observation times and these values were in classes II and III with thiacloprid (Table 2). 4 DISCUSSION Imidacloprid and thiacloprid as conventional neoni-cotinoid insecticides are effective against aphids and other AAS_vsebina_2C2C_115_1_koncna 18C32C.indd 1CC 26. C3. 2C2C 12:C5:46 Comparison of the toxicity and repellency of two conventional ... toward the parasitoid wasp Aphelinus mali Haldeman, 1851 Figure 2: Mean percentage mortality (± SE) of Aphelinus mali exposed to imidacloprid, thiacloprid and a coconut-derived insecticide soap after feeding on contaminated sugary water in filter papers within 72 h. Mortalities (%) compared with Tukey's test at p <0.05 in which same letters are not significantly different Table 2: Percentage of repellency (PR) of imidacloprid, thiacloprid and coconut-derived insecticide soap against Aphelinus mali at different exposure times at the Y-tube olfactometer using sprayed apple leaves as volatile sources Insecticide Time (h) 3 6 9 12 15 18 Imidacloprid 49.340 (III) 48.564 (III) 52.625 (III) 47.965 (III) 36.833 (III) 53.118 (III) Thiacloprid 43.593 (III) 33.359 (II) 38.121 (II) 35.034 (II) 40.551 (III) 38.039 (II) Insecticide soap 14.268 (I) 10.69 (I) 15.992 (I) 17.417 (I) 14.276 (I) 16.685 (I) Classification of PR values followed that cited in Juliana and Su (1983): class 0 (PR = 0.1 %), class I (PR = 0.1-20 %), class II (PR = 20.1-40 %) class III (PR = 40.1-60 %), class IV (PR = 60.1-80 %) and class V (PR = 80.1-100 %). sap-sucking insects. However, insect natural enemies and pollinators such as honeybees, Apis mellifera L., are the later reported to be affected by the exposure to imidacloprid and thiacloprid (Schmuck et al., 2001; Blacquiere et al., 2012; Fischer et al., 2014). Toxicity of imidacloprid and thiacloprid to eggs, N1 and N5 nymphs and adults of the predatory bug, Deraeocoris lutescens (Schilling, 1837) (He-miptera: Miridae), via residual contact was investigated and results showed that all stages were susceptible to these chemicals (Azimizadeh et al., 2012). Sublethal concentrations of imidacloprid reduced the longevity and the fecundity of females of the seven-spot ladybird beetle Coccinella septempuntacta L. (Coleoptera: Coccinellidae). Moreover, similar effects were noticed on the F1 generation, i.e., the progeny of the exposed individuals (Xiao et al., 2016). In the study of Martinou et al. (2014), the direct, residual and oral toxicity of thiacloprid were evaluated against Mac-rolophus pygmaeus (Rambur, 1839) (Hemiptera: Miridae) as a common generalist predator in Mediterranean agro-ecosystems. Along with lethal effects, thiacloprid caused an increase in resting and preening times of the predator. Based on the significant acute toxicity of imidacloprid and thiacloprid against A. mali, results of the mentioned studies are consistent with the results of the present work. Accordingly, the use of these chemicals cannot be compatible with the activity of this parasitoid and thus they should be used properly taking into consideration its effects on non-target organisms for successful integrated pest management (IPM) strategies. However, they clearly had less toxicity than some organophosphate insecticides such as chlorpyrifos, diazinon and carbaryl, which have been categorized as "highly toxic" compounds by Bradley et al. (1997). Further, Rogers et al. (2011) reported that thiacloprid had no or low-level toxicity on A. mali in the contact toxicity with filter papers, while in the present study it showed low and moderate levels of toxicity in the bioassays with apple leaves and filter papers, respectively. Different experimental conditions and materials might contribute to the variations in results. For example, contact toxicity in the study of Rogers et al. (2011) was carried out by filter paper but in the present study leaf discs were used. Furthermore, according to results of the present work, thiacloprid showed less toxicity in bioassays Acta agriculturae Slovenica, 115/1 - 2020 101 AAS_vsebina_2020_115_1_koncna 180320.indd 101 26. 03. 2020 12:05:46 A. EBADOLLAHI, R. SADEGHI with leaf discs than that with filter papers. It is worthy to mention that leaf spray pesticide application would provide more realistic orchard conditions, resulting in lower toxicity against A. mali. The insecticide soap (Palizin®), the coconut-derived pesticide, showed great potential in the management of insect pests and its toxicity and repellency against some insect pests have been recently acknowledged (Amiri-Esheli, 2009; Sadeghi et al., 2014; Sheiba-ni & Hassani, 2014). The side effects of the Palizin have not yet been reported before. Comparison of the Pali-zin® and imidacloprid toxicity was made against cotton aphid (Aphis gossypii Glover, 1877) and its natural enemy (Aphidius colemani Viereck, 1912) (Ketabi et al., 2014). In this research, imidacloprid had a higher level of toxicity against both the aphid and the wasp than Palizin® which consistent with the obtained findings. 5 CONCLUSION Generally, the insecticide soap showed significantly less toxicity than tested chemicals in all bioassays. Furthermore, the repellent effects of insecticide soap were significantly lower than imidacloprid and thiaclo-prid applications. 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Ecotoxicology, 25, 1782-1793. https://doi.org/10.1007/s10646-016-1721-z Acta agriculturae Slovenica, 115/1 - 2020 103 AAS_vsebina_2020_115_1_koncna 180320.indd 103 26. 03. 2020 12:05:46 AAS_vsebina_2020_115_1_koncna 180320.indd 104 26. 03. 2020 12:05:46 doi:10.14720/aas.2020.115.1.1336 Original research article / izvirni znanstveni članek Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid and hexaploid cultivars of wheat using stress tolerance indices Sara KHOSRAVI 1, Reza AZIZINEZHAD 1 2, Amin BAGHIZADEH 3, Mahmood MALEKI 3 Received November 8, 2019; accepted January 4, 2020. Delo je prispelo 8. novembra 2019, sprejeto 4. januarja 2020. Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid and hexaploid cultivars of wheat using stress tolerance indices Abstract: This study was carried out on grain yield in wheat genotypes with the aim of assessing genetic potential of drought tolerance. The experiment was performed as split plot in the form of randomized complete block design with three replications under normal and drought stress conditions with 32 genotypes. Based on grain yield, and under the condition of non-stress and drought stress, 5 drought tolerance indices are estimated including Tolerance Index (TOL), Stress Tolerance (STI), Mean Productivity (MP), Geometric Mean (GMP) and, Harmonic Mean (HM) for all kinds of genotypes. The analysis of yield correlation and drought tolerance indices in two environments indicated that STI, MP, GMP, HM indices were the most suitable parameters for screening wheat genotypes. Principal components analysis exhibited that the 83 % of first principal component and the 15 % of second one justified the variation of the initial data. Drawing bi-plot diagram declared that Sabalan, Shabrang, Aria, Azar, Azadi, and T2 genotypes were highly functional and resistant to drought stress. Key words: wheat; index; component analysis; cluster analysis Ovrednotenje in primerjava tolerance na sušo nekaterih divjih diploidnih populacij, tetraploidnih in heksaploidnih sort pšenice z uporabo indeksov tolerance na stres Izvleček: Raziskava je bila opravljena na pridelku zrnja med genotipi pšenice za oceno genetskega potenciala tolerance na sušo. Poskus je bil izveden kot popolni naključni bločni poskus z deljenkami s tremi ponovitvami na 32 genotipih pšenice v normalnih in sušnih razmerah. Na osnovi pridelka je bilo za vse genotype v nestresnih razmerah in v razmerah sušnega stresa določenih 5 indeksov tolerance na sušo in sice: indeks tolerance (TOL), stresna toleranca (STI), srednja produktivnost (MP), geometrična sredina (GMP) in harmonična sredina (HM). Analiza korelacije med pridelkom in indeksi tolerance na sušo je pokazala, da so bili v obeh okoljih za ovrednotenje genotipov pšenice primerni STI, MP, GMP, HM indeksi. Analiza glavnih komponent je pokazala, da je 83 % variabilnosti izvirnih podatkov pojasnila prva komponenta, 15 % pa druga. Bi-plot diagram je pokazal, da so genotipi pšenice Sabalan, Shabrang, Aria, Azar, Azadi, in T2 zelo produktivni in odporni na sušni stress. Ključne besede: pšenica; indeks; analiza component; kla-sterska analiza 1 Department of Plant Breeding and Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Corresponding author, e-mail: r.azizi@srbiau.ac.ir 3 Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran Acta agriculturae Slovenica, 115/1, 105-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 S. KHOSRAVI et al. 1 INTRODUCTION Wheat (Triticum aestivum L.) is the most important crop in the world (Sleper and Poehlman, 2006) and is cultivated in more than 250 million hectares of agricultural lands of all over the world (Royo and Di Fonzo, 2005). It is estimated that about 67 percent of the total Iranian wheat is produced in arid lands (Shamsi et al., 2011). So many breeding programs have been developed to improve drought tolerance of crop (Maazou et al., 2016). The crop rotation diversity is associated with a greater yield stability under abnormal conditions like drought stress (Gaudin et al., 2015). Selection of drought-resistant cultivars is the best solution to improve stability of crop yield in dry conditions (Farshadfar et al., 2012). Drought tolerance is a quantitative trait that is controlled by many genes (Fleury et al., 2010). Thus, detecting of drought-tolerance genotypes is very difficult (Takeda and Matsuoka, 2008). Evaluation of relative yield of genotypes under drought stress and non-stress conditions is a starting point for identification of drought-tolerant mechanisms and for screening of drought-tolerant genotypes (Fernandez, 1992; Mitra, 2001). Based on tolerance index (TOL) as difference in the yield of two genotypes under both normal and stress conditions, the higher ToL indicates more sensitivity of plants to stress and thus, the selection was mainly based on the low amounts of ToL, but high mean productivity (MP), the mean yield of each genotype in two conditions, shows more tolerance to stress (Rosielle and Hamblin, 1981). In 1992, on the base of yield, mung bean's genotypes were divided into four groups under both normal and stress environments: (A) high-yield under both conditions; (B) high-yield under normal conditions; (C) good-yield under drought stress and (D) low-yield under both normal and drought stress condition (Fernandez, 1992). It showed that selections based on ToL and MP would be able to separate genotypes of B and D groups. By MP index, high yielding genotypes under both normal and stress conditions could be selected but this index is not able to distinguish between A and B groups. The MP index also leads to choose high yielding genotypes but with a low stress tolerance (Fernandez, 1992; Rosielle and Hamblin, 1981). When there is a significant relative difference between the yield under stress and non-stress conditions, MP index is oriented toward the yield under normal conditions so to obviate this problem, geometric mean productivity (GMP) index that is based on the geometric mean of genotypes yield under normal and stress conditions was proposed by Fernandez (1992). Since this index is less sensitive to different values in normal conditions and drought stress, Fernandez introduced another index called stress tolerance index (STI), in order 106 Acta agriculturae Slovenica, 115/1 - 2020 to identify high-yield genotypes under two normal and stress conditions and also for identifying drought tolerance genotypes. This index considers the stress intensity (SI) that is estimated as mean ratio of genotypes yield in the stress environment to the normal environment from one (Fischer and Maurer, 1978; Schneider et al., 1997). High values of GMP and STI are subjected to determine stress tolerance genotypes. Harmonic mean index (HM) was also introduced by Fernandez. These indices are introduced as the best having a high correlation with grain yield in both normal and stress environments (Fernandez, 1992). In 2006, STI, GMP, and MP indices were evaluated for selecting of wheat genotypes under water deficit conditions (Golabadi et al., 2006; Mardeh et al., 2006). Also, in 2009, STI, GMP, and MP were introduced as the most effective indicators for evaluating and selecting wheat genotypes under drought stress (Talebi et al., 2009). The purpose of the present study is to determine the genotypes with yield stability under drought stress and non-stress conditions as well as introducing the appropriate indices for screening wheat genotypes under stress and non-stress conditions. 2 MATERIALS AND METHODS In this experiment, 7 population of wild diploid wheat (Triticum boeoticum Boiss.) with AA genome, 5 population of wild diploid wheat (Aegilops tauschii Coss.) with DD genome, 10 cultivars of tetraploid wheat (Triticum durum Desf.) with AABB genome and 10 cultivars of hexaploid wheat (Triticum aestivum L.) with AABBDD genome, (Table 1) have been used. Wheat seeds were implanted in research farm of Graduate University of Advanced Technology in Kerman province in October 2016. The experiment was designed as split plot in a randomized complete block design (RCBD), with three replications, assigning normal and stress levels as main plots and population and wheat cultivars as sub-plots. Each plot consisted of three lines keeping 2 m length and 20 cm distance within which on each line 15 seeds had been implanted. All plots of the normal and stress experiments were well watered (once every 7 days) by using an installed pipeline system. The volume of water input for each plot was controlled by using adjustable counter. Since April (the grain-filling period), drought stress treatment, irrigation at 30-day intervals, was imposed on the plants until the beginning of June (time of harvesting). After harvesting each plot at crop maturity, grain yield was recorded in both normal and stress conditions. Tolerance index, stress tolerance index, mean pro- AAS_vsebina_2020_115_1_koncna 180320.indd 106 26. 03. 2020 12:05:46 Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid ... of wheat using stress tolerance indices Table 1: Names of genotypes used in the study Genotype Species Genome Ploidy level Source B1 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Lorestan-10 km to Nurabad-On behalf of Alshatr B2 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Lorestan - Firouzabad-Khorramabad Road B3 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Kermanshah - Deh sefeed-Not-To-Dumbel Islamabad Kermanshah B4 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Kermanshah - 20 km from Paveh to Ravensar B6 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Kurdistan - Two Sanandaj Moths B7 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Kurdistan - 10 km after Ganji to Ghorveh B8 Triticum. boeticum Wild Diploid Genome AA 2n = 2x = 14 Kurdistan - Bolban abad city T1 Aegilops tauschii Wild Diploid Genome DD 2n = 2x = 14 Gilan - Isfahan-Rasht Road T2 Aegilops tauschii Wild Diploid Genome DD 2n = 2x = 14 Mazandaran-Amol T3 Aegilops tauschii Wild Diploid Genome DD 2n = 2x = 14 Karaj Road - Chalous (50 km before Chalous) T4 Aegilops tauschii Wild Diploid Genome DD 2n = 2x = 14 Ardebil-Moghan-Parsabad plain T5 Aegilops tauschii Wild Diploid Genome DD 2n = 2x = 14 East Azarbaijan - 10 km from Ahar-Kalibar road Aria Triticum durum Tetraploid AABB 2n = 4x = 28 Behrang Triticum durum Tetraploid AABB 2n = 4x = 28 Dehdasht Triticum durum Tetraploid AABB 2n = 4x = 28 Dena Triticum durum Tetraploid AABB 2n = 4x = 28 Shabrang Triticum durum Tetraploid AABB 2n = 4x = 28 Karkhe Triticum durum Tetraploid AABB 2n = 4x = 28 Yavarus Triticum durum Tetraploid AABB 2n = 4x = 28 DW/95/4 Triticum durum Tetraploid AABB 2n = 4x = 28 DW/90/8 Triticum durum Tetraploid AABB 2n = 4x = 28 WS Triticum durum Tetraploid AABB 2n = 4x = 28 Azar Triticum durum Hexaploid AABBDD 2n = 6x = 42 Azadi Triticum durum Hexaploid AABBDD 2n = 6x = 42 Pishtaz Triticum durum Hexaploid AABBDD 2n = 6x = 42 Roshan Triticum durum Hexaploid AABBDD 2n = 6x = 42 Sabalan Triticum durum Hexaploid AABBDD 2n = 6x = 42 Superhead Triticum durum Hexaploid AABBDD 2n = 6x = 42 Shiraz Triticum durum Hexaploid AABBDD 2n = 6x = 42 Hirmand Triticum durum Hexaploid AABBDD 2n = 6x = 42 Moghan Triticum durum Hexaploid AABBDD 2n = 6x = 42 mr-17 Triticum durum Hexaploid AABBDD 2n = 6x = 42 Acta agriculturae Slovenica, 115/1 - 2020 107 AAS_vsebina_2020_115_1_koncna 180320.indd 107 26. 03. 2020 12:05:46 S. KHOSRAVI et al. Table 2: Formulas drought tolerance indices Index Formula Source Tolerance Index (TOL) Stress Tolerance Index (STI) Mean productivity (MP) Geometric Mean Productivity (GMP) Harmonic Mean (HM) Y - Y p s (Y*YS) /(7p) 2 (Yp + Ys) / 2 (Yp*Ys) * [2(Yp x Ys)/ (Yp + Ys)] (Rosielle and Hamblin, 1981) (Fernandez, 1992) (Rosielle and Hamblin, 1981) (Fernandez, 1992) (Fernandez, 1992) ductivity (Rosielle and Hamblin, 1981) and the geometric mean productivity and harmonic mean (Fernandez, 1992) were calculated according to the following formulas (Table 2): In which, YP and YS are the yield of each genotype under non-stress and stress conditions, respectively, and yP is the mean yield of all genotypes. After calculating of different indices, correlation between grain yield under normal and stress conditions (YP, YS) and stress tolerance indices was calculated and the best index was determined. So that, the indices having high and significant correlation with grain yield under both conditions were introduced as the best indicators. Also, principal component analysis of the indices was performed for further evaluation of drought tolerant cultivars. To identify relatively tolerant cultivars, a biplot chart was drawn according to the relationship of the studied components and indices. Finally drought tolerant cultivars were identified for the weather condition of this experiment. In order to obtain data analysis, SPSS ver. 24 and Excel 2017 software were used. Also, yield stability analysis was done using biplot software. In order to do this, each one of the drought stress treatment levels were considered as an environment and the mean yield (per unit surface) of the studied cultivars at different levels of the less-watering stress was analyzed for stability. 3 RESULTS AND DISCUSSION For all genotypes, five indices of drought tolerance, yield potential (YP), and stress yield (YS) were calculated (Table 3). According to many of the researches these indices are the most applicant ones for selecting drought-tolerance genotypes (Mardeh et al., 2006; Pirayvatlou, 2001; Talebi et al., 2009). There was a significant difference among stress conditions for grain yield. The grain yield mean under normal and stress conditions was 2.36 g m-2 and 1.10 g m-2, respectively. The stress intensity index (SI) would get value between 0 and 1. The larger value of stress intensity indicates more severe stress conditions (Raman et al., 2012), in this study SI was equal to 0/54, that showed yield reduction was about more than one-half under stress conditions in comparison to yield under normal conditions. Indices, that have strong correlation with the grain yield under normal and stress condition, are suitable for selecting stress tolerance genotypes (Far-shadfar et al., 2012). The T5 wild diploid and Sabalan hexaploid genotypes possessed the highest value for grain yield under normal and stress conditions, respectively. The genotypes of B2 and B8 in normal condition and Moghan and B7 in stress condition had the least value for grain yield (Table 3). The highest difference in yield (YS-YP) was found in the population T1, T3, T4, T5, and superhead. Correlation coefficients between drought tolerance indices and seed yield in normal and stress conditions presented in Table 4. There was a significant positive correlation between YP and YS (r = 0.67, p < 0.01) showing that high yield under normal condition resulted in relatively high yield under stress conditions. Also, YP and YS with STI (r = 0.85 and 0.90), MP (r = 0.95 and 0.86), GMP (r = 0.89 and 0.92), and HM (0.83 and 0.95) have significant positive correlation (p < 0.01) (Table 4). These suggest that in selecting high yielding lines under stress and non-stress conditions, theses indices are very important. In fact, high correlation YS and YP with other indices is critical for selecting tolerance genotypes (Far-shadfar et al., 2012). There is a significant positive correlation between YP and TOL (r = 0.82, p < 0.01) but for YS and TOL, this correlation is negative (r = 0.14) (Table 4). It has been concluded that selecting based on TOL, results in reduced yield in normal condition. Khalili et al. (2012) reported that MP, GMP and STI indices had a positive and significant correlation with grain yield under normal and stress conditions. Also, Naghavi (2013) indicated that there was a positive correlation between MP, GMP, YS and YP. Drought resistance indices such as MP and GMP could be appropriate for identifying tolerant genotypes (Farshadfar et al., 2012; Khalili et al., 2014; Mardeh et al., 2006; Mirzaei et al., 2014; Naghavi et al., 2013). Principal component analysis (PCA) are represented in Table 5. Principal component analysis (PCA) showed that 83.77 % of variations resulted from YP, YS, 108 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 108 26. 03. 2020 12:05:46 Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid ... of wheat using stress tolerance indices Table 3: Yield potential (YP), stress yield (YS), and five indices of drought tolerance for 32 wheat genotypes Genotype YP YS TOL STI MP GMP HM B1 0.2 0.11 0.08 0 0.15 0.15 0.14 B2 0.18 0.15 0.03 0 0.16 0.16 0.16 B3 0.30 0.17 0.13 0.01 0.24 0.23 0.22 B4 0.44 0.17 0.26 0.01 0.30 0.27 0.25 B6 0.67 0.11 0.56 0.01 0.39 0.27 0.19 B7 0.20 0.1 0.1 0 0.15 0.14 0.13 B8 0.16 0.13 0.03 0 0.15 0.14 0.14 T1 4.6 1.4 3.20 1.25 3.03 2.57 2.18 T2 2.50 2.93 -0.4 1.39 2.71 2.70 2.70 T3 4.16 1.78 2.37 1.40 2.97 2.72 2.50 T4 5.14 1.81 3.23 1.76 3.47 3.05 2.68 T5 5.40 2.28 3.12 2.34 3.84 3.51 3.21 Dehdasht 2.15 0.55 1.6 0.22 1.35 1.08 0.87 Dena 1.97 0.43 1.54 0.16 1.20 0.92 0.70 DW/90/8 1.50 1.90 -0.3 0.54 1.70 1.69 1.68 DW/95/4 1.87 0.43 1.44 0.15 1.15 0.89 0.69 Karkhe 2.14 0.96 1.17 0.39 1.55 1.43 1.33 Yavarus 1.9 1.12 0.78 0.40 1.51 1.46 1.41 Shabrang 2.77 1.98 0.79 1.04 2.38 2.34 2.31 Behrang 3.17 0.23 2.94 0.13 1.70 0.85 0.42 WS 0.72 0.56 0.15 0.07 0.64 0.63 0.63 Aria 3.1 2.05 1.05 1.20 2.57 2.52 2.46 Azadi 3.09 1.53 1.56 0.89 2.31 2.17 2.04 Azar 3.58 1.90 1.68 1.29 2.74 2.61 2.48 Hirmand 3.98 1.68 2.30 1.26 2.83 2.58 2.36 Moghan 1.72 0.1 1.62 0.03 0.91 0.41 0.18 mr-17 1.14 1.16 -0.02 0.25 1.15 1.14 1.14 Pishtaz 2.60 0.53 2.07 0.26 1.57 1.17 0.88 Roshan 3.11 1.24 1.87 0.73 2.17 1.96 1/77 Sabalan 3.55 3.30 0.24 2.22 3.43 3.42 3.42 Shiraz 2.68 0.97 1.70 0.49 1.82 1.61 1.43 Superhead 4.75 1.42 3.33 1.27 3.08 2.59 2.18 STI, MP, GMP, and HM as first PCA (Table 5). Therefore, PCA 1, the first dimension, was named as a yield component and drought tolerance. The second principal component analysis (PCA) indicated 15.20 % of total variations. The highest positive factor in the second PCA was YP and TOL, and the highest negative factor was YS. Therefore, this component can be named as sensitivity component to stress. The genotypes with low values of the second PCA have the least sensitivity to stress conditions. Thus, for both non-stress and stress environment, selection of genotypes with high PCA1 and low PCA2 are recommended. Therefore, genotypes B1 and B8 have high PCA1 and low PCA2 that are preferable genotypes. It's been also reported that genotypes with larger PCA1 and lower PCA2 scores gave high yields (stable genotypes), and genotypes with lower PCA1 and larger PCA2 scores had low yields (unstable genotypes) (Kaya et al., 2002). The principal component analysis (PCA) also was described by many of researchers (Parchin et al., 2013; Zabet et al., 2003). Shafazadeh Acta agriculturae Slovenica, 115/1 - 2020 109 AAS_vsebina_2020_115_1_koncna 180320.indd 109 26. 03. 2020 12:05:46 S. KHOSRAVI et al. Table 4: Correlation coefficients between drought tolerance indices and seed yield in normal and stress conditions YP YS TOL STI MP GMP HM YP 1 YS 0.67** 1 TOL 0.82** 0.14 1 STI 0.85** 0.90** 0.45** 1 MP 0.95** 0.86** 0.62** 0.94** 1 GMP 0.89** 0.92** 0.94** 0.96** 0.98** 1 HM 0.83** 0.95** 0.39* 0.96** 0.95** 0.99** 1 * p < 0.0S, ** p < 0.01. Table 5: Linear composition coefficients of the main components of stress tolerance indexes Component Eigen values Variance Study Indicators YP YS TOL STI MP GMP HM 1 5.8 83.77 0.93 0.88 0.58 0.96 0.99 0.99 0.97 2 1.06 15.20 0.34 -0.45 0.81 -0.13 0.05 -0.10 -0.21 et al. (2004) in investigation of response of bread wheat genotypes to drought stress conditions, concluded that MP, GMP and STI indices in specification of drought tolerant genotypes provide an agreeable result than TOL and ssI indices. For comparison among genotypes, many researchers have used of biplot analysis (Nazari and Pakniyat, 2010). Biplot analysis was used to study the relationship between drought tolerance indices and grain yield under both normal and drought stress conditions (Figure 1). This diagram can be divided into four zones. Zone A includes the best genotypes such as Sabalan, T2, Aria, Azar and Azadi which have high-yield and are resistant to stress. Zone B includes T1, T3, T4, T5, Hirmanad, Shi-raz, Superhead and Roshan which have a high-yield but are sensitive to stress. Zone C does not possess desirable properties, since has low-yield and are sensitive to stress. Most of the genotypes of this group were wild diploids. The genotypes of zone D, unlike the low-yield, are resistant to drought stress. For soybean, applying genotype-trait (GT) biplot to the multiple trait data illustrated that, GT biplots graphically displayed the interrelationships among seed yield, oil content, protein content, plant height and days to maturity and facilitated visual cultivar comparisons and selection (Yan and Rajcan, 2002). Mollasadeghi et al. (2011) reported 4 zone with 12 wheat genotypes. The cluster analyses based on yield under normal and stress conditions, and the 5 mentioned indices were carried out, and the results are shown in Figure 2. UPG- Œ) m GO a Figure 1: Biplot for drought tolerance indices in 32 genotypes wheat based on first two components. 110 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 110 26. 03. 2020 12:05:46 Evaluation and comparison of drought tolerance in some wild diploid populations, tetraploid ... of wheat using stress tolerance indices Figure 2: Dendrogram of cluster analysis of the wheat population tested based on TOL, STI, MP, GMP, HM indices using UPGMA method MA dendrogram clustered the examined 32 wheat genotypes into two five clusters (Figure 2). Genotypes B1, B7, B2, B8, B3, B4, B6 and WS, which have the low yield and are resistant to drought stress, were put together. Second group are DW/90/8, mr-17, Karkheh, Yavarus, Shiraz, Dena, DW/95/4, Dehdasht, Pishtaz, Moghan, Behrang which have low yield. 'T2' and 'Sabalan' formed the third group with the highest yield, in normal and resistant to stress especially in stress conditions. Fourth group contains of genotypes are Aria, Shabrang, Azadi, Roshan, T3, Hirmand, Azar, T1, Superhead and T4, which are high in terms of yield and can be significant. 'T5' forms the 5th group and this genotype is high-yield and sensitive to stress. The results of cluster analysis completely agreed by those of principal component analysis and biplot analysis. Based on biplot analysis, genetic variations were observed in studied genotype under drought stress. This is also approved by cluster analysis. Other researchers, using cluster analysis based on stress tolerance indices and genotypic classification, indicated that the results obtained from this method are consistent with the principal components analysis (Ahmadizadeh et al., 2012; Mohammadi et al., 2011; Rad and Abbasian, 2011). The clusteranalysis of 30 genotypes of wheat were placed into three separate groups (Parchin et al., 2013). For tolerate adverse environmental conditions, there are different mechanisms. 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Effect of water stress on different traits and determination of the best water stress index in mung bean (Vigna radiata). Iranian Journal Agriculture Science, 34(4), 889-898. 112 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 112 26. 03. 2020 12:05:46 doi:10.14720/aas.2020.115.1.1145 Original research article / izvirni znanstveni članek Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions Seyyed Shahaboddin HOSEINI \ Asad ROKHZADI 1 2, Ezzat KARAMI 1 Received May 4, 2019; accepted January 15, 2020. Delo je prispelo 4. maja 2019, sprejeto 15. junija 2020. Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions Abstract: Adopting an appropriate planting method and application of soil amendments such as zeolite in dryland conditions could be highly effective in improving growth and productivity of crop plants. Therefore the response of two chickpea (Cicer arietinum L.) genotypes (Azad and Adel) to two planting methods (flat and furrow patterns) and different rates of zeolite (0, 10 and 20 t ha-1) was examined in a field study under dryland conditions. Zeolite application increased plant height, pods number/plant and seed yield, under flat planting pattern, while in furrow pattern, chickpea yield decreased with the application of zeolite. Under no zeolite condition, sowing in furrows improved plant height and pods number compared with flat sowing. Zeolite had no effect on Azad cultivar, but the pods number/plant of Adel was significantly increased by applying 20 t ha-1 zeolite compared with control. In general, the best result in terms of crop growth and yield was obtained from applying 20 t ha-1 zeolite in flat planting pattern. Positive response of the plant to zeolite application in flat planting pattern and, on the other hand, the yield reduction resulting from zeolite addition in furrow planting condition suggested that the influence of zeolite on the crop was dependent on planting method. Key words: flat planting; furrow planting; dryland farming; zeolite Medsebojni vpliv sejalnih metod in uporabe zeolita na komponente pridelka čičerke (Cicer arietinum L.) v sušnih razmerah Izvleček: Uporaba primernih metod setve in dodatkov zeolita v tla bi lahko bili zelo učinkoviti ukrepi za izboljšanje rasti in povečanja pridelka poljščin v sušnih razmerah. V ta namen je bil ovrednoten odziv dveh genotipov čičerke (Cicer arietinum 'Azad', 'Adel') v poljskem poskusu z dvema načinoma setve (setev na ravno površino in setev v brazde)) ob različnih dodatkih zeolita (0, 10 in 20 t ha-1) v sušnih razmerah. Uporaba zeolita je povečala višino rastlin, število strokov na rastlino in pridelek semena pri setvi na ravno površino medtem, ko se je pridelek čičerke pri setvi v brazde in dodatku zeolita zmanjšal. Pri dodatku zeolita se je pri setvi v brazde povečala višina rastlin in število strokov na rastlino v primerjavi s setvijo na ravno površino. Zeolit ni imel nobenega učinka na sorto Azad, pri sorti Adel pa se je število strokov na rastlino značilno povečalo pri dodatku zeolita 20 t ha-1 v primerjavi s kontrolo. Nasplošno so bili doseženi najboljši rezultati glede rasti in pridelka poljščine pri dodatku zeolita 20 t ha-1 in pri setvi na ravno površino. Pozitivni odziv rastlin ob dodatku zeolita pri setvi na ravno površino in upad pridelka ob setvi v brazde kažeta, da je vpliv zeolita na uspevanje poljščine odvisen od načina setve. Ključne besede: setev na ravno površino; setev v brazde; kmetovanje na sušnih območjih; zeolit 1 Islamic Azad University, Faculty of Agriculture, Sanandaj Branch, Department of Agronomy and Plant Breeding, Sanandaj, Iran 2 Corresponding author, e-mail: asadrokh@yahoo.com Acta agriculturae Slovenica, 115/1, 113-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 S. S. HOSEINI et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) is the second most important grain legume in the world with the harvest area of about 14.56 million ha, and in Iran is the most important food legume grown on 0.566 million ha with the annual production of 0.271 million tons (FAO, 2017). More than 97 % of chickpea in Iran is cultivated under rain-fed conditions and grown on stored soil moisture (Anonymous, 2017). Therefore, the application of any kind of technique or method that would increase the chickpea production efficiency in dryland conditions can be recommended. Selecting a proper planting pattern plays an important role in improving the growth and yield of crop plants especially in dryland farming and water deficit conditions. Different cultivation techniques including two methods of flat and furrow planting have been studied and compared by many researchers. Results of a study on the effects of furrowing the soil surface on barley yield and soil water storage revealed that the application of deep furrow techniques had a positive effect on water storage and availability in soil leading to a significant increase in grain and straw yield of barley as compared with the natural (not furrowed) soil surface treatment (Abu-Awwad & Kharabsheh, 2000). Comparison of three planting systems of RP (ridge planting), FRP (flat planting with ridging at early jointing stage) and FP (flat planting without ridging) in rain-fed conditions showed that corn growth and yield in FRP system were significantly higher than those in RP and FP systems due to the improvement of soil moisture and temperature under FRP system (Song et al., 2013). Implementation of ridge-furrow planting system along with mulching techniques in semiarid conditions led to a significant increase in dry matter, grain yield and water use efficiency of corn compared with conventional flat farming (Ren et al., 2010). In an experiment the effects of three seed bed patterns including flat sowing (T1), raised bed with pair row of crop at 20 cm spacing on one bed and 40 cm furrow width (T2) and raised bed with pair row of crop at 30 cm spacing on one bed with 50 cm furrow width (T3) on chickpea were studied and the results showed that treatment T3 was superior to T1 and T2 in terms of pods number per plant, grain yield and growth parameters such as plant height and nodulation (Bhargav et al., 2018). Pourghasemian and Zahedi (2009) reported that the flat planting method compared to furrow method significantly increased seed yield, yield components, harvest index and seed oil content of safflower. Also, in a study carried out by Montazar et al. (2014), the planting method did not have a significant effect on any of the studied traits in wheat. Preservation and storage of soil moisture in dry- land farming conditions is of great importance. Zeolites are a large group of aluminosilicate mineral compounds with useful properties including absorbing and releasing water, high porosity and high cation exchange capacity, which are applied in agriculture to improve soil conditions (Mumpton, 1999; Eroglu et al., 2017). Zeolites improve the water use efficiency of plants by increasing the water holding capacity of soil and facilitating the transfer of water to plant roots (Sangeetha & Baskar, 2016). Zeolites can improve plant growth and increase its yield by storing and maintaining nutrients and releasing them at the right time and in a gradual manner (Polat et al., 2004). The beneficial effects of zeolite application on crops, especially under water-limited conditions have been reported in many studies (Mahmoodabadi et al., 2009; Aghaalikhani et al., 2012; Gholamhosseini et al., 2013; Najafinezhad et al., 2015; Ozbahce et al., 2015). Although many studies have been done on the effects of planting techniques and zeolite application on different crops, there is no report on the interaction between planting method and application of zeolite in dryland conditions on chickpea. Therefore, the present study was conducted to investigate the growth and yield response of two chickpea cultivars to the application of different amounts of zeolite in two systems of flat and furrow planting under dryland conditions. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE DESCRIPTION This experiment was carried out during 2015-2016 growing season in Kamyaran county, Kurdistan province, west of Iran. The experimental farm is located at a latitude of 34° 47' N and longitude of 46° 53' E with an elevation of 1425 m above sea level. The long-term annual precipitation and mean temperature of the region are 464.5 mm and 14.4 °C respectively. The monthly precipitation and temperature of the site in the growing season and the characteristics of farm soil are shown in Fig. 1 and Table 1 respectively. 2.2 EXPERIMENT LAYOUT AND MANAGEMENT The experiment was conducted in a factorial arrangement with three factors based on a randomized complete block design in three replications under dryland conditions. The first factor was planting method including two patterns of flat and furrow, the second factor was zeolite application rate (0, 10 and 20 t ha-1 as Z0, Z10 and Z20 respectively) and two Kabuli type chickpea cul- 114 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 114 26. 03. 2020 12:05:47 Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions Figure 1: Precipitation and temperature during the growing season of 2015-2016 at the experimental site Table 1: Soil properties of the experimental site Clay Silt sand OC TNV EC N P K (%) (%) (%) (%) (%) pH (dS m-1) (%) (ppm) (ppm) 32.72 51.28 16 0.74 9.25 7.85 0.511 0.08 9.8 220 OC = Organic carbon, TNV = Total neutralizing value, EC = Electrical conductivity Figure 2: Schematic diagram of the two planting patterns tivars of Azad and Adel were studied as the third factor items. Each experimental plot contained four rows, 3 m in length, 0.25 m apart and the space between the plants on each row was 10 cm. The fertilizers of urea, triple superphosphate and potassium sulfate were used before planting at the rates of 60, 35 and 25 kg ha-1 respectively according to the soil analysis results. The natural zeolite of clinoptilolite in relevant rates was applied in the seed bed prior to sowing. The seeds of chickpea were treated with benomyl fungicide before sowing to prevent soil-borne diseases. In flat planting pattern the seeds were sown at a depth of 3-5 cm and in furrow planting pat- tern were sown at the same depth in the middle of the furrows. The width and depth of furrows were 25 and 12 cm respectively (Fig. 2). The operation of sowing was performed manually in mid-March. The experiment was done under dryland conditions with no irrigation and hand weeding was performed at different growth stages of the plant. 2.3 DATA COLLECTING Different crop traits including plant height, number Acta agriculturae Slovenica, 115/1 - 2020 115 AAS_vsebina_2020_115_1_koncna 180320.indd 115 26. 03. 2020 12:05:47 S. S. HOSEINI et al. of primary and secondary branches per plant, pods number per plant, seeds number per pod, 100-seed mass and seed yield were studied in the experiment. The morphological characteristics of the plant and yield components were measured using seven randomly harvested plants in each plot. The seed yield in each plot was determined by hand-harvesting the central two rows of the plot. 2.4 STATISTICAL ANALYSIS The measured data were subjected to analysis of variance (ANOVA) and when the ANOVA result was significant, the least significant difference (LSD) test was done for means separation. The statistical operations were performed using SAS software (SAS Institute Inc., Cary, NC, USA). 3 RESULTS AND DISCUSSION 3.1 PLANT HEIGHT AND NUMBER OF BRANCHES The plant height at maturity stage was significantly affected by the interaction of planting pattern and zeolite application but the number of primary and secondary branches per plant was not affected by the experimental factors and their interactions (Table 2). A significant increase in plant height was recorded by using 20 t ha-1 zeolite compared with Z0 (control) and Z10 treatments in flat planting pattern, whereas the plant did not respond to the application of zeolite in terms of plant height under furrow planting pattern (Fig. 3). Moreover, it can be seen that the plant height in furrow planting was higher than that in flat planting under the Z0 treatment (Fig. 3). Table 3: Impact of planting pattern, zeolite application and cultivar on growth and yield parameters Treatments Plant height (cm) Primary branches plant-1 Secondary branches plant-1 Pods no Seeds no 100-seed mass Seed yield plant-1 pod-1 (g) (kg ha-1) Planting pattern Flat Furrow LSDO05 Zeolite rate Z0 Z10 Z20 LSD0.05 Cultivar Azad Adel LSD0.05 Source of variation Planting pattern (P) Zeolite (Z) Cultivar (C) P x Z P x C Z x C P x Z x C CV% 35.0 ± 0.8 35.5 ± 0.4 1.43 34.4 ± 0.7 34.9 ± 0.8 36.5 ± 0.7 1.75 35.1 ± 0.6 35.5 ± 0.6 1.43 ns ns * ns ns ns 5.86 3.2 ± 0.09 3.1 ± 0.07 0.23 3.3 ± 0.13 3.0 ± 0.08 3.2 ± 0.09 0.28 3.2 ± 0.09 3.1 ± 0.07 0.23 ns ns ns ns ns ns 10.50 9.9 ± 0.68 10.4 ± 0.56 1.52 10.3 ± 0.72 9.8 ± 0.64 10.3 ± 0.93 1.86 10.3 ± 0.75 10.1 ± 0.46 1.52 ns ns ns ns ns ns 21.85 36.4 ± 1.8 33.6 ± 1.2 2.18 33.6 ± 1.9 33.5 ± 1.0 37.9 ± 2.4 2.67 36.2 ± 1.4 33.8 ± 1.7 2.18 ns 9.01 1.19 ± 0.05 1.17 ± 0.03 0.11 1.18 ± 0.06 1.18 ± 0.06 1.18 ± 0.04 0.14 1.17 ± 0.03 1.19 ± 0.05 0.11 ns ns ns ns ns ns 13.70 24.2 ± 0.9 1258.7 ± 48.4 24.4 ± 0.9 1052.4 ± 65.4 2.32 131.14 25.2 ± 1.5 23.9 ± 0.8 23.8 ± 0.6 2.84 25.7 ± 0.9 22.9 ± 0.9 2.32 ns * ns ns ns ns 13.82 1193.5 ± 82.8 1103.8 ± 54.4 1169.4 ± 89.6 160.62 1237.1 ± 67.5 1074.1 ± 50.3 131.14 ns * ns ns ns 16.42 ns: Not significant. *, ** and ***: Significant at the 0.05, 0.01 and 0.001 probability levels, respectively. Values are given as means ± standard errors. 116 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 116 26. 03. 2020 12:05:46 * ** ns ns ns ns ns ** * ** * Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions Figure 3: Interactive effect of planting pattern and zeolite application on plant height. Vertical bars indicate the standard error of the means Figure 4: Interactive effect of planting pattern and zeolite application on pods number plant '. Vertical bars indicate the standard error of the means An increased plant height in chickpea is a desirable trait for the ease of mechanical harvesting (Patil et al., 2014). According to the results of present study, plant height of chickpea can be increased through the application of zeolite in conventional flat planting system. The increase in plant growth due to zeolite application may be attributed to the positive effects of zeolite on the availability of water and essential nutrients for the plant (Rehakova et al., 2004; Mahmoodabadi et al., 2009; Zahedi et al., 2009). 3.2 YIELD COMPONENTS The interaction between planting method and zeolite application had a significant effect on pods number plant-1 (Table 2). In the case of no zeolite application, the number of pods per plant in furrow system was higher than that in flat system (Fig. 4). Under flat planting pattern a significant increase in pods number was recorded by 14 and 52 % through the application of 10 and 20 t ha-1 zeolite respectively compared with Z0, while in furrow system a decreasing trend was shown in pods number with the application of zeolite (Fig. 4). The interactive effect of planting pattern and cultivar on pods number demonstrated that under flat planting pattern there was no significant difference between two chickpea cultivars but in furrow system the number of pods per plant in Azad was significantly higher than that in Adel (Fig. 5). The pods number per plant was significantly affected by the zeolite x cultivar interaction (Table 2). The effect of zeolite application on pods number in Azad cultivar was not significant while Adel positively responded to zeolite, so that the pods number/plant in this genotype Acta agriculturae Slovenica, 115/1 - 2020 117 AAS_vsebina_2020_115_1_koncna 180320.indd 117 26. 03. 2020 12:05:48 S. S. HOSEINI et al. Flat Furrow Planting pattern Figure 5: Interactive effect of planting pattern and cultivar on pods number plant-1. Vertical bars indicate the standard error of the 50 o CL 0 -I-1-1-1 0 10 20 Zeolite rate (t ha1) Figure 6: Interactive effect of zeolite application and cultivar on pods number plant-1. Vertical bars indicate the standard error of the means was increased by 10 and 27 % in Z10 and Z20 treatments, respectively, compared with control (Z0) treatment (Fig. 6). Our result regarding the different reaction of genotypes to zeolite application is consistent with that of Za-hedi et al. (2009) who showed a significant interaction between zeolite and cultivar in canola. Different response of two chickpea cultivars to planting pattern and zeolite application in terms of pod production is an indication of different growth potential of genotypes under various agronomic practices. Analysis of variance results indicated that number of seeds per pod was not affected by the experimental factors of planting pattern, zeolite, cultivar and their interactions (Table 2). Planting method and zeolite application did not significantly affect the 100-seed mass, but it was significantly influenced by cultivar factor (Table 2). Azad cultivar was superior to Adel by 12 % in terms of 100-seed mass (Table 2). The non-significance of zeolite and planting method effects on seed mass was similarly reported by Sepaskhah and Barzegar (2010) and Bhargav et al. (2018). 3.3 SEED YIELD seed yield was affected by planting pattern, cultivar and the interaction between planting pattern and zeolite (Table 2). Azad cultivar with seed yield of 1237 kg ha-1 was significantly superior to Adel with 1074 kg ha-1 seed yield (Table 2). Seed yield response to the interaction of planting method x zeolite showed that under the flat planting pattern, the application of zeolite led to seed yield improvement. The highest seed yield (1407 kg ha-1) was obtained in Z20 treatment under flat pattern which 118 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 118 26. 03. 2020 12:05:47 Interactive effects of planting method and zeolite application on yield attributes of chickpea (Cicer arietinum L.) in dryland conditions was 21 % higher than that of Z0 treatment (Fig. 7). On the other hand, under furrow method, seed yield decreased as the result of zeolite application (Fig. 7). Our results regarding the positive effects of zeolite application on seed yield under conventional flat planting method are in line with those of previous studies. Zahedi et al. (2009) in a study on rapeseed showed that application of 10 t ha-1 zeolite under conditions that the crop is subjected to late season drought stress could improve the crop growth and yield. In another study, root and shoot dry mass and the content of micro and macro nutrients in roots and shoots of soybean were increased as the result of zeoilte application (Mahmoodabadi et al., 2009). The findings of Ozbahce et al. (2015) indicated that the application of zeolite significantly improved the concentration of different nutrients including nitrogen, potassium, zinc, manganese and copper in common bean leaves and increased the seed yield and yield components of the crop under water deficit stress conditions. Seed yield reduction resulted from zeolite application under furrow planting pattern in current study may be related to the interaction between zeolite function and topology of the soil surface. In other words, the behavior of zeolite and its positive effects on crop growth can be affected by the shape of soil surface and the level of tillage operation. However, in order to better understand the performance of zeolite in each planting system, further studies, such as moisture and temperature measurements in different soil profiles and evaluation of soil nutrients availability in both planting patterns under the various amounts of zeolite are needed. Under conditions of no zeolite application the response of chickpea to furrow sowing compared with flat planting pattern was more desirable in terms of plant height, pods number and seed yield (Fig. 3, 4 and 7), in- dicating the advantages of furrow planting over flat pattern with no zeolite application in dryland farming conditions. Several studies have shown that furrow sowing in dryland conditions has many benefits such as easily shedding of rain from ridges, accumulation of runoff in furrows, infiltration of rainwater below the furrow and better access of plant roots to sub-soil water (Yang et al., 1996; Feng et al., 2001; Ren et al., 2010; Roper et al., 2015) and improvement in growth and yield of various crops under furrow sowing compared with flat planting has been declared in many reports (Blackwell, 1993; Wang et al., 2011; Quanqi et al., 2012; Lian et al., 2016; Li et al., 2018). 4 CONCLUSIONS Under flat planting pattern, plant height, pods number/plant and seed yield increased, whereas under furrow system, these traits decreased as the result of zeolite application. On the other hand, we found that under no zeolite condition, the furrow sowing had a positive effect on plant height and pods production. 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Notulae Scientia Biologicae, 1(1), 73-80. http://dx.doi.org/10.15835/nsb113500 Acta agriculturae Slovenica, 115/1 - 2020 121 AAS_vsebina_2020_115_1_koncna 180320.indd 121 26. 03. 2020 12:05:48 AAS_vsebina_2020_115_1_koncna 180320.indd 122 26. 03. 2020 12:05:46 doi:10.14720/aas.2020.115.1.1258 Original research article / izvirni znanstveni članek Allelopathic interference of Sonchus oleraceus L. with wheat and the associated weeds: a field study Mahmoud O. HASSAN 1 2 Received September 2, 2019; accepted January 17, 2020. Delo je prispelo 2. septembra 2019, sprejeto 17. januarja 2020. Allelopathic interference of Sonchus oleraceus L. with wheat and the associated weeds: a field study Abstract: A field study was conducted to examine the allelopathic potential of Sonchus oleraceus L. residue against the weeds associated with wheat crop. Residue application was carried out under field conditions in two doses: 150 and 300 g m-2. Weed richness, density and above-ground biomass were assessed at 6 and 12 weeks after application to evaluate the potential effect of S. oleaceus manure on weed control. Some growth criteria and the total yield of the cultivated wheat crop were also measured. The residue-containing quadrates attained lower weed richness, density and biomass. Unlikely, residue application reduced the grain yield of wheat. The available nitrogen and phosphorus were increased in soil at the higher application dose. These results suggest that S. oleraceus could interfere most of winter weeds, but affect productivity of wheat. Weed suppression could be attributed to the allelopathic potential of S. oleraceus residue. These results suggest also that the manure of this weed could be used successfully in the integrated weed management programs to reduce weed infestation in winter crops. However, another crop species may be selected. Key words: organic agriculture; ecological weed management; allelopathy; Sonchus oleraceus Alelopatični učinek navadne škrbinke (Sonchus oleraceus L.) na pšenico in njene plevele: poljski poskus Izvleček: Za preučitev alelopatičnega potenciala ostankov navadne škrbinke (Sonchus oleraceus L.) na plevele povezane s pridelovanjem pšenice je bil izveden poljski poskus. Ostanki škrbinke so bili uporabljeni v dveh odmerkih, 150 in 300 g m-2. Pestrost plevelov, njihova gostota in nadzemna biomasa so bili ocenjeni 6 in 12 tednov po nanosu ostankov škrbinke za ocenitev njihovega potenciala za nadzor plevelov. Izmerjeni so bili tudi nekateri parametri rasti in pridelka pšenice. Kvadranti polja, ki so vsebovali ostanke škrbinke so imeli manjšo pestrost, gostoto in biomaso plevelov. Uporaba ostankov škrbinke je zmanjšala pridelek zrnja pšenice. Na območjih z večjim odmerkom ostankov škrbinke sta se povečali razpoložljivost dušika in fosforja v tleh. Rezultati kažejo, da ostanki škrbinke lahko vplivajo na večino ozimnih plevelov a hkrati vplivajo tudi na pridelek pšenice. Zaviranje rasti plevelov lahko torej pripišemo alelopatičnem učinku ostankov škrbinke. Ti rezultati tudi nakazujejo, da bi se ostanki škrbinke lahko uspešno uporabljali kot gnojilo pri integriranem upravljanju s pleveli pri ozimnih poljščinah, vendar bi v tem primeru namesto pšenice morali izbrati drugo vrsto poljščine. Ključne besede: organsko kmetijstvo; ekološko upravljanje s pleveli; alelopatija; Sonchus oleraceus 1 Beni-Suef University, Faculty of Science, Department of Botany and Microbiology, Egypt 2 Corresponding author, e-mail: dr_modyl983_science@yahoo.co.uk Acta agriculturae Slovenica, 115/1, 123-112, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 105 26. 03. 2020 12:05:46 M. O. HASSAN. 1 INTRODUCTION As common practices in modern agriculture, large quantities of synthetic fertilizers and agrochemi-cals are used to enhance crop productivity (Rüegg et al., 2007). The continuous use of synthetic herbicides in agriculture produces the emergence of herbicide-resistant weeds and leads to environmental pollution with impacts on both human health and ecosystems (Qasem, 2013). Thus, there is an increasing demand for alternative and sustainable practices and, thereby, the research institutions are currently applying innovative approaches to improve agriculture without synthetic herbicides (Khanh et al., 2007; Chauhan and Gill, 2014). The increased incidence of herbicide-resistant weed species, and the related biological consequences, poses a major threat to the potential sustainability of crop production. Natural weed management practices are truly looking for solutions to minimize environmental impacts related to the input of synthetic herbicides into the agroecosystems. Now, organic farming is a substantial aspect that gives the environmentally safe practices with respect to weed control and crop productivity. Organic agriculture provides several merits as an agriculture practice if compared with the conventional one. Briefly, organic agriculture represents an approach to land management that emphasizes preservation of the immediate environment, improves employment opportunities in the local communities as a social benefit, and finally has been positively correlated with economic growth (Luttikholt et al., 2007; Vaarst, 2010). Furthermore, it is considered as an environmentally safe tool for weed control (Lemessa & Wakjira, 2015). Within this context, there is an existing trend to incorporate ecological practices to the agroecosystems so as to design alternative and sustainable cropping systems (Hassan et al., 2018) either for weed management or crop safety. Allelopathic interactions generally involve the release of chemical compounds (i.e. allelochemicals) from living or dead plant parts in sufficient quantities that may suppress germination and/or establishment of weed seedlings in the agroecosystem (Qasem & Foy, 2001; Hassan et al., 2014a). Used as cover crops, some plants produce relevant amounts of allelochemicals which are released from living or dead plant tissues that can exert a strong influence on the target weeds (Cheng & Cheng, 2015). In this regard, some ecolo-gists pay attention to the use of allelochemicals as 'bioherbicides' in weed control, providing environmentally safe agriculture (Gomaa & Abd El-Gawad, 2012). Application of plant residues is a common practice that is frequently recurring in the agroecosystems, and it mostly offers a strategy for weed control (Campiglia et al., 2010). In this regard, the use of phytotoxic or allelo-pathic plants is gaining attention due to positive results in the potential weed management. Potential use of annual sowthistle residue as a weed species in this regard was still unknown. In this article, I used the residues of such common weed for this purpose. Annual sowthistle (Sonchus oleraceus L.) is an annual weed species native to Eurasia and North Africa. It has been introduced to a wide range of countries around the world and become a common weed causing a major problem in the agroecological systems (Peer-zada et al., 2019; Widderick et al., 2010). It also dominates weed communities in winter crops and other urban areas where water is available (Gomaa et al., 2012; Hassan & Hassan, 2019). Therefore, this weed may give heavy biomass. Moreover, it may provide a cheapest, highly available and environmentally safe material to be used in the agroecology in terms of potential weed management due to its allelopathic capacity (Gomaa et al., 2014; Hassan et al., 2014b) and natural biofertilizer to enhance crop yield due to its fertilizing agency (Hassan et al., 2018). During hoeing practices in cultivated fields, the farmers uproot this plant and mix it with the soil during ploughing, the phenomenon that may affect the incoming crops and its associated weed species due to the potential release of some phytotoxins from the plant residue. S. oleraceus was found to be allelopathic against some common weeds (Gomaa et al., 2014; Hassan et al., 2014a). Besides, the allelopathic compounds released from its residue were long persistent in soil (Hassan et al., 2014b). Nevertheless, the previous studies were performed under greenhouse conditions, and a field application to evaluate the allelopathic potential of this weed is still lacking. It was therefore necessary for wide-ranged researchers interested and specialized in this field to fill this gap. On the other hand, the amounts of residue application of such weed that were similar to those applied the current study stimulated growth and productivity of kidney bean crop (Hassan et al., 2018). In the view of these statements, I tested the hypotheses that (i) the strongly allelopathic S. oleraceus can display adverse effects on emergence and growth of the common weeds associated with wheat under field conditions and (ii) a potential stimulatory or, at least, no effect on the crop tested (i.e. wheat) could be obtained. The main objective of this field study was to assess the allelopathic potential of S. oleraceus residue against some common weeds associated with wheat (Triticum aestivum L.), i.e. its ability to suppress weeds, and the potent to use S. oleraceus as a bioherbicide. 124 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 124 26. 03. 2020 12:05:48 Allelopathic interference of Sonchus oleraceus L. with wheat and the associated weeds: a field study 2 MATERIALS AND METHODS 2.1 COLLECTION OF PLANT MATERIAL Fresh shoots of S. oleraceus L. were collected during the growing season, from different locations in the agroecosystems of Beni-Suef governorate, Egypt, (from January to April 2015). Plant collection was carried out during flowering-early fruiting stage in order to facilitate the distinction of S. oleraceous from S. asper (L.) Hill. Furthermore, at this stage, plants mostly pose the maximum amounts of bioactive metabolites. Plant material was placed in polyethylene bags and immediately carried to the laboratory for further processing. In the lab, fruits and inflorescences were completely removed and the plant material was air dried and stored in refrigerator at 2 °C until use. 2.2 THE SELECTED CROP Common wheat (Triticum aestivum L.) is one of the most important cereals in the world in terms of production and use for human and animal feeding under a wide range of climatic conditions and in many geographic regions (Feldman, 1995; Shewry and Hey, 2015). In addition, it was proved to be a major source of carbohydrates and energy, and it also provides substantial amounts of ingredients which are important or beneficial for health such as proteins, vitamins, dietary fibers, and other phyto chemicals (Shewry and Hey, 2015). Due to considerable land extension dedicated to wheat production through the world, there were substantial amounts of economic funds invested in weed management. It was necessary to provide a potential safe weed management strategy in wheat fields as weeds threaten the production of wheat worldwide (Oerke, 2006). Consequently, increasing the wheat crop yield will be expected. 2.3 FIELD WORK Field experiment was conducted at a crop farm land in Beni-Suef governorate; about 17 km north west of Beni-Suef University (lat. 29° 09.13 N, long. 031° 08.36 E, alt. 29 m a.s.l.), Egypt, in the period from the beginning of January to the mid of May 2016. This period was synchronized with the time of cultivation of this crop. Before cultivation soil surface was ploughed twice to homogenate the soil and simultaneously provide a high potential for the equal distribution for the weed seeds in the seed bank. The soil characteristics Table 1: Soil physicochemical properties (mean ± S.D.) of study field prior to cultivation Soil type Sandy clay loam Field capacity 39.08 ± 0.038 pH 7.97 ± 0.04 EC 0.42 ± 3.13 OC 1.69 ± 0.052 OM 2.975 ± 0.096 N (mg kg-1 soil) 99.0 ± 9.31 o P (mg kg-1 soil) 2.83 ± 0.61 rt K (mg kg-1 soil) 607.5 ± 92.78 3 Zn (mg kg-1 soil) 5.9775 ±1.07 Table 2: Mean average meteorological data of Beni-Suef gover-norate during the growing season Parameter Jan. Feb. Mar. Apr. May Mean average high 23 21.5 25.5 29.5 32.5 temperature (°C) Mean average low 10 11.5 14.5 16.5 19.5 temperature (°C) Rainfall (mm) 7.5 355.5 14.5 1 0 Relative Humidity (%) 61.5 53.5 48.5 40.5 37.5 and the surrounding climatic conditions of the study site are well illustrated in Table 1 and 2, respectively. Thereafter, under field conditions, S. oleaceus residues were amended in the study area previously divided as quadrate (2 x 2 m2 each) at the rates 150 and 300 g m-2, whereas the residue-free quadrates were left as control. The experiments were conducted in a complete randomized design (CRD) involving four replicates for each treatment. Weed control assessment was carried out twice: six and twenty weeks after residue application, during which I determined the emerging weed species, species richness, density and biomass of the total as well as individual weed species from each quadrat. Identification and nomenclature of the weed species detected were obtained using Boulos (1999, 2002 & 2005). The above-ground parts of the detected weeds were carefully cut and oven-dried at 70 °C till constant dry weight to obtain the biomass. The grains of wheat were immediately seeded after residue incorporation via manual spraying, as usual in Egyptian wheat fields, obtaining quantities around 200 grains/m2. After 3 weeks, the emerging individuals were thinned to the most similar 100 ones. No fertilization or herbicide regimes were applied, and irrigation process was carried out when need. At harvest, growth Acta agriculturae Slovenica, 115/1 - 2020 125 AAS_vsebina_2020_115_1_koncna 180320.indd 125 26. 03. 2020 12:05:49 M. O. HASSAN. parameters of such crop were measured using ten randomly selected plants per quadrate. These parameters comprised shoot height, above-ground biomass, leaf area and grain yield (expressed as total dry weight m-2). Besides, three soil samples were collected from each quadrate at 0-30 cm depth to form a single composite for each treatment. Soil properties comprising pH, soil electrical conductivity (EC), organic carbon, organic matter, available nitrogen, phosphorus, potassium, and zinc were determined using the standard methods (Allen, 1989). 2.4 STATISTICAL ANALYSIS The obtained field data were first tested for their normality and homogeneity of variances using Kolmog-orov-Smirnov and Levene's tests, respectively. If the data were normal and homogeneous, the data were analysed through one-way ANOVA followed by Tukey's test (p < 0.05) for post hoc multiple comparison of means. When the data exhibited non-normal distribution and heteroscedasticity of variances, Kruskal-Wallis H test (p < 0.05) was performed. A correlation analysis was performed between the total weed richness, density and biomass vs. the dose of the residue applied. All statistical analyses were carried out using the IBM SPSS Statistics 20.0 software package (IBM SPSS Inc., Chicago, IL, USA). 3 RESULTS 3.1 WEED COMPOSITION biomass of this species was not affected in the treated quadrates. With respect to the first harvest, three weed species, namely: Phalaris minor Retz., Polypogon mon-speliensis (L.) Desf. and P. vridis (Gouan) Breistr., were totally absent. Simultaneously, the density of Matricaria chamomilla L. was significantly reduced at the highest dose. Besides, its biomass was significantly supressed at both doses. The density of Chenopodium murale L. and Poa annua L. showed gradual decline with increasing the dose applied. At the end of the experiment (i.e. the second harvest), the density of Phalaris minor and Poa annua was substantially reduced. Additionally, Polypogon monspeliensis was completely absent at the higher application rate. On the contrary, the emergence and biomass of Polypogon viridis was significantly stimulated. Furthermore, the biomass of Phalaris minor was gradually increasing with the amount of residue applied. As a whole, the total number of weeds (weed richness) was maintained in the treated plots at the first harvest only. However, density and biomass of the weeds observed were significantly decreased at both harvests in the residue-amended plots. Furthermore, the reduction in these criteria increased gradually with the increment in the dose applied (Table 4). The correlation analysis between the measured criteria; weed richness, density and the above-ground biomass, with the amount of manure applied is shown in Table 5. Significant negative correlation was observed between the total weed density and biomass with the amount of residue applied at both harvest intervals. Besides, such correlation was also manifested between the weed richness and the dose applied at the second harvest. The weed species with their corresponding families detected in wheat field are listed in Table 3. A total of eight weed species belonging to five families were detected. Four of these species were monocots, whilst the remaining ones were dicot. 3.2 EFFECT OF S. OLERACEUS RESIDUE ON ON WEED DENSITY, RICHNESS AND BIOMASS The effects of S. oleraceus residue depended mostly upon the amount of residue applied, weed species detected and time of harvest (Table 3). Amongst the weed species, the density of Convolvulus arvensis L. was not affected at all under the influence of Sonchus residue. However, the highest dose significantly reduced the biomass of such weed at both harvests. On converse, the highest dose significantly inhibited the emergence of Coronopus niloticus (Del.) Spreng. at both harvests. The 126 Acta agriculturae Slovenica, 115/1 - 2020 3.3 EFFECT OF S. OLERACEUS MANURE ON YIELD COMPONENTS OF WHEAT In general, the application of S. oleraceus manure had not affected shoot length and the above-ground biomass of wheat crop (Table 6). However, the leaf area showed significant reduction in wheat plants at the higher dose (10 %, p < 0.05). Furthermore, the grain output of wheat was significantly reduced at both treatments (Table 6). 3.4 SOIL ANALYSIS The measured soil criteria after application of S. ol-eraceus residue are shown in Table 7. Clearly, most of the measured soil parameters were not affected on addition of the plant residue. However, the available soil nitrogen AAS_vsebina_2020_115_1_koncna 180320.indd 126 26. 03. 2020 12:05:48 Table 3: Effect of Sonchus oleraceus residue at the rates 150 and 300 g m 2 on density (no. quadrate ') and aboveground biomass (g quadrate ') (Mean ± SE) of the weed species detected in wheat crop at 6 and 12 weeks after application (WAA) Weed species Time after application weed density at the dose applied weed biomass at the dose applied 0 150 300 0 150 300 Chenopodium murale L. (Chenopodiaceae) 6 WAA 12 WAA 6.33a± 0.28 4.33b ± 0.27 2.0e ± 0.0 1.74a ± 0.05 1.27b ± 0.052 1.07e ± 0.0 Convulvulus arvensis L. (Convolvulaceae) 6 WAA 2.33a ± 0.29 2.66a ± 0.67 3.33a ± 0.27 1.53a ± 0.16 1.55a ± 0.21 0.92b ± 0.07 12 WAA 2.33a ± 0.28 2.33a ± 0.27 3.66a± 0.29 3.42a ± 0.18 2.93ab± 0.21 2.40b ± 0.19 Coronopus niloticus (Delile) Spreng.(Brassicaceae) 6 WAA 4.33a ± 0.29 4.33a ± 0.29 2.0b ± 0.0 1.01a ± 0.04 0.96a ± 0.1 0.84a ± 0.03 12 WAA 4.0a ± 0.29 3.33ab± 0.52 2.33b ± 0.29 1.12a ± 0.10 1.01a ± 0.2 0.95a ± 0.03 Matricaria chamomilla L. (Astraceae) 6 WAA 4.33a± 0.27 3.33ab ± 0.26 2.33b± 0.27 4.71a ± 0.28 3.63b ± 0.16 2.47b ± 0.14 12 WAA 3.66a± 0.0 3.33a ± 0.77 2.0a ± 0.27 1.83a ± 0.01 5.1b± 0.061 3.33c± 0.006 Phalaris minor Retz. (Poaceae) 6 WAA 12 WAA 9.0a± 0.56 4.33b ± 0.28 1.67c± 0.26 1.67a ± 0.01 2.67a± 0.007 6.70b ± 0.006 Poa annua L. (Poaceae) 6 WAA 7.33a ± 0.28 5.67b ± 0.27 4.33e ± 0.28 8.60a ± 0.48 7.88a± 0.38 5.43b± 0.16 12 WAA 6.33a ± 0.30 4.33b ± 0.29 2.33e ± 0.28 6.70a ± 0.36 2.67a ± 0.46 1.67b ± 0.27 Polypogon monspeliensis (L.) Desf. (Poaceae) 6 WAA - - - - - - 12 WAA 10.33a± 0.77 3.33b ± 0.27 0.0C±0.0 10.37a± 0.78 4.50b ± 0.28 0.0C ± 0.0 Polypogon viridis (Gouan) Breistr.(Poaceae) 6 WAA - - - - - - 12 WAA 2.33a ± 0.29 8.0b ± 0.51 8.0e ± 0.51 8.4a ± 0.42 13.67b± 0.44 19.34c± 0.38 Values in each row within the same parameter sharing the same letter are not significantly different at the 0.05 probability level according to Tukey's test. - = not detected. M. O. HASSAN. Table 4: Effect of Sonchus oleraceus residue at the rates 150 and 300 g m-2 on the species richness, total weed density and above-ground biomass (Mean ± SE) of the weed species detected in the wheat field at 6 and 12 weeks after application (WAA) Dose applied (g m-2) Parameter Time after application 0 150 300 Species richness (No. m-2) 6 WAA 5.0a ± 0.0 5.0a± 0.0 4.66a ± 0.29 12 WAA 6.67a ± 0.28 6.0b ± 0.0 5.67b ± 0. 29 Total weed density (No. m-2) 6 WAA 24.67a ± 0.76 21.0b ± 0.88 13.0c ± 0.29 12 WAA 37.0a ± 1.34 31.0b ± 0.88 20.0c ± 1.04 Total weed biomass (g m-2) 6 WAA 17.58a ± 0.76 15.3b ± 0.38 10.99c ± 0.066 12 WAA 40.6a ± 1.04 37.69a ± 1.30 30.02b ± 0.43 Values in each row within the same crop sharing the same letter are not significantly different at the 0.05 probability level according to Tukey's test. and phosphorus were significantly induced at the higher dose. 4 DISCUSSION The results of this study indicated that S. oleraceus residue significantly reduced the richness, emergence and biomass of most of the detected weeds associated with the studied wheat field. This observation was consistent with that of Hassan et al. (2014b) who indicated that Sonchus residue reduced emergence and growth of some tested weeds under greenhouse conditions. This result was also consistent with field observations monitored by Hassan et al. (2018) who indicated that the same amounts of the residues applied suppressed weed density, richness and biomass in a kidney bean field (unpublished data). This result substantially obeys the first hypothesis of this study. This result suggests also that the residue added had a role in weed interference and declining species richness. In general, the decaying plant residues release phenolic compounds into the rhizosphere part of the soil (Djurdjevic et al., 2011). Furthermore, these phenolic allelo chemicals were found in the soils amended Table 5: Correlation coefficients (r) between the measured weed criteria and the amount of Sonchus oleraceus residue applied in wheat field at both time intervals after residue application Parameter Time after application (weeks) r value Total weed richness 6 - 0.55 12 - 0.71* Total weed density 6 - 0.96** 12 - 0.96** Total weed biomass 6 - 0.96** 12 - 0.91** * Correlation is significant atp < 0.05. "Correlation is significant atp < 0.01. 128 Acta agriculturae Slovenica, 115/1 - 2020 with S. oleraceus (Hassan et al., 2014b). Accumulation of phytotoxins in soil leads to inhibition of seed germination, seedling growth and uptake of mineral nutrients (Rice, 1984), the circumstances that decrease density of individual plant species from the plant communities as suggested by several authors (Barritt and Facelli, 2001; Djurdjevic et al., 2011). Therefore, weed suppression could be related to the phenolic compounds released from the plant residues, i.e. allelopathic interaction. This notion may be consistent with that of Foy and Inderjit (2001) who reported that allelopathy had an important role in weed interference and declining weed diversity. Within this side, the dry matter of S. oleraceus shoots has been reported to be rich in saponins, alkaloids and total phenols (Gomaa et al., 2014). On the other hand, most of the measured soil criteria were not affected by the residue applied. However, the higher dose-amounts enhanced the available soil nitrogen and phosphorus. The increase in the available N and P could be attributed to the residue applied. May be, Sonchus residue is rich in nitrogenous and phosphorus compounds. This result obeys that obtained by Hassan et al. (2014b) and Hassan et al. (2018) who indicated that the residue of this species induced soil nutrients. Therefore, it was so difficult to claim that the reduction in weed diversity and growth was attributed to the change in soil criteria. This result confirms the phyto-toxic effect of the residue applied. The field observations obtained by Hassan et al. (2018) showed that the same amounts of the incorporated residue had a stimulatory effect of on growth, productivity and several metabolites of the kidney beans. However, the effect of these amounts on wheat was contradictory. S. oleraceus produced a negative effect on the total seed yield of wheat. This result substantially does not obey the second hypothesis of this study. Such effect could be also attributed to the smaller seed size of wheat when compared with kidney bean. In this regard, larger seed-sized species were more resistant/tolerant for the released allelochemicals from plant litter (Hassan, 2018). AAS_vsebina_2020_115_1_koncna 180320.indd 128 26. 03. 2020 12:05:48 Allelopathic interference of Sonchus oleraceus L. with wheat and the associated weeds: a field study Table 6: Growth and yield parameters (mean ± SE) of wheat in response to Sonchus oleraceus residue at the rates 150 and 300 g m-2 Dose applied (g m-2) Parameter Control 150 300 Shoot length (cm) 85.0a ± 3.85 82.6a ± 3.60 86.0a ± 2.62 Shoot biomass (g individual-1) 14.4a ± 0.70 13.44a ± 0.59 13.28a ± 0.64 Leaf area individual-1 (cm2) 162.92a ± 5.50 153.15* ± 5.95 146.31b ± 5.24 Number of spikes tiller-1 4.27a ± 0.32 4.43a ± 0.27 4.20a ± 0.20 Spike length (cm) 8.60a ± 0.47 6.65a ± 0.41 8.72a ± 0.51 Seed/Grain yield (g m-2) 703.0a ± 37.23 548.8b ± 22.61 570.6b ± 26.36 Values in each row within the same crop sharing the same letter are not significantly different at the 0.05 probability level according to Tukey's test. Table 6: Influence of Sonchus oleraceus residue at the rates 150 and 300 g m-2 on the measured soil properties (Mean ± SE) at harvest of wheat Dose applied (g m-2) Soil Properties control 150 300 pH 7.95a ± 0.015 7.95a ± 0.017 7.88a ± 0.035 EC (mS cm-1) 0.38a ± 0.025 0.39a ± 0.004 0.46a ± 0.027 OC (%) 1.54a ± 0.028 1.77a ± 0.095 1.48a ± 0.16 OM (%) 2.7a ± 0.048 3.08a ± 0.16 2.60a ± 0.28 N (mg kg-1 soil) 91.0a ± 7.50 113.0a ± 6.80 139.0b ± 2.20 le P (mg kg-1 soil) 1.77a ± 0.11 2.25ab ± 0.13 3.09b ± 0.07 a li ia K (mg kg-1 soil) 590.0a ± 50.68 647.0a ± 45.53 562.0a ± 82.53 3 Zn (mg kg-1 soil) 4.75a ± 0.46 4.71a ± 0.43 4.38a ± 0.76 Values in each row within the same crop sharing the same letter are not significantly different at the 0.05 probability level according to Tukey's test. Besides, wheat germination and early growth displayed a degree of sensitivity to phytotoxic species (Tamak et al., 1994; Al-Sherif et al., 2013). Therefore, the dose of the applied residue should be further explored and adjusted to add benefits in weed control and, at the same time, avoid undesirable phytotoxicity on the selected crop. As proved by Hassan et al. (2014b), the phenolic compounds released from S. oleraceus residues have been detected during 60 days after residue incorporation into the soil, the result that may explain the extending effect of the residue. In this study, the bioactivity of S. oleraceus manure seems to be extended in time since herbicidal effects were still evident 12 weeks after manure application. Progressive and long-term effects collected suggest that S. oleraceus manure gradually releases phytotoxic compounds, showing a strong suppressive effect on weed emergence. The extending bioactivity of the phytotoxic residue applied is highly needed because weeds in the soil seed bank are not synchronized, but germinate gradually all along the establishment of the crop (Mohler et al., 2001; Puig et al., 2013). In this study, as expected, the weed suppression seemed to be related to the dose of S. oleraceus manure applied. In general, the magnitude of weed suppression is quantitatively proportional to the applied dose in the studies related to phytotoxicity (Hassan et al., 2014b; Hassan, 2018). While the emergence and biomass of Phalaris minor, Poa annua, Polypogon monspeliensis was significantly reduced through the phytotoxicity of S. oleraceus manure, Polypogon viridis was notably increasing with respect to its density and biomass in the long term. Different responses to chemical compounds can be associated to the amplitude of weed sensitivity to phytotoxicity (Latif et al., 2017), even those weed species are closely related. Moreover, selective inhibition of weed growth may be related to concentration and distribution of allelochemicals in the rhizosphere soil (Blum et al., 1999; Aslam et al., 2017). However, rather than indicating phytotoxic resistance, the emergence of P. viridis could be also associated with seed germination dynamics or better competitive abilities that allows this weed to rapidly occupy its own niche after the decline of other monocot species. The results also indicate a punctual weed increase in the case of Matricaria chamomilla at the second harvest and mainly Polypogon viridis at both. None of them Acta agriculturae Slovenica, 115/1 - 2020 129 AAS_vsebina_2020_115_1_koncna 180320.indd 129 26. 03. 2020 12:05:49 M. O. HASSAN. was considered as a highly problematic weed. In this sense, weed stimulation can be related with differential responses as plant residues are able to exhibit positive or negative effects on target species based on chemical concentration or environmental conditions (Cheng and Cheng, 2015). Also, punctual weed stimulation could be related to the nutritional benefits provided by the residue incorporated (Hassan et al., 2014b). 5 CONCLUSION The present study represents evidence that S. olera-ceus residue showed remarkable weed suppression. This investigation had the merit of field application. Apparently, reduction of richness, density and biomass of the weeds associated with wheat crop was manifested. The inhibitory effect of the residue was not attributed to soil properties, but it could be related to some phytotoxins released from the residue that reduce or, perhaps, completely inhibit the emergence and growth of the detected weeds. The undesirable result obtained in this study was the reduction of grain yield of wheat. Therefore, application of such residue in another crop species, probably with larger seeds, may be recommended. Moreover, the dose of the manure applied should be adjusted in order to attain the desired weed management with a potential stimulatory or, at least, no effect on the cultivated crop. The experimental approach described was adequate to demonstrate the efficacy of S. oleraceus residues as a bio-active green manure for future weed management practices. 6 ACKNOWLEDGEMENTS This study was financed by the post-graduate affairs in the faculty of Science, Beni-Suef University, with the grant number S-2-2015. Dr. Mahmoud O. Hassan is so gratefully thanking his wife and Dr. Nasr H. Gomaa, Faculty of Science, Beni-Suef University, for their help and guidance during this study. 7 REFERENCES Allen, S. E. (1989). Chemical Analysis of Ecological Materials. Blackwell Scientific Publishers, London. Al-Sherif E., Hegazy, A. K., Gomaa, N. H., Hassan, M. O. (2013). 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Weed Biology and Management, 10, 102-112. https://doi.org/10.1111/ j.1445-6664.2010.00370.x Acta agriculturae Slovenica, 115/1 - 2020 131 AAS_vsebina_2020_115_1_koncna 180320.indd 131 26. 03. 2020 12:05:49 AAS_vsebina_2020_115_1_koncna 180320.indd 132 26. 03. 2020 12:05:49 doi:10.14720/aas.2020.115.1.1299 Original research article / izvirni znanstveni članek Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. King & H. Rob. extracts on some field insect pests of okra (Aabelmoschus esculentus (L.) Moench) Emmanuel T. NNEJI 1 2, Robert O. UDDIN II \ Abdulrasak K. MUSA 1 Received October 7, 2019; accepted January 20, 2020. Delo je prispelo 7. oktobra 2019, sprejeto 20. januarja 2020. Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. King & H. Rob. extracts on some field insect pests of okra (Aabelmoschus esculentus (L.) Moench) Abstract: A field study was carried out to assess the efficacy of some botanicals in the control of field insect pests in two varieties of okra. Dry leaf extract of Chromolaena odorata (Siam weed) and fresh and dry root extracts of Boerhavia diffusa served as treatments which were compared with Lambda-cyhalothrin and a control. The experiment was fitted into a randomized complete block design with three replications. Data collected were subjected to Spearman's correlation analysis and a two-way analysis of variance and significant different means were separated using Fishers Least Significant Difference (LSD) test at 5 % level of probability. The results revealed that treated plants generally performed better than the untreated plants as they were taller, had more leaves, branches, wider stem girths and gave higher fruit yield. Fresh root extract of B. diffusa performed better in most parameters measured. A significant positive correlation was observed between insect pest populations, and between growth and yield parameters, while there was a significant negative correlation between overall yield and insect pest population. NHAe 47-4 okra variety performed better than F1-Lucky variety. Fresh root extract of B. diffusa is recommended for management of B. tabaci (Genadius, 1889) and Amrasca biguttula Ishida, 1912 in okra. Key words: botanicals; Boerhavia diffusa; Chromolaena odorata; Bemisia tabaci; Amrasca biguttula; sticky traps Učinki izvlečkov iz rastlin Boerhavia diffusa L. nom. cons. in Chromolaena odorata (L.) R.M. King & H. Rob. na nekatere škodljive žuželke na jedilnem oslezu (Aabelmoschus esculentus (L.) Moench) Izvleček: Za ocenitev učinkovitosti izvlečkov izbranih rastlin pri uravnavanju škodljivih žuželk na dveh sortah jedilnega osleza (okre, bamije) je bil izveden poljski poskus. Suhi listni izvlečki vrste Chromolaena odorata (L.) R.M. King & H. Rob. in sveži ter suhi izvlečki korenin vrste Boerhavia diffusa L. nom. cons. so bili uporabljeni za obravnavanja v primerjavi z lamda cihalotrinom in kontrolo. Poskus je bil izveden kot popolni naključni bločni poskus s tremi ponovitvami. Zbrani podatki so bili analizirani s Spearmanovo korelacijo in dvosmerno analizo variance, značilno različna poprečja so bila ločena s Fisherjevim testom najmanjše značilne razlike (LSD) pri 5 % verjetnosti. Rezultati so pokazali, da so tretirane rastline na splošno rastle bolje kot netretirane, bile so višje, imele so več listov in stranskih poganjkov, večji obseg stebla in večji pridelek plodov. Sveži izvlečki korenin vrste B. diffusa so se izkazali kot boljši v vseh merjenih parametrih. Opažena je bila značilno večja pozitivna korelacija med populacijami škodljivih žuželk in med parametri rasti in pridelka medtem, ko je bila korelacija med celokupnim pridelkom in populacijami škodljivih žuželk značilno negativna. Sorta okre NHAe 47-4 je uspevala bolje kot sorta F1-Lucky. Za uravnavanje tobakovega ščitkarja (B. tabaci (Genadius, 1889)) in vrste Amrasca biguttula Ishida, 1912 na jedilnem oslezu priporočamo izvlečke svežih korenin vrste B. diffusa. Ključne besede: botanična sredstva za zatiranje škodljivcev; Boerhavia diffusa; Chromolaena odorata; Bemisia tabaci; Amrasca biguttula; lepljive pasti sticky traps 1 University of Ilorin, Faculty of Agriculture, Department of Crop Protection, Ilorin, Nigeria 2 Corresponding author, e-mail: emmanuelnneji@gmail.com Acta agriculturae Slovenica, 115/1, 133-139, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 133 26. 03. 2020 12:05:49 E. T. NNEJI et al. 1 INTRODUCTION Okra, Abelmoschus esculentus (L.) Moench, is a commercially grown vegetable crop widely cultivated in Africa and Asia. Within 2009 and 2010, a global area of 0.43 million hectares was cultivated with total production standing at 4.54 million tons (Varmudy, 2011). India is the largest producer (67.1 %), with Nigeria following at (15.4 %) and Sudan (9.3 %) (Varmudy, 2011). Okra plays a vital role in human diet (Kahlon et al. 2007, Saifullah and Rabbani 2009) providing phosphorus, calcium, sulphur, iron, fibre, fats, proteins, carbohydrates and vitamins (Lamont 1999, Owolarafe and Shotonde, 2004, Gopalan et al., 2007, Arapitsas 2008, Dilruba et al, 2009). Okra fruit is normally boiled in water to give slimy soup sauces that is relished. The seeds are nutritious, can be dried roasted and ground for use as additive or coffee substitute (Moekchantuk and Kumar 2004) while the fruits are also used to thicken soups and prepare vegetable curds. In industries, mucilage from okra is often used to produce glace papers and confectioneries, also serving as a replacement for blood plasma as well as blood volume expander (Lengsfeld et al., 2004, Adetuyi et al., 2008, Kumar et al., 2010), it is also a useful remedy against genito-urinary problems, chronic dysentery and spermatorrhoea (Nadkarni, 1927). Okra has also been reported to cure ulcers and hemorrhoids (Adams, 1975). Results of tests conducted in China holds that alcoholic extracts from okra leaves have a potential to remove free radicals, reduce proteinuria, and generally improve renal functions (Liu et al., 2005, Kumar et al., 2009). The cultivation of okra in Nigeria has indeed known success, however, it has also been greeted with a handful of constraints which include but may not be limited to insect pest infestations, disease incidence and poor soil nutrient level (Onunkun, 2012). In response to the constraints from insect pests, man has continued to search out eco-friendly strategies and methods for the management of these setbacks to okra production and agriculture in general (Praveen and Dhandapani, 2001). Some of these methods are; cultural, physical and biological control (Praveen and Dhandapani, 2001). Chemical control is also used and has become the most commonly used control method among farmers because of its almost immediate rate of success in the eradication of these insects, there has however been serious concerns about the long-term negative effect of continued or excessive use of synthetic formulations (Praveen and Dhandapani, 2001). More so, there is a serious need to enlighten farmers on safer, yet effective measures which are useful with almost no side effects or residual negative effects as those experienced while using chemicals. This study was therefore carried out to evaluate the effects of Boerhavia diffusa and Chromolaena odorata in control of insect pest infestations of two varieties of okra. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE The experimental site was located behind the Faculty of Agriculture greenhouse, University of llorin, Kwara State, Nigeria, located in the Southern Guinea Savannah agro-ecological zone of Nigeria, between Latitude 8°29'N and Longitude 4°35'E. The experiment was carried out in 2018 cropping season. The climate of Ilorin includes rainy season which starts in April and ends in September while the dry season commences in October and ends in March. The dry spell is experienced in August of every year. The site has an average temperature of 35 0C and is characterized by sandy loam soil. 2.2 FIELD PREPARATION, AND LAYOUT OF EXPERIMENTAL PLOT The land was cleared using cutlass and then mapped out into a plot size of 30 m x 15 m. Ridges were made at intervals of 50 cm spacing within plots. The field already mapped out for the experiment was set out using a Randomized Complete Block Design (RCBD). A spacing of 3 m was maintained between the two subplots of varieties, there were three replicates per treatment and 1 m was maintained between replicates, and 2 m from the boundaries of the plot. Each plot measured 2m x 3m (6 m2) with three (3) ridges in each plot. 2.3 OKRA VARIETIES AND PLANTING Two varieties of okra common to the farmers in Kwara State were used for the study, they are F1 Lucky and NHAe 47-4 varieties purchased from an Agro-input store in Ilorin. Seeds were planted at five (5) seeds per hole and 50 cm intra-row spacing on 6th October 2018. Two weeks after emergence, thinning was carried out leaving two plants per stand. There were 24 plants in each plot. 2.4 TREATMENTS, PREPARATION, AND APPLICATION Treatments used for the study were; ethanol leaf 134 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 134 26. 03. 2020 12:05:49 Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. ... pests of okra (Aabelmoschus esculentus (L.) Moench) extracts of Siam weed, Chromolaena odorata (L.) R.M. King & H. Rob. ethanol extracts of fresh and dried Red Spiderling, Boerhavia diffusa L. nom. cons. Laraforce ® (a.i. lambda-cyhalothrin) as standard check; and untreated control. Chromolaena odorata leaf and B. diffusa root were sourced from the forest of University of Ilorin. These plant parts were harvested and air dried in the shade for three (3) weeks, the roots were crushed using mortar and pestle while the leaves were ground using a kitchen size electric blender. They were further sieved using a sieve of 2 mm mesh to obtain a uniform powder. Plant extracts were prepared by maceration. 50 g of each powder were measured into 400 ml of 90 % ethanol and allowed to stand for twenty-four hours while shaking from time to time to ease extraction. The resulting suspension was filtered using muslin cloth, and the filtrate was made up to 2 litres (1:40 or 2.5 % w/v ratio) by adding distilled water, 2 ml of liquid soap per litre of solution was used as surfactant (Anjarwalla, 2016). Lambda-cyhalothrin 2.5 % emulsifiable concentrate was used as a standard check at 0.5 g per litre. Treatment application commenced four (4) weeks after planting and was done at one-week interval to the 7th week after planting (WAP). 2.5 CULTURAL PRACTICES Plots were weeded using hoe at 3 WAP (Adigun, 2005). Selective spraying using herbicide (Glyphosate) was done at 6 WAP at the recommended rate. 2.6 INSTALLATION OF YELLOW STICKY TRAPS One (1) yellow sticky trap was installed per plot, they were placed at the center of the plots at 4 WAP. They were 50 cm above the ground and were left on the plots for two weeks after which they were collected and taken to the Crop Protection Department Laboratory for insect identification and counting. Insects identified include whiteflies (Homoptera) and leafhoppers (Hemiptera). Physical counting was done using hand lens of 10 x magnification and Gordon's dichotomous key to the order of insects (Gordon, 2019) as aid. 2.7 DATA COLLECTION Five plants per plot were selected at random and tagged. Collection of data commenced at 5 WAP and continued at a week interval to fruiting and harvest. Data were collected on plant height, number of branches, whitefly and leafhopper population, number of days to flowering, number of fruits, and mass of fruits. 2.8 DATA ANALYSIS Data collected were subjected to a two-way analysis of variance (ANOVA) and Spearman's Correlation Analysis using Genstat 17th edition, and significantly different means were separated using Fishers Least Significant Difference (LSD) test at 5 % level of significance. 3 RESULTS AND DISCUSSION The results of the study revealed that the various treatments used in the experiment had significant effects on both growth and yield parameters of F1-Lucky and NHAe 47-4 varieties of okra. Plants treated with fresh root extracts of B. diffusa generally performed better than untreated plants. The treated plots produced plants which were taller and had more branches compared to those of untreated plots (Tables 1 and 2). This may be because treated plants suffered less infestation from whitefly (Bemisia tabaci (Gennadius, 1889) and leaf-hopper (Amrasca biguttula Ishida, 1912) compared to untreated plots (Table 4). This corroborates the findings of Bindhu et al. (2015) who reported that B. diffusa and some other plants contained various compounds which are known to have repellent and toxic effects on insects. It also confirms the reports from several studies which have shown C. odorata to have pesticidal properties, among these reports are Owolabi et al. (2010) who reported that C. odorata has been used to manage Sitophilus zeamais (Motschulsky), 1855 which belongs to the order (Coleoptera). Studies have revealed that B. diffusa and C. odorata contain phytochemicals that are effective in insect pest control (Afolabi et al., 2007 and Deepti et al., 2013). The better growth performance recorded in treated plants may also be because the plants had lesser infection from diseases vectored by the insect pests identified during the study. Okra is susceptible to at least nineteen plant viruses (Brunt et al., 1990; Swanson and Harrison, 1993). These viruses seriously affect its growth and yield. In several parts of Africa, okra leaf curl disease (OLCD) is considered to be the most serious disease threatening okra production (N'Guessan et al., 1992; Swanson and Harrison, 1993; Bigarré et al., 2001). OLCD is transmitted by whitefly (Bemisia tabaci). The incidence and abundance of whiteflies will always be directly proportional to the incidence and severity of OLCD (Bigarré et al., 2001). Therefore, the treated plants facing lesser infestation of whiteflies may have also had lesser incidence of Acta agriculturae Slovenica, 115/1 - 2020 135 AAS_vsebina_2020_115_1_koncna 180320.indd 135 26. 03. 2020 12:05:49 E. T. NNEJI et al. Table 1: Main effect of treatments and variety on the plant height of okra Plant height (cm) WAP Treatments 5 6 7 8 9 Fresh Root extract of B. diffusa 25.29 39.92 54.53a 59.70a 70.70a Dry root extract of B. diffusa 23.15 35.33 47.83ab 55.75ab 62.71ab Dry leaf extract of C. odorata 20.74 28.61 38.00b 43.68ab 49.10b Lambda-cyhalothrin 21.82 31.66 41.22ab 47.14ab 53.76ab Control 19.90 28.47 36.79b 41.33b 46.47b SEM 2.67 4.39 5.47 6.00 7.07 LSD 7.81 12.84 15.99 17.57 20.69 Variety F1-Lucky (kousko) 18.55b 28.40b 39.10 45.00 51.50 NHAe 47-4 25.81a 37.10a 48.20 54.00 61.50 SEM 1.34 2.61 3.52 3.88 4.69 LSD 3.88 7.59 10.22 11.27 13.63 Values in the same column followed by the Difference (LSD) Test Key: WAP: Weeks after Planting same letter(s) are not significantly different at p = 0.05 according to Fisher's protected Least Significant Table 2: Main effect of treatments and variety on number of branches of okra Number of branches (WAP) Treatments 6 7 8 9 Fresh root extract of B. diffusa 4.18a 5.43a 6.48a 7.48a Dry root extract of B. diffusa 3.02b 4.27b 5.24b 6.24b Dry leaf extract of C. odorata 2.28b 3.53b 4.96b 5.96b Lambda-cyhalothrin 3.35* 4.60ab 5.40ab 6.40ab Control 2.75b 4.00b 4.48b 5.48b SEM 0.50 0.50 0.50 0.50 LSD 1.10 1.10 1.10 0.70 Variety F1-Lucky (kousko) 2.00b 4.00b 5.00b 6.00b NHAe 47-4 4.00a 5.00a 6.00a 7.00a SEM 0.30 0.30 0.30 0.30 LSD 0.70 0.70 0.70 0.70 Values in the same column followed by the same letter(s) are not significantly different at p = 0.05 according to Fisher's protected Least Significant Difference (LSD) Test Key: WAP: Weeks after Planting Okra Leaf Curl Virus which translated to better growth and yield (Table 6). Fidèle (2010) reported a significant decrease in plant height and some other yield-contributing parameters in four accessions of okra which had symptoms of OLCD. The NHAe 47-4 variety flowered earlier than the F1-Lucky variety (Table 3). Furthermore, treated plants in 136 Acta agriculturae Slovenica, 115/1 - 2020 the plots for fresh root extracts of B. diffusa gave higher fruit yield in both varieties studied (Table 6). This may be because treated plants which were less infested were able to carry out photosynthesis without disruption. Edward and Abdelaziz (2007) reported that any impairment of photosynthetic efficiency by insect pests such as leafhop-pers is irreversible and hopper-burn affects plant biomass AAS_vsebina_2020_115_1_koncna 180320.indd 136 26. 03. 2020 12:05:49 Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. ... pests of okra (Aabelmoschus esculentus (L.) Moench) Table 3: Main effect of treatments and variety on days to flowering of okra Treatment Days to flowering Fresh root extract of B. diffusa 45.76 Dry root extract of B. diffusa 46.37 Dry leaf extract of C. odorata 46.97 Lambda-cyhalothrin 48.07 Control 48.70 SEM 1.69 LSD 4.17 Variety F1-Lucky (kousko) 49.38b NHAe 47-4 44.96a SEM 0.85 LSD 5.90 Values followed by the same letter(s) are not significantly different at p = 0.05 according to Fisher's protected Least Significant Difference (LSD) Table 4: Main effect of treatments and variety on the population of adult whitefly and leafhopper per yellow sticky trap Whitefly Leafhopper Treatment population population Fresh root extract of B. diffusa 298.20b 2.33a Dry root extract of B. diffusa 404.20d 9.00d Dry leaf extract of C. odorata 289.30a 5.00b Lambda-cyhalothrin 301.50c 5.50bc Control 653.30e 7.00c SEM 1.12 0.66 LSD 3.32 1.96 Variety F1-Lucky (kousko) 416.00b 6.00 NHAe 47-4 363.00a 5.53 SEM 0.71 0.42 LSD 2.10 1.24 Values in the same column followed by the same letter(s) are not significantly different at p = 0.05 according to Fisher's protected Least Significant Difference (LSD) Test accumulation by reducing green leaf area and efficiency of solar radiation capture. Aishwarya (2018) also submitted that both nymph and adults cause damage to okra by sucking the cell sap from the lower surface of leaf, twigs and other tender parts of the plant, causing curling of leaves and leading to stunted growth of the plants. They further reported that leafhoppers excrete honey dew which causes the black sooty mold which has an adverse effect on the photosynthetic ability of the plants. Several reports including Kedar et al. (2014) and Aishwarya Ray (2018) have revealed that whiteflies and their nymphs suck cell sap from plant leaves and affected leaves curl and dry with the affected plants showing a stunted growth. Whiteflies are also responsible for transmitting yellow vein mosaic virus (YVMV), an economically important disease of okra, the symptoms being interwoven networks of yellow veins surrounded by islands of green tissues on the leaves which also turn yellow with time (Kedar et al., 2014; Aishwarya, 2018). The control of incidence of these insect pests and the consequent reduction or elimination of the occurrence of diseases mediated by the insect pests identified on the plots may have translated to the better yield obtained from the treated plots. The study also revealed a strong positive correlation exists between leafhopper and whitefly population, plant height and number of fruits, plant height and overall yield, and number of fruits and overall yield (Table 5). This suggests that the leafhopper population increased with the whitefly population, while the yield was directly proportional to the growth of the plants. 4 CONCLUSION The findings of this study revealed that ethanol extracts of Boerhavia diffusa and Chromolaena odorata had significant positive effects on growth and yield of okra and were also effective in reducing the population of leafhoppers and whiteflies in treated plots. There was a significant positive correlation between leafhopper and whitefly population, plant height and number of fruits, plant height and overall yield, and number of fruits and overall yield. While there was a significant negative correlation between overall yield and whitefly population. There was a non-significant negative correlation between whitefly population and plant height, whitefly population and number of fruits, leafhopper population and plant height, leafhopper population and number of fruits, and leafhopper population and overall yield. Of the two botanicals evaluated fresh root extract of B. diffusa on NHAe 47-4 okra variety performed better than other treatments from germination and emergence, through growth parameters to yield. 5 REFERENCES Adams, C. F. (1975). Nutritive value of American foods in common units, U.S. Department of Agriculture, Agric Handbook. 425-29. Adetuyi, F.O., Osagie, A.U. and Adekunle, A. T. (2008). Effects of postharvest storage techniques on the nutritional prop- Acta agriculturae Slovenica, 115/1 - 2020 137 AAS_vsebina_2020_115_1_koncna 180320.indd 137 26. 03. 2020 12:05:49 E. T. NNEJI et al. Table 5: Spearman's correlation matrix for various parameters in the study Whitefly population Leafhopper population Plant height Number of fruits/ha Leafhopper population 0.469** Plant height -0.287 -0.191 Number of fruits/ha -0.249 -0.117 0.688** Overall yield (kg ha-1) -0.364* -0.215 0.746** 0.966** **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed). Table 6: Main effect of treatments and variety on number of fruits per plant, number of fruits per plot, and overall yield (kg ha 1) of okra Treatments Number of fruits/plant Number of fruits/plot Fruit yield (kg ha 1) Fresh root extract of B. diffusa 13.76 165.10 2029.00a Dry root extract of B. diffusa 9.48 113.80 1231.00ab Dry leaf extract of C. odorata 9.50 114.10 1304.00ab Lambda-cyhalothrin 8.81 106.10 1223.00ab Control 7.66 91.20 980.00b SEM 2.20 26.30 334.20 LSD 6.40 76.90 977.60 Variety F1-Lucky (kousko) 7.00b 80.00b 878.00b NHAe 47-4 13.00a 155.90a 1829.00a SEM 1.10 13.50 178.30 LSD 3.30 39.25 518.40 Values in the same column followed by the same letter(s) are not significantly different at p = 0.05 according to Fisher's protected Least Significant Difference (LSD) Test erties of Benin indigenous Okra Abelmoschus esculentus (L.) Moench). Pakistan Journal of Nutrition, 7, 652-657. https://doi.org/10.3923/pjn.2008.652.657 Adigun, J.A. (2005). 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Identification and quantification of poly-phenolic compounds from okra seeds and skins. Food Chemistry, 110, 1041-1045. https://doi.org/10.1016/j.food-chem.2008.03.014 Bigarré, L., Chazly, M., Salah, M., Ibrahim, M., Padidam, M., Nicole, M., Peterschmitt, M., Fauquet, C., Thou-venel, J.C. (2001). Characterization of a new Begomo virus from Egypt infecting hollyhock (Althea rosea). European Journal of Plant Pathology, 107, 701-711. https://doi. org/10.1023/A:1011967232319 Bindhu, V.R., Ganga, S., Dayanandan, S. (2015). Mortality effects of some medicinal plants on the pulse beetle Cal-losobruchus chinensis (Coleoptera: Bruchidae). Journal of Fertilizer & Pesticides, 6, 150. https://doi.org/10.4172/2155-6202.1000150 Brunt, A., Crabtree, K., Gibbs, A. (1990). Viruses of Tropical Plants. CAB International, Wallingford, UK. Deepti Malhotra, Amir Khan and Fouzia Ishaq (2013). Phytochemical screening and antibacterial effect of root extract of Boerhavia diffusa L. (Family Nyctaginaceae). Journal of Applied and Natural Science, 5(1), 221-225. https://doi. org/10.31018/jans.v5i1.310 Dilruba, S., Hasanuzzaman, M., Karim, R., Nahar, K. (2009). Yield response of okra to different sowing time and application of growth hormones. Journal of Horticultural Science. Ornamental Plants, 1, 10-14. Edward, B. Radcliffe, Abdelaziz Lagnaoui (2007). Potato Biology and Biotechnology. 138 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 138 26. 03. 2020 12:05:49 Effects of Boerhavia diffusa L. nom. cons. and Chromolaena odorata (L.) R. M. ... pests of okra (Aabelmoschus esculentus (L.) Moench) Fidèle Tiendrébéogo, V.S. Edgar Traoré, Jean-Michel Lett, Nicolas Barro, Gnissa Konaté, Alfred S. Traoré, Oumar Traoré (2010). Impact of okra leaf curl disease on morphology and yield of okra. Crop Protection, 29. (2010)712e716. https://doi.org/10.1016/jxropro.2010.02.007 Gopalan, C., Sastri, S. B. V., Balasubramanian, S. (2007). Nutritive value of Indian foods, National Institute of Nutrition (NIN), ICMR, India. P1. Gordon Ramel (2019). Key to the Identification of Insects to Order, A Few Words About KEYS, Adapted from Harold Ol-droyd 1958. https://www.earthlife.net/insects/orders-key. html#key Kedar, S. C., K. M. Kumaranag, D. S., Bhujbal and N. H. Thod-sare4 (2014). Insect Pests of Okra and Their Management. Popular Kheti, 2(3), 112-119. Kumar, R., Patil, M. B., Patil, S. R., Paschapur, M. S. (2009). Evaluation of Abelmoschus esculentus mucilage as suspending agent in paracetamol suspension. International Journal of Pharmaceutical and Technology Research, 1, 658-665. Lamont, W. (1999). Okra a versatile vegetable crop. Horticultural Technology; 9: 179-184. Lee K, Cho CY, Park SK (1990). The effect of nitrogen fertilizer, plant density and sowing date on the yield of okra. Korean Journal of Crop Science, 35(8), 179-183. https://doi.org/10.21273/H0RT-TECH.9.2.179 Lengsfeld, C., Titgemeyer, F., Faller, G., Hensel, A. (2004). Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. Journal of Agriculture and Food Chemistry, 52, 1495-1503. https://doi. org/10.1021/jf030666n Liu, I. M., Liou, S. S., Lan, T. W., Hsu, F. L., Cheng, J. T. (2005). Myricetin as the active principle of Abelmoschus moscha-tus to lower plasma glucose in streptozotocin-induced diabetic rats. Planta Medica, 71, 617-621. https://doi. org/10.1055/s-2005-871266 Moekchantuk, T., Kumar, P. (2004). Export okra production in Thailand. Inter-country programme for vegetable IPM in South and SE Asia phase II Food and Agriculture Organization of the United Nations, Bangkok, Thailand. P1. Nadkarni, K. M. (1927). Indian Meteria Medica. Nadkarni and Co Bombay. P1. N'Guessan, K.P., Fargette, D., Fauquet, C., Thouvene, L.J.C. (1992). Aspects of the epidemiology of okra leaf curl virus in Côte d'Ivoire. Tropical Pest Management, 38, 122-126. https://doi.org/10.1080/09670879209371668 Onunkun, O. (2012). Evaluation of aqueous extracts of five plants in the control of flea beetles on okra (Abelmoschus esculentus (L.) Moench). JBiopest, 5(supplementary), 62-67. Owolarafe, O. K., Shotonde, H. O. (2004). Some physical properties of fresh okra fruit. Journal of Food Engineering, 63, 299-302. Journal of the Ghana Science Association, 3(3), 136-144. https://doi.org/10.1016Zj.jfoodeng.2003.08.002 Praveen, P.M., & N. Dhandapani (2001). Eco-rriendly management of major pests of okra (, (L.) Moench), Journal of Vegetable Crop Production, 7(2), 3-12. https://doi.org/10.1300/ J068v07n02_02 Saifullah, M., Rabbani, M. G. (2009). Evaluation and characterization of okra (Abelmoschus esculentus L. Moench.) genotypes. SAARC Journal of Agriculture, 7, 92-99. Swanson, M.M., Harrison, B.D. (1993). Serological relationships and epitope profiles of isolates of okra leaf curl gemi-nivirus from Africa and the Middle-East. Bio-chimie, 75, 707-711. https://doi.org/10.1016/0300-9084(93)90101-W Varmudy, V. (2011). Marketing Survey needed to boost okra exports. Department of economic Vive Kananda College, Put-tur Karnataka. P1. Acta agriculturae Slovenica, 115/1 - 2020 139 AAS_vsebina_2020_115_1_koncna 180320.indd 139 26. 03. 2020 12:05:49 AAS_vsebina_2020_115_1_koncna 180320.indd 140 26. 03. 2020 12:05:49 doi:10.14720/aas.2020.115.1.1407 Original research article / izvirni znanstveni članek Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylis glomerata L.): primerjava in agronomska vrednost Jure COP 1 2, Klemen ELER 1 Received December 27, 2019; accepted January 11, 2020. Delo je prispelo 27. decembra 2019, sprejeto 11. januarja 2020. Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): primerjava in agronomska vrednost Izvleček: Opisovanje in vrednotenje akcesij, na primer ekotipov kulturnih rastlin, je temeljna naloga kmetijske rastlinske genske banke. S poljskim poskusom smo v letih 2015-2019 raziskovali agro-biološko raznolikost 15 slovenskih ekotipov (skupina 1) in 7 standardnih sort (skupina 2) navadne pasje trave (Dactylis glomerata L.) s poudarkom na primerjavi teh dveh skupin. ocenili smo tudi agronomsko vrednost ekotipov glede na sorte. Zasnova poskusa je bila naključni blok s 3 ponovitvami. Vsak ekotip oziroma sorta je bila zastopana z 20 posameznimi rastlinami na ponovitev. Med obravnavanimi entitetami so bile ugotovljene značilne razlike v vseh preučevanih agro-bioloških lastnostih (p < 0,001). Značilne razlike so bile ugotovljene tudi pri primerjavi skupin (p < 0,001) razen v okuženosti z listnimi boleznimi (p = 0,113). Pri tem so bile sorte z agronomskega vidika boljše kot ekotipi. Raznolikost znotraj populacij je bila pri slovenskih ekotipih večja kot pri standardnih sortah, na njo je odpadel tudi večji del skupne variance ekotipov. Analiza glavnih komponent (AGK) na vseh preučevanih lastnostih je pokazala izrazito razlikovanje ekotipov od sort in večjo podobnost med sortami kot med ekotipi. Pri slednjih se kot rezultat AGK kažeta dve podskupini, kar pa ne moremo razložiti z izvorom ekotipov. Ključne besede: genska banka; populacije; opisovanje; vrednotenje Agro-biological diversity of Slovene ecotypes and standard varieties of cocksfoot (Dactylisglomerata L.): comparison and agronomic value Abstract: Characterisation and evaluation of accessions, e.g. ecotypes of cultivated plants, are the primary task of each agricultural gene bank. In a field experiment, agro-biological diversity and agronomic value of 15 Slovene ecotypes (group 1) and 7 standard varieties (group 2) of cocksfoot (Dactylis glomerata L.) were investigated during the 2015-2019 period. A particular emphasis was given to the comparison of the two groups. The spaced plant experiment with 20 single plants of each treatment entity per replicate was arranged in a randomized complete block design with three replicates. Significant differences among investigated entities were confirmed for all agro-biological traits (p < 0.001). The same holds when the groups were compared (p < 0.001) except for the infection with leaf fungal diseases (p = 0.113).Considering these differences varieties possessed higher agronomic value than ecotypes. In general, the intra-population diversity of Slovene ecotypes was higher than that of standard varieties and represented a higher portion of the ecotype complete variance. Principal component analysis (PCA) of all investigated traits showed a distinctive difference between ecotypes and varieties and higher similarity within the variety group than within the ecotype group. PCA also showed that the ecotypes can be separated into two subgroups, which however cannot be explained by the characteristics of ecotype origin. Key words: gene bank; populations; characterization; evaluation 1 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenija 2 Korespondenčni avtor, e-naslov: jure.cop@bf.uni-lj.si Acta agriculturae Slovenica, 115/1, 141-149, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 141 26. 03. 2020 12:05:50 J. ČOP, K. ELER 1 UVOD Navadna pasja trava (Dactylis glomerata L.) je trajnica zmernega podnebnega pasu, ki jo sestavlja več različno ploidnih podvrst. Na travinju v Evropi in širše prevladuje antropofilna, tetraploidna (2n = 4x = 28) oblika (Lindner in Garcia, 1997; Tuna in sod., 2004). Enako velja tudi za situacijo z navadno pasjo travo v Sloveniji (Martinčič in sod., 2007). Navadna pasja trava je konkurenčna in na stres tolerantna vrsta, zato je pomembna za pridelavo krme na rodovitnih travniških tleh, v travno-deteljnih mešanicah na njivah in na manj rodovitnih travniških tleh, kjer se redno pojavlja suša. Priporoča se za setev v dveletnih do štiriletnih mešanicah z ljuljkami, ker pripomore k boljši in zanesljivejši pridelavi krme v drugi polovici pridelovalnega obdobja (Suter in sod., 2013; AGFF, 2017). Krmna vrednost navadne pasje trave je v zgodnjih razvojnih fazah zelo velika. V primerjavi z drugimi kakovostnimi travami izstopa z veliko vsebnostjo surovih beljakovin (DLG, 1997). Tako je na primer v zadnji dekadi aprila povprečna dveletna vsebnost surovih beljakovin v zelinju navadne pasje trave znašala 219 g kg-1 sušine, pri trpežni ljuljki pa le 171 g kg-1 sušine (Čop in sod., 2009). slabšanje kakovosti zelinja s staranjem poganjkov je agronomska pomanjkljivost navadne pasje trave, ki se ji lahko delno izognemo s primerno zgodnjo rabo. Uspešnost pridelave krme z navadno pasjo travo, tako v smislu prilagajanja okoljskim in pridelovalnim razmeram kot zagotavljanja ustrezne kakovosti krme, je v veliki meri odvisna od sort, pa tudi od ekotipov, ki se uporabljajo za žlahtnjenje. Agro-biološke lastnosti trav, s katerimi se ukvarjamo pri ocenjevanju ekotipov, sort v potrjevanju (test razločljivosti, izenačenosti in nespremenljivosti - RIN-test) in žlahtnjenju, so pomembne tako s stališča količine in kakovosti pridelka kot trajanja pridelave (trpežnosti). Ocenjujemo agro-biološke lastnosti, kot na primer višina stebel, pokončnost rasti (habitus), začetek latenja, tvorba socvetij med rastno sezono, regeneracija po defolia-ciji, okuženost z boleznimi, trpežnost idr. (IPGRI, 1985; UPOV, 2002). Kakovostna sorta navadne pasje trave ne sme biti previsoka. Mora biti pokončne rasti in pozna v razvoju, imeti mora slabo izraženo determinantno rast ter dobro regeneracijo, odpornost proti boleznim in tr-pežnost. Z raziskavo smo želeli ugotoviti: (1) kakšno je va-riiranje preučevanih agro-bioloških lastnosti med in znotraj ekotipov in standardnih sort, (2) kakšna je agronomska vrednost ekotipov glede na preučevane lastnosti v primerjavi s standardnimi sortami in (3) ali izvor ekotipov (geografska lega, talne lastnosti, pridelovalne razme- 142 Acta agriculturae Slovenica, 115/1 - 2020 re) oziroma žlahtnjenje sort povzroči njihovo grupiranje ob upoštevanju vseh preučevanih lastnosti obenem. 2 MATERIALI IN METODE 2.1 EKOTIPI IN STANDARDNE SORTE V raziskavo je bilo vključenih 15 slovenskih ekotipov in 7 standardnih sort navadne pasje trave. Ekotipe smo nabirali v obliki semena na trajnih travnikih v nižinskih, gričevnatih in hribovitih predelih Slovenije v letih od 2007 do 2014. Vsi so vključeni v rastlinsko gensko banko Slovenije. Sorte smo pridobili neposredno od žlahtniteljev oziroma pooblaščenih semenarskih hiš. Pri izboru sort smo upoštevali priporočili inštitucij, ki izvajata RIN-teste, tj. Centralnega inštituta za kontrolo in testiranje v Bratislavi (Ustredny kontrolny a skušobny ustav pol'nohospodarsky v Bratislave, Slovaška) in Raziskovalnega centra za testiranje sort v Slupia Wielka (Centralny Osrodek Badania Odmian Roslin Uprawnych, Poljska). Upoštevali smo tudi pridelovalno vrednost in ekološke lastnosti sort. Podatki o izvoru ekotipov in sort so v Preglednici 1. 2.2 OPIS POSKUSA Poljski poskus je potekal po standardiziranem postopku na poskusnem polju Oddelka za agronomijo Biotehniške fakultete v Ljubljani od leta 2015 do leta 2019. Zasnovan je bil v naključnem bloku s tremi ponovitvami. Vsak ekotip oziroma sorto v posamezni ponovitvi je sestavljalo 20 rastlin, ločeno posajenih na razdaljo 50 x 70 cm. Vseh rastlin v poskusu je bilo 1320. Letna oskrba poskusa je zajemala spomladansko gnojenje z NPK 15:15:15, dve dognojevanji s kalcijevim amonijevim nitratom (KAN, 27 % N), tri košnje in dve okopavanji. Letni odmerek hranil je znašal 200 kg N v treh obrokih, 80 kg P2O5 in 80 kg K2O ha-1. Višina rezi ob košnji je bila od 5 do 7 cm. Na poskusnem polju se nahajajo rjava aluvialna tla na karbonatnem pesku in produ. V zgornji 30 cm plasti je meljasta ilovica, pod njo je do globine 110 cm zmerno oglejena meljasto glinasta ilovica. Na poskusnem polju je postavljena cevna drenaža, po kateri odteka padavinska voda, ki pronica skozi tla. Na začetku poskusa so bila tla v zgornji 22 cm plasti nevtralna (pH 6,9 v CaCl2) ter dobro preskrbljena s fosforjem (137 mg P2O5 kg-1 tal) in srednje dobro s kalijem (172 mg K2O kg-1 tal). Vremenske razmere so bile v poskusnem obdobju razmeroma ugodne za rast krmnih trav. Poletna suša, ki je običajna v pretežnem delu Slovenije, je bila izrazitejša AAS_vsebina_2020_115_1_koncna 180320.indd 142 26. 03. 2020 12:05:50 Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): ... vrednost Preglednica 1: Podatki o izvoru ekotipov in standardnih sort navadne pasje trave, vključenih v raziskavo. Dodani so podatki o rastišču in rabi za ekotipe in podatki o zgodnosti za sorte. Table 1: Data on the origin of ecotypes and standard varieties of cocksfoot included in the research. Data on the habitat and number of harvests for ecotypes and development earliness for the varieties are added. Ekotip/ Pokrajina/ Lokacija/ Zemljepisna Zemljepisna Nadmorska Vlažnost Košnja Sorta Država Zlahtnitelj širina (S) dolžina (V) višina (m) rastišča (število) Dg 01/14 Bela krajina Špeharji 45°26' 15°8' 278 suho 1 Dg 02/14 Bela krajina Marindol 45°30' 15°19' 238 suho 2 Dg 03/14 Bela krajina Semič 45°39' 15°6' 446 polsuho 1-2 Dg 04/14 Zasavje Padež 46°3' 15°0' 748 suho 1 Dg 01/13 Dolenjska Veliko Mlačevo 45°56' 14°40' 328 sr. vlažno 2-3 Dg 04/13 Dolenjska Zdenska vas 45°49' 14°42' 441 polsuho 1-2 Dg 05/13 Dolenjska Kompolje 45°48' 14°44' 463 polsuho 1-2 Dg 08/13 Dolenjska Ambrus 45°49' 14°48' 355 sr. vlažno 2-3 Dg 09/13 Zasavje Ribče 46°5' 14°46' 272 polsuho 1-2 Dg 13/13 Gorenjska Jamnik 46°16' 14°12' 830 polsuho 1-2 Dg 14/13 Osrednja Slovenija Črnuče 46°56' 14°26' 298 sr. vlažno 2-3 Dg 16/13 Osrednja Slovenija Gorenja Brezovica 45°56' 14°26' 481 suho 1 Dg 19/13 Zasavje Podkum 46°4' 15°2' 750 suho 1 Dc 06/07 Notranjska Travnik 45°41' 14°35' 703 sr. vlažno 2 Dg 03/07 Notranjska Travna Gora 45°44' 14°38' 891 polsuho 2 Trerano Nemčija Feldsaaten Freu-denberger zelo zgodna sorta Beluga Švica DSP AG zelo pozna sorta Reda Švica DSP AG zgodna sorta Intensiv Nizozemska Barenbrug srednje zgodna do pozna sorta Dascada Nizozemska Barenbrug zgodna do srednje zgodna sorta Barlegro Nizozemska Barenbrug zelo pozna sorta Padania Italija CREA-ZA Lodi srednje zgodna sorta v letih 2016, 2017 in 2019. Najbolj je na to vplivala neugodna časovna razporeditev padavin med rastno sezono, manj pa tudi povečana temperatura zraka. Tako je bilo v skrajnih primerih 151 mm manj padavin med majem in avgustom 2017 in 4,4 °C višja povprečna temperatura zraka v juniju 2019 kot v referenčnem obdobju 19812010. 2.3 MERITVE IN OCENJEVANJE AGRO-BIOLOŠKIH LASTNOSTI V posameznih letih smo opravili meritve višine ra- stlin in šest ocenjevanj agro-bioloških lastnosti. Pri tem smo upoštevali navodila Mednarodnega inštituta za rastlinske genske vire (IPGRI, 1985) in Mednarodne zveze za zaščito novih rastlinskih sort (UPOV, 2002). Višino poganjkov po posameznih šopih smo merili v maju, ko je bila večina poganjkov v fazi zorenja plodov. Upoštevali smo dolžino od osnove do vrha socvetja večine poganjkov v šopu. Ocenjevali smo ob pomoči petstopenjske lestvice, razen pri prisotnosti generativnih poganjkov v jeseni in okuženosti z listnimi boleznimi. V prvem primeru smo uporabili dvostopenjsko lestvico, v drugem pa devetstopenjsko. Habitus pomeni izraženost pokončne rasti poganjkov in sicer je 1 ležeča rast (prostratum) in Acta agriculturae Slovenica, 115/1 - 2020 143 AAS_vsebina_2020_115_1_koncna 180320.indd 143 26. 03. 2020 12:05:50 J. ČOP, K. ELER 5 pokončna rast (erektum). Začetek latenja je razvojna faza trav, ki jo je najlažje določiti in se uporablja za oceno zgodnosti. To ocenjevanje smo izvedli v časovnem obdobju dveh mesecev v petih terminih (pred 15. aprilom, 15. in 22. aprila, 9. in 23. maja). Faza začetka latenja je nastopila, ko so se v šopu pojavili trije poganjki s socvetjem. Regeneracijo po košnji smo ocenili v juliju, ko visoka temperatura zraka in pogosto tudi suša poslabšata tvorbo novih listov, ki pomeni začetek nove rasti [ocene: 1 (slaba) do 5 (zelo dobra regeneracija)]. Tendenco tvorbe generativnih poganjkov izven sezone generativne rasti, smo ocenili konec septembra z binarnim popisom prisotnosti oziroma odsotnosti le-teh po posameznih šopih. Vigor rastlin smo ocenili konec pete rastne sezone, s čimer smo dobili posredno oceno trpežnosti [ocene: 1 (slab) do 5 (zelo dober vigor)]. Okuženost listov z glivičnimi boleznimi, med katerimi so prevladovale okužbe z glivami rodu Puccinia, smo ocenili v začetku avgusta [ocene: 1 (< 10 %) do 9 (75-100 % okuženost)]. 2.4 STATISTIČNA OBDELAVA Raznolikost preučevanih agro-bioloških lastnosti med in znotraj ekotipov in standardnih sort je prikazana z grafom za razpršenost oziroma z grafi za frekvenčno porazdelitev podatkov. Okvir z ročaji je bil uporabljen pri zvezni spremenljivki za višino rastlin, stolpčni grafi pa pri ordinalnih spremenljivkah za vse druge lastnosti. Razlika v višini rastlin med posameznimi obravnavanimi entitetami (ekotipi in standardne sorte) ter med skupino ekotipov in skupino sort je bila testirana z analizo variance, razlike v drugih lastnostih pa s Kruskal-Wallisovim H testom. Primerjava ekotipov in standardnih sort po vseh parametrih hkrati je bila narejena z analizo glavnih komponent, pri čemer smo zaradi večinoma ordinal-nih spremenljivk uporabili polihorično korelacijo med spremenljivkami. Analize smo opravili v statističnem programskem okolju R (R Core Team, 2019). Za izris stolpčnih grafov ordinalnih spremenljivk smo uporabili knjižnico »likert«. 3 REZULTATI Z RAZPRAVO 3.1 AGRO-BIOLOŠKA RAZNOLIKOST EKOTIPOV IN STANDARDNIH SORT Primerjava obravnavanih entitet navadne pasje trave je pokazala, da so med njimi statistično značilne razlike v vseh preučevanih agro-bioloških lastnostih (p < 0,001). Enako velja za primerjavo skupine slovenskih ekotipov s 144 Acta agriculturae Slovenica, 115/1 - 2020 skupino standardnih sort (p < 0,001) razen pri okuženosti listov z boleznimi, kjer razlika ni značilna (p = 0,113). V agronomskem smislu so bile sorte izrazito boljše od ekotipov v treh lastnostih, ki so zelo pomembne za količino in kakovost pridelka ter trajanje pridelave. V primerjavi z ekotipi so sorte rastle izrazito bolj pokončno, se počasneje razvijale in dalj časa ohranjale rastno kondicijo (Slike 2, 3 in 6). Rastline standardnih sort so tudi bolje regenerirale po defoliaciji in razvile manj ge-nerativnih poganjkov (Sliki 4 in 5), vendar prednost sort pred ekotipi v teh dveh lastnostih ni bila tako izrazita kot pri prvih treh. V preostalih dveh lastnostih - višini generativnih poganjkov in okuženosti listov z glivičnimi boleznimi - so si bile sorte in ekotipi podobni (Sliki 1 in 7). Za višino poganjkov to velja kljub značilni razliki med skupinama, ki je posledica zgolj treh značilno manjših ekotipov od povprečne višine sort. Navedene razlike med ekotipi in sortami so pričakovane, zlasti zato, ker smo za standard izbrali najboljše sorte. Te pa so praviloma boljše od ekotipov oziroma izvornega selekcijskega materiala (Fehr, 1991). Pri naši primerjavi ekotipov in standardnih sort ni bilo nobenega izjemnega ekotipa pri katerikoli lastnosti, čeprav tak primer navajajo Boller in sod. (2009). Pri mnogocvetni ljuljki so namreč ugotovili večjo pridelovalno vrednost izbranih švicarskih ekotipov v primerjavi s sortami. 3.2 ZNOTRAJ POPULACIJSKA RAZNOLIKOST EKOTIPOV IN STANDARDNIH SORT Znotraj populacijska raznolikost v preučevanih lastnostih je bila izražena tako pri ekotipih kot sortah. Vendar so bile populacije ekotipov izrazito bolj raznolike glede habitusa, regeneracije po defoliaciji, izvensezonske tvorbe generativnih poganjkov in vigorja (Slike 2, 4, 5 in 6). V primerjavi z večino sort je bila znotrajpopulacijska raznolikost ekotipov tudi večja glede zgodnosti razvoja in okuženosti z listnimi boleznimi (Sliki 3 in 7). Znotraj-populacijska raznolikost v višini generativnih poganjkov je bila podobna pri obeh skupinah (Slika 1). Ker je navadna pasja trava alogamna vrsta, so populacije ekotipov in sort genetsko - s tem pa tudi fenotipsko - precej raznolike. Vendar je ta raznolikost izrazitejša pri ekotipih, ker naravna selekcija poteka manj usmerjeno kot odbira pri žlahtnjenju. Raznolikost v preučevanih lastnostih znotraj ekotipov je bila na splošno tudi večja od raznolikosti med samimi ekotipi. Enake ugotovitve glede genetske in fenotipske raznolikosti navajajo Last in sod. (2011) ter Xie in sod. (2012) za ekotipe navadne pasje trave, Bolaric in sod. (2005) za ekotipe trpežne ljuljke ter Pagnotta in sod. (2011) za ekotipe črne detelje. Ve- AAS_vsebina_2020_115_1_koncna 180320.indd 144 26. 03. 2020 12:05:50 Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): ... vrednost E" 150 Slika 1: Razpršenost višine 20 rastlin navadne pasje trave po posameznih ekotipih (označeni so z lokacijo) in standardnih sortah, prikazana z okvirji z ročaji. Meritev je bila opravljena 30. maja 2017. Opomba: Simbol * označuje ekotipe, ki se značilno razlikujejo od povprečja standardnih sort. Figure 1: Box-plot for the height distribution of 20 cocksfoot plants by individual ecotypes (denoted by location name) and standard varieties. The measurement was conducted on May 30 2017. Note: Symbol * marks ecotypes which differed significantly from the average height of varieties. 'Dascada' 0% 0 'Beluga' 0% 0 100°/ 'Barlegro' 0% 0 100°/ 'Reda' 0% 2 'Intensiv' 0% 2 'Trerano' 0% 'Padanja' 0% Dg 14/13 Črnuče 0% Dg 09/13 Rlbče 0% Dg 01/13 Vel. Mlačevo 0% 25% 1 ■ 75% Dg 03/14 Semič 0% 32% 68% Dg 04/13 Zdenska vas 0% 32 % I 68% Dg 03/07 Travna Gora 6% ■ 34% 60% Dg 08/13 Ambrus 5% 38% 57% Dg 02/14 Marlndol 4% 1 44% 53% Dg 05/13 Kompolje 4% 1 45% 51% Dg 04/14 Padež 4% 1 50% 46% Dg 19/13 Podkum 3% 1 53% 1 44% Dg 06/07 Travnik 16% 44% 40% Dg 16/13 Gor. Brezovica 9% 1 61 % 1 30% Dg 01/14 Špeharjl 6% ■ 65% 29% Dg 13/13 Jamnik 11% 84% ■ 5% 100 50 0 50 100 Odstotek Slika 2: Pogostnostna porazdelitev ocen habitusa po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Ocenjevanje je bilo opravljeno 23. maja 2016. Ocene: 1 (ležeča rast) do 5 (pokončna rast). Figure 2: Frequency distribution of the habitus estimates by individual ecotypes and standard varieties of cocksfoot (n = 20). The estimation was conducted on May 23 2016. Estimates: 1 (prostrate growth) to 5 (erect growth). lika genetska raznolikost med ekotipi je bolj redka kot znotraj ekotipov in je predvsem pogojena z geografsko oddaljenostjo izvora ekotipov (McGrath, 2008; Last in sod., 2013), kar povečuje razlike v rastnih razmerah in zmanjšuje prenašanje genov med oddaljenimi skupinami rastlin. Genetska raznolikost vrst, ki se na travinju izraža tudi v raznolikosti ekotipov, je bistvenega pomena za vitalnost ekosistemov, pri kulturnih rastlinah pa tudi za žlahtniteljski napredek pri vzgoji novih sort. Pri sortah namreč hitro pride do izgube redkih alelov, prisotnih v naravnih ali polnaravnih populacijah, zato vključevanje samo teh v žlahtnjenje zmanjša možnosti za uspeh (Boller in Greene, 2010). 3.3 MULTIVARIATNA PRIMERJAVA EKOTIPOV IN STANDARDNIH SORT Analiza glavnih komponent, v katero so bile vključene vse preučevane spremenljivke, je pokazala, da prvi dve glavni komponenti pojasnjujeta večino (79,8 %) variabilnosti obravnavanih entitet. Pokazala je tudi, da obstaja zelo jasno razlikovanje med populacijami ekotipov in Acta agriculturae Slovenica, 115/1 - 2020 145 AAS_vsebina_2020_115_1_koncna 180320.indd 145 26. 03. 2020 12:05:50 J. ČOP, K. ELER Slika 3: Pogostnostna porazdelitev ocen zgodnosti po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Zgodnost je bila ocenjena z razvojno fazo »začetek latenja«, pomladi 2016. Ocene: 1 (zelo zgodnja) do 8 (zelo pozna). Figure 3: Frequency distribution of the earliness estimates by individual ecotypes and standard varieties of cocksfoot (n = 20). The development earliness was estimated by means of development stage »ear emergence« in spring 2016. Estimates: 1 (very early) to 8 (very late). 'Barlegro' 'Beluga' 'Reda' 'Dascada' 'Intensiv' 'Trerano' Dg 01/13 Vel. Mlačevo Dg 04/13 Zdenska vas Dg 19/13 Podkum Dg 03/14 Semič Dg 14/13 Črnuče 'Padania' Dg 03/07 Travna Gora Dg 08/13 Ambrus Dg 09/13 Rlbče Dg 04/14 Padež Dg 06/07 Travnik Dg 05/13 Kompolje Dg 16/13 Gor. Brezovica Dg 13/13 Jamnlk Dg 02/14 Marlndol Dg 01/14 Špeharji 0% 0% 2% 0% 0% 2% 5% 78% 3% 1 22% 1 75% 2% 24% 75% 3% 1 22% 75% 2% 25% I 73% 5% 1 23% 72% 5% 1 25% 70% 12% 30% 58% 9% H 34% 57% 12% 1 35% 53% 7% ■ 49% 18% 40% 42% 20% 38% 42% 15% 46% 39% 14% II 47% 1 39% 22% 52% 27% Odstotek Slika 4: Pogostnostna porazdelitev ocen regeneracije po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Ocenjevanje regeneracije rastlin po poletni defoliaciji je bilo opravljeno 10. julija 2017. Ocene: 1 (zelo slaba) do 5 (zelo dobra regeneracija). Figure 4: Frequency distribution of the regeneration estimates by individual ecotypes and standard varieties of cocksfoot (n = 20). The estimation of plant regeneration after summer harvest was conducted on July 10 2017. Estimates: 1 (very poor) to 5 (very good regeneration). sortami (Slika 8), kar smo glede na rezultate preučevanih lastnosti tudi pričakovali. Velika podobnost sort je posledica žlahtnjenja, katerega cilji so bili v vseh primerih enaki - vzgoja sort, primernih za zmerno celinsko podnebje in srednje intenzivno rabo. Podobnost ekotipov je znatno manjša od podobnosti sort. Predvsem odstopajo v smeri druge glavne komponente štirje ekotipi, za katere pa ni bila ugotovljena neka skupna posebnost glede izvora. Za to skupino, ki jo sestavljajo ekotipi 'Ambrus', 'Črnuče', 'Kompolje' in 'Ribče, je bila v celoti značilna poudarjena generativna rast izven njene običajne sezone. Poleg tega sta bila ekotipa 'Kompolje' in 'Ambrus' zelo občutljiva za listne bolezni, ekotipa 'Črnuče' in 'Ribče' pa najbolj pokončne rasti med vsemi ekotipi. Vse te tri lastnosti so povzročile odmik navedenih ekotipov od drugih, kar ponazarjata smer in dolžina vektorjev. Pri primerjavi ekotipov je zanimivo, da selekcijski pritisk, vezan na rodovitnost tal, ni bil tako velik, da bi povzročil grupacijo glede na vlažnost rastišča - vsaj med skrajnima skupinama na svežih in izrazito suhih tleh. Geografska oddaljenost izvornih lokacij ekotipov, ki v našem primeru ni bila velika, je ključnega pomena za na- 146 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 146 26. 03. 2020 12:05:50 Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): ... vrednost Slika 5: Pogostnostna porazdelitev prisotnosti generativnih poganjkov po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Popis je bil opravljen 26. septembra 2016. Oceni: 0 (brez), 1 (z generativnimi poganjki). Figure 5: Frequency distribution of the presence of reproductive tillers by individual ecotypes and standard varieties of cocksfoot (n = 20). The estimation was conducted on September 26 2016. Estimates: 0 (without), 1 (with reproductive tillers). 'Barlegro' 'Padania' 'Intensiv' 'Dascada' 'Trerano' 'Beluga' 'Reda' Dg 06/07 Travnik Dg 01/13 Vel. Mlačevo Dg 03/07 Travna Gora Dg 02/14 Marindol Dg 03/14 Semič Dg 05/13 Kompolje □g 04/14 Padež Dg 19/13 Podkum Dg 09/13 RibCe Dg 04/13 Zdenska vas Dg 01/14 Špeharji Dg 14/13 Črnuče Dg 08/13 Ambrus Dg 16/13 Gor Brezovica Dg 13/13 Jamnik 0% 0% 0% 0% 0% 0% 15% I I 85% 0% 15% 85% 0% 43% 57% 3% 1 52% 45% 15% 55% 30% 5% 71% 24% 21% 65% 14% 10% 77% 13% 40% 47% 13% 24% 66% H 10% 27% 65% ■ 8% 20% 72% ■ 8% 22% 72% ■ 7% 30% 67% 1 3% 27% 70% 1 3% 27% 72% 2% 33% 67% 0% Odstotek Slika 6: Pogostnostna porazdelitev ocen vigorja po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Ocenjevanje je bilo opravljeno ob koncu 6. rastne sezone, 18. novembra 2019. Ocene: 1 (slab) do 5 (zelo dober rastni vigor). Figure 6: Frequency distribution of the vigour estimates by individual ecotypes and standard varieties of cocksfoot (n = 20). The estimation was conducted at the end of the 6th growth season on the November 18 2019. Estimates: 1 (poor) to 5 (very good growth vigour). stanek genetsko in fenotipsko različnih ekotipov. Izjema se pri tem lahko pojavi, če so pridelovalne razmere zelo različne. Tak primer so švicarske populacije navadne pasje trave, ki so izvirale iz razmeroma bližnjih lokacij trajnega travinja (velikost območja 12 km2), a je bila na njih pridelava krme različno intenzivna (Last in sod., 2014). 4 ZAKLJUČKI Preučili smo raznolikost in agronomsko vrednost na novo nabranih slovenskih ekotipov navadne pasje trave. Pri tem smo kot standard uporabili kakovostne evropske sorte, namenjene za gojenje v zmerno celinskem podnebnem pasu, kamor spada tudi večji del Slovenije. Med obravnavanimi entitetami so bile ugotovljene značilne razlike v vseh preučevanih agro-bioloških lastnostih. Značilne razlike so bile ugotovljene tudi pri primerjavi skupine ekotipov s skupino sort razen v okuženosti z listnimi boleznimi. Pri tem so bile z agronomskega vidika sorte boljše kot ekotipi. Znotrajpopulacijska raznolikost ekotipov v preučevanih lastnostih je bila večja kot tista pri sortah. Na njo je tudi odpadel večji del skupne variabilnosti ekotipov. Žlahtnjenje navadne pasje trave se je odrazilo v manjši medsebojni raznolikosti sort v primerjavi z med- Acta agriculturae Slovenica, 115/1 - 2020 147 AAS_vsebina_2020_115_1_koncna 180320.indd 147 26. 03. 2020 12:05:50 J. ČOP, K. ELER Dg 05/13 Kompolje Dg 03/07 Travna Gora Dg 04/13 Zdenska vas Dg 08/13 Ambrus 'Trerano' Dg 06/07 Travnik Dg 03/14 Semič Dg 14/13 Črnuče Dg 09/13 Ribče 'Reda' Dg 19/13 Podkum Dg 04/14 Padež Dg 16/13 Gor. Brezovica Dg 13/13 Jamnik 'Dascada' Dg 02/14 Marindol Dg 01/13 Vel. Mlačevo Dg 01/14 Špeharji 'Padanja' 'Intensiv' 'Beluga' 'Barlegro' 100 50 0 50 100 Odstotek Slika 7: Pogostnostna porazdelitev ocen okuženosti z listnimi boleznimi po posameznih ekotipih in standardnih sortah navadne pasje trave (n = 20). Ocenjevanje je bilo opravljeno 1. avgusta 2016. Ocene: 1 (1-5 %) do 9 (76-100 % pokritost listnih ploskev s simptomi). Figure 7: Frequency distribution of the estimates of the infection with leaf fungal diseases by individual ecotypes and standard varieties of cocksfoot (n = 20). The estimation was conducted on the August 1 2016. Estimates: 1 (1-5 %) to 9 (76-100 % leaf blade cover by symptoms). Slika 8: Rezultati analize glavnih komponent. Prikaz 15 slovenskih ekotipov in 7 standardnih sort navadne pasje trave skupaj z vektorsko težo obravnavanih lastnosti v dvorazsežnem prostoru glavnih komponent. Figure 8: Results of principal component analysis. Arrangement of 15 Slovene ecotypes and 7 standard varieties together with the weight vectors for all investigated traits in 2D dimensional space of two main components sebojno raznolikostjo ekotipov. Sorte so kot skupina tudi izrazito odstopale od skupine ekotipov. Čeprav sta kot rezultat analize glavnih komponent nastali dve podskupini ekotipov, za to nismo ugotovili nobenega z izvorom povezanega vzroka. Velika znotraj populacijska raznolikost ekotipov navadne pasje trave bogati biotsko raznolikost travniških ekosistemov in izboljšuje njihovo funkcionalnost. To velja tudi za druge vrste. Genetsko razno- liki ekotipi so nujni tudi za žlahtniteljski napredek pri vzgoji novih sort, saj so posamezni genotipi vir potencialno koristnih alelov, ki pri sortah več niso prisotni. Raziskava je bila izvedena v okviru »Javne službe nalog rastlinske genske banke«, ki jo financira Ministrstvo za kmetijstvo, gozdarstvo in prehrano R Slovenije. Tehnično izvedbo poljskega poskusa je vodil Boštjan Medved Karničar. 148 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 148 26. 03. 2020 12:05:50 Agro-biološka raznolikost slovenskih ekotipov in standardnih sort navadne pasje trave (Dactylisglomerata L.): ... vrednost 5 VIRI AGFF. (2017). Standardmischungen für den Futterbau Revision 2017-2020. Agrarforschung Schweiz, 8, 1-16. Bolaric, S., Barth, S., Melchinger, A. E., & Posselt, U. K. (2005). Molecular genetic diversity within and among German ecotypes in comparison to European perennial ryegrass cultivars. Plant Breeding, 24, 257-262. https://doi. org/10.1111/j.1439-0523.2005.01108.x Boller, B., & Greene, S. L. (2010). Genetic Resources. In: B. Boller, U. K. Posselt & Veronesi F. (Eds.), Fodder Crops and Amenity Grasses (pp. 13-37). New York, Springer-Verlag. https://doi.org/10.1007/978-1-4419-0760-8_2 Boller, B., Peter-Schmid, M. K. I., Tresch, E., Tanner, P., & Schubiger, F. X. (2009). Ecotypes of Italian ryegrass from Swiss permanent grassland outperform current recommended cultivars. Euphytica, 170, 53-65. https://doi. org/10.1007/s10681-009-9963-y Cop, J., Lavrencic, A., & Kosmelj, K. (2009). Morphological development and nutritive value of herbage in five temperate grass species during primary growth: analysis of time dynamics. Grass Forage Science, 64,122-31. https://doi. org/10.1111/j.1365-2494.2008.00676.x DLG (Deutsche Landwirtschafts-Gesellschaft). (1997). DLG-Futterwerttabellen - Wiederkäuer. 7., erweiterte und überarbeitete Auflage. Frankfurt am Main, DLG-Verlag. Fehr, W. (1991). Principles of cultivar development: Theory and technique. Agronomy Books. 1. URL https://lib.dr.iastate. edu/agron_bo oks/1 IPGRI (International Plant Genetic Resources Institute). (1985). Forage grass descriptors. Rome. URL https://www. bioversityinternational.org/fileadmin/user_upload/De-scriptors_Forage_Grass.pdf Last, L., Herzog, F., Boller, B., Widmer, F., & Kölliker, R. (2011). Influence of habitat and management on genetic diversity of Dactylis glomerata in Switzerland. In: E. M. Pötsch, B. Krautzer & Hopkins A. (Eds.), Grassland farming and land management systems in mountainous regions (Grassland Science in Europe, 16) (pp. 562-564). Gumpenstein, Austria. https://doi.org/10.1016/j.ecolind.2013.11.004 Last, L., Lüscher, G., Widmer, F., Boller, B., & Kölliker, R. (2014). Indicators for genetic and phenotypic diversity of Dactylis glomerata in Swiss permanent grassland. Ecological Indicators, 38, 181-191. https://doi.org/10.1186/1471-2156-14-102 Last, L., Widmer, F., Fjellstad, W., Stoyanova, S., & Kölliker, R. (2013). Genetic diversity of natural orchardgrass (Dactylis glomerata L.) populations in three regions in Europe. BMC Genetics, 14, 1-13. https://doi.org/10.1186/1471-2156-14-102 Lindner, R., & Garcia, A. (1997). Genetic differences between natural populations of diploid and tetraploid Dactylis glomerata ssp. izcoi. 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URL https://www.R-project.org/ Suter, D., Hirschi, H., Frick, R., & Aebi, P. (2013). Knaulgras: Prüfergebnisse von 31 Sorten. Agrarforschung Schweiz, 4, 324-329. https://doi.org/10.1007/978-3-531-19729-6_4 Tuna, M., Khadka, D. K., Shrestha, M. K., Arumuganathan, K., & Golan-Goldhirsh, A. (2004). Characterization of natural orchardgrass (Dactylis glomerata L.) populations of the Thrace Region of Turkey based on ploidy and DNA polymorphisms. Euphytica, 135, 39-46. https://doi.org/10.1023/ B:EUPH.0000009537.08697.4e UPOV (The International Union for the Protection of New Varieties of Plants). (2002). Guidelines for the conduct of tests for distinctness, uniformity and stability. Cocksfoot (Dactylis glomerata L.). Geneva. URL https://www.upov. int/edocs/tgdocs/en/tg031.doc Xie, W. G., Lu, X. F., Zhang, X. Q., Huang, L. K., & Cheng, L. (2012). Genetic variation and comparison of orchardgrass (Dactylis glomerata L.) cultivars and wild accessions as revealed by SSR markers. Genetics and Molecular Research, 11, 425-433. https://doi.org/10.4238/2012.February.24.1 Acta agriculturae Slovenica, 115/1 - 2020 149 AAS_vsebina_2020_115_1_koncna 180320.indd 149 26. 03. 2020 12:05:50 AAS_vsebina_2020_115_1_koncna 180320.indd 150 26. 03. 2020 12:05:49 doi:10.14720/aas.2020.115.1.1314 Original research article / izvirni znanstveni članek Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Hajar SABET \ Mahmood MALEKI 2 3, Maryam ABDOLI NASAB 2, Saeid MIRZAEI 2 Received September 27, 2019; accepted January 20, 2020. Delo je prispelo 27. septembra 2019, sprejeto 20. januarja 2020. Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Abstract: Optimization of tissue culture and regeneration conditions of eggplant is necessary for achieving different goals such as gene transformation and the development of soma-clonal variations. In this study, hypocotyl explants ware used to produce callus in a medium containing different concentrations of NAA and BAR Moreover, the concentration of the elements Ca, Mn, Mg, Fe and K were measured and analysed between embryogenic and non-embryogenic calli. For shoot elongation, embryogenic calli were transferred to a new culture medium containing 3.5, 4 and 4.5 mg l-1 BAR plus 2 mg l-1 GA3. Finally, produced shoots were rooted in a culture medium containing 1, 1.5 and 2 mg l-1 NAA. Results showed that the best treatment for the embryogenic callus induction was MS medium containing 0.5 mg l-1 BAR plus 0.25 mg l-1 NAA. Two elements, Fe and K, had the highest amount in non-embryogenic calli compare to the embryogenic one. For plant regeneration, Ms medium containing 4.5 mg l-1 BAR plus 2 mg l-1 GA3 and 2 mg l-1 NAA were the best treatments for shooting and rooting, respectively. In this study, the best treatments for plant regeneration produced 35 shoots from an explant with 92 % shooting. This regeneration protocol could be useful for gene transformation and micro-propagation studies. Key words: eggplant; tissue culture; somatic embryogenic regeneration; BAR; NAA Abbreviations: NAA Naphthaleneacetic acid BAR 6-Benzylaminopurine GA3 Gibberellic acid MS medium Murashige & Skoog Medium IBA Indole-3-butyric acid IAA Indole acetic acid Somatska embriogeneza hipokotilnih kalusov izbrane sorte jajčevca Izvleček: Optimizacija tkivnih kultur jajčevca in regener-acijskih razmer sta potrebni za doseganje različnih ciljev kot sta genska transformacija in razvoj somaklonalne variabilnosti. V tej raziskavi so bili uporabljeni hipokotilni izsečki za pridobitev kalusa v gojiščih, ki so vsebovala različne koncentracije NAA in BAR Med embriogenimi in neembriogenimi kalusi so bile izmerjene koncentracije elementov kot so Ca, Mn, Mg, Fe in K. Za dolžinsko rast poganjkov so bili embriogeni kalusi premeščeni v drugo gojišče, ki je vsebovalo 3,5, 4 in 4,5 mg l-1 BAR in 2 mg l-1 GA3. Na koncu so bili poganjki ukoreninjeni v gojišču, ki je vsebovalo 1, 1,5 in 2 mg l-1 NAA. Rezultati so pokazali, da je bilo za indukcijo embriogenih kalusov najboljše MS gojišče, ki je vsebovalo 0,5 mg l-1 BAR in 0,25 mg l-1 NAA. Dva elementa, Fe in K , sta imela največjo vsebnost v ne-em-briogenih kalusih v primerjavi z embriogenimi. Za regeneracijo rastlin, za nastanek in vkoreninjenje poganjkov, je bilo MS gojišče, ki je vsebovalo 4,5 mg l-1 BAR, 2 mg l-1 GA3 in 2 mg l-1 NAA najboljše. V raziskavi je najboljše regeneracijsko obravnavanje dalo 35 poganjkov iz enega izsečka z 92 % vkoreninjen-jem. Ta regeneracijski protocol bi lahko bil koristen za gensko transformacijo in raziskave mikro propagacije. Ključne besede: jajčevec; tkivna kultura; regeneracija s somatsko embriogenezo; BAR; NAA Okrajšave: NAA Naftalen ocetna kislina BAR 6-Benzil aminopurin GA3 Giberelinska kislina MS gojišče Murashige & Skoog gojišče IBA Indol-3-maslena kislina IAA Indol ocetna kislina 1 MSc student of Rlant Breeding, Graduate University of Advanced Technology, Kerman, Iran 2 Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran 3 Corresponding author, e-mail: maleki.li@gmail.com Acta agriculturae Slovenica, 115/1, 151-159, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 151 26. 03. 2020 12:05:55 H. SABET et al. 1 INTRODUCTION Eggplant (Solanum melongena L., 2n = 2x = 24) is one of the important plants of the Solanaceae family (Por-tis, et al., 2018), which grown for its often purple edible fruit. Different cultivars of eggplant have different sizes, shapes and colours ranging from oval or egg-shaped to long club-shaped; and from white, yellow, green through degrees of purple pigmentation to almost black (Amini-fard, et al., 2010). Eggplant after potatoes and tomatoes represents the third most important solanaceous crop species, and it is believed that eggplant is native to India and China (Magioli & Mansour, 2005). Its bulk production is concentrated in China, India, Iran, Turkey, Egypt, Italy and Spain (Portis, et al., 2018). Although the eggplant has a low calorie content, it is a rich source of magnesium, calcium, potassium and iron (Michalojc & Buczkowska, 2008). In addition, in traditional medicine, eggplant is used to treat diabetes, arthritis, asthma and bronchitis (Magioli & Mansour, 2005). Also, nasunin, as the major component of anthocyanin of eggplant, prevents lipid peroxidation (Igarashi, et al., 1993). The tissue culture technique allows researchers to grow and manipulate plants under in-vitro sterile conditions and can be very useful to plant breeders (Bridgen et al., 2018). In fact, this technique can provide the diversities needed for selection the desired traits by inducing somaclonal variation. On the other hand, by optimizing the complete regeneration of callus, it is possible to transfer the desired genes to the desired plants (Chakravarthi, et al., 2010). Due to the reasons mentioned above, studies of the full plant regeneration from explants are of special importance in plants. The fruit of the eggplant is found in the diet of many people in the world. For this reason, several studies have been carried out on regeneration of this plant through somatic embryogenesis using different explants such as leaf and cotyledon (Foo, et al., 2018), root (Franklin, et al., 2004) and hypocotyl (Statish, et al., 2015). However, it has been shown that plant regeneration can be affected by the cultivar. For example, Gandonou et al. (2005) showed that there is a significant difference between 9 genotypes of sugar beet in terms of callus induction capacity, embryogenic response and plant regeneration ability (Gandonou, et al., 2005), which indicates that the overall regeneration rate of the plant is genotype dependent. Hoque and Mansfield (2004), Schween and Schwen-kel (2003) and Zale et al. (2004), working respectively on Oryza sativa L., Primula ssp. and wheat came to the same conclusions (Hoque & Mansfield, 2004; Schween & Schwenkel, 2003; Zale, et al., 2004). For this reason, in this study, regeneration of a native cultivar of Iran, Ghal- 152 Acta agriculturae Slovenica, 115/1 - 2020 ami Varamin, which is commonly cultivated, was studied using somatic embryogenesis using hypocotyl explants. 2 MATERIALS AND METHODS 2.1 SEED SURFACE STERILIZATION AND IN- VITRO GERMINATION This research was carried out on an Iranian eggplant variety called 'Ghalami Varamin. Seeds of this variety were purchased from Pakan Bazr Esfahan Company (http://www.pakanbazr.com/en/). The seeds were surface-sterilized in 70 % ethanol for 30 seconds and then 2 % sodium hypochlorite for 10 minutes and rinsed 3 times with sterile distilled water. The MS medium was used for germination and seedlings growth of eggplant (Park & Facchini, 2000). The MS medium (Duchefa Company) was prepared as 4.43 g l-1. After adjusting to pH 5.7 and adding agar (7.5 g l-1) and sucrose (3 %), the culture medium was autoclaved. The MS medium was poured into autoclaved glass containers under sterile laminar hood. Seeds were cultured on the medium for germination, and placed in a growth chamber at 25 ± 2 °C with a 16/8 h light/dark period, light intensity of 32.38 ^mol s-1 m2-1. 2.2 CALLUS INDUCTION CULTURE MEDIUM After 15 to 20 days, hypocotyl explants were placed in MS culture medium containing different concentrations of auxin (NAA) and cytokinin (BAP) to produce callus (Table 1). All treatments were carried out in 3 replicates. Then, the percentage of callus induction and embryogenic calluses, and fresh mass and dry mass of calluses were measured. All data were analysed by ANOVA as a factorial experiment based on a completely randomized design. BAP as first factor and NAA as second factor had seven and three levels, respectively. The mean comparisons were done based on the Duncan's Multiple Range Tests (Duncan, 1955). 2.3 NUTRIENTS MEASUREMENT Embryogenic and non-embryogenic calli were washed in distilled water, oven dried at 70 °C for 48 h and grounded after being weighed. Different ions concentration was taken from the chloride acid (2 N) extract of the samples that were measured using atomic absorption spectrometer (Varian Spectra aa220; made in Italy). All data were analysed by ANOVA based on a completely AAS_vsebina_2020_115_1_koncna 180320.indd 152 26. 03. 2020 12:05:55 Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Table 1: Various hormonal treatments used to induce callus Hormonal combination code BAP( mg l-1 ) NAA(mg l-1 ) A 0 0 B 0 0.25 C 0 0.5 D 0.25 0 E 0.25 0.25 F 0.25 0.5 G 0.5 0 H 0.5 0.25 I 0.5 0.5 J 3.5 0 K 3.5 0.25 L 3.5 0.5 M 4 0 N 4 0.25 o 4 0.5 P 4.5 0 Q 4.5 0.25 R 4.5 0.5 s 5 0 T 5 0.25 U 5 0.5 randomized design with three replications. The mean comparisons were done based on the Duncan's Multiple Range Tests. 2.4 SHOOT PROLIFERATION MEDIUM The embryogenic calli isolated from the whole callus masses and transferred to the MS medium culture with different concentrations of BAP (3.5, 4 and 4.5 mg l-1) in order to shooting. Then, the percentage of shoot induction, shoot length, number of shoots and leaves were measured. In facts, the only treatments in this section were different concentrations of BAP that were analysed by ANOVA based on a completely randomized design with three replications. The mean comparisons were done based on the Duncan's Multiple Range Tests. 2.5 ROOT PROLIFERATION MEDIUM For further prolongation and development of stem and leaf system, shoots were transferred to MS medium supplemented with 2 mg l-1 gibberellic acid (GA3). For root induction, shoot lengths of 3 to 4 cm were transferred to MS culture media containing different concentrations of NAA (1, 1.5 and 2 mg l-1). The studied traits included root induction percentage and root length. The only treatments in this section were different concentrations of NAA that were analysed by ANOVA based on a completely randomized design with three replications. The mean comparisons were done based on the Duncan's Multiple Range Tests. 3 RESULTS AND DISCUSSIONS 3.1 CALLUS INDUCTION In this study, callus induction from hypocotyl explant was successfully performed. Callus initiation and appearance of embryogenic calli were occurred after 7 and 16 days, respectively (Figure 1 and 5). Embryogenic calli were seen in white, frangible and sponge-like forms so that after a while, the formations of shoot primordia in these calli were observed (Figure 1). But, non-embry-ogenic types appeared as compact, non-frangible, and sometimes greenish callus masses (Figure 1), similar to what was reported by other researchers (Corral-Martinez & Segui-Simarro, 2012; Rivas-Sendra, et al., 2015). Eventually, these types of calli were necrosed and died on the medium. The measured traits were the percentage of callus induction, percentage of embryogenic callus induction and fresh and dry mass of calluses. Variance analysis of data showed that there is a significant difference between different concentrations of BAP, NAA and their interactions at 1 % probability level (Table 2). The mean comparison of interaction effects of NAA*BAP for induction of callus showed that the highest percentage of callus induction (85 %) observed in the MS media supplemented with the combination of 0.5 mg l-1 NAA + 4 mg l-1 BAP, and the lowest percentage of callus induction (zero) observed in combinations of 0 mg l-1 BAP + 0 mg l-1 NAA, 4.5 mg l-1 BAP + 0 mg l-1 NAA and 5 mg l-1 BAP + 0.5 mg l-1 NAA (Figure 2). Zayova et al., (2008) reported the highest induction of callus using hypocotyls (63.3%) in the treatment MS media supplemented with 0.5 mg l-1 BAP + 2.0 mg l-1 NAA (Zayova, et al., 2008). In another study, callus induction using cotyledons as explant, the highest callus induction (100%) occurred in some concentrations of Kinetin, BAP and combinations of both (Foo, et al., 2018). In this study, the highest percentage of embryogenic calli (52 %) was related to 0.5 mg l-1 BAP + 0.25 mg l-1 NAA. In the treatment with BAP alone, induction of cal- Acta agriculturae Slovenica, 115/1 - 2020 153 AAS_vsebina_2020_115_1_koncna 180320.indd 153 26. 03. 2020 12:05:55 H. SABET et al. Figure 1: A: non-embryogenic callus. B: embryogenic callus Table 2: Variance analysis of traits related to the callus induction section Mean Square Percentage of embryo- Fresh mass Sources of variation Degrees of freedom Callusing percentage genic calluses (g) Dry mass (g) a (BAP) b (NAA) ab 6 2 12 42 0.35*" 0.17*" 0.19*" 0.02 0.11** 0.067*" 0.06** 0.01 0.027** 0.029** 0.018** 0.002 0.01** 0.017** 0.009** 0.002 7 Significant differences between the treatments at the level of 1 % lus in Ghalami Varamin cultivar occurred in all BAP concentrations except for the 4.5 mg l-1 BAP, and 0.5 mg l-1 was the best concentration (Figure 2). However, embryogenic calli occurred in 0.25, 0.50, 3.5 and 4 mg l-1 treatments, and no embryogenic calli were recorded in the other two concentrations (4.5 and 5 mg l-1). In the study of Foo et al., (2018), the highest shoot formation from the cotyledon explants was induced on Kinetin (2 mg l-1) alone (Foo, et al., 2018). In our study, in addition to the best hormone composition selected, BAP alone induced embryogenic calli too while Foo et al. (2018), showed that kinetin alone is sufficient to induce shoots from the cotyledon explants (Foo, et al., 2018). It is likely, different concentrations of auxin and cytokinin hormones in the two tissues caused different results. Therefore, it seems that in the eggplant, depending on the type of explant, certain concentrations of auxin or cytokinin alone have the potential to induce embryogenic calli. In our study, the NAA hormone (as a type of auxin) alone induced calli, but at a much lower rate than the BAP alone. In addition, the NAA hormone did not induce the embryogenic calli (Figure 2). Foo et al. (2018) 154 Acta agriculturae Slovenica, 115/1 - 2020 revealed that all concentrations of another type of auxin, Kinetin, (0.5, 1, 1.5 and 2 mg l-1) induced calli (Foo, et al., 2018). However, only two concentrations (1.5 and 2 mg l-1) induced embryogenic calli (Foo et al., 2018). Therefore, depending on the type of auxin, explant and the concentration used, this hormone alone can induce calli, especially embryogenic calli. In the case of fresh and dry weight of callus, the concentrations of 5 mg l-1 BAP + 0.25 mg l-1 NAA and 4.5 mg l-1 BAP + 0.25 mg l-1 NAA showed the highest results respectively (Figure 3). 3.2 COMPARISON OF IONS IN EMBRYOGENIC AND NON-EMBRYOGENIC CALLUS Concentration of five elements, calcium, magnesium, iron, potassium and manganese were measured in embryogenic and non-embryogenic calli. The results of variance analysis showed that there is no significant difference between the two embryogenic and non-embryo-genic calli for calcium, magnesium, and manganese. But AAS_vsebina_2020_115_1_koncna 180320.indd 154 26. 03. 2020 12:05:55 Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Figure 2: Mean comparison of the interaction effects of NAA*BAP for the percentage of callus induction and embryogenic callus induction. Black columns are percentage of callus induction and others are related to the percentage of embryogenic callus induction Figure 3: Mean comparison of the interaction effects of NAA*BAP for fresh and dry mass of calli. Black columns are fresh mass of calli trait and others are related to the dry mass of calli iron and potassium were dissimilar between embryogenic and non-embryogenic calli (Table 3). Our results revealed that the amount of both iron and potassium were more in non-embryogenic calli than the embryogenic ones (Fig. 4). Unfortunately, there is no report so far on the difference between the embryogenic and non-embry-ogenic calli in terms of the amount of these elements. 3.3 PLANT REGENERATION 3.3.1 Shooting Shooting was initiated by transferring embryogenic calli to MS medium containing 3.5, 4 and 4.5 mg l-1 BAP. For shooting regeneration, some traits including shooting percentage, number of shoots, Shoots length and number of leaves were measured. Variance analysis for these traits showed that there are significant differences between them (Table 4). The mean comparisons showed that the highest percentage of shooting (94%), number of shoots (35 shoots), shoot length (25 cm) and leaf number (14 leaves) were observed with a MS medium containing 4.5 mg l-1 BAP + 2 mg l-1 GA3 (Table 5). Different explants have been used for eggplant regeneration including hypocotyl (Mallaya & Ravishankar, 2013), leaf (Ray et al., 2011) and cotyledon (Rahman et al., 2006; Shivaraj & Rao, 2011), epicotyl and stem node, root (Ray et al., 2011). The highest percentages of shooting in various studies were different based on types of explant. Kaur et al. (2013) showed that cotyledon induced significantly the highest somatic embryogenesis on MS media fortified with 1.5 mg l-1 IBA + 1.0 mg l-1 BAP (89.62 %). In Acta agriculturae Slovenica, 115/1 - 2020 155 AAS_vsebina_2020_115_1_koncna 180320.indd 155 26. 03. 2020 12:05:56 H. SABET et al. Table 3: Variance analysis of iron and potassium amounts between embryogenic and non-embryogenic calli Mean Square Sources of variation Degrees of freedom K Fe treatment 1 81.99 ** 0.133 ** error 1 1.47 0.002 ** Significant differences at the level of 1 % 5 0 --I Non-embryonic embryonic calluses calluses Figure 4: Mean comparison of iron and potassium amounts between embryogenic and non-embryogenic calli Table 4: Analysis of variance of shooting traits Mean square Sources of variation Degrees of freedom shooting percentage number of shoots treatment 2 0.172** 540.7** error 6 0.001 10.33 Shoots length (cm) number of leaves 352.44** 121.33** 3.77 1.22 Significant differences at the level of 1 % Table 5: Mean comparison of different concentrations of BAP on traits related to shoot elongation number of leaves Shoots length (cm) number of shoots shooting percentage GA3(mg l-1) BAP(mg l-1) 2b 3b 14a 5c 9b 25a 9b 15b 35a 0.47c 0.62b 0.94a 3.5 4 4.5 Table 6: Analysis of variance of rooting traits Mean Square Sources of variation Degrees of freedom rooting percentage root length (cm) Treatment 2 0.06** 30.33** Error 6 0.001 1.22 ** Significant differences at the level of 1 % 156 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 156 26. 03. 2020 12:05:55 Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Table 7: Mean comparison of different concentrations of NAA on traits related to root induction NAA(mg l-1) rooting percentage root length (cm) 1 0.6b 4.5b 1.5 0.71b 5b 2 0.9a 10a leaf expiant, it was also the maximum on 1.5 mg l-1 IBA + 1.0 mg l-1 BAP (69.60 %). However, hypocotyl achieved the maximum of 38.41 % somatic embryogenesis on 0.5 mg l-1 IBA + 1.0 mg l-1 BAP (Kaur, et al., 2013). Mir et al., (2011) studied plant regeneration from different cultivars of eggplants with different explants. Their results showed that shoot induction percentage were different among the cul-tivars. The highest shoot induction percentage using hypocotyl (88.31 %), cotyledon (90 %) and root (60 %) were observed from PBSR-11 cultivar on MS medium containing 2.5 mg l-1 IAA + 0.5 mg l-1 BAP (Mir et al., 2011). The number of shoots and shoot length in the most studies were low. For example, Ray et al. (2011) used stem, root and leaf explants for eggplant regeneration. They observed that the highest number of shoot regenerated through callus from stem containing 2.0 mg l-1 BAP and 0.5 mg l-1 NAA was 3.4 (Ray, et al., 2011). Shivaraj and Rao (2011) used cotyledonary leaf as explants for eggplant regeneration. Their results showed the highest number of shoots was 23.3 at 2 mg l-1 BAP + 0.5 mg l-1 Kinetin (Kn), and the highest shoot length was 11.2 cm at 2 mg l-1 BAP + 1.5 mg l 1 Kn from cultivar Pusa Purple long (Shivaraj & Rao, 2011). Mallaya & Ravishankar (2013) showed that the maximum number of shoot buds elongated and shoot length using hypocotyl explant were 19 and 3.3 cm respectively on 0.5 mg l-1 TDZ + 0.1 mg l-1 GA3 (Mallaya & Ravishankar, 2013). By comparing the study of other researchers with this study, it can be concluded that the use of 4.5 mg l-1 BAP and 2 mg l-1 GA3 can provide a better result. 3.3.2 Rooting Variance analysis of data showed that there was a significant difference between treatments for rooting percentage and root length traits (table 6). In this study, rooting percentage and root length were 92 % and 10 cm respectively at 2 mg l-1 NAA (table 7; Figure 5). For root regeneration, the results of Shivaraj and Rao (2011) showed that the highest frequency and number of roots were 100% and 89.3 respectively on 3 mg l-1 IBA (Shivaraj & Rao, 2011). Mallaya, & Ravishankar (2013) reported that the highest number of root and root length were 4 and 5.6 cm respectively on 1 mg l-1 IBA (Mallaya & Ravishankar, 2013). 5 CONCLUSION In this study, the best treatments for plant regenera- Figure 5: A: embryogenic callus. B: production of multiple shoots from embryogenic callus. C: production of roots in elongated shoots. D: production of roots. E: production of adult eggplant Acta agriculturae Slovenica, 115/1 - 2020 157 AAS_vsebina_2020_115_1_koncna 180320.indd 157 26. 03. 2020 12:05:56 H. SABET et al. tion resulted in 35 shoots and 90 % rooting from embry-ogenic calli. With our protocol, we could produce adult eggplant from embryogenic calli and then transferred to the pots (Figure 5). It seems that this protocol could be an efficient protocol for plant regeneration of eggplant specially Ghalami Varamin cultivar. 6 ACKNOWLEDGEMENTS We are grateful to Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran. 7 REFERENCES Aminifard, M. H., Aroiee, H., Fatemi, H., Ameri, A. & Karim-pour, S. (2010). Responses of eggplant (Solanum melongena L.) to different rates of nitrogen under field conditions. Journal of Central European Agriculture, 11(4), 453-458. https:// doi.org/10.5513/JCEA01/11.4.863 Bridgen, M. P., Van Houtven, W., & Eeckhaut, T. (2018). Plant Tissue Culture Techniques for Breeding. Ornamental Crops. Springer, 127-144. https://doi.org/10.1007/978-3-319-90698-0_6 Corral-Martinez, P., & Segui-Simarro, J. M. (2012). Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). 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Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture (PCTOC), 122, 759-765. https://doi.org/10.1007/ s11240-015-0791-6 Satish, L., Rameshkumar, R., Rathinapriya, P., Pandian, S., Rency, A. S., Sunitha, T., & Ramesh, M. (2015). Effect of seaweed liquid extracts and plant growth regulators on in vitro mass propagation of brinjal (Solanum melongena L.) through hypocotyl and leaf disc explants. Journal of Applied Phycology, 27, 993-1002. https://doi.org/10.1007/s10811-014-0375-6 Schween, G., & Schwenkel, H. G. (2003). Effect of genotype on callus induction, shoot regeneration, and phenotypic stability of regenerated plants in the greenhouse of Primula ssp. Plant Cell, Tissue and Organ Culture, 72, 53-61. https://doi. org/10.1023/A:1021227414880 158 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 158 26. 03. 2020 12:05:55 Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar Shivaraj, G., & Rao, S. (2011). Rapid and efficient plant regeneration of eggplant (Solanum melongena L.) from cotyle-donary leaf explants. Indian Journal of Biotechnology, 10, 125-129. Zale, J. M., Borchardt-Wier, H., Kidwell, K. K., & Steber, C. M. (2004). Callus induction and plant regeneration from mature embryos of a diverse set of wheat genotypes. Plant Cell, Tissue and Organ Culture, 76, 277-281. https://doi. org/10.1023/B:TICU.0000009248.32457.4c Zayova, E., Nikova, V., Ilieva, K., & Philipov, P. (2008). Cal-lusogenesis of eggplant (Solanum melongena L.). Comptes Rendus De L Academie Bulgare Des Sciences, 61, 1485-1490. Acta agriculturae Slovenica, 115/1 - 2020 159 AAS_vsebina_2020_115_1_koncna 180320.indd 159 26. 03. 2020 12:05:56 AAS_vsebina_2020_115_1_koncna 180320.indd 160 26. 03. 2020 12:05:49 doi:10.14720/aas.2020.115.1.1300 Original research article / izvirni znanstveni članek Association of traits in Ethiopian fenugreek (Trigonella foenum-graecum L.) genotypes regarding to seed yield by using phenotypic data Yimam ALI ABTEW 1 2, Alemu ABATE 3 Received October 8, 2019; accepted January 26, 2020. Delo je prispelo 8. oktobra 2019, sprejeto 26. januarja 2020. Association of traits in Ethiopian fenugreek (Trigonella foe-num-graecum L.) genotypes regarding to seed yield by using phenotypic data Abstract: Shortage of information on association of traits is one of the problems in fenugreek productivity. Field experiment was implemented at Jamma district of South Wollo Administrative Zone of Amhara National Regional State, in 2018/19 main rainy season to examine the nature and extent of correlation, direct and indirect effects among yield and yield related traits. Sixty-two nationally collected fenugreek genotypes along with standard and local checks were evaluated in simple lattice design. Seed yield plot-1 was significantly and positively correlated to biomass yield-1 (r = 0.5) and harvest index (r = 0.6***) at genotypic level. Seed yield was also significantly and positively correlated with harvesting index (r = 0.6***) and weakly and negatively (r = -0.01) correlated to biomass yield at phenotypic level. Path coefficient analysis revealed that biomass (0.951), harvesting index (0.283) and pod length (-0.163) had contributed the maximum positive and negative direct effect on seed yield respectively, at phenotypic level. At genotypic level biomass yield ha-1 (0.816) and harvesting index (0.930) had contributed strong positive direct effect and plant height (-0.004) had revealed weak negative direct effect. Key words: correlation; path analysis; simple lattice Povezava med fenološkimi značilnostmi pri različnih genotipih sabljastega triplata (Trigonella foenum-graecum L.) Izvleček: Pomanjkanje informacij o povezanosti med fenološkimi lastnostmi je eden izmed problemov pri žlahtnenju sabljastega triplata. Za preučitev neposrednih in posrednih povezav med pridelkom in fenološkimi lastnostmi pri različnih genotipih je bil na območju Jamma, južno od upravnega območja Wollo, države Amhara, Etiopija, v glavnem deževnem delu sezone 2018/19 izveden poljski poskus. 62 genotipov sabljastega triplata, zbranih po vsej državi, je bilo ob standardnih in lokalnih preverbah ovrednoteno v nepopolnem bločnem poskusu. Pridelek semena na ploskev je bil statistično značilno pozitivno povezan s pridelkom biomase (r = 0,5) in žetvenim indeksom (r = 0,6***) na ravni genotipa. Pridelek semena je bil statistično značilno pozitivno povezan z žetvenim indeksom (r = 0,6***) in v šibki negativni korelaciji (r = -0,01) s pridelkom biomase na ravni fenotipov. Analiza neposrednih in posrednih vplivov nabora neodvisnih spremenljivk na odvisno spremenljivko je pokazala, da so biomasa (0,951), žetveni indeks (0,283) in dolžina stroka (-0,163) prispevali največji pozitivni in negativni neposredni učinek na pridelek zrnja na fenotipski ravni. Na genotipski ravni sta imela pridelek biomase na hektar (0,816) in žetveni indeks (0,930) močan, pozitivni neposredni učinek, višina rastlin je imela šibek neposredni negativni učinek (-0,004) na pridelek. Ključne besede: korelacija; analiza posrednega in neposrednega vpliva; nepopolni bločni poskus 1 Amhara Agricultural Research Institute, Sirinka Agricultural Research Center, Woldia, Ethiopia 2 Corresponding author, e-mail: yimamali121102@gmail.com 3 Bahir Dar University, College of Agriculture and Environmental Science, Bahir Dar, Ethiopia Acta agriculturae Slovenica, 115/1, 161-169, Ljubljana 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 161 26. 03. 2020 12:05:56 Y. ALI and A. ABATE 1 INTRODUCTION Fenugreek (Trigonella foenum-graecum L.) is an annual plant in the family Fabaceae, with leaves consisting of three small obovate to oblong leaflets. It is believed that fenugreek is native to Mediterranean region (Petropou-los, 2002). Correlation measures the mutual association between two variables but does not indicate the cause and effect relationship of traits contributing directly or indirectly towards economic yield (Shivanna et al., 2007). The value of correlation coefficient, which is a ratio of the covariance between the two variables and the geometric mean of their variances, ranges from -1 to +1, the extreme values indicating perfect negative and positive association, respectively (Gomez and Gomez, 1984). The correlations between characters indicate that the gene for the traits are either linked or are influenced by the same differences of environmental conditions (Falconer and Mackay, 1996). Phenotypic correlation is the observable correlation between two variables; it includes both genotypic and environment effects and genotypic correlation on the other hand is the inherent association between two variables; it may be either due to a pleiotropic action of genes, linkage or, more likely both (Singh, 1993). Environment plays an important role in correlation. In some cases, environment affects two traits simultaneously in the same direction or sometimes in different directions. Genetic and environmental causes of correlation combine together and give phenotypic correlation. The dual nature of phe-notypic correlation makes it clear that the magnitude of genetic correlation cannot be determined from pheno-typic correlation (Usman et al., 2006). Correlation coefficient analysis helps to determine the nature and degree of relationship between any two measurable characters. Knowledge of the correlations that exist between important characters may facilitate the interpretations of the results that are already obtained, and provides the basis for planning more efficient breeding program. However, as the number of independent variables influencing a particular dependent variable, a certain amount of interdependence is expected (Aryo et al., 1973). Mevlut et al. (2008) stated that correlation analyses are being widely used in many crop species by plant breeders to understand the nature of complex interrelationships among traits and to identify the sources of variation in yield. Yield is a quantitatively inherited trait; its expression is an outcome of complex interaction of several genes and environment. Therefore, proper understanding of association of different traits provides more reliable criterion for selection to achieve the goal of high yield (Mohammad et al., 2001). High yield through yield attributes, as primary interest in crop improvement, re- 162 Acta agriculturae Slovenica, 115/1 - 2020 quires understanding the magnitude of correlations among various yield traits (Tadele Taddese et al., 2009). Yield is the result of yield-correlated characters and some other undefined factors. Yield contributing traits can be ranked and specific characters producing a given correlation can be observed through path coefficient analysis (Ariyo et al., 1973). Therefore, the use of path coefficient analysis is important to come up with meaningful results of cause and effect. Therefore, this research is conducted to examine the nature and extent of correlation, direct and indirect effects among yield and yield related traits. 2 MATERIALS AND METHODS 2.1 DESCRIPTION OF THE EXPERIMENTAL SITE The experiment was conducted at Jamma research site of Sirinka Agricultural Research Center (SARC) at Jamma district during the growing season of 2018. Jamma is located at 10°27'N and 39°16'E on an altitude of 2622 meters above sea level, South Wollo, Amhara National Regional State, Ethiopia. Based on the last ten years (2008-2017) meteorological data obtained from Ethiopian Meteorological Agency, Kombolcha station, Jamma receives an average annual rainfall of 1047 mm and minimum and maximum temperature of 9.2 °C and 26.2 °C, respectively. Jamma is 120 km and 320 km away from Dessie and Addis Ababa, respectively. 2.2. EXPERIMENTAL MATERIALS, DESIGN AND PROCEDURE Sixty-two fenugreek accessions collected from De-bre-Zeit Agricultural Research Center (DZARC) along with local and standard checks were evaluated at Jamma testing site of SARC. The experiment was laid out using simple lattice design (8 x 8) on plot size of 1.6 m2, with an inter-row of 20 cm and intra-row spacing of 5 cm. The accessions were collected from different parts of the country. Clean fenugreek seeds were sown at distances of 20 and 5 cm between rows and plants, respectively, as per the national recommendation. Each genotype grew on a gross plot size of 1.6 m2 (0.8 m width x 2 m length). The distances between plots and blocks were 0.5 m and 1 m, respectively. Being fenugreek is leguminous crop, fertilizer was not applied at all. Weeding and thinning were carried out at the appropriate time. Data were obtainerd from the central two rows with net plot size of 0.8 m2 (0.4 m x 2 m). AAS_vsebina_2020_115_1_koncna 180320.indd 162 26. 03. 2020 12:05:56 Association of phonological traits in Ethiopian fenugreek (Trigonella foenum-graecum L.) genotypes [for seed yield] Table 1: Passport data of accessions S.no Accession Number Region Zone S.no Accession Number Region Zone 1 53003 Oromiya N/Shewa 33 201627 NA 2 53008 Amhara S/ Gondar 34 201632 NA 3 53009 Amhara S/Gondar 35 202121 NA 4 53014 Amhara S/ Wollo 36 202122 NA 5 53016 Oromiya W/ Harerge 37 202124 NA 6 53021 Amhara E/Gojam 38 202125 NA 7 53023 Oromiya N/ Shewa 39 202126 NA 8 53026 Amhara E/Gojam 40 202127 NA 9 53027 Amhara E/Gojam 41 202129 NA 10 53028 Amhara E/Gojam 42 202132 NA 11 53035 Amhara E/Gojam 43 202133 NA 12 53037 Amhara E/Gojam 44 207361 Amhara S/ Gondar 13 53039 Amhara E/Gojam 45 207362 Amhara N/ Gondar 14 53040 Amhara E/Gojam 46 207363 Amhara N/Gondar 15 53041 Amhara E/Gojam 47 207364 Amhara N/ Gondar 16 53042 Amhara E/Gojam 48 207365 Amhara N/ Gondar 17 53045 Amhara E/Gojam 49 207390 Amhara N/Gondar 18 53055 Amhara E/Gojam 50 207391 Amhara S/ Gondar 19 53056 Amhara E/Gojam 51 207394 Amhara S/ Gondar 20 53057 Amhara E/Gojam 52 208680 Oromiya E/ Harerge 21 53058 Amhara E/Gojam 53 210864 NA 22 53059 Amhara E/ Gojam 54 212549 Amhara N/ Shewa 23 53080 Amhara E/ Gojam 55 212552 Amhara N/ Shewa 24 53085 Oromiya Bale 56 212777 Amhara E/ Gojam 25 53086 Oromiya N/Shewa 57 213115 Amhara S/ Wollo 26 53094 SNNP S/Omo 58 213116 Amhara S/ Wollo 27 53097 Amhara E/ Gojam 59 214942 Amhara N/ Shewa 28 53098 Amhara E/ Gojam 60 215056 Oromiya Borena 29 53099 Amhara E/ Gojam 61 216898 Oromiya Arssi 30 53106 Amhara N/ Shewa 62 216899 Oromiya Arssi 31 53108 Amhara N/Gondar 63 Jamma 32 201577 NA 64 Local NA = not identified 2.3 DATA COLLECTION The following data were recorded from the central two rows, leaving a guard row from both sides of the plot either plot or plant basis's. Days to 50 % flowering: days to 50 % flowering was recorded as the number of days from planting to the time when 50 % of the plants in the plots produced flower. Days to 90 % maturity: was recorded as number of days from planting to the time when 90 % of the plants in the plot reach physiological maturity. Pod filling period: number of days from flowering or exertion of pods to the time when 50 % of the pod forms seeds. Biomass yield (above ground): it was taken as the total above-ground biomass weight of the plants from the central two rows. Total above-ground biomass was harvested and sun-dried and weighed using spring balance Acta agriculturae Slovenica, 115/1 - 2020 163 AAS_vsebina_2020_115_1_koncna 180320.indd 163 26. 03. 2020 12:05:56 Y. ALI and A. ABATE Seed yield: it was taken from the central two rows. Entire plants were harvested, threshed and winnowed. Clean seed were measured using electronic sensitive balance. Thousand seeds mass: thousand seeds were counted and weighed using electronic sensitive balance for each replication. Harvesting index: it was calculated as the ratio of seed yield to biomass yield in percent Plant height: plant height was measured from the main stem, measured from the ground level to the tip of the plant using measurement tape at 90 % physiological maturity. Pod length: pod length was measured from the tip to petiole of the pod at 90 % physiological maturity. Number of branches per plant: the total number of branches arising from the main stem was counted at 90 % physiological maturity. Number of pods per plant: the total number of pods per plant was counted at physiological maturity. Number of seeds per pod: the total number of seeds per pod was counted at physiological maturity. 2.4 DATA ANALYSES Correlations (genotypic and phenotypic) and path coefficient analysis were analyzed using SAS statistical software package, version 9.0 as per Gomez and Gomez (1984). 2.5 PHENOTYPIC AND GENOTYPIC CORRELATION COEFFICIENTS Phenotypic correlation, the observable correlation between two variables, which includes both genotypic and environmental components between two variables, was estimated using the formula suggested by Miller et al. (1958). The calculated value was compared with the tabulated 't' value at 5 % or 1 % level of significance for both phenotypic and genotypic correlations. 2.6 PATH COEFFICIENT ANALYSIS Associations of yield with its components were estimated using correlation and path analysis. The use of path analysis requires a cause and effect situation among the variables. Path coefficient analysis is calculated using the formula suggested by Dewey and LU (1959) to assess direct and indirect effects of different traits on seed yield as: r = p + Z rikPkj Where r.. is mutual association between the inde- i) pendent traits (i) and the dependent trait ()) as measured by the correlation coefficient, pi) is component of direct effect of the independent trait (i) on the dependent variable ()); and rik p^ is the components of indirect effect of a given independent trait (i) on the dependent traits ()) via all other independent traits (k). 3 RESULT AND DISCUSSION 3.1 CORRELATION COEFFICIENT ANALYSIS Yield is a complex character governed by several other yield attributing characters. Since, most of the yield attributing characters are quantitatively inherited and highly affected by environment, it is difficult to judge whether the observed variability is heritable or not. Correlation studies are helpful in determining the components of complex traits like yield. The correlation coefficient is the measures of degree of symmetrical association between two traits and helps us in understanding the nature and magnitude of association among yield and yield components. Association between any two traits or among various traits is of immense importance to make desired selection of combination of traits (Ahmad et al., 2003). Therefore, phenotypic and genotypic correlation coefficients were estimated for all pairs of traits and results are presented in Table 2. The magnitudes of genotypic correlation coefficients for most of the characters were higher than their corresponding phenotypic correlation coefficients, except few cases, which indicate the presence of inherent or genetic association among various characters. The degree of association between two characters was measured by the correlation coefficient. Phenotypically seed yield had weak to strong positive correlation with days to flowering, pod filling period, plant height, number of branch per plan, number of pod per plant, harvesting index and thousand seed mass (r = 0.18, 0.119, 0.129, 0.115, 0.222, 0.6*** and 0.158 respectively). While weak and negatively correlated with days to maturity, biomass yield, number of seed per pod and pod length (r = -0.04, -0.02, -0.08 and -0.08 respectively). Harvesting index revealed very highly significance strong negative and positive phenotypic correlation with biomass yield and seed yield (r = -0.6*** and 164 Acta agriculturae Slovenica, 115/1 - 2020 AAS_vsebina_2020_115_1_koncna 180320.indd 164 26. 03. 2020 12:05:56 Association of phonological traits in Ethiopian fenugreek (Trigonella foenum-graecum L.) genotypes [for seed yield] 0.6***) respectively. It also strongly negative correlated with biomass (r = -0.8***) and weakly correlated with the rest traits. Biomass yield had only weak positive pheno-typic correlation with pod filling period and plant height, while it correlates negatively and weakly with the rest traits except harvesting index. Pod length revealed significance positive and negative phenotypic correlation with plant height and number of seed per plant and date of maturity (r = 0.31*, 0.43** and -0.26*) respectively. Plant height had positive significance positive phenotypic correlation with number of branches (r = 0.3*). Therefore, phenotypically traits like number of pods plant-1, plant height, pod length and harvesting index could be utilized in breeding program to improve fenugreek genotypes for higher yield. Selection simultaneously based on these characters would be effective and a worthwhile for increasing seed yield and can be taken as primary selection criteria in fenugreek. Genotypically seed yield revealed positive and significance correlation with biomass and harvesting index (r = 0.5** and 0.6***). It is also weak negatively correlated with days to maturity, pod filling period and number of seed per plant, while weak and positive correlation with the rest of the traits. Pod length had positive significance genotypic correlation with days to flowering and negative significance with days to maturity and plant height (r = 0.4**, -0.29* and -0.25*). Number of seed per plant had strong significance negative correlation with thousand seed mass (r = -0.4**), while number of branches had strong positive correlated with plant height and negatively significant correlated with pod filling period (r = 0.5** and-0.28*). Biomass yield revealed positively correlated with all traits except days to maturity. Generally genotypic correlation was higher magnitude than their phenotypic correlation and most traits weakly correlated to each other. Therefore, genotypically improving of biomass yield and harvesting index will leads to develop high yielding fenugreek genotypes. Generally, in the present study, seed yield revealed strong positive correlations with harvesting index both at genotypic and phenotypic levels, implying pleiotropic effect of harvesting index with seed yield. Hence, selection genotypes having high harvesting index would improve seed yield of fenugreek. Biomass yield also affected seed yield positively and significantly at genotypic level. In agreement with Mahendra (2015) reported, negative and weakly phenotypic correlation was achieved between days to flowering to number of branches per plant, number of seeds per plant, pod length and thousand seed mass, while opposite in level of significance and relation of days to flowering with plant height and harvesting index. Krishan et al. (2013) reported seed yield had positive correlation with plant height, number of seed plant-1 and number of branch per plant at both levels. Betelhiem Belete (2018) also reported similarly in days to flowering to days to maturity, thousand seed mass to days to flowering, days to flowering to seed yield, seed yield to thousand seed mass and days to flowering to number of seed plant-1 both at phenotypic and genotypic level. An-buha et al. (2013) also reported similar result that positive correlation with days to flowering to number of pods plant-1 at genotypic level, significant negative and positive correlation with days to flowering to pod length and plant height to number of pods plant-1 at genotypic and phenotypic level respectively and number of seed plant-1 had positive correlation to plant height both at genotypic and phenotypic level. 3.2. PATH COEFFICIENT ANALYSIS Path coefficient analysis was computed to estimate the contribution of individual characters to seed yield. It is performed to understand the causes and effects of chain relationships of different yield contributing characters with yield. The path coefficient analysis was conducted using seed yield as dependent variable and all other traits studied as independent variables. The results of phenotypic and genotypic path coefficient analysis are presented in Table 3 and 4 below. 3.3 PHENOTYPIC DIRECT AND INDIRECT EFFECTS OF OTHER TRAITS ON SEED YIELD Correlation coefficients were further partitioned into direct and indirect effects. Biomass yield (0.95) had exerted the highest positive direct effects on seed yield. Plant height (0.15), number of branches per plant (0.05), thousand seed mass (0.07) and harvesting index (0.28) exerted positive direct effects on seed yield (Table 3). High values of direct effects suggested that the true relationship and direct selection for these traits may also increase and give better response for improvement of seed yield and can be major selection criteria in fenugreek breeding programs. Number of pods per plant (-0.14), number of seeds per pod (-0.01) and pod length (-0.16) on the other hand, exerted negative phenotypic direct effect and unfavourable effect on seed yield (Table 3). The negative phenotypic direct effects on seed yield indicated that selection for these traits would not be rewarding for yield improvement. The direct effect of biomass yield (0.95) and number of pods per plant (-0.014) but their association to seed yield was (r= -0.14 and 0.12) opposite in direction. Indicating that fenugreek genotypes having maximum biomass yield and number of pods would Acta agriculturae Slovenica, 115/1 - 2020 165 AAS_vsebina_2020_115_1_koncna 180320.indd 165 26. 03. 2020 12:05:56 Y. ALI and A. ABATE -o 3 tg M a c g c o c o g -a Oi -d c c o g jy o c QJ 0