A Spletna izdaja – Online: iSSn 1854-1941 Biotehniška fakulteta Univerze v Ljubljani Biotechnical Faculty University of Ljubljana Acta agriculturae Slovenica iSSn 1581-9175 · letnik / Volume 115 · številka / number 2 · 2020 Acta agriculturae Slovenica • eiSSn 1854-1941 • 117 – 1 • Ljubljana, marec 2021 A c t A A g r ic U L t U r A e S L O V e n ic A 117•1 2021 115 2 2020 VSeBinA / cOntentS ActA AgricULtUrAe SLOVenicA Antonio AGUILAR-LOPEZ, Salvador GONZÁLEZ-ANDRADE, Aleš KUHAR 215 Estimation of Engel curves for household expenditure on dry bean and processed bean in Mexico Ocena Engelovih krivulj za izdatke gospodinjstev za suhi in predelani fižol v Mehiki Mojca KOROŠEC, Jasna BERTONCELJ 223 Pomen čebeljih pridelkov v humani prehrani The importance of bee products in human nutrition Vaibhav Bhagwan AWACHAT, Arumbackam Vijayarangam ELANGOVAN, Olajide Mark SOGUNLE, Corbon Godfrey DAVID, Jyotirmoy GHOSH, Shivakumar Nisarani Kollurappa GOWDA, Subrat Kumar BHANJA, Samir MAJUMDAR 237 Influence of in ovo and pre-starter zinc and copper supplementation on growth performance and gastrointestinal tract development of broiler chickens Vpliv dodatka cinka in bakra v jajce in v krmo po izvalitvi na rast in razvoj prebavil pri brojlerskih piščancih Babasola Daniel ADEWALE and Beatrice Abanum NDUKA 247 Genotype and within-pod bean position microenvironment effect on seed choice for raising cocoa (Theobroma cacao L.) seedlings Genotip in mikrookolje glede na položaj semena znotraj ploda vplivata na izbor semen kakavovca (Theobroma cacao L.) za vzgojo sadik Ildar GANEEV, Khasan KARIMOV, Shamil FAYZRAKHMANOV, Ilgam MASALIMOV, Valeri PERMYAKOV 261 Intensification of the drying process of small seed oilseeds using microwave electromagnetic radiation Pospeševanje sušenja majhnih semen oljnih poljščin z mikrovalovnim elektromagnetnim sevanjem Khaled A.A. ABDELAAL, Sahar H. RASHED, Adel RAGAB, Akbar HOSSAIN, Ayman EL SABAGH 273 Yield and quality of two sugar beet (Beta vulgaris L. ssp. vulgaris var. altissima Döll) cultivars are influenced by foliar application of salicylic acid, irrigation timing, and planting density Vpliv foliarnega dodajanja salicilne kisline, časa namakanja in gostote setve na pridelek in kakovost dveh sort sladkorne pese (Beta vulgaris L. ssp. vulgaris var. altissima Döll) Insha YOUSUF and Abdul A. BUHROO 283 Seasonal incidence and bionomics of rose aphid, Macrosiphum rosae (Linnaeus, 1758), (Hemiptera: Aphididae) in Kashmir, India Sezonsko pojavljanje in bionomika vrtnične uši (Macrosiphum rosae (Linnaeus, 1758), Hemiptera: Aphididae) v Kašmirju, Indija Seda TUNÇAY ÇAĞATAY, Gülşah ÇALIK KOÇ, Fereshteh REZAEİ, Özlem DARCANSOY İŞERI, Feride İffet ŞAHIN, Mehmet HABERAL 297 Evaluation of production conditions of tomato grafted with different tobacco rootstocks and determining nicotine content and quality of fruit Ovrednotenje pridelovalnih razmer paradižnika cepljenega na različne podlage tobaka in določitev vsebnosti nikotina in kakovosti plodov Asgar EBADOLLAHI 307 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Na estragolu bogato eterično olje vrtnega šetraja (Satureja hortensis L.) kot okolju prijazna alternativa sintetičnim insekticidom pri zatiranju dveh vrst skladiščnih škodljivih žuželk (Nadaljevanje na notranji strani zadnje platnice / Continued on inside back cover) Acta agriculturae Slovenica Letnik / Volume 117 · Številka / Number 1 · 2021 eISSN 1854-1941 Glavna in odgovorna urednika / Editors-in-Chief Franc BATIČ, rastlinska pridelava / plant production Peter DOVČ, živalska prireja / animal production Področni uredniki / Section Editors Franc BATIČ (botanika in ekologija rastlin / botany and plant ecology), Majda ČERNIČ-ISTENIČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural development), Jure ČOP (pridelovanje krme / fodder production), Zalika ČREPINŠEK (agrometeorolologija / agrometeorology), Marko FLAJŠMAN (poljedelstvo / field crops), Matjaž GLAVAN (urejanje kmetijskih zemljišč / agricultural land management), Helena GRČMAN (pedologija / soil science), Andrej GREGORI (gojenje gob / mushrooms growing), Metka HUDINA (hortikultura / horticulture), Anton IVANČIČ (genetika in biotehnologija / genetics and biotechnology), Jernej JAKŠE (genetika in biotehnologija / genetics and biotechnology), Damjana KASTELEC (statistika / statistics), Aleš KOLMANIČ (poljedelstvo / field crops), Zlata LUTHAR (genetika in biotehnologija / genetics and biotechnology), Andrej LAVRENČIČ (pridelovanje krme / fodder production), Marina PINTAR (urejanje kmeti- jskih zemljišč / agricultural land management), Andrej SIMONČIČ (varstvo rastlin / plant protection), Stanislav TRDAN (var- stvo rastlin / plant protection), Andrej UDOVČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural de- velopment), Andreja URBANEK-KRANJC (fiziologija rastlin / plant physiology), Rajko VIDRIH (živilstvo / food technology), Dominik VODNIK (fiziologija rastlin / plant physiology), Filip VUČANJK (kmetijsko strojništvo / agricultural machinery) Peter DOVČ (živalska biotehnologija / animal biotechnology, populacijske študije / population studies, genomika / genomics), Milena KOVAČ (selekcija in biometrija / selection and biometry), Janez SALOBIR (prehrana / nutrition) Mednarodni uredniški odbor / International Editorial Board Dunja BANDELJ (Koper, Slovenia), Michael BLANKE (Bonn, Germany), Ivan N. FESENKO (Orel, Russia), Marko FLAJŠMAN (Ljubljana, Slovenia), Jürg FUHRER (Liebefeld-Bern, Switzerland), Helena GRČMAN (Ljubljana, Slovenia), Metka HUDINA (Ljubljana, Slovenia), Anton IVANČIČ (Maribor, Slovenia), Lučka KAJFEŽ BOGATAJ (Ljubljana, Slovenia), Damijana KASTELEC (Ljubljana, Slovenia), Iztok Košir (Žalec, Slovenija), Chetan KESWANI (Varanasi, India), Ivan KREFT (Ljubljana, Slovenia), Jaromír LACHMAN (Prague, Czech Republic), Mario LEŠNIK (Maribor, Slovenia), Zlata LUTHAR (Ljubljana, Slovenia), Senad MURTIĆ (Sarajevo, Bosnia and Herzegovina), Alessandro PERESSOTTI (Udine, Italy), Hardy PFANZ (Essen, Germany), Slaven PRODANOVIĆ (Belgrade, Serbia), Naser SABAGHNIA (Maragheg, Iran), Olalekan Sulei- man SAKARIYAWO (Abeokuta, Nigeria), Andrej SIMONČIČ (Ljubljana, Slovenia), Giuseppe SORTINO (Palermo, Italy), Bojan STIPEŠEVIĆ (Osijek, Croatia), Massimo TAGLIAVINI (Bolzano, Italy), Željko TOMANOVIĆ (Beograd, Serbia), Stanislav TRDAN (Ljubljana, Slovenia), Andrej UDOVČ (Ljubljana, Slovenia), Rajko VIDRIH (Ljubljana, Slovenia), Dominik VODNIK (Ljubljana, Slovenia), Alena VOLLMANNOVA (Nitra, Slovak Republic) Drago BABNIK (Ljubljana, Slovenia), Tomaž BARTOL (Ljubljana, Slovenia), Michel BONNEAU (Saint Gilles, Belgium), Milena KOVAČ (Ljubljana, Slovenia), Amarendra Narayan MISRA (Balasore, Orissa, India), Zdenko PUHAN (Zürich, Switzerland), Dejan ŠKORJANC (Maribor, Slovenia), Jernej TURK (Maribor, Slovenia) Tehnični uredniki / Technical Editors Karmen STOPAR, Maja MIHIČINAC KRISTAN, Jože STOPAR Oblikovanje / Graphic art and design Milojka ŽALIK HUZJAN Jezikovni pregled / Proofreading Avtorji v celoti odgovarjajo za vsebino in jezik prispevkov / The authors are responsible for the content and for the language of their contributions. Založnik in izdajatelj / Publisher and Issuer Univerza v Ljubljani, Biotehniška fakulteta / University of Ljubljana, Biotehnical Faculty Za izdajatelja / For the Issuer: Nataša POKLAR ULRIH, dekanja Biotehniške fakultete UL / the Dean of the Biotehnical Faculty UL Naslov Uredništva / Editorial Office Address Univerza v Ljubljani, Biotehniška fakulteta, Acta agriculturae Slovenica Jamnikarjeva ulica 101, SI-1000 Ljubljana E-naslov / E-mail Acta.Agriculturae.Slovenica@bf.uni-lj.si Spletni naslov / Web address http://ojs.aas.bf.uni-lj.si Avtorske pravice in licenca / Copyright and licence Licenca Creative Commons / Open access licence CC BY-NC-ND 4.0 Dovoljeno je kopiranje in razširjanje vsebin v kakršnemkoli mediju in obliki, če se upoštevajo pogoji licence. Priznanje av- torstva – avtor mora biti primerno naveden. Nekomercialno – prepovedana je uporaba objavljenih člankov v komercialne namene. 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Acta agriculturae Slovenica izhaja s finančno pomočjo / is published with the financial support: Javne agencije za raziskovalno dejavnost Republike Slovenije / Slovenian Research Agency. Acta agriculturae Slovenica je vključena v / is included into: Scopus (SJR, SNIP), DOAJ, WOS Zoological Records, CrossRef, CAB Abstracts, FSTA, Google Scholar, dLib, COBISS. Ovitek: Gomolj krompirja, poškodovan od strun (Agriotes spp., Elateridae); (foto: Stanislav Trdan, 1–11) Cover: Potato tuber damaged by wireworms (Agriotes spp., Elateridae); (photo: Stanislav Trdan, 1–11) Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Comperative study of aboveground biomass and carbon storage between Tembawang and conventional rubber agroforestry in West Kalimantan Indonesia Primerjalna raziskava nadzemne biomase in shranjevanja ogljika med Tembawangom in konvencionalnim pridobivanjem kavčuka v Zahodnem Kalimantanu, Indonezija Rafdinal RAFDINAL, Ramadanil PITOPANG, Riza LINDA, Adityo RAYNALDO, Eko SUBRATA 1–7 Nutritional indices and biochemical profile of Helicoverpa armigera [Hübner (1808)] on different groundnut genotypes Prehranjevalni indeksi in biokemični profil južne plodovrtke, Helicoverpa armigera [Hübner (1808)], na različnih genotipih arašidov Abdul Rasheed WAR, Barkat HUSSAIN, Suraj Prashad MISHRA, Rajendra S MUNGHATE, Hari C SHARMA 1–9 Assessement of control strategy by spraying low doses of sugars on apple orchard against Cydia pomonella (Linnaeus, 1758.) Ocenitev nadzorovanja jabolčnega zavijača (Cydia pomonella (Linnaeus, 1758)) s škropljenjem z majhnimi odmerki sladkorjev Abdelkader Nabil TIFFRENT, Nadia LOMBARKIA 1–6 Exploiting heterosis and combining ability in two-line hybrid rice Uporaba heteroze in kombinacijske sposobnosti pri dveh linijah hibridnega riža Mahmoud M. GABALLAH, Aziz F. EL-EZZ, Adel M. GHONEIM, Baochang YANG, Langtao XIAO 1–16 Bionomics of slender burnished brass (Thysanoplusia orichalcea [Fabricius, 1775], Lepidoptera: Noctuidae) on potato (Solanum tuberosum L.) in Kashmir Bionomija sovke Thysanoplusia orichalcea (Fabricius, 1775) (Lepidoptera: Noctuidae) na krompirju (Solanum tuberosum L.) v Kašmirju Gousul NISA, Abdul A. BUHROO 1–8 Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage Učinek solnega stresa na fiziološki in biokemični odziv treh genotipov koruze v zgodnji razvojni stopnji semenke Ali DOĞRU 1–11 Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new records Pestrost listnih uši (Hemiptera: Aphididae) v nasadih krompirja na območju Tizi-Ouzou (Severna Alžirija) z novimi najdbami Samia AIT AMAR, Karima BENOUFELLA-KITOUS 1–9 Acta agriculturae Slovenica Volume / Letnik 117 · Number / Številka 1 · 2021 Effect of selected Rwandan wheat varieties on the physicochemical characteristics of whole wheat flour Vpliv izbranih ruandskih sort pšenice na fizikalno-kemijske lastnosti polnozrnate pšenične moke Bernard RWUBATSE, Michael Wandayi OKOTH, Angela Adhiambo ANDAGO, Sophia NGALA, Anastase KIMONYO, Clement BITWAYIKI 1–9 Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria Vplivi datuma setve na rast in razvoj koruze (Zea mays L.) v območjih deževnega gozda jugozahodne Nigerije Chris Adegoke FAYOSE, Morakinyo Abiodun Bamidele FAKOREDE 1–9 Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) Solni in sušni stres se izražata kot oksidacijski stres in vplivata na vzorce beljakovin v koreninah in listih dateljeve palme (Phoenix dactylifera L.) Hussein J. SHAREEF, Jameel M. AL-KHAYRI 1–10 Analysis of gliadin patterns and diversity in Triticum polonicum L. accessions from Ethiopia Analiza vzorcev gliadinov in raznolikosti akcesij poljske pšenice (Triticum polonicum L.) v Etiopiji Eleni SHIFERAW 1–6 The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) Učinki biofortifikacije semen s cinkom in uporaba NPK gnojil na rast in pridelek sezama (Sesamum indicum L.) Ehiokhilen Kevin EIFEDIYI, Grace Adejoke ILORI, Henry Emeka AHAMEFULE, Abduquadir Yusuf IMAM 1–11 Induction of defence related enzymes and biocontrol efficacy of Trichoderma harzianum in tomato plants infected with Fusarium oxysporum and Fusarium solani Vzpodbuditev aktivnosti encimov povezanih z obrambo in učinkovitost biokontrole z glivo Trichoderma harzianum Rifai v paradižniku okuženem z glivama Fusarium oxysporum Schlecht. emend. Snyder & Hansen in Fusarium solani (Mart.) Sacc. Tavga Sulaiman RASHID, Sirwa Anwar QADIR, Hayman Kakakhan AWLA 1–6 The role of exogenous silicon to mitigate Al2O3 nanoparticle-induced toxicity in barley (Hordeum vulgare L.) Vloga dodajanja silicija za preprečevanje strupenosti nano delcev Al2O3 pri ječmenu (Hordeum vulgare L.) Ghader HABIBI, Maryam SHAHINFAR 1–9 Variability of some isolates of Prunus necrotic ringspot virus and Prune dwarf virus infecting sour and sweet cherry in Ukraine Variabilnost izolatov virusa nekrotične obročkaste pegavosti breskve (PNRSV) in virusa pritlikavosti slive (PDW) na višnji in češnji v Ukrajini Liliia PAVLIUK, Kateryna UDOVYCHENKO, Iryna RIABA, Mykola BUBLYK 1–9 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices Odkrivanje tolerance na sušo v mednarodni zbirki genotipov žafranike (Carthamus tinctorius L.) na osnovi izbranih indeksov Pooran GOLKAR, Esmaeil HAMZEH, Seyed Ali Mohammad MIRMOHAMMADY MAIBODY 1–11 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria Ali ima enakopravnost žensk v gospodinstvih vpliv na dobrobit otrok? Primeri iz kmečkih gospodinjstev iz države Ogun, Nigerija Chioma Patricia ADEKUNLE, David Alaba ALORI, Adebayo Augustine KUTU 1–13 Review Article / Pregledni znanstveni članek Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae) Comparison of traditional and modern methods for identification of economically important wireworm species (Coleoptera: Elateridae) Eva PRAPROTNIK, Jaka RAZINGER, Stanislav TRDAN 1–11 Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) Management methods for marmorated stink bug (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) Žiga LAZNIK, Stanislav TRDAN 1–11 Acta agriculturae Slovenica, 117/1, 1–7, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1859 Original research article / izvirni znanstveni članek Comperative study of aboveground biomass and carbon storage between Tembawang and conventional rubber agroforestry in West Kalimantan Indonesia Rafdinal RAFDINAL 1, 2, Ramadanil PITOPANG 3, Riza LINDA 1, Adityo RAYNALDO 4, Eko SUBRATA 5 Received September 01, 2020; accepted December 08, 2020. Delo je prispelo 01. septembra 2020, sprejeto 08. decembra 2020. 1 Tanjungpura University, Faculty of Mathematics and Natural Sciences, Department of Biology, Pontianak, West Kalimantan, Indonesia 2 Corresponding author, e-mail: rafdinal@fmipa.untan.ac.id 3 Tadulako University, Faculty of Mathematics and Natural Sciences,Department of Biology, Palu, Central Sulawesi, Indonesia 4 OSO University, Department of Marine Science, Pontianak, West Kalimantan, Indonesia 5 Muhammadiyah Sumatera Barat University, Faculty of Forestry, Department of Forestry, Padang, West Sumatra, Indonesia Comperative study of aboveground biomass and carbon stor- age between Tembawang and conventional rubber agrofor- estry in West Kalimantan Indonesia Abstract: In the era of intensive oil palm and rubber plantations in Kalimantan, some local communities of Dayak’s tribe in West Kalimantan preserved the traditional agroforestry system “Tembawang”. In the last two decades, rubber has been planted traditionally by local communities since the expansion of rubber industries. This study aimed to compare tree above ground biomass (AGB) distribution and carbon storage in dif- ferent DBH (diameter at breast height) classes between Tem- bawang and conventional rubber plantation in West Kaliman- tan, Indonesia. Vegetation transect analysis was carried out on two types of traditional agroforestry namely Tembawang and conventional rubber. AGB estimation was based on the exist- ing allometric, carbon storage was estimated from the percent- age of biomass. Total AGB of Tembawang was higher than conventional rubber plantation and significantly different (p < 0.01). The highest AGB accumulation both Tembawang and conventional rubber was found at above 50 cm diameter class. The aboveground carbon storage from Tembawang and con- ventional rubber plantation were 90.26 and 42.01 Mg C ha-1, respectively. The highest contribution to carbon storage was found at above 50 cm diameter class, estimated 62.58 % from Tembawang and 49.24 % from conventional rubber. AGB and carbon storage at traditional agroforestry in West Kalimantan were greater than varied different agroforestry system, also the estimated value was closed to tropical secondary forests. Tem- bawang agroforestry has good potential contribution to carbon storage and conservation of native fruit trees of Kalimantan. Key words: carbon storage; Dayak’s tribe; ethnoecology; plantation; Tembawang Primerjalna raziskava nadzemne biomase in shranjevanja ogljika med Tembawangom in konvencionalnim pridobivanjem kavčuka v Zahodnem Kalimantanu, Indonezija Izvleček: V obdobju intenzivnega gojenja oljne palme in plantaž kavčukovca v Kalimantanu so nekatere lokalne skupnosti plemena Dajakov v Zahodnem Kalimantanu ohranile tradicionalni kmeti- jsko gozdarski sistem (agroforestry), imenovan “Tembawang”. Kljub povečevanju industrije kavčuka v zadnjih desetletjih so nekatere lo- kalne skupnosti ohranile tradicionalno gojenje kavčukovca. V raziska- vi sta bili primerjani nadzemna biomasa dreves (AGB) in porazdelitev ogljika v različnih debelinskih razredih dreves (DBH= premer drevesa na prsni višini) med sistemom Tembawang in konvencialno pridelavo kavčuka na plantažah v Zahodnem Kalimantanu, Indonezija. Analiza vegetacije je bila opravljena v dveh transektih tradicionalnega pri- dobivanja kavčuka imenovenega Tembawang in v konvencionalnih plantažah kavčukovca. Določitev nadzemne biomase je temeljila na alometričnih enačbah, zaloge ogljika so bile ocenjene iz odstotkov biomase. Celokupna nadzemna biomasa je bila v sistemu Tembawa- ng večja kot v konvencionalnih plantažah kavčukovca in značilno različna (p < 0,01). Največja akumulacija nadzemne biomase je bila v obeh sistemih, Tembawangu in pri konvencionalni pridelavi kavčuka v debelinskih razredih nad 50 cm. Nadzemna zaloga ogljika je bila v sistemu Tembawang 90,26 Mg C ha-1 in 42,01 Mg C ha-1pri konven- cionalni pridelavi. Največji delež shranjenega ogljika je bil v debelin- skih razredih dreves nad 50 cm, ocenjen na 62,58 % pri Tembawangu in 49,24 % pri konvencionalni pridelavi kavčuka. Nadzemna biomasa in zaloga ogljika sta bili v tradicionalnem kmečkem gozdarstvu Za- hodnega Kalimantana večji kot v različnih konvencionalnih sistemih, tudi ocenjene vrednosti so bile blizu tistim v drugotnih tropskih goz- dovih. Tembawang sistem kmečkega gozdarstva ima dober potencial za shranjevanje ogljika in ohranjanja samoniklih sadnih dreves v Ka- limantanu. Ključne besede: shranjevanje ogljika; pleme Dayak; etnoekolo- gija; plantaža; Tembawang Acta agriculturae Slovenica, 117/1 – 20212 R. RAFDINAL et al. 1 INTRODUCTION Forest ecosystems are an important component of the terrestrial ecosystem. They contain more than half of biodiversity, and highly contribute to stored carbon. Trees are the main component in forest ecosystems con- taining about 677 Pg or 80 % of the total forest biomass (Kinderman et al., 2008). Forest ecosystems absorb car- bon dioxide (CO2) from the atmosphere through pho- tosynthesis and increase in the average concentration of CO2 in the atmosphere can be reduced through carbon sequestration (C) (IPCC, 2007). Carbon sequestration in forest ecosystems has be- come an important issue both in global climate change, climate discussions and in forest ecosystem studies. For- est conversion into other land-uses such as agriculture, settlement, and also forest degradation can pose a serious threat to sustaining carbon storage. Carbon storage in a forest ecosystem is mainly determined by growth factors, mortality, decomposition, disturbance, forest succession, and climate variations (Gough et al., 2008). Indonesia has the third-largest area of tropical for- est after Brazil and Congo, with an area of 92.133 M ha (FAO, 2020). Furthermore, the potential for carbon stocks in natural forests in Indonesia is large, ranging from 7.5-264.70 t C ha-1 (Ministry of Forestry, 2010). In the period of 2000 to 2015, the rate of deforestation of primary forests in Indonesia is also very high (FAO, 2015). Deforestation and forest degradation in Indonesia are generally caused by the settlement expansion driven by population growth, forest logging, and conversion to plantation / agricultural land (Prasetyo et al., 2011). Kalimantan (Borneo) is the second-largest island in Indonesia, with a vast expanse of natural forests with high diversity and endemicity (Rhee et al., 2004). Among 109 tree families found in Kalimantan, Dipterocarpace- ae family was dominated by the forest composition and more than half species in this group were endemic spe- cies in Kalimantan (Soepadmo and Wong, 1995). De- forestation in tropical forests in Kalimantan from 2000- 2017 reached 6.04 M ha, of which half was caused by land conversion to the plantation industry (Gaveau et al., 2017). According to Carlson et al. (2012), by 2020 the ex- pansion of palm oil industries is projected to contribute about 0.12-0.15 Gt C year-1 CO2 equivalent emissions. Tembawang represents the term of agroforestry management practices by Dayak’s tribe in Kalimantan. This term also represents the other management systems by local communities such as forests, plantations, rice fields, and settlements (Wahyuni, 2002). Tembawang and rubber agroforestry are part of the tropical rainforest in West Kalimantan. This area is a very important natural resource to provide oxygen and preserving carbon. Over the past few decades, the expansion of plantations, ag- riculture, and settlements in West Kalimantan has been a serious threat to Tembawang and rubber forests. Re- quired anticipation of land-use changes to provide a bet- ter climate from assessing population and biomass in Tembawang and traditional rubber agroforestry for en- hancing carbon storage. Preliminary research has been conducted in this traditional agroforestry, a total of 43 species and 8 dominant species were found (Rafdinal and Pitopang, 2019). This study aims to compare the standing biomass of Tembawang and conventional rubber agro- forestry related to carbon storage and mitigation of cli- mate change. 2 MATERIALS AND METHODS 2.1 DESCRIPTION OF RESEARCH AREA This study was located at Kayu Lapis Road, Kilom- eter 21, Nanga Pemubuh Village, Sekadau Hulu District, Sekadau Regency, West Kalimantan Province, Indonesia (Figure 1). Sekadau Hulu is one of 7 districts in Seka- dau Regency, has an area of ± 869.7 km2. Sekadau Hulu District is geographically located in the southern part of Sekadau Regency, consists of 15 villages including Nanga Pemubuh. Nanga Pemubuh village has an area of 89.92 km2 and is located 55 km from the central district. In general, this region’s has relatively high annual rainfall and wet tropical climate. Based on the record of Meteoro- logical, Climatological, and Geophysical Agency of Indo- nesia (Indonesian Statistics, 2016), the highest rainfall in 2011 occurred in November (826 mm) and the lowest in May (118 mm). The average monthly temperature ranged from 24.8-27.3 oC, and there was no dry season. Soil con- ditions are generally very heterogeneous, with the pH of soil H2O in horizon-A ranging from 3.69 to 5.55. 2.2 FOREST INVENTORY Analysis of vegetation in each sampling site was car- ried out by a belt transect method. The transects were further divided into 20 m x 20 m plots. In each plot, all individuals with ≥ 10 cm DBH (Diameter at Breast Height) were tagged, measured, and identified. The girth and height of each individual were measured. The plant specimens were identified. Frequency, density, basal area, and Importance Value Index (IVI) were calculated fol- lowing Misra (1968), Mueller-Dombois and Ellenberg (1974). Trees were grouped into five DBH classes i.e. 10- 20 cm, 20-30 cm, 30-40 cm, 40-50 cm, and > 50 cm and the density and Aboveground biomass (AGB) distribu- tion under each DBH class were analyzed. Acta agriculturae Slovenica, 117/1 – 2021 3 Comperative study of aboveground biomass and carbon storage ... and conventional rubber agroforestry in West Kalimantan Indonesia 2.3 ESTIMATION OF ABOVEGROUND BIOMASS The AGB in different tree diameter classes in each site was estimated using the following mentioned model (Ketterings et al., 2001): Where: ρ = wood density D = diameter at breast height The aboveground biomass for all diameter class- es was summed to calculate the total aboveground biomass in each agroforestry type. The values for the Tembawang and the traditional rubber were compared using t-test analysis. 2.4 ESTIMATION OF CARBON The aboveground biomass carbon storage was calculated by assuming that the carbon content is 50 % of the total aboveground biomass (Brown, 1997; Can- nel and Dewar, 1995; Dixon et al., 1994; Ravindranath et al., 1997; Richter et al., 1995; Schroeder, 1992). 3 RESULTS AND DISCUSSION 3.1 STAND CHARACTERISTICS Twenty-eight species were recorded from the Tembawang agroforestry, and 31 species were record- ed from the conventioal rubber plantation. The den- sity of woody species (≥ 10 cm DBH) was greater in Tembawang (195 trees ha-1) than in traditional rubber agroforestry (190 trees ha-1), but not statistically dif- ferent (p > 0.5). Based on density, Hevea brasiliensis Müll. Arg. (57.50 trees ha-1) and Durio zibethinus L. (26.25 trees ha-1) were the dominant species in the Tembawang agroforestry and these two species ac- counted for 31 % of ha-1) and Durio zibethinus (18.33 trees ha-1) were the dominant and codominant species, respectively. The basal area was greater in the Tem- bawang agroforestry (1,560.97 m2 ha-1) than in the conventional rubber plantation (811.27 m2 ha-1) (Table 1). Tembawang and traditional rubber agroforestry in Dayak Tribe were implemented for decades. Both Tembawang and conventionalrubber agroforestry were managed traditionally from generation to gen- eration. The differences in these systems is based on the constituent trees species of each agroforestry type (Rafdinal and Pitopang, 2019). Tembawang is former forest land that is used for planting fruit trees for pro- viding food or traditional ceremonies need by local people, therefore the 3 main constituent species are Durio zibethinus, Nephelium lappaceum, and Artocar- pus sp. However, in the recent decades, Tembawang agroforestry has began to be planted rubber trees by local people, and the latex is sold to industries or com- panies in the city, therefor now the rubber trees domi- nated Tembawang agroforestry. Rubber plantation was generally formed from land clearing by local communities in the decade 2000-2010 in West Kalimantan to support the national rubber industry, rubber latex had high selling value at that time (Indonesia-Investment, 2018). However, some conventional rubber plantations still preserved trees used for daily needs such as wood and fruit trees. Thus, Durio zibethinus and Shorea species still exist besides the dominant rubber trees. Figure 1: Location and land use of the study site Acta agriculturae Slovenica, 117/1 – 20214 R. RAFDINAL et al. 3.2 ABOVEGROUND BIOMASS DISTRIBUTION Although the young individuals belonging to 10- 20 cm DBH class dominated both forests for tree den- sity (Figure 2), the AGB accumulation was greater in the up 50 cm diameter class in Tembawang agroforestry forest and traditional rubber plantation (Figure 3). The contribution of AGB by higher diameter classes gener- ally increases in both agroforestry. The contribution of > 50 cm diameter trees to AGB is greater in Tembawang agroforestry (62.58 %) than in traditional rubber planta- tions (49.24 %). Rubber growth is faster than in other trees in these agroforestry systems, latex harvesting of rubber trees was carried out when entering 5-6 years vegetation ages with 45 cm girth or about 15 cm trunk diameter for 25- 30 years production period (Damanik et al., 2010). After production age, rubber trees were usually felled and re- planted, so that the highest density of rubber agroforestry was dominantly distributed in the lower diameter class, whereas tree density in Tembawang agroforestry was mainly distributed in the higher diameter class, covered by old fruit trees that have been down through genera- tions. Further, the difference in density distribution from diameter class caused the basal area and overall AGB val- ue of Tembawang were greater than rubber plantations. 3.3 TOTAL ABOVEGROUND BIOMASS AND CARBON STORAGE The total AGB of Tembawang agroforestry (180.51 Mg ha-1) was significantly greater (p < 0.01) than tradi- tional rubber plantation (85.01 Mg ha-1). A primary for- est can store AGB of up to ± 350 Mg ha-1 while a second- ary forest varies considerably between 59 - 140 Mg ha-1 (Stas, 2014). The value of AGB in a forest ecosystem depends on the composition and structure of the forest. The AGB value of rubber agroforestry was in the range of secondary forest values and was still in the inten- sive tree crop plantation value range of 60-120 Mg ha-1 (Hairiyah et al., 2011). The ABG value of Tembawang agroforestry was higher than the average AGB value of the secondary forest. The composition and structure of Tembawang agroforestry were quite varied and the conditions had been preserved for generations so that it had large AGB potential, but not as large as primary forest. The aboveground carbon stored by Tembawang and traditional rubber forests was 90.26 and 42.01 Mg C ha-1, respectively and were significantly different (p < 0.01). Carbon organic was mostly stored in up 50 cm DBH class in Tembawang (62.58  %) and traditional rubber (49.24 %). The younger (10-20 cm DBH class) trees, which had the highest density in both forests, stored only 3.52  % of total carbon in the Tembawang agroforestry and 5.52 % of traditional rubber (Table 2). Aboveground carbon storage of Tembawang agro- forestry was two times higher than rubber plantations, in terms of carbon storage Tembawang agroforestry was better, but the age of the community and vegetation cycle also to be considered. When compared to other agroforestry systems, the Tembawang agroforestry sys- tem stored more carbon. In a comprehensive report Variables Agroforestry Tembawang Conventional Rubber Species richness (number of species) 28 31 Stand density (trees ha-1) 195 190 Density of dominant tree species: Durio zibethinus L. 26.25 18.33 Hevea brasiliensis Müll. Arg. 57.50 38.33 Nephelium lappaceum L. 11.25 10.00 Artocarpus sp. 13.75 10.00 Shorea stenoptera Burck. 12.50 10.00 Artocarpus integer Merr. 10.00 - Anacardium occidentale L. - 10.00 Shorea leprosula Miq. - 13.33 Shorea macrophylla (de Vr.) Ashton - 10.33 Basal area (m2 ha-1) 1,560.97 811.27 Table 1: Stand characteristics of Tembawang and conventional rubber agroforestry of West Kalimantan Acta agriculturae Slovenica, 117/1 – 2021 5 Comperative study of aboveground biomass and carbon storage ... and conventional rubber agroforestry in West Kalimantan Indonesia bon storage values of 14.35 to 37.88 Mg C ha-1 (Awang Besar et al., 2020), the carbon storage value of rubber plantation had higher value. In the same DBH range (≥ 10 cm), the carbon storage value in Tembawang agro- forestry was in the aboveground carbon storage range of secondary tropical forests in Maluku, Indonesia with an average value of 70.3 Mg C ha-1 (Stas, 2014). from Schroeder (1984), agroforestry systems in humid areas had an average value of 50 Mg C ha-1, whereas in another report, Takimoto et al. (2008) reported the value of biomass C storage ranged from 0.7 to 54.0 Mg C ha-1 in traditional and improved agroforestry in West Africa. Compared to the dominating palm oil and agar- wood industries in Indonesia and Malaysia, with car- Figure 3: Aboveground biomass at different diameter classes from Tembawang forest and traditional rubber of West Kalimantan Figure 2: Tree density in different diameter classes in Tembawang forest and traditional rubber of West Kalimantan Diameter Size (cm) Tembawang Forest Conventional Rubber Forest Density (%) Carbon Storage (%) Density (%) Carbon Storage (%) 10-20 41.03 3.52 44.49 5.52 20-30 21.79 8.39 23.68 12.19 30-40 12.18 10.02 17.54 20.94 40-50 11.54 15.49 5.26 12.07 >50 13.46 62.58 7.02 49.24 Table 2: Carbon storage in different tree diameter classes in Tembawang forest and conventional rubber of West Kalimantan Acta agriculturae Slovenica, 117/1 – 20216 R. RAFDINAL et al. 4 CONCLUSIONS The aboveground carbon storage value of the tradi- tional Tembawang and Rubber agroforestry system was estimated at 90.26 and 42.01 Mg C ha-1. The traditional agroforestry system of the Dayak tribe community of Sekadau Regency, West Kalimantan had better impact and potential to preserve carbon than an intensive agro- forestry system. The preservation of several species of fruit trees in the agroforestry system was major contribu- tion to carbon storage. Tembawang agroforestry has high potential for car- bon storage compared to rubber plantations and varied different agroforestries, also the value was almost similar to carbon storage potential in secondary tropical forests. Local people’s knowledge in traditional agroforestry sys- tems need to be reported. 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Delo je prispelo 20. avgusta 2020, sprejeto 09. decembra 2020. 1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, India 2 Corresponding author, e-mail: abdulwar2@gmail.com 3 Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST-K), Shalimar, Srinagar, Jammu and Kashmir, India The study in this manuscript is part of the Ph.D. research work of the Abdul Rasheed WAR (Mentor-Hari C SHARMA) Nutritional indices and biochemical profile of Helicoverpa armigera [Hübner (1808)] on different groundnut genotypes Abstract: Nutritional indices and biochemical profile of Helicoverpa armigera in response to feeding on different groundnut genotypes was studied. The moderately resistant genotypes were ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697. JL 24 was used as the susceptible check. Consumption index (CI), approximate digestibility (AD), efficiency of con- version of ingested food (ECI) and efficiency of conversion of digested food (ECD) were recorded. In addition, the activities of digestive and defensive enzymes of H. armigera were stud- ied. H. armigera larvae showed significantly lower CI, AD, ECI and EDI when fed on moderately resistant genotypes than the insects fed on JL 24. Serine protease and trypsin activities were low in insects fed on resistant genotypes than the ones fed on JL 24. Further, insects fed on resistant genotypes showed sig- nificantly greater glutathione-S-transferase activity than the insects fed on JL 24. A reverse trend was observed for esterase activity. Similar trend was observed for total protein content of the insects. Thus, nutritional quality of host plants affects in- sect’s physiology and could be used as an important indicator of host plant resistance against insect pests and to understand the adaptation of insect pests, if any, to various genotypes/host plants. Key words: Host plant resistance; nutritional indices; di- gestive enzymes; groundnut; Helicoverpa Prehranjevalni indeksi in biokemični profil južne plodovrtke, Helicoverpa armigera [Hübner (1808)], na različnih genotipih arašidov Izvleček: V raziskavi so bili preučevani prehranjevalni indeksi in biokemični profil južne plodovrtke (Helicoverpa ar- migera) kot odziv na prehranjevanje na različnih genotipih ara- šidov. Zmerno odporni genotipi arašidov so bili ICGV 86699, ICGV 86031, ICG 2271 in ICG 1697. Genotip JL 24 je bil up- orabljen kot občutljiva kontrola. Določeni so bili prehrambeni indeks (CI), navidezna prebavljivost (AD), učinkovitost pret- vorbe pojedene hrane (ECI) in učinkovitost pretvorbe pre- bavljene hrane (ECD). Dodatno so bile v škodljivcu preučene aktivnosti prebavnih in obrambnih encimov. Gosenice južne plodovrtke so imele značilno manjše vrednosti parametrov kot so CI, AD, ECI in EDI, kadar so se hranile na zmerno odpornih genotipih v primerjavi s tistimi, ki so se hranile na občutljivem genotipu JL 24. Aktivnosti serin proteaze in trip- sina so bile manjše pri žuželkah, ki so se hranile na odpornih genotipih v primerjavi s tistimi, ki so se hranile na občutljivem ‘JL 24’. Žuželke, ki so se hranile na odpornih genotipih so imele značilno večjo aktivnost glutation-S-transferaze kot žuželke, ki se hranile na ‘JL 24’. Nasproten trend je bil opažen v aktivnosti esterase. Podoben trend je bil ugotovljen v vsebnosti celokupnih beljakovin v žuželkah. Hranilna kakovost gostiteljskih rastlin vpliva na fiziologijo škodljivih žuželk in bi jo lahko uporabili kot pomemben kazalnik odpornosti gostiteljskih rastlin proti škodljivim žuželkam. S tem bi razumeli prilagoditve škodljivih žuželk na različne genotipe gostiteljskih rastlin. Ključne besede: odpornost gostiteljskih rastlin; prehran- jevalni indeksi; prebavni encimi; arašidi; Helicoverpa Acta agriculturae Slovenica, 117/1 – 20212 A. R. WAR et al. 1 INTRODUCTION Plants face innumerable challenges from biotic and abiotic stresses, however, biotic stress by insect pests is one of the major stresses the plants face and take a heavy toll on crop yields. Though synthetic insecticides are the main insect pest controlling methods in many crops, they pose a great threat to the non-target organ- isms. For example, pesticide resistance is developed by insects, pest-resurgence, pesticide residues in food and health hazards in human beings (Isman, 2006; Sharma, 2007). Therefore, there is a need for an alternative en- vironmentally safe crop protection technologies for safe and sustainable crop production. Breeders and en- tomologists have been developing insect resistant crop cultivars that could withstand insect pressure (Sharma et al., 2003; Smith, 2005; Sharma, 2009; Nair et al., 2020). Groundnut (Arachis hypogaea L.) is one of the important oilseed crops across the tropical and subtropical regions. Groundnut crop is affected by a number of biotic and abi- otic stresses. Insect pests are the major biotic constraints of groundnut. The economically important insect pests of groundnut include western flower thrips (Franklini- ella occidentalis Pergande, 1895 and melon thrips (Thrips palmi Karny, 1925); leaf miners (Aproaerema modicella [Deventer, 1904]); aphids (Aphis craccivora Koch, 1854); leafhoppers (Empoasca dolichi Paoli, 1930); white grubs (Holotrichia consanguinea Blanchard, 1850); pod borer, Helicoverpa armigera (Hübner(1808)) and armyworm, Spodoptera litura (Fabricius, 1775) (Sharma et al., 2003). Helicoverpa. armigera (Hübner,[1808]) is a polyph- agous lepidopteran pest with wide distribution across Asia, Africa, Australia and southern Europe (Sharma et al., 2003; Sharma, 2005). It causes severe damage to cere- als, fruit crops, cash crops, vegetables including ground- nut. Host plant resistance plays an important role to ward of insect pests by plants (Howe & Jander, 2008; War et al., 2011). It is a simple, inbuilt and eco-friendly method of managing insect pests (Sharma & Ortiz, 2002; War et al., 2012). Plant defensive traits interfere with host plant selection by the insect pest, deter the insects by produc- ing volatile compounds or by averting oviposition by the insects. Plant defense against insect pests is mani- fested through morphological (surface wax, lignification, spines, hairs and sclerophylly) and biochemical traits (Dwivedi et al., 1986; Sharma et al., 2009; He et al., 2011; War et al., 2012; Bohinc et al., 2013). Biochemical traits constitute toxic secondary metabolites as a major com- ponent of plant defese against insect pests. They are di- rectly toxic to insect pests or recruit the natural enemies of the insect pests (Howe & Jander, 2008; Karban, 2011; War et al., 2011, 2013). In groundnut, toxic secondary metabolites have been reported to hamper growth and development of insect pests (Stevenson et al., 1993; Seng- uttuvan & Sujatha, 2000; War et al., 2013, 2014). Several reports have shown the role of plant toxic metabolites affecting insect growth and development. For example, Rao et al. (1998) showed that polyphe- nols in groundnut plants provide resistance against leaf miner A. modicella. Further, plant secondary metabolites such as dihydroxybenzoic acid, vanillic acid, caffeic acid and umbelliferone have been suggested to be involved in resistance against insect pests (War et al., 2016). In groundnut, structural (trichomes) and biochemical traits (phenols, tannins and defensive proteins) are involved in defense against insect pests including H. armigera (War et al., 2013, 2016; War & Sharma, 2014). Similarly, Steven- son et al. (1993) reported that in groundnut, plant toxins such as caffeoylquinic acids, quercetin, and diglycosides are the main contributors of insect resistance. They found that chlorogenic acid and rutin are also involved in resis- tance against S. litura. To test the hypothesis that insect resistant plants contain plant defensive traits that affect the insect growth and development by interfering with the nutritional indices of the insects, consumption, di- gestion and utilization of food and also the biochemical traits of H. armigera larvae were studied after feeding on the insect resistant and susceptible groundnut genotypes. 2 MATERIALS AND METHODS 2.1 CHEMICALS The chemicals used were of analytical grade. Ethyl- ene diamine tetra acetic acid (EDTA), tannic acid, tryp- sin inhibitor, bovine serum albumin, 1-napthol, glycine, 4-chloronapthol, disodium hydrogen phosphate, sodium dihydrogen phosphate, N-α-Benzoyl-DL-arginine p- nitroanilide (BApNA), glucose, GSH, sodium hydroxide, and sodium carbonate (Na2CO3) were procured from Sigma Aldrich (St Louis, Missouri). Acetic acid and 1-chloro-2, 4-dinitrobenzene (CDNB) were obtained from Sisco Research Laboratory (India) and HiMedia Pvt. Ltd (India), respectively. 2.2 HElicovErpA ArmigErA Helicoverpa armigera larvae were collected from the field at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, India (17° 25’N latitude, 78° 00’E longitude and 545 m.a.s.l.). The field collected insects were reared for one generation on the natural host under laboratory condi- tions before mixing with the laboratory culture. Under Acta agriculturae Slovenica, 117/1 – 2021 3 Nutritional indices and biochemical profile of Helicoverpa armigera [Hübner (1808)] on different groundnut genotypes laboratory conditions, insects were reared on chickpea based artificial diet (Table 1, Armes et al., 1992). The pu- pae were disinfected in 2 % sodium hypochlorite solu- tion before transferring them to plastic jars containing Vermiculite for adult emergence. Newly emerged adults were immediately transferred to the wooden oviposition cages (30 x 30 x 30 cm) containing 10 % honey or sucrose solution in a cotton swab as food. Eggs were collected on rough surfaces of diaper liners (5 x 15 cm) and thin cot- ton wool sheets hung inside the cage. The eggs laid on the liners sterilized in 2 % sodium hypochlorite solution. The liners were dried and placed inside the plastic cups. The newly emerged larvae were reared initially in groups of 200 to 250 for five days in 200 ml plastic cups containing 2 to 3 mm layer of artificial diet on the bottom and sides. To avoid cannibalism, the larvae were reared individu- ally in six cells well plates, of which each cell well was 3.5 cm in diameter and 1.5 cm in depth. The cell wells were filled with 7 ml diet for larval development until pupa- tion. After every six months, the laboratory culture was mixed with the field-collected insects to maintain culture heterogeneity. Newly emerged larvae were used for the experiments. 2.3 GROUNDNUT PLANTS The groundnut genotypes used in this study in- cluded four moderate to high levels of insect resistant cultivars (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697) and a susceptible check (JL 24) (Sharma et al., 2003). The groundnut plants were grown in plastic pots (30 cm diameter and 40 cm deep) containing soil, sand, and farmyard manure (2:1:1 ratio) in a greenhouse at ICRISAT, Patancheru, Telangana, India. The plants were watered as needed and were maintained as per good ag- ricultural practices. Five seeds were sown and later two seedlings were retained in each pot for the experimental purpose at 10 days after seedling emergence. The tem- perature and relative humidity were maintained at 26 ± 5 ºC and 65 + 5 %, respectively, using desert coolers. Leaves (first fully expanded tetrafoliates) from 20-days old plants were used for the experiments. 2.4 CONSUMPTION, DIGESTION AND UTILIZA- TION OF FOOD BY HElicovErpA Armig- ErA The detached leaf assay technique described by Sharma et al. (2005) was followed to study consumption, digestion and utilization of food by H. armigera. The leaves were brought from glasshouse to lab in ice box. In each 100 ml plastic cup, a single tetrafoliate was em- bedded in 3  % agar-agar. Third-instar larvae of similar size were starved for 4 h before releasing on the leaves and a single larva was released on one tetrafoliate. After 5 days of feeding, larval mass, leaf damage rating, and dry mass of the residual food was recorded. To calculate the dry mass of the introduced food, fresh mass of the Diet component Ingredients Quantity (g) per 1,000 ml diet Part A Chickpea flour 300 Sorbic acid 3.0 Methyl-p-hydroxybenzoate 5.0 Ascorbic acid 4.7 Yeast 48 Aureomycin powder 11.5 Cholesterol 1.5 Formaldehyde (1%) 20 ml Multivitamin solution (A,B,D,E,C) drops 10 μl Water 450 ml Part B Agar-agar 17.3 Water 800 ml Table 1: Composition of semi-synthetic diet for Helicoverpa armigera Diet preparation: The diet was prepared as follows: 1. Measured quantities of part A were mixed. 2. Agar-agar was added to water in a separate container and boiled for 5 min (Part B). 3. Part A and Part B were mixed thoroughly in a blender to get an even consistency. 4. The diet was poured into small plastic cups and allowed to cool under a laminar flow for 1 to 2 h. Acta agriculturae Slovenica, 117/1 – 20214 A. R. WAR et al. food remaining after larval consumption was multiplied by a standard factor determined by maintaining an ali- quot of the food under similar conditions in the absence of larvae, weighing it, then drying and reweighing it. The dry and fresh mass of aliquots were used to determine the percentage dry matter. The dry mass was expressed as the percentage dry matter in each genotype. The uncon- sumed food and frass from each detached leaf assay were removed, weighed and dried at 65 ºC for 72 h in a hot-air oven. The dry mass of the food unconsumed by the insects was calculated as the difference between the dry mass of the unconsumed food and the calculated dry mass of the offered food. The difference between the mass of the larvae before and after the feeding period was taken as the larval mass gain. The nutritional indices such as food consumption, digestion, and efficiency of conversion of the ingested food into body matter were calculated as per Waldbauer (1968) and Sharma & Franzmann (2000). The consumption index (CI) was calculated as: Approximate digestibility (AD) of food was calcu- lated as follows: Efficiency of conversion of ingested food into body matter (ECI) was calculated as follows: Efficiency of conversion of digested food (ECD) was calculated as: 2.5 BIOCHEMICAL TRAITS OF INSECT PESTS 2.5.1 Total serine protease assay Insects from the bioassay cups were collected after 5 days of the infestation and dissected. The midguts of the larvae were extracted in in 0.2 M sodium phosphate buf- fer (pH 7.5) and homogenized in 0.1 M glycine-NaOH buffer (pH 10), containing 1 mM EDTA. The filtrate was passed through three-layered cheese cloth and centrifuged at 10,000 rpm for 20 min at 4 °C. A separate tube was used to collect the supernatant to be used as source for deter- mining the enzyme activity. For the estimation of serine protease activity, azocasein was used as a substrate (He- gedus et al., 2003). To midgut supernatant (0.04 ml), 0.3 ml of 1 % azocasein solution that was prepared in 0.05 M glycine-NaOH buffer (pH 10) was added. The solution was incubated at 28 °C for 15 min. To the reaction mixture, 0.34 ml of 10 % TCA was added and then incubated for 1 h at room temperature. After centrifugation at 12,000 rpm for 10 min, 0.68 ml of 1 M NaOH was added to the super- natant. The absorbance was read at 495 nm and total mid- gut serine protease activity (SP) was calculated as follows: The total serine protease activity was expressed as tryptic activity (mU) per min of incubation per mg insect protein (mU min-1 mg-1 protein). 2.5.2 Trypsin assay Trypsin activity was determined as per Perlmann & Lorand (1970). To the midgut extract of 0.15 ml, 1 ml of 1 mM BApNA (in 0.2 M glycine–NaOH buffer, pH 10), was added. The reaction mixture was incubated at 37 °C for 10 min. The reaction was terminated by adding 0.2 ml of 30 % acetic acid and the absorbance was read at 410 nm. The unit l mol min-1 mg-1 protein was used to express the trypsin activity. 2.5.3 Esterase (est) assay For determination of esterase (EST) and glutathione- S-transferase assay (GST) activities, similar procedure was followed for extraction by dissecting the larvae in 0.1 M sodium phosphate buffer (pH 7.5). Homogenization of midguts was carried out in 0.1 M sodium phosphate buffer (pH 7.5) containing 1 mM EDTA. The filtrate was passed through three-layered cheese cloth and centrifuged for 15 min (4 °C) at 12,000 rpm. The supernatant was used for the estimation of EST and GST activities. The EST activ- ity was estimated by adding 0.1 ml enzyme sample diluted 10 times with 0.1 M sodium phosphate buffer to 1.5 mM 1-naphthyl acetate solution. The reaction mixture was in- cubated at for 30 min at 25 °C and the reaction was stopped by adding Fast Blue B (in 5 % SDS) staining solution. The absorbance was read at 490 nm after 15 min of addition of the stopping solution. The hydrolysed substrate concentra- tion was determined from the standard curve of 1-naph- thol. The EST specific activity was expressed as l mol of 1-naphthol formed min-1 mg-1 protein. 2.5.4 Glutathione-s-transferase (gst) assay The GST activity was estimated by using 1-chlo- Acta agriculturae Slovenica, 117/1 – 2021 5 Nutritional indices and biochemical profile of Helicoverpa armigera [Hübner (1808)] on different groundnut genotypes ro-2,4-dinitrobenzene (CDNB) and reduced GSH as sub- strates (Habig et al., 1974). To 1 ml of phosphate buffer (pH 7.5), 0.1 ml of CDNB (25 mM) and 1.6 ml of distilled water were added. To this mixture, 0.1 ml of 10 fold en- zyme solution diluted with 0.1 M sodium phosphate buffer (pH 7.5) was added. The reaction mixture was incubated for 5 min at 37 °C and 0.1 ml of 20 mM GSH was added. Absorbance was read for 3 min at 30 s interval at 340 nm. The CDNB extinction coefficient of 9.6 mM cm-1 was used in calculating the enzyme activity. The specific activity of GST was expressed as nmol of CDNB conjugate formed min-1 mg-1 protein. 2.5.5 Estimation of protein content Total protein content of the insects fed on groundnut genotypes was determined by Lowery’s method (Lowry et al., 1951). The bovine serum albumin was used as a stan- dard and the protein content was expressed as mg g-1 body mass. 2.6 STATISTICAL ANALYSIS The data was analysed through analysis of variance (ANOVA) using SPSS v15.1 (SPSS, Inc., Chicago, IL, USA). The significant effects of the treatment (p ≤ 0.05) were separated by the Tukey’s test. 3 RESULTS 3.1 FOOD CONSUMPTION AND UTILIZATION BY Helicoverpa armigera The insects fed on genotypes ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697 showed significantly lower CI per unit body mass than the insects on the susceptible check, ‘JL 24’ (Table 2). Similarly, the insects fed on insect- resistant genotypes, ICGV 86699, ICGV 86031, ICG 1697 and ICG 2271 (36.5 – 45.4%) had lower AD than those fed on ‘JL 24’ (67.5 %). Further, ECI was significantly lower in larvae fed on genotypes ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697 (21.3 – 28.2 %) than those fed on the susceptible check, ‘JL 24’ (54.1  %). The ECD in insects fed on the insect-resistant genotypes varied from 23.6 - 30.2 %, while the larvae fed on ‘JL 24’ showed a high ECD (45.7 %). 3.2 ENZYMES Genotypes Nutritional indices CI (mg/mg/day) AD (%) ECI (%) ECD (%) ICGV 86699 2.3 ± 0.01bc 36.5 ± 3.8c 21.3 ± 1.5b 27.1 ± 1.3bc ICGV 86031 2.6 ± 0.03bc 41.2 ± 2.3b 25.5 ± 1.2b 23.6 ± 1.4bc ICG 2271 3.5 ± 0.02b 44.3 ± 2.9b 28.2 ± 1.8b 30.2 ± 2.5b ICG 1697 2.9 ± 0.01b 45.4 ± 3.0b 24.7 ± 1.9b 29.3 ± 2.2b JL 24 4.1 ± 0.04a 67.5 ± 3.7a 54.1 ± 2.3a 45.7 ± 2.7a Table 2: Nutritional indices of Helicoverpa armigera larvae fed on groundnut genotypes Within columns, (means ± SD) followed by same letter(s) do not differ significantly (Tukey‘s HSD test, p < 0.05). CI = Consumption index, AD = Approximate digestibility, ECI = Efficiency of conversion of ingested food and ECD = Efficiency of conversion of digested food. Genotypes Serine protease (mU min-1 mg-1 protein) Trypsin (μmol min-1 mg-1 protein) ICGV 86699 2.32 ± 0.07ab 0.22 ± 0.002b ICGV 86031 2.11 ± 0.05ab 0.19 ± 0.002bc ICG 2271 1.78 ± 0.02bc 0.20 ± 0.003b ICG 1697 1.94 ± 0.04bc 0.18 ± 0.002bc JL 24 3.26 ± 0.08a 0.41 ± 0.005a Table 3: Total serine protease and trypsin activities of Helicoverpa armigera larvae fed on resistant and susceptible groundnut geno- types Values (Mean ± SD) with similar letters within a column do not differ significantly at p ≤ 0.05 (Tukey‘s HSD test). Acta agriculturae Slovenica, 117/1 – 20216 A. R. WAR et al. 3.2.1 Total serine protease and trypsin activity Lower total serine protease activity was observed in insects fed on genotypes ICGV 86031 (F4,14 = 21.7), ICG 2271 (F4,14 = 23.1) and ICG 1697 (F4,14 = 12.9) than those fed on ‘JL 24’ (F4,14 = 21.3) (p < 0.05) (Table 3). Similarly, the trypsin activity was significantly lower in insects fed on insect resistant genotypes (F4,14 =10.2, 11.1, 13.8 and 6.2, respectively, for ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697, p < 0.05) than in the insects fed on the susceptible check, ‘JL 24’. 3.2.2 GST and EST activity Significantly greater GST activity was observed in insects fed on genotypes ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697 (F4,14 = 22.1, 18.2 and 12.8, respec- tively, p < 0.05) than those fed on ‘JL 24’ (Table 4). The EST activity in insects fed on ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697 was significantly lower (F4,14 = 9.5, 9.0 and 11.5, respectively, p < 0.05) than the insects fed on the JL 24. 3.2.3 Total protein content Insect pests exhibited differential protein in insect resistant and susceptible genotypes. The protein con- tent of the insects fed on genotypes ICGV 86699, ICGV 86031, ICG 2271, and ICG 1697 did not differ signifi- cantly (7.43, 8.22, 8.70 and 9.01 mg ml−1, respectively), however, it was significantly lower in the insects fed on the susceptible check, ‘JL 24’ (12.7 mg ml−1) (Table 5). 4 DISCUSSION The toxic plant secondary metabolites are important weapons employed by plants against insects. The main mode of action of these metabolites is through the anti- biosis mechanism, when ingested, these metabolites have detrimental effects on insects and reduce insect growth and development. The low nutritional quality of plant tissues, proteinase inhibitors and other metabolites are some of the antibiosis factors (Bhonwong et al., 2009; Barbehenn et al., 2010; War et al., 2013). These factors affect the insect food intake and its consumption and utilization by insect pests. The antibiosis and antixenosis plant defences against insect pests can be determined by studying the consumption, digestion and utilization of insects fed with specific host plants (Devetak et al., 2013). Imbalance in insect’s food constituents will have drastic effects on its growth and development. Nitrogen content of plant tissues is an important limiting factor for growth and development of insect herbivores (Zhong-xian et al., 2007). Since plant tissues are the main source of nutrients for insects, their availability depends on the amount of food ingested and how efficiently it has been converted to body matter. Genotypes GST (μmol CDNB min-1 mg-1 protein) EST (μmol 1-napthol min-1 mg-1 protein) ICGV 86699 1.91 ± 0.01a 0.29 ± 0.004a ICGV 86031 1.88 ± 0.03a 0.27 ± 0.004a ICG 2271 1.86 ± 0.04a 0.31 ± 0.006ab ICG 1697 1.52 ± 0.07a 0.30 ± 0.003a JL 24 1.02 ± 0.03a 0.39 ± 0.006a Table 4: GST and EST activities of Helicoverpa armigera larvae fed on resistant and susceptible groundnut genotypes Values (Mean ± SD) followed by same letter(s) within a column are not significantly different at p ≤ 0.05 (Tukey‘s HSD test). Genotypes Protein content (mg ml-1) ICGV 86699 7.43 ± 0.4a ICGV 86031 8.22 ± 0.2a ICG 2271 8.70 ± 0.3a ICG 1697 9.01 ± 0.2a JL 24 12.7 ± 1.3b Table 5:Total protein content (mg ml-1) of Helicoverpa armigera larvae fed on fed on resistant and susceptible groundnut genotypes Values (mean±SD) followed by same followed by letter(s) within a column are not significantly different at p ≤ 0.05 (Tukey’s HSD test). Acta agriculturae Slovenica, 117/1 – 2021 7 Nutritional indices and biochemical profile of Helicoverpa armigera [Hübner (1808)] on different groundnut genotypes Our results showed that AD, CI, ECI, and ECD were reduced in H. armigera larvae fed on insect-resistant groundnut genotypes and were considerably lower than the insects fed on JL 24. The reduced consumption and utilization of food by H. armigera can be attributed to the antibiosis effect of the constitutively produced second- ary metabolites including flavonoids, tannins and some defensive proteins (Grayer et al., 1992; Stevenson et al., 1993; Senguttuvan & Sujatha, 2000; Rao, 2003; War et al., 2013). Antibiosis is one of the important modes of host plant resistance against insects that affects the oviposi- tion, survival and growth and development of the target pests (Sharma & Norris, 1991; Sharma et al., 2005; Su- jana et al., 2008; Ansari et al., 2011). We observed that even though the consumption index of H. armigera was high in some of the insect resistant genotypes such as ICG 1697, ICG 2271 and ICGV 86031, the ECI and ECD were significantly less. This clearly showed that the plant defensive traits pertaining to antibiosis such as toxic sec- ondary metabolites occur in these genotypes. The ingest- ed plant allelochemicals affect the insect’s physiological and biochemical traits that in turn impact the post-in- gestive nutrient utilization by the pest (Sharma & Norris, 1991; Hasan & Ansari, 2011; Ansari et al., 2011; War et al., 2013). Further, insects excrete some of the plant toxic chemicals along with the faecal matter, which in turn, re- sults in reduced efficiency of food utilization. The results showing the differential responses of insects in ingesting the food material, digestion efficiency and conversion of the ingested food into body matter can be used as impor- tant indicators for identifying the host plant resistance against insect pests (Sharma & Norris, 1991; Yazdanfar et al., 2015). The digestive and the detoxifying enzymes in insect pests showed a differential response on insect resistant and susceptible groundnut genotypes. Serine protease and trypsin activities were reduced in insects fed on the resistant genotypes. This could be attributed to the higher amounts of toxic plant secondary metabolites and/or protease inhibitors in insect resistant genotypes than the susceptible genotype. Reduced serine protease and trypsin activities in insects fed on insect resistant genotypes can be attributed to the antibiosis mechanism of resistance in plants mediated by the toxic secondary metabolites. Further, some plant antioxidative enzymes such as peroxidases are directly toxic to insect pests (Barbehenn et al., 2010). It has also been reported that H. armigera larvae when fed on a diet containing plant secondary metabolites show reduced serine protease and trypsin activities (War et al., 2013). Reduced protein di- gestion, thereby, low levels of amino acids result in re- duced growth and developments in insects (Lawrence & Koundal, 2002; Azzouz et al., 2005). Insect detoxifying enzymes such as GST and EST convert plant allelochemicals into non-toxic or low toxic compounds (Leszczynski & Dixon, 1992; Yang et al., 2005; War et al., 2013). These enzymes are induced in insects in response to plant metabolites. Increased ac- tivities of GST were observed in insects fed on resistant genotypes as compared to the ones fed on the susceptible genotype JL 24. The higher toxicity of the compounds is attributed to the higher toxicity of the plant toxic sec- ondary metabolites in the insect resistant genotypes (War et al., 2013). In barley, aphid Sitobion avenae (Fa- bricius, 1775) showed increased levels of GST when fed on plants with greater phenolic content (Leszczynski & Dixon, 1992). The EST activity did not differ significantly in insects fed on the insect resistant genotypes but was significantly higher in the insects fed on the susceptible genotype, JL 24. The higher levels of toxic secondary metabolites in resistant genotypes than the susceptible genotype might have led to the reduced activity of the EST in insects. ESTs are directly involved in the hydrox- ylation of toxic plant secondary metabolites and insecti- cides to less toxic compounds (Yang et al., 2005). Positive correlation has been observed in insect midgut serine proteases, trypsin and GST and larval mass and survival (War et al., 2013). Therefore, plant toxic compounds in resistant genotypes that led to the decrease in the levels of these enzymes could be used as important biochemi- cal markers for plant resistance. myzus persicae (Sulzer, 1776) exhibited higher levels of GSTs when fed on bras- sicaceous host plants containing toxic plant metabolites such as glucosinolates and isothiocyanates (Francis et al., 2005). H. armigera fed on artificial diet containing plant toxic metabolites showed higher levels of GST, which has been attributed to the fact that the insect is trying to adapt to the plant toxins (War et al., 2013). They further reported a negative correlation between EST activity and larval growth in H. armigera. The total protein content of the larvae fed on insect resistant plants was significantly lower than the larvae fed on the susceptible genotype, JL 24. Reduced protein levels could have resulted from the toxic effects of the plants metabolites on serine proteases, trypsin and other enzymes. Further, plant protease in- hibitors and other antinutritional components are some of the important factors that affect protein synthesis in insects (War et al., 2013). Further, essential amino acid reduction drastically affects the insect growth and devel- opment (Chen et al., 2005). It has been reported that S. litura larvae fed on ba- nana leaves showed reduced growth and development, digestibility and consumption rate of plant tissues, how- ever, they exhibited a high conversion efficiency of the ingested food with a high rate of conversion of the digested food. This shows that that the insects compensated for the Acta agriculturae Slovenica, 117/1 – 20218 A. R. WAR et al. nutrient intake from the limited plant tissues by more ef- ficiently utilizing the host plant tissues (Zhu et al., 2005). The digestion of the plant tissues by insect pests depends on the activities of various enzymes including serine prote- ases, trypsin, and others. Several factors affect the AD, CI, ECI, and ECD in insect-plant interaction and it is practi- cally difficult to determine the exact “cause” and “effect” re- sponses of these parameters. The questions that arise here are, is it because of low digestibility that the insects eat low or is it that the digestibility is low as the insect eats more? The efficiency parameters involved in host plant feeding by insect pests are physiologically closely related. Though the factors responsible for the efficient conversion of the di- gested food are still largely unknown, role of shifts in food selection, insect digestive physiology, body composition and metabolic rates can’t be ruled out. It is very important to understanding the basic principles of nutritional ecolo- gy to identify the host plant resistance against insect pests. 5 CONCLUSION This study determined that the AD, CI, ECI, and ECD in H. armigera larvae were significantly reduced when fed with the insect-resistant groundnut genotypes. 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Delo je prispelo 20. junija 2020, sprejeto 10. decembra 2020. 1 ISVSA, University, Agronomy Department, Laboratory of Improvement of the Phytosanitary Protection Techniques in Mountanious Agrosystems, Algeria 2 Corresponding author, e-mail: abdelkader@univ-batna.dz Assessement of control strategy by spraying low doses of sugars on apple orchard against Cydia pomonella (Linnaeus, 1758.) Abstract: Codling moth (Cydia pomonella [L.]) is the most serious pest of apple worldwide. Its control still relies largely on insecticide applications. To deal with this situation, it becomes essential to design eco-friendly control systems to minimize chemical treatments. In this context, the effect of spraying of fructose (100 ppm), glucose (100 ppm) and insecti- cide (Deltamethrin), on the Golden Delicious variety against C. pomonella larval damages, was studied in an orchard located in Batna province (Algeria). The results of this study showed that codling moth own four generations in the study area. It is a very important pest with about 59.19 ± 1.15 % of damaged fruits at harvest. The spraying of glucose alone, fructose alone and the chemical insecticide alone causes a significant increase in the percentages of healthy fruit at harvest compared to the untreat- ed control. The use of fructose and glucose has significantly re- duced the percentage of damaged fruits at harvest followed by the spraying of the insecticide which produces the lowest per- centage. The Abbott’s efficacy of glucose treatments was 23.75 ± 2.6 % compared to the insecticide 37.6 ± 2.55 %; and fructose 15.54 ± 3.01 %. The use of sugars is a completely innovative way in the field of plant protection. These first results demonstrate a promising alternative to conventional programs. Key words: codling moth; glucose; fructose; Deltame- thrin; ‘Golden Delicious’ Ocenitev nadzorovanja jabolčnega zavijača (Cydia pomonella (Linnaeus, 1758)) s škropljenjem z majhnimi odmerki slad- korjev Izvleček: Jabolčni zavijač (Cydia pomonella [L., 1758]) je v svetovnem merilu najpomembnejši škodljivec jablan. Nje- govo uravnavanje še vedno v največji meri temelji na uporabi insekticidov. Za odpravo tega je bistveno najti okolju prijazen način uravnavanja, ki bi zmanjšal obravnavanje s kemikalija- mi. V tem smislu je bil preučevan učinek škropljenja s fruktozo (100 ppm), glukozo (100 ppm) in insekticidom (Deltamethrin) na obseg poškodb zlatega delišesa (Golden Delicious) zaradi ličink jabolčnega zavijača v sadovnjaku, v provinci Batna, Alži- rija. Rezultati so pokazali, da ima jabolčni zavijač na tem obmo- čju štiri generacije. Je zelo pomemben škodljivec, ki povzroči 59,19 ± 1,15 % poškodovanih plodov ob obiranju. Škropljenje samo z glukozo, fruktozo ali samo s kemijskim incekticidom je značilno povečalo odstotek zdravih plodov v primerjavi s kon- trolo brez obravnavanj. Uporabi glukoze in fruktoze sta zna- čilno zmanjšali odstotek poškodovanih plodov ob obiranju, a pri škropljenju z insekticidom je bil njihov odstotek najmanjši. Abbottove učinkovitosti obravnavanj so bile: z glukozo 23,75 ± 2.6 %, z insekticidom 37,6 ± 2.55 %; in s fruktozo 1554 ± 3.01 %. Uporaba sladkorjev je popolnoma nov način zaščite rastlin na prostem. Ti prvi izsledki nakazujejo obetajočo alter- nativo konvencionalnim programom zatiranja tega škodljivca. Ključne besede: jabolčni zavijač; glukoza; fruktoza; Del- tametrin; ‘Golden Delicious’ Acta agriculturae Slovenica, 117/1 – 202116 A. K. TIFFRENT and N. LOMBARKIA 1 INTRODUCTION The economic impact of C. pomonella (L., 1758). (Lepidoptera; Tortricidae) on apple trees is continuous- ly increasing. For reasons, the appearance of resistant populations to different insecticides (Charmillot et al., 2007). In addition to its high cost, insecticides present a potential danger to the environment and both the nega- tive side effects on beneficials and occasionally resulting in outbreaks of secondary phytophagous pests. The con- cept of “Sweet Immunity” postulates that sugar metabo- lism and signalling influence plant immune networks (Tarkowski et al., 2019). Water-soluble metabolites (sugars and sugar-alcohols) have been already identi- fied from surfaces of apple tree organs that they influ- ence plant site acceptance after alighting and stimulate C. pomonella egg-laying (Derridj et al., 1999). Fructose, sorbitol, and myo-inositol are then especially concerned within a blend of six metabolites (Lombarkia & Derridj, 2002). Soluble carbohydrates and sugar-alcohols on the leaf surface should, however, provide information both about plant physiology and host plant specificity. The concept of exogenous application of sugars every 20 days on apple trees, to modify the egg-laying of C. pomonella to reduce the damage it causes, was tested in commer- cial orchards of several countries (in France, in Italy, in Greece and in Algeria) and over several years it showed an Abbott efficiency of 40 to 59 % (Derridj et al., 2011). The research hypothesis was that exogenous ap- plication of sugars on apple trees would provide satis- factory efficacy and treatments with fructose alone and glucose alone would have less fruit damages than the untreated control. Codling moth larvae bore deep into the fruit mak- ing it unmarketable, it often leads to the fall of the fruit and may go through several generations in a growing season. However, it is often difficult to cope with large yield losses and the problem of reducing codling moth damage is therefore associated with challenges econom- ic benefits for the study area. Thus, the aim of the present paper is to evaluate the efficacy of sugars in reducing the damages caused by the codling moth. 2 MATERIAL AND METHODS 2.1 STUDY SITE The experiment was performed in a ‘Golden Deli- cious’ apple orchard in the Batna region (eastern Algeria) (35°21ʹ12,4" N, 006°01ʹ 19,1" E) during 2019 with four different treatments against C. pomonella. The treated or- chard was managed under common practices of the zone; it had a surface of 0.8 ha with 17 rows (19 trees each). The trees were 9 years old, and the plant spacing is 4 m × 4 m, whereas the height of the trees is approximately 3 m. 2.2 SEXUAL TRAPPING OF ADULTS Attractive sex pheromone trap (produced and sup- plied by Russell IPM) was used to monitor the progress of flight (qualitative forecasting), follow the pest dynam- ics and to estimate the level of pest population of C. pomonella. The sexual trap consisted of a plate coated with glue, which was deposited on a capsule containing the specific pheromone ([E, E]-8, 10-dodecadien-1-ol), which attracts the males entering the delta trap and be- come trapped in the sticky area. The device was attached by a wire to the tree foliage, placed at eye level. The pher- omone trap was installed between 17th March and the harvest date (25th September), and it was replaced at 6 weeks intervals. The observations were carried out every 3 days for 6 months (from March until September 2019). For each output, the date of catches and the total number of captured moths were mentioned. 2.3 TREATMENTS The treatments were adjusted in a randomized Latin square with 4 repetitions. All the modalities (4) are then distributed within each of the four blocks and each block has 3 trees. The efficacy was measured by the extent to which fruit damage attributable to C. pomonella cater- pillars was reduced by each treatment, compared to the unsprayed control. The applications were performed us- ing electrical pressure sprayer (12 V-12 Ah), capacity of Modalities Doses Treatments 1 Control (Untreated) Control (Untreated) 2 Fructose (Fluka Biochemika) 10 g 100 l-1 (100 ppm) 3 Glucose (Fluka Biochemika) 10 g 100 l-1 (100 ppm) 4 Decis 25 EC containing 25g l-1 Deltamethrin (Bayer) (0.5 l) 1000 l-1 Table 1: Tested Modalities and their doses Acta agriculturae Slovenica, 117/1 – 2021 17 Assessement of control strategy by spraying low doses of sugars on apple orchard against Cydia pomonella (Linnaeus, 1758.) 16 l. The modalities tested and their doses are reported in Table 1. The morning treatments (sugars and insecticide) were carried out every 20 days throughout the season from the flowering end until harvest (Derridj et al., 2012). 2.4 DAMAGE ASSESSMENTS The variable ‘percentage of infested and healthy fruits at harvest’ is based on the ratio of the total number of infest- ed fruits (damaged) and the total number of healthy fruits per plot. Abbott’s formula is very commonly used in field trials. Its efficacy at harvest measures the percentage of C. pomonella caterpillars infested fruits versus untreated con- trols according to the formula T0  –  Tt /T0  × 100; where T0 is the percentage of infested fruits in the untreated plots and Tt is the percentage of infested fruits in the treated plots. 2.5 STATISTICAL ANALYSIS The means between each variable ‘percentage of in- fested fruits, healthy fruits at harvest’, and Abbott’s effica- cy for the four modalities were compared by ANOVA on ranks test, followed by post hoc analysis using Fisher’s and Tukey’s tests (Table 3). A p-value of 0.05 was used to es- tablish significance in all tests. All analyses were performed using SPSS software v. 2016. 3 RESULTS AND DISCUSSION 3.1 SEXUAL TRAPPING OF ADULTS Considering the entire trapping season, 708 males were captured between March 20 and September 25, the captures per trap per three days varied between 1 and 73, that is on average 25.82 males /trap/week. The pace of the annual flight is shown quad-modal with the first tip of 32 males on April 22, and the second of 73 individuals from July 03, the third of 22 individuals on August 20 and the fourth 18 individuals on September 10 (Figure 1). The gates of the developed generations would then present the following sequences, the first (14.26 % of annual catches) taked place between March 20 (milestone date of the start of the insect’s activity) and May first. The second has a sig- nificant importance of 56.35 %, from May 4 to July 18. The third (15.96 %) took place between July 21 and August 26. The fourth, scanty (13.41 %) due to diapauses, fully covered the month of September (Table 2). The determination of generations is based on the fol- lowing principle: the division a significant and stable in- crease in catches, followed by a sufficiently long inter-flight (± 30 days) with few catches, indicates a nascent or finish- ing flight comparable to the start or the end of a generation (Hmimna & El Iraqui, 2015). 3.2 DAMAGE ASSESSMENTS AND TREATMENTS EFFICACY The spraying of glucose alone, fructose alone and the insecticide induced a significant increase in the per- centages of healthy fruit at harvest compared to the un- treated control. The analysis of variance (ANOVA) fol- lowed by the Tukey test (p < 0.05) identified three groups, control (40.80 %, group a), glucose and fructose 55.03 %, 50.29 % respectively (group b) and insecticide 63.19 % (group c) (Table 3). Similarly, foliar sprays of fructose and glucose made it possible to significantly reduce the percentage of fruit Figure 1: Seasonal flights of adult males of C. pomonella in ‘Golden Delicious’ orchard Acta agriculturae Slovenica, 117/1 – 202118 A. K. TIFFRENT and N. LOMBARKIA damaged at harvest compared to the control, the analy- sis of variance (ANOVA) followed by the Tukey test (p < 0.05) revealed three groups, control (59.19 %, group a), glucose and fructose 44.96 % and 49.70  % respectively (group b), followed by the spraying of the insecticide which produces the lowest percentage of 36.80 % (group c), (Table 3). The Abbott’s efficiency of glucose treatments is 23.75  % compared to the insecticide with 37.6  % and fructose generating an average percentage efficiency of 15.54 % (Table 3). Previous studies in Algeria and in France from 2009 to 2014 summarized by Arnault et al. (2016) have dem- onstrated the potential of foliar application of sucrose in micro-doses to control C. pomonella, and revealed a de- gree of infested fruits at harvest of the untreated control varied from 12 to 46.8 % in nine experiments were con- ducted in apple orchards. Compared with this study, the infestation was more important with 59.19 % at harvest which represent a very high pressure of the pest. The mentioned studies showed that sucrose can in- duce partial resistance to C. pomonella by foliar applica- tions at a very low dose of 0.01  %. The mean Abbott’s efficacy obtained for sucrose 100 ppm alone was 41.0 ± 10.0 %, while sucrose in combination with thiacloprid in- secticide; enhanced the efficacy of thiacloprid treatment by 18 %. The researchers have concluded that the effect of this combination is not additive but synergistic and rather potentiating. Furthermore, Arnault et al. (2015) confirmed, in their study conducted in an integrated fruit protection orchard on the Granny Smith variety, the advantage of applying fructose 100 ppm in combination with Or- ganoPhosphorus OP or Insect Growth Regulator (IGR) insecticides against codling moth. Sprays of fructose at 100 ppm in combination with OP and IGR have signifi- cantly reduced the percentage of damaged fruits at har- vest compared to the OP and IGR alone (6.5 % and 10 % respectively); efficiency was therefore improved by 35 % when fructose combined with OP and IGR. The same author has specified that in studies leading during 2013 and 2014 in an organic orchards, the foliar applications of fructose at 100 ppm could reduce codling moth damage by 55 % on the Gala variety, knowing that the use of fruc- tose at 100 ppm has significantly reduced the percentage of damaged fruits at harvest compared to the control (3,6 %, 14,9 %) and (2,5 %, 3,9 %) respectively, and the Abbott effectiveness was 76 % (in 2013) and 36 % in 2014). These researches are in good agreement with the results of the present study; this is consistent that the exogenous foliar application of a single sugar can induce plant resistance to the pest. Derridj et al. (2012) argued that foliar appli- cation of sucrose alone and/or other compounds from the formulation such as sugars are introduced into the commercial granulovirus products as phago-stimulants for more effective ingestion by larvae can induced modi- fication of the cuticular permeability and, therefore, of the composition of the leaf surface blend and its effects on pest behavior. Burges and Jones (1998) presented the sugars advantages in the formulation of microbial biope- sticides; act as phagostimulants, preservatives and dis- persants. It was the main purpose of the paper to draw atten- tion to primary metabolites, in particular, soluble carbo- hydrates. The sugars applied on the leaves in the morn- ing (best diffusion through the cuticle while the apoplast concentrations are low) are those usually found on apple leaves in the evening. This knowledge of sweet immunity here is even more complex. It seems to be resulted from an induction of partial antixenosis at the C. pomonella Generations G1 G2 G3 G4 Total number of catches 101 399 113 95 % of catches 14.26 % 56.35 % 15.96 % 13.41 % Table 2: The number of catches and their percentage of C. pomonella during the four generations Treatments % damaged fruits (Mean + SEM*) % healthy fruits (Mean + SEM*) % Abbott effectiveness (Mean + SEM*) Control (Untreated) 59,19 ± 1,15 a 40,80 ± 1,15 a fructose 100 ppm 49,70 ± 1,20 b 50,29 ± 1,19 b 15,54 ± 3,0 a glucose 100 ppm 44,96 ± 1,34 b 55,03 ± 1,33 b 23,75 ± 2,6 a Insecticide (Deltamethrin) 36,80 ± 1,40 c 63,19 ± 1,39 c 37,60 ± 2,55 b Table 3: Percentage of damaged and healthy fruits in apple orchards (n = 12) on different modalities (control, fructose, glucose, insecticides) * Values indicated with different letters are significantly different at p < 0.05 Acta agriculturae Slovenica, 117/1 – 2021 19 Assessement of control strategy by spraying low doses of sugars on apple orchard against Cydia pomonella (Linnaeus, 1758.) egg-laying linked to changes in chemical signals of the leaf surface induced by applications of these sugars, confirmed by the findings of Lombarkia (2002) and Lombarkia and Derridj (2008) that have been clearly investigated the im- portant role of primary metabolites and particularly sug- ars on C. pomonella. Egg-laying site preference within the apple tree and its intensity is related to a blend of three soluble carbohydrates (sucrose, D-fructose, and glucose) and three sugar alcohols (sorbitol, quebrachitol, myo-in- ositol) present at the surface of the apple tree. Quantities and ratios of the six primary metabolites found on the leaf surface may influence host preference of C. pomonella as well as their egg-laying behavior, thus they may play a role in the trees’ resistance to the codling moth (Lombarkia & Derridj, 2008). On the other hand, the resistance effect on C. pomo- nella female egg-laying is predominant in the reduction of larval damage, but this does not exclude effects on the neonate larvae before penetrating into the fruit. Derridj et al. (1999) found that soluble carbohydrates and sugar- alcohols present on apple tree surface discriminates apple tree sites such as leaves and leaf side, among the present substances, six were studied in laboratory in their study, fructose, sorbitol and myo-inositol stimulate C. pomonella egg-laying and enhance examination of the support by the neonate larvae. Glucose showed more deterrent effect on females and larvae, whereas quebrachitol and myo-inosi- tol arrested neonate larvae at the highest concentrations. In addition, we know that in the formulation of micro- bial biopesticides, there are simple sugars used for many reasons. It is probable that sugar has physiological effects on the plant. Stacey et al. (1977, 1980), suggested that in- creases in cotton yield recorded in the presence of sugars may have been due to the physiological effects of the sugar on the plant. 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Sweet Immunity: Inulin Boosts Resistance of Lettuce (Lactuca sa- tiva) against Grey Mold (Botrytis cinerea) in an Ethylene- Dependent Manner, International Journal of. Molecular. Sciences, 20, 1052. https://doi.org/10.3390/ijms20051052 Acta agriculturae Slovenica, 117/1, 1–16, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1847 Original research article / izvirni znanstveni članek Exploiting heterosis and combining ability in two-line hybrid rice Mahmoud M. GABALLAH 1, Aziz F. Abu EL-EZZ 1, Adel M. GHONEIM 2, 3, Baochang YANG 4, Langtao XIAO 5 Received August 22, 2020; accepted December 16, 2020. Delo je prispelo 22. avgusta 2020, sprejeto 16. decembra 2020. 1 Agricultural Research Center, Field Crops Research Institute, Rice Research and Training Center, Sakha, Kafr Elsheikh, Egypt 2 Agricultural Research Center (ARC), Field Crops Research Institute, Giza, Egypt 3 Corresponding author, e-mail: adelrrtc.ghoneim@gmail.com 4 Winall Thriving Seed Co., ltd. Hefei, Anhui, China 5 Hunan Agricultural University, Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Changsha, China Exploiting heterosis and combining ability in two-line hybrid rice Abstract: Twenty hybrids were developed from crossing, four environmental genic male sterile (EGMS) lines with five testers in line × tester mating design to magnitude of hetero- sis over better parent for grain yield and contributing traits in rice (Oryza sativa L.). Five hybrids ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9039’ × ‘Sakha108’ and ‘WTSC9039’ × ‘Sakha101’ ex- press superior value for number of panicles, fertility percentage, 100-grain mass, grain yield, apparent heterosis and phenotypic acceptance. The top three heterotic combinations identified for grain yield/ha were ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha105’ which exhibited 100.00, 71.51 and 66.61  % heterobeltiosis, respectively. The lines ‘WTSC9059’ and ‘‘Longping’’ and testers ‘Sakha101’, ‘Sakha102’ and ‘Sakha108’ was found to be good general com- biner for most of the characteristics and could be extensively used in future hybrid rice breeding program. The grain yield was correlated highly significant and positive with panicle exer- tion, panicle mass, fertility percentage and appearance of het- erosis, otherwise the negative correlation and significant was found with flag leaf area. Key words: heterosis; combining ability; line × tester; cor- relation; hybrid rice Uporaba heteroze in kombinacijske sposobnosti pri dveh lini- jah hibridnega riža Izvleček: Dvajset križancev je nastalo iz križanj štirih okoljsko gensko moško sterilnih (EGMS) linij s petimi testerji po principu linija × tester za povečanje heteroze v primerjavi z boljšo starševsko linijo za pridelek zrnja in z njim povezanimi lastnostmi pri rižu (Oryza sativa L.). Pet križancev ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sa- kha108’, ‘WTSC9039’ × ‘Sakha108’ in ‘WTSC9039’ × ‘Sakha101’ ima veliko boljše vrednosti za znake kot so število latov, odsto- tek plodnosti, masa 100 zrn, pridelek zrnja, aparentna heteroza in fenotipska sprejemljivost. Najboljše tri heterotične kombi- nacije za pridelek zrna na hektar so bile ‘WTSC9059’ × ‘Sa- kha101’, ‘WTSC9039’ × ‘Sakha102’ in ‘Longping’ × ‘Sakha105’, ki so pokazale 100,00; 71,51 in 66,61  % heterobeltioze. Liniji ‘WTSC9059’ in ‘Longping’ in testerji ‘Sakha101’, ‘Sakha102’ in ‘Sakha108’ so bili prepoznani kot dobri splošni kombinatorji za večino lastnosti in bi lahko bili na široko uporabljeni v bodo- čih križanjih pri žlahtnenju riža. Pridelek zrnja je zelo značilno pozitivno koreliral z močjo lata in njegovo maso, odstotkom fertilnosti in pojavom heteroze, nasprotno je bila negativna ko- relacija s površino najvišjega, zastavnega lista. Ključne besede: heteroza; kombinacijska sposobnost; li- nija × tester; korelacija; hibridni riž Acta agriculturae Slovenica, 117/1 – 20212 M. M. GABALLAH et al. 1 INTRODUCTION Rice (Oryza sativa L.) is one of the world’s three ma- jor food crops and represents an important component of the world’s food supply. Hybrid rice technology has con- tributed significantly toward food security, environmen- tal protection, and employment opportunities (Gramaje et al., 2020). Generating hybrid rice, which presents a higher grain yield than inbred rice varieties, is one of the most important applications of heterosis in agricul- ture (Liu et al., 2020). Rice is a self-pollinating crop, and nearly all traditional rice cultivars are inbred lines. Since the 1970s, hybrid seed production has mainly used two- line or three-line hybrid systems (Chang et al., 2016). The three-line system is based on a cytoplasmic male sterile (CMS) line, a restorer line (to produce F1 hybrid seeds) and a CMS maintainer line (to maintain the CMS line). The two-line hybrid system is based on environmentally sensitive genetic male sterility (Yu et al., 2020)” and it usually uses photoperiod- and thermo-sensitive genic male sterile (PTGMS) lines as maternal parents to pro- duce hybrid seeds. PTGMS lines are sterile under restric- tive conditions (high temperatures and long days) but become fertile under permissive conditions such as low temperatures and short days (Lan et al., 2019). Environ- ment-sensitive genic male sterility is controlled by nucle- ar gene expression, which is influenced by environmental factors such as temperature, day length, or both (Chang et al., 2016). In the two-line system, certain lines, referred to S lines, can be either male sterile (functionally female) or male (produces viable pollen) depending upon tem- perature and day length. Under temperature / day length combined condition, the S lines are crossed as females to fertile inbred lines to produce hybrid seed, while under separate temperature/day length combination, the same lines are allowed to self-pollinate and produce viable seeds to maintain a source. The success of hybrid rice technology primarily depends on genetic purity, timely availability and the affordability of hybrid seed costs to the farmers (Singh and Ram, 2012). The line × tester analysis is a powerful tool to dis- criminate the good and poor combiners for choosing ap- propriate parental material in successful hybrid breeding program. It gives reliable information about the nature and degree of gene action and combining ability effects existent in the genetic materials (Akter et al., 2010). Breeding value can be deduced from the general and spe- cific combining abilities for key desirable traits, as well as from the phenotypic and genotypic value of the parental forms for highly inheritable traits. The general combin- ing ability (GCA) of a given parent for a particular trait is defined by the mean value of the trait in the half–sibling progeny of this parent. The specific combining ability (SCA) reflects how a particular pair of parents contrib- utes to the presence of a particular trait level in the hybrid progeny. SCA is defined as interaction between a pair of the parents for a progeny trait. Studies on the genetic ba- sis of heterosis for polygenic traits in various crops have shown that heterosis is the result of partial to complete dominance, overdominance, and epistasis and it may be a combination of all these (Gokulakrishnan and Kumar, 2013). Evidence of real overdominance for quantitative traits is hard to find. However, apparent overdominance caused by nonallelic interaction and linkage disequilib- rium is a common contributor to heterosis (Dan et al., 2019 and Hijam et al., 2019). The present study was con- ducted with objective to determine the heterosis levels in generated hybrids, estimate GCA of the pollinator tester and EGMS lines, determine SCA of the generated hybrid crosses, and estimate the nature and degree of gene ac- tions and heritability. 2 MATERIALS AND METHODS 2.1 GENETIC MATERIALS A total of 20 testcrosses were developed by ap- plying a line × tester mating design using four EGMS lines (S) and five pollinators tester (P) as described by Kempthorne (1957). The nine parents were cultivated thrice at an interval of 10 days to ensure synchroniza- tion in flowering for the purpose of hybridization in San- ya, Hainan Province, China, 2017 rice growing season. The hybrid crosses and their parents were evaluated at the experimental field of the Winall Thriving Seed Co., (WTSC) Farm in 2018 rice growing season in Hefei, An- hui Province, China. The origin, pedigree, salience and feature of nine rice genotypes were illustrated in Table 1. At 2017 rice growing season thirty days after sowing, the seedlings were transplanted by hand into the paddy field with each plot containing 5 rows with 10 plants per row at a space of 20 cm × 20 cm. Twenty-five days af- ter pollination, the F1 seeds were harvested, dried by air, threshed by hand and stored at room temperature. The F1 hybrids’ seeds and their parents (pollinator lines and EGMS lines) were nursed in the May 1ST 2018. Thirty days later, the seedlings were transplanted into the paddy field with one seedling per hill using a randomized com- plete block design and three replications for each geno- type. Each plot contained 15 rows with 20 plants per row at a space of 20 cm × 15 cm, each test entry consisted 15 rows of 2 m length, the plant area for each genotype was 6 m2. All practices recommended package for planting, transplanting, N, P, K and Zn fertilizers, water manage- ment and plant protection were followed. The soil of the Acta agriculturae Slovenica, 117/1 – 2021 3 Exploiting heterosis and combining ability in two-line hybrid rice experimental site was clay, with organic matter contents of 17.65 %. The geographical location and climate of the experimental site were given in Fig. 1. 2.2 FIELD EVALUATION After harvest maturity, all the panicles of randomly selected 10 plants were harvested manually, put them into a Nylon mesh bag, sun-dried and threshed. Grain yield (t ha-1, GY) for each plot was recorded from a 33- hill sample, adjusted to a moisture content of 14 % and expressed in kg ha-1. Important agronomic traits includ- ing days to 50 % heading (DTH), plant height (cm; PH), SPAD-chlorophyll values (SPAD; CC), flag leaf angle(o; FAG) and area (cm2; FAR), panicle length (cm; PL), num- ber of panicles per plant (NP), panicle mass (g; PW), fer- tility percentage (%; FP) and 100-grain mass (g; HGW) according to standard evaluation system (IRRI, 2002). Panicle exertion (PE) refers to the proportion of the pan- icle that is exerted from the flag leaf sheath to the total panicle length after the full blooming, which is expressed in percentage. Grain type (GT) as per 1-9 scale, where 1, 3, 5, 9 scale based on length-to-width ratio: slender, > 3.0; me- dium, 2.1 to 3.0; bold 1.1 to 2.0; and round, < 1.0, respec- tively. Apparent heterosis (AH) subjective superiority of a hybrid over its parents or a check variety based on visual observation and expressed as vigorous vegetative growth in the field. Phenotypic acceptability (PA), pollen sterility/fertility were monitored and lines were evaluated on a single plant basis for phenotypic acceptability (on a 1–9 scale, where 1 is unacceptable, 3 is poor, 5 is fair, 7 is good and 9 is excellent) according to (Khush et al., 2003). 2.3 STATISTICAL ANALYSIS 2.3.1 Analysis of variance and correlation coefficient of traits Analysis of variance (ANOVA) was performed us- ing a general linear model Whereas, Yijk = the observed value, µ = the popu- lation mean, Pij = the mean effect of the ijth genotype, Rk = the kth replication effect and eijk is the experimen- tal error assumed with ijkth observation and is assumed to be normally and independently distributed with a mean of zero and variance of σ2. Mean comparisons among genotypes were evaluated using Tukey’s HSD test (p 0.05). Correlation among values of the different traits investigated were performed based on Pearson’s product- moment correlation as implemented. 2.3.2 Estimation of heterosis Heterobeltiosis or better parent heterosis (BPH) was estimated in terms of percent increase or decrease of the F1 hybrid over its better parent (Fehr, 1987). Significance of better parent heterosis was deter- mined following the “t” test suggested by (Wynne et al., 1970). Figure 1: Illustrate of the geographical location and average of temperature and humidity of the experimental site. Acta agriculturae Slovenica, 117/1 – 20214 M. M. GABALLAH et al. Where, F1 = Mean of the F1 hybrid for a trait, BP = Mean of better parent in the cross, and EMS = Error mean square. 2.3.3 Combining ability analysis Estimates of GCA and SCA effects and their vari- ances were computed using line × tester analysis accord- ing to (Singh & Chaudhary, 1985). The analysis was done using the Agrobase software statistical package. Additive and dominance genetic variances (σ2A and σ 2 D) were cal- culated by taking inbreeding coefficient (F) equal to one; that is, F = 1 because both lines and testers were inbred. Significance test for general and specific combining abil- ity effects were performed using t-test. The relative mass of additive versus non-additive type of gene actions was calculated as described by (Verma & Srivastra, 2004). 2.3.4 Heritability Broad (H2) and narrow sense (h2) heritability for the measured traits were estimated based on Griffiths et al.) (2000). 3 RESULTS 3.1 MEAN PERFORMANCE Means of the parental genotypes and relative F1 crosses for the fifteen studied characters showed that the earlier heading was obtained in genotypes ‘Longping’ × ‘Sakha108’, ‘Sakha107’, ‘WTSC9059’ × ‘Sakha105’ and ‘Longping’ × ‘Sakha105’ while the delayed heading geno- types were ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha108’, ‘WTSC9039’ × ‘Sakha108’ and ‘WTSC9369’ × ‘Sakha107’. The tallest plants were observed for ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha108’ and ‘WTSC9369’ × ‘Sakha108’ crosses while minimum for ‘WTSC9059’, ‘WTSC9039’ and ‘Longping’ lines. The highest chlorophyll content values were observed for parents ‘Sakha105’, ‘WTSC9039’, ‘Sakha107’ and cross ‘WTSC9059’ × ‘Sakha101’, otherwise the lowest values for ‘WTSC9369’ × ‘Sakha107’, ‘WTSC9039’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha105’ and ‘WTSC9369’, respectively. With respect to flag leaf angle (o) the erect flag leaves were found for crosses ‘Longping’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha108’ and ‘Longping’ × ‘Sakha105’ while the wid- est flag leaf angle was found with genotypes ‘WTSC9059’ No. Entries Pedigree Origin Salience and feature 1 ‘WTSC9039’ 18925/815 2011SH7 China Indica/Japonica type, EGMS line, short grain, plant height 90 cm, grain yield 7-7.20 t ha-1, resistance to blast, sensitive to drought and salinity. 2 ‘WTSC9059’ Y585/18925 2011SH8 China Indica/Japonica type, EGMS line, short grain, plant height 82 cm, grain yield 4-4.50 t/ha, resistance to blast, sensitive to drought and salinity. 3 ‘WTSC9369’ 18925/9451/958/9054 China Indica/Japonica type, EGMS line, short grain, plant height 88 cm, grain yield 5-5.20 t/ha, resistance to blast, sensitive to drought and salinity. 4 ‘Longping’ Long ping seed company ltd. China Indica/Japonica type, EGMS line, short grain, plant height 80 cm, grain yield 4.30-4.50 t/ha, resistance to blast, sensitive to drought and salinity. 5 ‘Sakha101’ Giza 176 / Milyang 79 Egypt Japonica type, pollinator, short grain, plant height 90 cm, high yield 10.5 t/ha, susceptible to blast and sensitive to drought and salinity 6 ‘Sakha102’ GZ 4096-7-1 / Giza 177 Egypt Japonica type, pollinator, short grain, plant height 110 cm, high yield 10.0 t/ha, resistance to blast and sensitive to drought and salinity 7 ‘Sakha105’ GZ 5581-46-3 / GZ 4316- 7-1-1 Egypt Japonica type, pollinator, short grain, plant height 103 cm, high yield 10.0 t/ha, resistance to blast and sensitive to drought and salinity 8 ‘Sakha107’ Giza 177 / BL1 Egypt Japonica type, pollinator, short grain, plant height 106 cm, high yield 10.0 t/ha, resistance to blast and tolerance to drought and moderate tol- erance to salinity 9 ‘Sakha108’ ‘Sakha101’/ HR5824- B-3-2-3 //’Sakha101’ Egypt Japonica type, pollinator, short grain, plant height 90 cm, high yield 10.5 t/ha, resistance to blast and sensitive to drought and salinity Table 1: Origin, pedigree, salience and feature of nine rice genotypes WTSC , Winall Thriving Seed Company ltd. Acta agriculturae Slovenica, 117/1 – 2021 5 Exploiting heterosis and combining ability in two-line hybrid rice Ta bl e 2: M ea n pe rf or m an ce fo r g ra in y ie ld a nd it s c on tr ib ut in g tr ai ts fo r l in es , t es te rs a nd th ei r c ro ss es G en ot yp e D ay s t o he ad in g (d ay ) Pl an t he ig ht (c m ) C hl or op hy ll co nt en t (S PA D ) Fl ag le af an gl e (o ) Fl ag le af ar ea (c m 2 ) Pa ni cl e ex er tio n (% ) Pa ni cl e le ng th (c m ) N um be r of p an ic le s/ pl an t Pa ni cl e m as s (g ) Fe rt ili ty Pe rc en ta ge (% ) 10 0- gr ai n m as s ( g) G ra in y ie ld t h a-1 G ra in Ty pe Ap pa re nt he te ro sis Ph en ot yp ic A cc ep ta bi lit y ‘W TS C 90 39 ’ 88 .2 8 69 .6 7 45 .0 0 27 .5 0 37 .4 0 81 .0 1 22 .3 3 8. 67 7. 67 87 .2 2 3. 63 7. 02 2. 52 4. 50 8. 00 ‘W TS C 90 59 ’ 82 .2 6 64 .0 0 41 .0 0 42 .5 0 42 .2 2 86 .0 8 25 .6 7 11 .6 7 4. 00 59 .6 3 2. 77 4. 51 2. 62 6. 50 8. 25 ‘W TS C 93 69 ’ 88 .2 8 78 .6 7 38 .0 0 37 .5 0 30 .4 0 81 .0 1 20 .6 7 11 .3 3 5. 33 58 .1 5 3. 00 5. 02 3. 52 5. 00 7. 90 ‘L on gp in g’ 80 .2 5 75 .0 0 40 .0 0 17 .0 0 23 .6 7 91 .1 4 20 .3 3 8. 17 4. 47 63 .8 4 2. 83 3. 51 1. 01 5. 50 6. 25 ‘S ak ha 10 1’ 94 .2 9 10 0. 00 40 .5 0 35 .5 0 45 .6 7 10 0. 00 22 .0 0 7. 33 2. 17 87 .7 9 2. 73 7. 02 1. 21 4. 50 8. 30 ‘S ak ha 10 2’ 79 .2 5 12 1. 00 41 .0 0 35 .0 0 57 .5 3 10 0. 00 22 .0 0 11 .3 3 2. 90 86 .0 1 2. 97 6. 52 1. 31 4. 50 8. 35 ‘S ak ha 10 5’ 80 .2 5 10 7. 33 43 .5 0 27 .5 0 45 .2 3 10 0. 00 24 .3 3 11 .6 7 2. 77 89 .5 8 3. 57 6. 02 1. 11 5. 50 8. 25 ‘S ak ha 10 7’ 76 .2 4 98 .3 3 45 .5 0 42 .5 0 52 .5 0 10 0. 00 20 .3 3 12 .3 3 2. 53 97 .0 0 2. 57 7. 52 0. 91 5. 50 8. 50 ‘S ak ha 10 8’ 90 .2 8 10 1. 00 40 .5 0 35 .5 0 46 .5 0 10 0. 00 23 .6 7 10 .6 7 2. 43 94 .6 7 3. 30 8. 03 1. 11 5. 50 7. 05 ‘W TS C 90 39 ’ × ‘S ak ha 10 1’ 88 .2 8 11 2. 67 41 .0 0 20 .5 0 72 .3 7 10 0. 00 25 .3 3 12 .0 0 8. 07 94 .2 2 3. 70 11 .0 3 1. 21 7. 50 8. 25 ‘W TS C 90 39 ’ × ‘S ak ha 10 2’ 80 .2 5 11 8. 67 40 .5 0 22 .5 0 65 .6 3 70 .8 9 29 .3 3 16 .6 7 3. 03 51 .0 0 3. 07 12 .0 4 1. 21 9. 00 6. 25 ‘W TS C 90 39 ’ × ‘S ak ha 10 5’ 81 .2 5 10 8. 67 40 .5 0 25 .0 0 81 .2 0 10 0. 00 27 .6 7 9. 33 8. 70 93 .0 3 3. 70 3. 01 0. 80 7. 50 6. 35 ‘W TS C 90 39 ’ × ‘S ak ha 10 7’ 82 .2 6 10 8. 00 38 .5 0 35 .0 0 50 .9 3 60 .7 6 25 .6 7 11 .6 7 2. 97 20 .0 0 2. 70 4. 01 0. 91 7. 50 5. 25 ‘W TS C 90 39 ’ × ‘S ak ha 10 8’ 11 4. 36 13 1. 00 40 .5 0 37 .5 0 73 .3 3 10 0. 00 27 .3 3 9. 00 8. 70 93 .6 2 3. 97 11 .0 3 1. 21 8. 00 6. 40 ‘W TS C 90 59 ’ × ‘S ak ha 10 1’ 10 0. 31 11 7. 00 44 .5 0 34 .5 0 63 .8 2 10 0. 00 25 .3 3 10 .6 7 10 .3 3 95 .0 0 4. 13 14 .0 4 1. 11 9. 00 8. 35 ‘W TS C 90 59 ’ × ‘S ak ha 10 2’ 78 .2 4 11 7. 67 41 .5 0 40 .0 0 49 .7 7 10 0. 00 25 .3 3 9. 67 5. 67 95 .6 7 2. 86 10 .0 3 1. 01 5. 50 5. 30 ‘W TS C 90 59 ’ × ‘S ak ha 10 5’ 76 .2 4 10 6. 67 40 .5 0 40 .0 0 55 .8 3 10 0. 00 26 .8 3 12 .6 7 5. 97 82 .6 7 2. 72 7. 52 1. 11 7. 50 8. 20 ‘W TS C 90 59 ’ × ‘S ak ha 10 7’ 10 7. 34 10 7. 33 41 .0 0 26 .5 0 61 .2 8 70 .8 9 23 .8 3 12 .3 3 3. 77 50 .6 7 2. 65 8. 03 1. 21 7. 50 7. 00 ‘W TS C 90 59 ’ × ‘S ak ha 10 8’ 10 0. 31 12 0. 67 42 .5 0 24 .5 0 72 .0 0 10 0. 00 27 .5 0 9. 00 5. 40 95 .0 0 2. 48 12 .0 4 1. 01 8. 50 9. 10 ‘W TS C 93 69 ’ × ‘S ak ha 10 1’ 11 0. 34 12 7. 67 40 .5 0 25 .5 0 62 .6 7 10 0. 00 24 .3 3 13 .3 3 6. 17 92 .9 3 2. 76 11 .0 3 1. 01 7. 50 8. 50 ‘W TS C 93 69 ’ × ‘S ak ha 10 2’ 86 .2 7 12 0. 00 40 .5 0 30 .0 0 54 .1 3 65 .8 2 24 .3 3 9. 67 2. 20 41 .0 0 2. 00 4. 01 2. 42 7. 25 5. 50 ‘W TS C 93 69 ’ × ‘S ak ha 10 5’ 85 .2 7 10 9. 67 39 .5 0 30 .0 0 66 .4 0 67 .8 5 29 .6 7 7. 00 2. 03 45 .0 0 1. 73 3. 70 1. 31 6. 00 5. 50 ‘W TS C 93 69 ’ × ‘S ak ha 10 7’ 11 4. 36 11 5. 33 39 .5 0 26 .5 0 39 .6 2 66 .8 4 25 .5 0 11 .0 0 2. 93 35 .0 0 2. 65 4. 01 2. 62 8. 25 7. 50 ‘W TS C 93 69 ’ × ‘S ak ha 10 8’ 11 1. 35 13 1. 33 39 .5 0 15 .5 0 91 .0 0 10 0. 00 27 .3 3 10 .6 7 3. 23 71 .0 0 3. 07 8. 03 0. 80 7. 30 6. 50 ‘L on gp in g’ × ‘S ak ha 10 1’ 10 1. 32 11 1. 67 41 .5 0 27 .5 0 41 .6 7 10 0. 00 22 .0 0 11 .0 0 4. 30 91 .0 0 2. 20 10 .0 3 0. 91 7. 15 6. 85 ‘L on gp in g’ × ‘S ak ha 10 2’ 79 .2 5 12 4. 00 40 .5 0 40 .0 0 65 .6 5 10 0. 00 24 .8 3 9. 67 4. 40 97 .3 3 2. 55 9. 53 1. 01 8. 25 5. 50 ‘L on gp in g’ × ‘S ak ha 10 5’ 76 .2 4 10 1. 67 41 .5 0 17 .5 0 49 .2 7 10 0. 00 23 .1 7 11 .0 0 3. 30 94 .0 0 2. 45 10 .0 3 1. 11 8. 20 7. 50 ‘L on gp in g’ × ‘S ak ha 10 7’ 10 7. 34 11 3. 33 41 .0 0 20 .0 0 62 .6 8 65 .8 2 24 .5 0 12 .6 7 1. 50 28 .0 0 3. 00 3. 01 1. 11 7. 30 7. 00 ‘L on gp in g’ × ‘S ak ha 10 8’ 75 .2 9 37 .3 3 33 .1 7 9. 17 14 .9 3 10 0. 00 8. 00 10 .6 7 4. 20 80 .3 3 0. 70 9. 03 1. 01 2. 40 2. 33 LS D 0 .0 5 18 .9 0 19 .8 8 7. 00 9. 25 15 .6 4 1. 95 4. 73 3. 33 0. 93 9. 23 0. 43 0. 08 0. 03 1. 45 1. 33 LS D 0 .0 1 26 .7 2 28 .1 1 9. 90 13 .0 8 22 .1 1 2. 76 6. 69 4. 71 1. 32 13 .0 5 0. 61 0. 11 0. 04 2. 05 1. 88 Acta agriculturae Slovenica, 117/1 – 20216 M. M. GABALLAH et al. × ‘Sakha105’ (40 o), ‘Longping’ × ‘Sakha102’(40 o), ‘WTSC9059’ (42.5 o) and ‘Sakha107’(42.5 o). Regard- less the small flag leaf area was found for ‘Longping’ × ‘Sakha108’, ‘Longping’, ‘WTSC9369’ and ‘WTSC9039’ crosses and the largest area for crosses ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9039’ × ‘Sakha105’ and ‘WTSC9369’ x ‘Sakha108’. The crosses ‘Longping’ × ‘Sakha101’, ‘Longping’ × ‘Sakha102’, ‘Long- ping’ × ‘Sakha105’ and ‘Longping’ × ‘Sakha108’ had the complete panicle exertion percentage while the par- tial and lowest values were obtained from hybrid com- binations ‘WTSC9039’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha102’, ‘Longping’ × ‘Sakha107’ and ‘WTSC9369’ × ‘Sakha107’. The highest panicle length (cm) was observed with crosses ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha102’ and ‘WTSC9369’ × ‘Sakha105’, respectively. However, the lowest pani- cle length found for genotypes ‘Longping’ × ‘Sakha108’, ‘Longping’, ‘Sakha107’ and ‘WTSC9369’. The maxi- mum number of panicles/ plants were obtained from crosses ‘WTSC9059’ × ‘Sakha105’ (12.67), ‘Longping’ × ‘Sakha107’ (12.67), ‘WTSC9369’ × ‘Sakha101’ (13.33) and ‘WTSC9039’ × ‘Sakha102’ (16.67) compared to gen- otypes ‘WTSC9369’ × ‘Sakha105’ (7), ‘Sakha101’ (7.33), ‘Longping’ (8.17) and ‘WTSC9039’ (8.67) with smaller panicles number. The heaviest panicle mass were found for crosses ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha108’ and ‘WTSC9059’ × ‘Sakha101’ that ranged from 8.07 g and 10.33 g while the lowest panicle mass was found for genotypes ‘Longping’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha105’, ‘Sakha101’ and ‘WTSC9369’ × ‘Sakha102’ which ranged between 1.5 g to 2.2 g. Maximum fertility percentage was found for geno- types ‘WTSC9059’ × ‘Sakha108’ (95 %), ‘WTSC9059’ × ‘Sakha102’ (95.67 %), ‘Sakha107’ (97 %) and ‘Longping’ × ‘Sakha102’ (97.33 %) while the minimum fertility was noted with ‘WTSC9039’ × ‘Sakha107’ (20 %), ‘Longping’ × ‘Sakha107’ (28 %), ‘WTSC9369’ × ‘Sakha107’ (35 %) and ‘WTSC9369’ × ‘Sakha102’ (41  %). The highest 100-grain mass (g) was found for crosses ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha108’ and ‘WTSC9059’ × ‘Sakha101’ which ranged from 3.7 g to 4.13 g. While, lowest 100-grain mass was gained from crosses ‘Longping’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha105’, ‘WTSC9369’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha101’. The highest grain yield was found for crosses ‘WTSC9369’ × ‘Sakha101’ (11.03 t/ha), ‘WTSC9039’ × ‘Sakha102’ (12.04 t/ha), ‘WTSC9059’ × ‘Sakha108’ (12.04 t/ha) and ‘WTSC9059’ × ‘Sakha101’ (14.04 t/ha), while the lowest grain yield was found with genotypes ‘WTSC9039’ × ‘Sakha105’ (3.01 t/ha), ‘Long- ping’ × ‘Sakha107’ (3.01 t/ha), ‘Longping’ (3.51 t/ha) and ‘WTSC9369’ × ‘Sakha105’ (3.7 t ha-1). The short grain type was found with genotype ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9369’ × ‘Sakha108’, ‘Sakha107’ and ‘WTSC9039’ × ‘Sakha107’, while the longest grain type was found for genotypes ‘WTSC9039’, ‘WTSC9059’, ‘WTSC9369’ × ‘Sakha107’ and ‘WTSC9369’. The highest apparent het- erosis was obtained from crosses ‘Longping’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9039’ × ‘Sakha102’ and ‘WTSC9059’ × ‘Sakha101’ which ranged from 8.25 to 9, however the lowest mean values were observed with gen- otypes ‘Longping’ × ‘Sakha108’, ‘WTSC9039’, ‘Sakha101’ and ‘Sakha102’ that ranged between 2.4 to 4.5. Regardless phenotypic acceptability, the desired mean values 8.35, 8.5, 8.5 and 9.1 were found with genotypes ‘WTSC9039’ × ‘Sakha108’, ‘Sakha105’, ‘WTSC9059’ × ‘Sakha108’ and ‘WTSC9059’ × ‘Sakha107’, respectively, otherwise the lowest mean values were obtained from crosses ‘Long- ping’ × ‘Sakha108’, ‘Longping’ × ‘Sakha107’, ‘WTSC9039’ × ‘Sakha105’ and ‘WTSC9059’ × ‘Sakha101’, respectively. Pearson’s correlation revealed that all yield com- ponent traits were positively correlated with GY except for FAR and highly significant for traits PE, PW, FP and AH otherwise the negative correlation and significant was found with AH (Table 3). DTH was correlated sig- nificant and positive with FAG and AH. PH correlated significant and positive with CC, FAG, PL, FP, HGMW and AH while negative and significant with GT. CC was correlated significant and positive with FAR, FP, HGWM and PA. The positive and significant correlation was found among FAG and PL, HGW and AH otherwise the correlated negative and significant with GT. PE has positive correlation and significant with PW, FP and GY while negative and significant with GT. The positive cor- relation and significant was found between PL and HGW, AH and PA. NPP has significant and positive correlation with AH. The significant correlation and positive was found among PW and FP, HGW and GY. FP has posi- tive and negative significant correlation with GY and GT, respectively. The positive and significant correlation was found with HGW, AH and PA. 3.2 HETEROSIS PERCENTAGE The degree of heterosis showed over better parent (Table 4). Days to heading; heterosis percent over bet- ter parent showed highly significant and negative which ranged from -5.00 (‘Longping’ × ‘Sakha105’) to 6.18% (‘Longping’ × ‘Sakha108’). Three hybrids out from twen- ty crosses exhibited negative significant heterosis for days to heading. The plant height, desirable highly significant and negative heterosis over better parent was found for cross ‘Longping’ × ‘Sakha108’. Four hybrids exhibited Acta agriculturae Slovenica, 117/1 – 2021 7 Exploiting heterosis and combining ability in two-line hybrid rice positively significant heterosis over better parent the de- gree of heterosis that varied from 1.22 % (‘WTSC9059’ × ‘Sakha102’) to 8.54 % (‘WTSC9059’ × ‘Sakha101’). For flag leaf angle, heterosis percent over better par- ent ranged from -2.82  % (‘WTSC9059’ × ‘Sakha101’) to -56.34 % (‘WTSC9369’ × ‘Sakha108’). Eleven hy- brids exhibited desirable significant negative heterosis for this trait over better parent. For flag leaf area, four- teen hybrids had desired significant positive heterosis over better parent. ‘WTSC9369’ × ‘Sakha108’ (95.70 %) recorded the highest positive heterosis over better par- ent followed by ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha108’ with 79.53  %, 58.46 % and 57.70 %, respectively. Significant and posi- tive heterosis ranged 8.93 % to 95.70 %. Thirteen crosses out from twenty crosses have complete panicle exertion and the heterosis over better parent equal 0.00 %. For panicle length (cm), fourteen out from twenty crosses showed highly significant and positive heterosis accord- ing to over better parent which ranged 4.52 % to 31.35 %. The crosses ‘WTSC9039’ × ‘Sakha101’, ‘Longping’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha105’ and ‘Longping’ × ‘Sakha107’ gave significant and positive heterosis over better parent that ranged be- tween 2.76 % to 38.41 % for number of panicles/plant. The panicle mass had significant and positive heterosis due to better parent found with eight hybrid crosses ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sakha105’ and ‘WTSC9059’ × ‘Sakha101’ were values 5.22 %, 13.43 %, 13.43 %, 15.76 %, 35.00 %, 41.75  %, 49.25  % and 158.25  %, respectively. Among the 20 hybrids, four hybrids showed significantly posi- tive heterosis for fertility percentage were ‘WTSC9039’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha102’ which ranged between 7.32 % to 13.16 %. Three hybrids out of twenty crosses showed significant and positive heterosis over better parent including ‘Longping’ × ‘Sakha107’ (6.01%), ‘WTSC9039’ × ‘Sakha108’(9.37%) and ‘WTSC9059’ × ‘Sakha101’(49.10%) for 100-grain mass. Eleven out of twenty crosses had significant and positive heterosis over better parent for grain yield with the best hybrids includ- ing ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha101’, ‘Longping’ × ‘Sakha105’ and ‘WTSC9039’ × ‘Sakha102’ with 57.12  %, 66.61  %, 71.51  % and 100.00  %, respec- tively. For grain type fifteen from twenty crosses showed significant and negative heterosis over better parent which ranged between -25.57 % to -77.27 %. For appar- ent heterosis, eighteen crosses recorded desired signifi- cant positive heterosis over better parent. Better parent heterosis ranged between 9.09 and 100.00 %. Only one cross ‘WTSC9059’ × ‘Sakha108’ had positive and signifi- cant heterosis over better parent for phenotypic accept- ability. 3.3 COMBINING ABILITY ANALYSIS Analysis of variance of combining ability revealed   DTH PH CC FAR FAG PE PL NPP PM FP HGM GY GT AH PH 0.44** CC 0.05 0.32* FAR -0.16 0.16 0.35* FAG 0.35* 0.78** 0.27 0.02 PE -0.14 0.03 0.18 0.06 0.09 PL 0.26 0.71** 0.40* 0.27 0.74** -0.21 NPP -0.01 0.13 0.01 -0.07 0.09 -0.15 0.06 PW 0.13 0.04 0.21 0.07 0.23 0.40* 0.12 -0.12 FP -0.15 0.06 0.36* 0.14 0.12 0.93** -0.14 -0.18 0.49** HGW 0.24 0.36* 0.57** 0.24 0.41* 0.19 0.49** 0.09 0.55** 0.27 GY 0.16 0.28 0.22 -0.03 0.26 0.53** 0.03 0.24 0.45** 0.61** 0.17 GT 0.05 -0.35* -0.06 0.24 -0.40* -0.44** -0.06 -0.04 -0.02 -0.36* 0.01 -0.36* AH 0.39* 0.68** 0.27 0.01 0.64** -0.16 0.72** 0.31* 0.24 -0.12 0.33* 0.32* -0.16 PA 0.23 0.24 0.68** 0.30 0.20 0.23 0.34* 0.16 0.17 0.29 0.53** 0.15 0.18 0.22 Table 3: Correlation between studied agronomic traits *p = 0.05; **p = 0.01; ***p = 0.001 Acta agriculturae Slovenica, 117/1 – 20218 M. M. GABALLAH et al. Ta bl e 4: H et er os is ov er b et te r p ar en t f or g ra in y ie ld a nd a ss oc ia te d tr ai ts C ro ss es D ay s t o he ad in g Pl an t he ig ht (c m ) C hl o- ro ph yl l co nt en t Fl ag le af an gl e Fl ag le af ar ea (c m 2 ) Pa ni cl e ex er tio n Pa ni cl e le ng th (c m ) N um be r o f pa ni cl es / pl an t Pa ni cl e m as s (g ) Fe rt ili ty Pe rc en t- ag e 10 0- gr ai n m as s (g ) G ra in yi el d t h a-1 G ra in Ty pe Ap pa re nt he te ro sis Ph en ot yp ic ac ce pt ab ili ty ‘W TS C 90 39 ’ × ‘S ak ha 10 1’ 0. 00 n s 61 .7 2* * -8 .8 9* * -2 5. 45 ** 58 .4 6* * 0. 00 n s 13 .4 3* * 38 .4 1* * 5. 22 ** 7. 32 * 1. 93 n s 57 .1 2* * -5 1. 98 ** 66 .6 7* * -0 .6 0 ns ‘W TS C 90 39 ’ × ‘S ak ha 10 2’ 1. 26 n s 70 .3 3* * -1 0. 00 ** -1 8. 18 ** 14 .0 8* * -2 9. 11 ** 31 .3 5* * 47 .1 3* * -6 0. 50 ** -4 1. 53 ** -1 5. 43 ** 71 .5 1* * -5 1. 98 ** 10 0. 00 ** -2 5. 15 ** ‘W TS C 90 39 ’ × ‘S ak ha 10 5’ 1. 25 n s 55 .9 8* * -1 0. 00 ** -9 .0 9* * 79 .5 3* * 0. 00 n s 13 .7 3* * -2 0. 05 ** 13 .4 3* * 3. 85 n s 1. 93 n s -5 7. 12 ** -6 8. 25 ** 36 .3 6* * -2 3. 03 ** ‘W TS C 90 39 ’ × ‘S ak ha 10 7’ 7. 90 ** 55 .0 2* * -1 5. 38 ** 27 .2 7* * -2 .9 9* -3 9. 24 ** 14 .9 6* * -5 .3 5* * -6 1. 28 ** -7 9. 38 ** -2 5. 62 ** -4 6. 68 ** -6 3. 89 ** 36 .3 6* * -3 8. 24 ** ‘W TS C 90 39 ’ × ‘S ak ha 10 8’ 29 .5 4* * 88 .0 3* * -1 0. 00 ** 36 .3 6* * 57 .7 0* * 0. 00 n s 15 .4 6* * -1 5. 65 ** 13 .4 3* * -1 .1 1 ns 9. 37 ** 37 .3 6* * -5 1. 98 ** 45 .4 5* * -2 0. 00 ** ‘W TS C 90 59 ’ × ‘S ak ha 10 1’ 21 .9 4* * 82 .8 1* * 8. 54 ** -2 .8 2* * 39 .7 4* * 0. 00 n s -1 .3 2* -8 .5 7* * 15 8. 25 ** 8. 21 ** 49 .1 0* * 10 0. 00 ** -5 7. 63 ** 38 .4 6* * 0. 60 n s ‘W TS C 90 59 ’ × ‘S ak ha 10 2’ -1 .2 7 ns 83 .8 6* * 1. 22 * 14 .2 9* * -1 3. 49 ** 0. 00 n s -1 .3 2* -1 7. 14 ** 41 .7 5* * 11 .2 3* * -3 .7 0 ns 53 .8 3* * -6 1. 45 ** -1 5. 38 ** -3 6. 53 ** ‘W TS C 90 59 ’ × ‘S ak ha 10 5’ -5 .0 0* * 66 .6 7* * -6 .9 0* * 45 .4 5* * 23 .4 4* * 0. 00 n s 4. 52 ** 8. 57 ** 49 .2 5* * -7 .7 1* -2 3. 81 ** 24 .9 2* * -5 7. 63 ** 15 .3 8* * -0 .6 1 ns ‘W TS C 90 59 ’ × ‘S ak ha 10 7’ 40 .7 9* * 67 .7 0* * -9 .8 9* * -3 7. 65 ** 16 .7 2* * -2 9. 11 ** -7 .1 7* * 0. 00 n s -5 .7 5* * -4 7. 76 ** -4 .3 3* * 6. 78 n s -5 3. 82 ** 15 .3 8* * -1 7. 65 ** ‘W TS C 90 59 ’ × ‘S ak ha 10 8’ 21 .9 4* * 88 .5 5* * 3. 66 ** -3 0. 99 ** 54 .8 4* * 0. 00 n s 7. 13 ** -2 2. 88 ** 35 .0 0* * 0. 35 n s -2 4. 85 ** 49 .9 4* * -6 1. 45 ** 30 .7 7* * 10 .3 0* * ‘W TS C 93 69 ’ × ‘S ak ha 10 1’ 24 .9 9* * 62 .2 9* * 0. 00 n s -2 8. 17 ** 37 .2 2* * 0. 00 n s 10 .5 9* * 17 .6 5* * 15 .7 6* * 5. 85 n s -8 .0 0* * 57 .1 2* * -7 1. 31 ** 50 .0 0* * 2. 41 n s ‘W TS C 93 69 ’ × ‘S ak ha 10 2’ 8. 86 ** 52 .5 4* * -1 .2 2* -1 4. 29 ** -5 .9 1* * -3 4. 18 ** 10 .5 9* * -1 4. 65 ** -5 8. 72 ** -5 2. 33 ** -3 3. 33 ** -3 8. 50 ** -3 1. 25 * 45 .0 0* * -3 4. 13 ** ‘W TS C 93 69 ’ × ‘S ak ha 10 5’ 6. 26 ** 39 .4 1* * -9 .2 0* * 9. 09 ** 46 .8 1* * -3 2. 15 ** 21 .9 5* * -4 0. 02 ** -6 1. 91 ** -4 9. 77 ** -5 1. 54 ** -3 8. 54 ** -6 2. 78 ** 9. 09 ** -3 3. 33 ** ‘W TS C 93 69 ’ × ‘S ak ha 10 7’ 50 .0 0* * 46 .6 0* * -1 3. 19 ** -2 9. 33 ** -2 4. 53 ** -3 3. 16 ** 23 .3 7* * -1 0. 79 ** -4 5. 03 ** -6 3. 92 ** -1 1. 67 ** -4 6. 68 ** -2 5. 57 * 50 .0 0* * -1 1. 76 ** ‘W TS C 93 69 ’ × ‘S ak ha 10 8’ 26 .1 3* * 66 .9 4* * -2 .4 7* * -5 6. 34 ** 95 .7 0* * 0. 00 n s 15 .4 6* * -5 .8 3* * -3 9. 40 ** -2 5. 00 ** -6 .9 7* * 0. 00 n s -7 7. 27 ** 32 .7 3* * -1 7. 72 ** ‘L on gp in g’ × ‘S ak ha 10 1’ 26 .2 6* * 48 .8 9* * 2. 47 ** 61 .7 6* * -8 .7 6* * 0. 00 n s 0. 00 34 .6 4* * -3 .8 0* * 3. 66 n s -2 2. 26 ** 42 .8 8* * -2 4. 79 n s 30 .0 0* * -1 7. 47 ** ‘L on gp in g’ × ‘S ak ha 10 2’ 0. 00 n s 65 .3 3* * -1 .2 2* 13 5. 29 ** 14 .1 1* * 0. 00 n s 12 .8 6* * -1 4. 65 ** -1 .5 7 ns 13 .1 6* * -1 4. 14 ** 46 .1 7* * -2 2. 90 n s 50 .0 0* * -3 4. 13 ** ‘L on gp in g’ × ‘S ak ha 10 5’ -5 .0 0* * 35 .5 6* * -4 .6 0* * 2. 94 ** 8. 93 ** 0. 00 n s -4 .7 7* * -5 .7 4* * -2 6. 17 ** 4. 93 n s -3 1. 37 ** 66 .6 1* * 0. 00 n s 49 .0 9* * -9 .0 9* * ‘L on gp in g’ × ‘S ak ha 10 7’ 40 .7 9* * 51 .1 1* * -9 .8 9* * 17 .6 5* * 19 .3 9* * -3 4. 18 ** 20 .5 1* * 2. 76 ** -6 6. 44 ** -7 1. 13 ** 6. 01 ** -5 9. 97 ** 9. 90 n s 32 .7 3* * -1 7. 65 ** ‘L on gp in g’ × ‘S ak ha 10 8’ -6 .1 8* * -5 0. 23 ** -1 8. 10 ** -4 6. 06 ** -6 7. 89 ** 0. 00 n s -6 6. 20 ** 0. 03 n s -6 .0 4* * -1 5. 15 ** -7 8. 79 ** 12 .4 5 ns -9 .0 1 ns -5 6. 36 ** -6 6. 95 ** * n s, * a nd ** : n on -s ig ni fic an t a nd si gn ifi ca nt e ffe ct at 0 .0 5 an d 0. 01 p ro ba bi lit y Acta agriculturae Slovenica, 117/1 – 2021 9 Exploiting heterosis and combining ability in two-line hybrid rice significant differences among genotypes, crosses, lines, testers and line × tester interactions. The significant dif- ferences among the lines, testers and lines × testers indi- cated that the genotypes had wide genetic diversity for all traits except days to heading, chlorophyll content, panicle length and apparent heterosis for parents (Table 5). The mean sum of squares for crosses was portioned into lines, testers and line × tester components. In case of lines and testers, significant variances were observed in all traits except number of panicles/ plants. All the line × tester interactions were significant for all traits studied. Variances of SCA were higher than the GCA variances for all traits. This was further supported by low magni- tude of σ2GCA/ σ2SCA ratios (Table 6). The ratio of GCA and SCA variances were found to be less than unity for all the characters. H2 ranged from 64.00 % (NPP) to 98.00 % (PE) However, all traits exhibited low h2, rang- ing from 1  % with PH, CC and FAN to 14  % with PE and FP. The relative contribution of lines, testers and inter- action of line × tester on expression fifteen traits stud- ied, three traits including panicle length, panicle mass and 100-grain mass showed high contribution of lines ranged from 34.19 to 39.18. Four traits, panicle exertion, panicle mass, fertility percentage and grain yield showed contributed tester ranged from 38.01 to 63.41 % in their hybrids. In other eight traits viz., grain type, phenotypic acceptability, days to heading, flag leaf angle, chlorophyll content, plant height, flag leaf area, number of panicles/ plant, apparent heterosis line × tester interaction con- tributed the highest depending on the respective cross showing mean percentage of 1.82, 52.72, 53.43, 54.9, 56.55, 60.98, 64.01, 69.28, 70.51, 73.67 and 79.73 % re- spectively. 3.4 COMBINING ABILITY ESTIMATED There were significant differences among the geno- types for traits (Table 5), which lead to the combining ability analysis. The increase and decrease in the values of traits desired, positive and negative values of gi were considered. Plant height, days to heading and flag leaf angle negative GCA and SCA effects were desirable, while in other characters positive GCA and SCA effects were desirable. 3.5 GENERAL COMBINING ABILITY (GCA) EF- FECTS GCA effects of fifteen traits showed that ‘WTSC9039’ was produced highly significant GCA for Ta bl e 5: A na ly sis o f v ar ia nc e of th e lin e × te st er m at in g de sig n fo r g ra in y ie ld a nd y ie ld co m po ne nt s So ur ce o f va ria nc e df D ay s t o he ad in g Pl an t he ig ht (c m ) C hl or o- ph yl l co nt en t Fl ag le af an gl e Fl ag le af ar ea (c m 2 ) Pa ni cl e ex er tio n Pa ni cl e le ng th (c m ) N um be r o f pa ni cl es / pl an ts Pa ni cl e m as s (g ) Fe rt ili ty Pe rc en ta ge 10 0- gr ai n m as s (g ) G ra in y ie ld t h a-1 G ra in Ty pe Ap pa re nt he te ro sis Ph en ot yp ic A cc ep ta bi lit y Re pl ic at io ns 2 55 .2 4 ns 23 1. 80 n s 19 .1 7 ns 23 4. 93 ** 13 8. 79 n s 23 .7 4* * 10 .2 2 ns 3. 88 2 ns 0. 32 1 ns 25 .2 4 ns 0. 03 n s 0. 40 ** 0. 05 ** 0. 77 n s 2. 08 ** Tr ea tm en ts 28 79 6. 34 ** 13 92 .1 9* * 89 .4 3* * 23 2. 93 ** 84 7. 79 ** 61 7. 13 ** 47 .1 6* * 11 .5 1* * 16 .1 0* * 17 17 .3 2* * 1. 52 ** 29 .3 ** 1. 37 ** 7. 63 ** 6. 35 ** Pa re nt s 8 11 0. 54 n s 11 22 .0 0* * 18 .3 7 ns 19 9. 64 ** 33 2. 39 ** 21 8. 69 ** 10 .4 5 ns 9. 80 * 9. 67 ** 71 1. 20 ** 0. 81 ** 6. 75 ** 2. 64 ** 1. 33 n s 1. 65 ** C ro ss es 19 10 86 .6 4* * 11 27 .8 4* * 11 9. 05 ** 22 3. 86 ** 81 3. 96 ** 79 4. 74 ** 58 .8 7* * 12 .4 3* * 18 .6 0* * 21 66 .8 8* * 1. 86 ** 35 .8 9* * 0. 66 ** 6. 23 ** 7. 22 ** Pa r. vs . c ro ss es 1 76 6. 97 ** 85 76 .3 0* * 94 .9 6* * 67 1. 58 ** 56 13 .8 1* * 43 0. 08 ** 11 8. 37 ** 7. 42 n s 19 .9 8* * 12 24 .4 9* * 0. 83 * 84 .4 2* * 4. 59 ** 84 .7 0* * 27 .5 1* * Li ne s 3 11 95 .7 1* * 15 13 .6 1* * 11 3. 00 ** 28 7. 21 ** 12 81 .7 5* * 64 6. 08 ** 13 2. 91 ** 4. 99 n s 40 .2 7* * 20 22 .6 0* * 4. 62 ** 43 .5 9* * 1. 22 ** 4. 24 ** 7. 85 ** Te st er s 4 13 46 .0 0* * 51 0. 55 ** 11 8. 75 ** 19 9. 47 ** 17 8. 94 23 93 .8 9* * 32 .5 2* * 11 .8 0* 33 .5 8* * 64 65 .3 6* * 0. 79 ** 93 .8 9* * 0. 55 ** 2. 81 ** 9. 57 ** Li ne s × te st er s 12 97 2. 92 ** 12 37 .1 5* * 12 0. 67 ** 21 6. 16 ** 90 8. 70 ** 29 8. 85 ** 49 .1 4* * 14 .5 0* * 8. 19 ** 77 0. 13 ** 1. 52 ** 14 .6 3* * 0. 56 ** 7. 86 ** 6. 27 ** Er ro r 56 13 3. 47 14 7. 69 18 .3 1 31 .9 5 91 .4 7 1. 42 8. 37 9 4. 15 0. 32 31 .8 7 0. 15 0. 00 2 0. 00 1 0. 78 0. 65 To ta l 86 C V % 13 .0 1 11 .5 4 10 .6 7 19 .3 3 17 .6 1 1. 33 12 .0 1 18 .8 8 12 .6 0 7. 54 13 .9 6 0. 65 10 .2 8 13 .2 2 11 .5 3 * n s, * a nd ** : n on -s ig ni fic an t a nd si gn ifi ca nt e ffe ct at 0 .0 5 an d 0. 01 p ro ba bi lit y Acta agriculturae Slovenica, 117/1 – 202110 M. M. GABALLAH et al. number of panicles/ plants, days to heading, flag leaf area, panicle length, panicle mass, 100-grain mass and apparent heterosis. Therefore, this line was considered as the best general combiner for the respective traits. Simi- larly, ‘WTSC9059’ was identified as good general com- biner for chlorophyll content, panicle exertion, panicle mass, fertility percentage, 100-grain mass, grain yield, apparent heterosis and phenotypic acceptability. Otherwise ‘WTSC9369’ was good combiner for panicle length, flag leaf angle, flag leaf area and grain type. The line ‘Longping’ was good combiner for panicle exertion, plant height, flag leaf angle, days to heading, fertility percentage and grain yield. The tester ‘Sakha101’ identified as good combiner for chlorophyll content, panicle exertion, number of panicles per plant, panicle mass, fertility percentage, 100-grain mass, grain yield, apparent heterosis and phenotypic acceptability. There- fore, this line was considered as the best general com- biner for the respective traits. The tester ‘Sakha102’ was good combiner for days to heading, panicle length, num- ber of panicles/ plants, chlorophyll content, grain yield and grain type while’’Sakha105’’ found good combiner for days to heading, plant height, chlorophyll content, flag leaf area, panicle exertion, panicle length and fertil- ity percentage. The tester ‘Sakha107’ identified as desired combiner for number of panicles/ plants, plant height, grain type and apparent heterosis therefore the tester ‘Sakha108’ was a good combiner for flag leaf area, pani- cle exsertion, flag leaf angle, plant height, panicle mass, fertility percentage and grain yield. However, none of parents was observed significant and positive GCA effect for relative water content and number of panicles/plant. 3.6 SPECIFIC COMBINING ABILITY (SCA) EF- FECTS The results of SCA effect of crosses showed that out of twenty hybrid combinations, two of them viz., ‘WTSC9039’ × ‘Sakha107’ and ‘Longping’ × ‘Sakha108’ produced significant and negative SCA effect for days to heading (Table 8). One and five crosses out from twenty hybrid combination showed significant and negative SCA effects in desirable direction for plant height and flag leaf angle, respectively. For chlorophyll content, five crosses, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha105’ and ‘Longping’ × ‘Sakha107’ showed significant and posi- tive values for SCA effect. Three hybrid combinations ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha107’ showed significant and positive SCA effect of flag leaf area. Ten out from twenty crosses were significant and positive SCA for panicle exertion. Four out of twenty crosses ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha107’ showed significant and positive SCA effects for panicle length two hybrid combinations ‘WTSC9039’ × ‘Sakha102’ and ‘WTSC9059’ × ‘Sakha105’ reported significant and positive SCA effect for num- ber of panicles/plant. For panicle mass, five out from twenty crosses ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha107’ and ‘Longping’ × ‘Sakha102’ had significant and positive SCA effect. Eight hybrid crosses showed significant and positive SCA effect for fertility percent- age. Six combinations possessed significant and posi- Traits DTH PH CC FAN FAR PE PL NPP PM FP HGM GY GT AH PA Cov H.S (lines 14.85 18.43 -0.51 4.74 24.87 23.15 5.59 -0.63 2.14 83.50 0.21 1.93 0.04 -0.24 0.11 Cov H.S (tester) 31.09 -60.55 -0.16 -1.39 -60.81 174.59 -1.38 -0.23 2.12 474.60 -0.06 6.61 0.00 -0.42 0.28 Cov F.S 342.70 316.32 33.10 67.22 235.53 356.03 20.77 2.16 8.71 977.28 0.71 16.28 0.26 1.45 2.39 σ2 GCA 3.32 -3.19 -0.05 0.23 -2.77 14.47 0.28 -0.06 0.30 40.77 0.01 0.62 0.00 -0.05 0.03 σ2 SCA 279.82 363.15 34.12 61.40 272.41 99.14 13.59 3.45 2.62 246.09 0.46 4.88 0.19 2.36 1.87 σ2GCA/ σ 2 SCA 0.01 -0.01 0.00 0.00 -0.01 0.15 0.02 -0.02 0.12 0.17 0.02 0.13 0.02 -0.02 0.01 σ2A 6.64 0.001 0.09 0.45 0.01 28.95 0.57 0.002 0.61 81.53 0.02 1.24 0.01 0.001 0.06 σ2D 279.82 363.15 34.12 61.40 272.41 99.14 13.59 3.45 2.62 246.09 0.46 4.88 0.19 2.36 1.87 (σ2D /σ 2 A) 1/2 42.15 -56.91 -362.90136.40 -49.26 3.43 23.92 -28.54 4.31 3.02 22.90 3.93 31.17 -24.85 34.05 H2% 83.00 89.00 80.00 86.00 89.00 98.00 82.00 64.00 97.00 93.00 90.00 67.00 75.00 90.00 90.00 h2% 3.00 1.00 1.00 1.00 1.00 14.00 4.00 1.00 11.00 14.00 4.00 13.00 2.00 1.00 3.00 Table 6: Estimates of genetic variances and heritability for grain yield and yield components σ2 GCA, Variance due to general combining ability (GCA); σ2 SCA, variance due to specific combining ability (SCA); σ2GCA/ σ2SCA, GCA variance ratio; σ2A, additive genetic variance; σ2D, dominance genetic variance; (σ2D /σ2A)1/2, degree of dominance; H2, Broad-sense heritability; h2 narrow-sense heritability. Acta agriculturae Slovenica, 117/1 – 2021 11 Exploiting heterosis and combining ability in two-line hybrid rice tive SCA effect for 100-grain mass with combinations of ‘WTSC9039’ × ‘Sakha105’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha107’ trait. Significant and positive SCA effects were observed in nine hybrid combinations for grain yield including ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha105’. Among the nine crosses which depicted highly significant positive SCA effects for grain yield showed high heterosis. Ten and seven out from twenty crosses showed significant and positive SCA ef- fect for grain type and apparent heterosis. The crosses Days to heading Plant height (cm) Chlorophyll content Flag leaf angle Flag leaf area (cm2) Panicle exertion Panicle length (cm) Number of panicles/ plant Line ‘WTSC9039’ -1.51 ns 3.78 ns 0.79 ns 0.72 ns 8.98** -2.11** 2.18** 0.75 ns ‘WTSC9059’ 1.70 ns 1.85 ns 2.59 ns 5.72** 0.83 ns 5.73** 0.88 ns -0.12 ns ‘WTSC9369’ 10.72** 8.78** 0.49 ns -1.88 ns 3.05 ns -8.34** 1.34* -0.65 ns ‘Longping’ -10.91** -14.42** -3.88** -4.55** -12.86** 4.72** -4.39** 0.02 ns LSD0.05 4.91 5.16 1.82 2.40 4.06 0.51 1.23 0.87 LSD0.01 6.94 7.30 2.57 3.39 5.74 0.72 1.74 1.22 Tester ‘Sakha101’ 9.27** 5.23 ns 2.47* -0.38 ns 0.42 ns 11.56** -0.64 ns 0.77 ns ‘Sakha102’ -9.79** 8.07* 1.34 ns 5.74** -0.91 ns -4.27** 1.07 ns 0.43 ns ‘Sakha105’ -11.04** -5.35 ns 1.09 ns 0.74 ns 3.47 ns 3.52** 1.94** -0.98* ‘Sakha107’ 12.03** -1.02 ns 0.59 ns -0.38 ns -6.08** -22.36** -0.02 ns 0.93 ns ‘Sakha108’ -0.47 ns -6.93* -5.49** -5.72** 3.11 11.56** -2.35** -1.15* LSD0.05 5.49 5.77 2.03 2.68 4.54 0.57 1.37 0.97 LSD0.01 7.76 8.16 2.87 3.80 6.42 0.80 1.94 1.37 Line Panicle mass (g) Fertility Per- centage 100-grain mass (g) Grain yield t ha-1 Grain Type Apparent heterosis Phenotypic Acceptability Line ‘WTSC9039’ 1.45** -1.95 ns 0.67** -0.03 ns -0.14** 0.55** -0.16 ns ‘WTSC9059’ 1.38** 11.48** 0.21* 2.07** -0.12** 0.25 ns 0.93** ‘WTSC9369’ -1.53** -15.34** -0.31** -2.10** 0.43** -0.10 ns 0.04 ns ‘Longping’ -1.30** 5.81** -0.57** 0.07* -0.18** -0.70** -0.82** LSD0.05 0.24 2.40 0.17 0.06 0.04 0.38 0.34 LSD0.01 0.34 3.39 0.24 0.08 0.06 0.53 0.49 Tester ‘Sakha101’ 2.37** 20.96** 0.44** 3.28** -0.15** 0.43* 1.33** ‘Sakha102’ -1.02** -1.07 ns -0.14 ns 0.64** 0.21** 0.15 ns -1.02** ‘Sakha105’ 0.16 ns 6.35** -0.10 ns -2.19** -0.12** -0.06 ns 0.23 ns ‘Sakha107’ -2.05** -38.91** 0.00 ns -3.49** 0.26** 0.28 ns 0.03 ns ‘Sakha108’ 0.54** 12.67** -0.20* 1.77** -0.20** -0.81** -0.57** LSD0.05 0.27 2.68 0.19 0.07 0.05 0.42 0.39 LSD0.01 0.38 3.79 0.26 0.09 0.07 0.59 0.54 Table 7: General combining ability estimates of lines and testers for traits studied ns, and * and **: nonsignificant and significant effect at 0.05 and 0.01 probability. Acta agriculturae Slovenica, 117/1 – 202112 M. M. GABALLAH et al. Ta bl e 8: S pe ci fic co m bi ni ng a bi lit y es tim at es o f c ro ss es fo r t ra its st ud ie d C ro ss es D ay s t o he ad in g Pl an t he ig ht (c m ) C hl or o- ph yl l co nt en t Fl ag le af an gl e Fl ag le af ar ea (c m 2 ) Pa ni cl e ex er tio n Pa ni cl e le ng th (c m ) N um be r o f pa ni cl es / pl an t Pa ni cl e m as s (g ) Fe rt ili ty Pe rc en t- ag e 10 0- gr ai n m as s ( g) G ra in yi el d t h a-1 G ra in Ty pe Ap pa re nt he te ro sis Ph en ot yp ic A cc ep ta bi lit y ‘W TS C 90 39 ’ × ‘S ak ha 10 1’ -1 0. 27 n s -8 .3 7 ns -1 .6 7 ns -7 .2 2* 3. 25 n s 2. 11 ** -1 .0 9 ns -0 .5 0 ns -0 .6 0* 2. 88 n s -0 .1 7 ns -0 .4 7* * 0. 29 ** -0 .8 3 ns 0. 42 n s ‘W TS C 90 39 ’ × ‘S ak ha 10 2’ 0. 76 n s -5 .2 0 ns -1 .0 4 ns -1 1. 34 ** -2 .1 5 ns -1 1. 18 ** 1. 20 n s 4. 50 ** -2 .2 4* * -1 8. 30 ** -0 .2 2 ns 3. 17 ** -0 .0 7* * 0. 96 * 0. 77 * ‘W TS C 90 39 ’ × ‘S ak ha 10 5’ 3. 02 n s -1 .7 8 ns -0 .7 9 ns -3 .8 4 ns 9. 04 n s 10 .1 5* * -1 .3 4 ns -1 .4 2 ns 2. 25 ** 16 .3 0* * 0. 38 * -3 .0 2* * -0 .1 4* * -0 .3 5 ns -0 .3 8 ns ‘W TS C 90 39 ’ × ‘S ak ha 10 7’ -1 9. 05 ** -6 .7 8 ns -2 .2 9 ns 7. 28 * -1 1. 68 * -3 .2 0* * -1 .3 8 ns -1 .0 0 ns -1 .2 8* * -1 1. 47 ** -0 .7 2* * -0 .7 2* * -0 .4 2* * -0 .6 8 ns -1 .2 8* * ‘W TS C 90 39 ’ × ‘S ak ha 10 8’ 25 .5 4* * 22 .1 3* * 5. 79 ** 15 .1 2* * 1. 53 n s 2. 11 ** 2. 62 n s -1 .5 8 ns 1. 87 ** 10 .5 8* * 0. 74 ** 1. 04 ** 0. 34 ** 0. 91 * 0. 47 n s ‘W TS C 90 59 ’ × ‘S ak ha 10 1’ -1 .4 5 ns -2 .1 0 ns 0. 03 n s 1. 78 n s 2. 86 n s -5 .7 3* * 0. 21 n s -0 .9 7 ns 1. 73 ** -9 .7 6* * 0. 72 ** 0. 44 ** 0. 16 ** 0. 97 * -0 .5 7 ns ‘W TS C 90 59 ’ × ‘S ak ha 10 2’ -4 .4 6 ns -4 .2 7 ns -1 .8 4 ns 1. 16 n s -9 .8 6* 10 .0 9* * -1 .5 0 ns -1 .6 3 ns 0. 46 n s 12 .9 4* * 0. 03 n s -0 .9 4* * -0 .2 9* * -2 .2 5* * -1 .2 7* * ‘W TS C 90 59 ’ × ‘S ak ha 10 5’ -5 .2 1 ns -1 .8 5 ns -2 .5 9 ns 6. 16 ** -8 .1 7 ns 2. 30 ** -0 .8 8 ns 2. 78 ** -0 .4 2 ns -7 .4 8* -0 .1 5 ns -0 .6 1* * 0. 14 ** -0 .0 5 ns 0. 38 n s ‘W TS C 90 59 ’ × ‘S ak ha 10 7’ 2. 82 n s -5 .5 2 ns -1 .5 9 ns -6 .2 2* * 6. 82 n s -0 .9 2 ns -1 .9 2 ns 0. 53 n s -0 .4 1 ns 5. 77 * -0 .3 1 ns 1. 19 ** -0 .1 3* * -0 .3 8 ns -0 .6 2 ns ‘W TS C 90 59 ’ × ‘S ak ha 10 8’ 8. 29 n s 13 .7 3* 5. 99 ** -2 .8 8 ns 8. 35 n s -5 .7 3* * 4. 08 ** -0 .7 2 ns -1 .3 7* * -1 .4 7 ns -0 .2 9 ns -0 .0 7* * 0. 12 ** 1. 71 ** 2. 08 ** ‘W TS C 93 69 ’ × ‘S ak ha 10 1’ -0 .4 4 ns 1. 63 n s -1 .8 7 ns 0. 38 n s -0 .5 2 ns 8. 34 ** -1 .2 6 ns 2. 23 * 0. 48 n s 14 .9 8* * -0 .1 3 ns 1. 60 ** -0 .4 8* * -0 .1 9 ns 0. 47 n s ‘W TS C 93 69 ’ × ‘S ak ha 10 2’ -5 .4 6 ns -8 .8 7 ns -0 .7 4 ns -1 .2 4 ns -7 .7 2 ns -1 0. 01 ** -2 .9 7* -1 .1 0 ns -0 .1 0 ns -1 4. 91 ** -0 .3 1 ns -2 .7 9* * 0. 58 ** -0 .1 6 ns -0 .1 8 ns ‘W TS C 93 69 ’ × ‘S ak ha 10 5’ -5 .2 1 ns -5 .7 8 ns -1 .4 9 ns 3. 76 n s 0. 17 n s -1 5. 77 ** 1. 49 n s -2 .3 5* -1 .4 4* * -1 8. 34 ** -0 .6 1* * -0 .2 6* * -0 .2 0* * -1 .2 1* * -1 .4 3* * ‘W TS C 93 69 ’ × ‘S ak ha 10 7’ 0. 81 n s -4 .4 5 ns -0 .9 9 ns 1. 38 n s -1 7. 07 ** 9. 10 ** -0 .7 2 ns -0 .2 7 ns 1. 67 ** 16 .9 2* * 0. 21 n s 1. 35 ** 0. 73 ** 0. 71 n s 0. 77 * ‘W TS C 93 69 ’ × ‘S ak ha 10 8’ 10 .3 0 ns 17 .4 7* * 5. 09 ** -4 .2 8 ns 25 .1 3* * 8. 34 ** 3. 45 * 1. 48 n s -0 .6 2* 1. 35 n s 0. 83 ** 0. 10 ** -0 .6 3* * 0. 85 * 0. 37 n s ‘L on gp in g’ × ‘S ak ha 10 1’ 12 .1 6* 8. 83 n s 3. 50 n s 5. 05 n s -5 .5 9 ns -4 .7 2* * 2. 14 n s -0 .7 7 ns -1 .6 1* * -8 .1 0* * -0 .4 2* -1 .5 7* * 0. 03 * 0. 06 n s -0 .3 2 ns ‘L on gp in g’ × ‘S ak ha 10 2’ 9. 15 n s 18 .3 3* * 3. 63 n s 11 .4 3* * 19 .7 2* * 11 .1 0* * 3. 27 * -1 .7 7 ns 1. 88 ** 20 .2 7* * 0. 51 * 0. 56 ** -0 .2 3* * 1. 45 ** 0. 68 n s ‘L on gp in g’ × ‘S ak ha 10 5’ 7. 40 n s 9. 42 n s 4. 88 * -6 .0 8* -1 .0 4 ns 3. 32 ** 0. 73 n s 0. 98 n s -0 .4 0 ns 9. 52 ** 0. 37 n s 3. 90 ** 0. 20 ** 1. 60 ** 1. 43 ** ‘L on gp in g’ × ‘S ak ha 10 7’ 15 .4 2* 16 .7 5* * 4. 88 * -2 .4 5 ns 21 .9 2* * -4 .9 7* * 4. 02 ** 0. 73 n s 0. 01 n s -1 1. 23 ** 0. 82 ** -1 .8 2* * -0 .1 8* * 0. 36 n s 1. 13 ** ‘L on gp in g’ × ‘S ak ha 10 8’ -4 4. 13 ** -5 3. 33 ** -1 6. 88 ** -7 .9 5* * -3 5. 01 ** -4 .7 2* * -1 0. 15 n s 0. 82 n s 0. 12 n s -1 0. 47 ** -1 .2 8* * -1 .0 7* * 0. 18 ** -3 .4 6* * -2 .9 3* * LS D 0 .0 5 10 .9 7 11 .5 4 4. 06 5. 37 9. 08 1. 13 2. 75 1. 93 0. 54 5. 36 0. 37 0. 04 0. 03 0. 84 0. 77 LS D 0 .0 1 15 .5 1 16 .3 2 5. 75 7. 59 12 .8 4 1. 60 3. 89 2. 74 0. 77 7. 58 0. 53 0. 06 0. 04 1. 19 1. 09 ns , * a nd ** : n on sig ni fic an t a nd si gn ifi ca nt e ffe ct at 0 .0 5 an d 0. 01 p ro ba bi lit y. Acta agriculturae Slovenica, 117/1 – 2021 13 Exploiting heterosis and combining ability in two-line hybrid rice ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sakha108’, ‘WTSC9369’ × ‘Sakha107’, ‘Longping’ × ‘Sakha105’ and ‘Longping’ × ‘Sakha107’ reported significant and positive SCA effect for phenotypic acceptability. 4 DISCUSSION Grain yield, being a complex quantitative trait, is con- trolled and influenced by yield contributing components, such as spikelet number, grain filling and grain mass. The present study the hybrid combinations ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9059’ × ‘Sakha108’ and ‘WTSC9059’ × ‘Sakha101’had more than 11 t ha-1 grain yield/ha-1 superior than all parents were used and also the highest in grain yield components, panicle length, fertility percentage, panicle mass and 100-grain mass. These combinations can be exploited as anew hybrids for high yield potentials and good ag- ronomic traits. Overall, hybrids performed better for GY and yield-related traits compared with the parental lines and also showed evident hybrid vigor. The days to heading is important trait that needs to be investigated in every generation and days to maturity and panicle length that needs to be selected in early segregating generation (Ganapati et al., 2020). Jaiswal and Jaiswal 2019 men- tioned that the genotype Hubr 16 showed the highest mean value for grain yield and yield contributing traits like panicle length and seeds / panicle followed by Bas- mati 370. Super hybrid varieties have increased rice yield potential by 12 % compared with ordinary hybrid and in- bred varieties. The higher grain yield of super hybrid va- rieties was attributed to improvement in both source and sink (Zhang et al., 2009). The hybrid varieties produced grain yield of 12 t ha-1 in on-farm demonstration fields, 8–15 % higher than the hybrid check varieties (Peng et al., 2008). Agronomic characteristics that are useful for rice breeding, such as large panicles and flowering habit have good panicle uniformity, concentrated flowering periods and good panicle exertion rates, which would be more conducive to hybrid seed production (El-namaky, 2018). Correlation coefficients  are used to measure rela- tionship between two variables as evident from positive correlation of yield components with GY except for FAR and highly significant for PE, PM, FP and AH traits, how- ever, the negative correlation and significant was found with AH (Table 3). (Yu et al., 2020 found the correlation between the introgression rate of the Habataki pedigree and yield related traits, whereas the introgression rate was significantly negatively correlated to 1000-grain mass. Li et al. (2019) found that correlations between agronomic traits and yield depend on the rice variety. The improve- ment in GY should focus on increasing the FGN, TGW, GP, PH, PL, GPP, SS, decreasing PN and LW. Gramaje et al. (2020) showed that the GCA effects were poorly cor- related with specific combining ability (SCA) effects and hybrid performance, while SCA was positively correlated to heterosis estimates for all traits although it was only correlated with Gy for per se performance. Genetic parameter analysis indicated the presence of significant differences among the lines, testers and lines × testers refer the genotypes had wide genetic di- versity among themselves for all traits except days to heading, chlorophyll content, panicle length and appar- ent heterosis for parents. The significance of variance due to lines and testers confirmed a prevalence of addi- tive variance while significant differences for line × tester among all the characters, indicating the importance of both additive and non-additive types of gene action oc- cur within parents. Additive effects are important for the fixation of the trait and for early selection of the plants. The dominance to additive variance ratio (D/A) could be used for quantitative evaluations at each single QTL through genetic analysis in the heterotic populations (Liu et al., 2020). GCA analysis seeks to facilitate breeding through effective and efficient selection of inbred lines for a cross based on additive and additive × additive gene effects. Moreover, GCA analysis maximize additive gene effect that increases the selection efficiency of breeders in selecting elite inbred parents with better performance (Ullah Zaid et al., 2019)we calculated the GCA effect val- ues of 33 parents of hybrid rice and sequenced them to identify genome-wide single nucleotide polymorphisms (SNPs. The RILs from the cross between the maintainer line Zhenshan97B and the restorer line Minghui63 (both parents of the elite hybrid Shanyou63, the classical hybrid of three-line system) was used to create the subsequent immortalized F2 population, which enabled the compre- hensive evaluations of the genetic contributions of domi- nance, over-dominance and epistatic effects across the whole rice genome (Liu et al., 2020). Otherwise, domi- nance effects are not fixable due to segregation and late selection may be fruitful for the selection of genotypes for the next generation of the future breeding studies to develop new rice varieties (Ashfaq et al., 2012). Uthar- asu & Anandakumar (2013) found that both additive and non- additive gene action were found to control the ex- pression of the traits under study. The magnitude of com- bining ability revealed non-additive genetic variance was higher than the additive variance for all the studied traits. Gaballah and Abdallah (2015) illustrated non-additive gene effects for grain yield and its components. Hijam et al. (2019) observed importance of both additive and non- additive gene effects for grain yield and yield contribut- ing characters studied. Acta agriculturae Slovenica, 117/1 – 202114 M. M. GABALLAH et al. The variances of SCA were higher than the GCA for all traits (Table 6) and ratio of GCA/ SCA variances were less than unity which reported preponderance of non-additive gene action in the inheritance of respective traits. It interprets greater importance of non-additive gene action in its expression and exploitation for traits through hybrid breeding (Tan et al., 2018). The ratios of GCA/SCA were less than unity in all studied characters except days to maturity and no. of panicles/plant, indi- cating that dominance gene effects were more important than additive gene effects in the expression of most traits (Zaazaa and Anis, 2014). Ganapati et al. (2020) reported the plant height, number of filled grain panicle−1, number of grain pani- cle−1 and dominant effects for yield hill−1, yield tiller−1 and 1000 grain mass have association to yield, governed by additive gene effect. Bano and Singh (2019) showed that both the additive and non-additive genetic variance ex- hibited importance for expression of days to 50 % flower- ing, days to maturity, plant height and effective tillers/ plant. The relative contribution of lines, testers and their interaction on expression of fifteen traits studied (Fig.3) showed the prevalence of additive gene action in pani- cle length, panicle mass and 100-grain mass refers to the lines contributed by more positive alleles while the testers contributed by more positive alleles in panicle exertion, panicle mass, fertility percentage and grain yield. Days to heading, flag leaf angle, chlorophyll content, plant height, flag leaf area, number of panicles/ plant, grain type, phe- notypic acceptability and apparent heterosis traits con- tributed by line × tester interaction depending on the respective crosses were subjective to non-additive gene action. Istipliler et al. (2015) revealed the line × tester interaction contributed to combinations variances was found much more than lines and testers, individually. The relative contribution of line, tester and com- binations of line × tester interaction of ten traits were calculated and found that panicle mass contributed the highest (69.53 %) followed by thousand grain mass (63.62 %), yield per plant (54.76 %), panicle per meter square 51.52 % in their hybrids (Akter et al., 2010). The parents, ‘WTSC9059’, ‘Sakha101’, ‘Sakha102’ and ‘Sakha108’ were recognized as good general combin- ers due to their maximum positive GCA effect values for most traits and could be utilized in breeding program to enhance grain yield and its related traits. Eltahawy et al. (2020)but also performance of grain quality traits of F2 bulk population which is the commodity consumed by humans. In order to make GCA improvement for qual- ity traits in parents of hybrid rice by molecular marker assisted selection feasible, genome-wide GCA loci for quality traits in parents were detected through associa- tion analysis between the effects of GCA and constructed single nucleotide polymorphism linkage disequilibrium blocks (SNPLDBs reported the general combining ability (GCA) of parents in hybrid rice affects playing vital role in both heterotic level of grain yield and other important agronomic traits. Considering the exhibition of useful GCA effects by the testers were identified as good general combiners for the traits concerned (Hossain et al., 2018). The crosses ‘WTSC9039’ × ‘Sakha102’ and ‘WTSC9059’ × ‘Sakha105’ found to be highly signifi- cant and positive SCA for number of panicle/plant and could be selected for further improving high heterosis. The hybrid combinations ‘WTSC9039’ × ‘Sakha102’, ‘WTSC9039’ × ‘Sakha108’, ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9059’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha107’, ‘WTSC9369’ × ‘Sakha108’, ‘Longping’ × ‘Sakha102’ and ‘Longping’ × ‘Sakha105’ de- scribed highly significant positive SCA effects for grain yield and showed high heterosis. Therefore, could be utilized for future breeding program for development of high yielding genotype. The SCA effects of the crosses for yield and its contributing characters indicated that most of the good specific cross combinations for different characters involved parents with either one or both good GCA effects. Bano and Singh (2019) reported that the three specific combiners for seed yield/plant were ‘Kas- turi Basmati’ × ‘Pusa-2517- 2-51-1’, ‘Pusa Sugandh-5’ × ‘Type-3’ and ‘Pusa Sugandh-5’ × ‘Pusa Sugandh-2’. These crosses were good specific combiners for grain yield / plant. Ganapati et al. (2020) revealed the cross combina- tions, which expressed high SCA effects for grain yield have invariably exhibited positive SCA effects for one or more yield related traits also. It appears that heterosis for yield may be through heterosis for individual yield components or alternatively due to multiplication effects of non-additive gene effects of component characters. Generally, high × high, low × high and high × low general combiner parents produced good specific cross combinations. In these crosses ad- ditive × additive, dominance × additive and additive × dominance type of gene action was found. In cases, high × high general produced inferior cross combinations in- dicating epistatic type of gene action for these traits (Anis et al., 2016). The discovery of male sterile lines plays a crucial role in the utilization of rice heterosis and het- erosis percent was estimated to know the possible gene action, exploit heterosis for high grain yield and as- sociated traits. The crosses ‘WTSC9059’ × ‘Sakha101’, ‘WTSC9369’ × ‘Sakha101’, ‘Longping’ × ‘Sakha105’ and ‘WTSC9039’ × ‘Sakha102’ express superior heterosis val- ues for grain yield. Thus, hybrids could be used for future breeding program for development grain yield and its as- sociated traits. Acta agriculturae Slovenica, 117/1 – 2021 15 Exploiting heterosis and combining ability in two-line hybrid rice Gokulakrishnan and Kumar (2013) found the cross combinations ‘IR58025A’ × ‘ASD19’, ‘IR62829A’ × ‘ASD16’, ‘PUSA3A’ × ‘IR42’ promising for seed yield and had high percentage of standard heterosis. (Yu et al., 2020) showed that heterosis in F1 involved grain number/panicle. The back cross inbreeding lines F1s showed an increase in grain number/panicle but a decrease in plant height com- pared with the back cross inbreeding lines. Abdullah et al. (2020) developed a neo-tetraploid rice line Huaduo8 with long panicles and harboring wide compatibility genes for pollen and embryo sac fertility. All the hybrids generated by Huaduo8 produced significant high parent yield heterosis and displayed long panicles. 5 CONCLUSION The combining ability of five pollinator and four EGMS lines through line × tester analyses for GY and yield related traits. Based on the results could be consid- ering improving the DTH, NOP, Pl, FAR, HGM, FP and GT to increase GY. Specific parents with the potential to produce superior hybrids and improve the existing breeding pool were identified. The variances of SCA were higher than the GCA for all traits and ratio of GCA/ SCA variances were less than unity which reported prepon- derance of non-additive gene action in the inheritance of respective traits. The prevalence of additive gene action in panicle length, panicle mass and 100-grain mass refer to the lines contributed by more positive alleles while the testers contributed by more positive alleles in panicle ex- ertion, panicle mass, fertility percentage and grain yield. Days to heading, flag leaf angle, chlorophyll content, plant height, flag leaf area, number of panicles/ plants, grain type, phenotypic acceptability and apparent het- erosis traits contributed by line × tester interaction de- pending on the respective crosses were subjective to non- additive gene action. Heterosis was also evident among the crosses although SCA effects did not automatically translate to better hybrid performance. 6 REFERNCES Abdullah, M., Ghaleb, A., Li, C., Shahid, M. Q., & Yu, H. (2020). Heterosis analysis and underlying molecular regulatory mechanism in a wide- compatible neo-tetraploid rice line with long panicles. BMC Plant Biology, 1–15. https://doi. org/10.1186/s12870-020-2291-z Akanksha, & H. K. Jaiswal. (2019). Combining ability studies for yield and quality parameters in basmati rice (Oryza sa- tiva L .) genotypes using diallel approach Combining abil- ity studies for yield and quality parameters in basmati rice. 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Field Crops Research, 114, 91–98. https://doi.org/10.1016/Journalfcr.2009.07.008. Acta agriculturae Slovenica, 117/1, 1–8, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1435 Original research article / izvirni znanstveni članek Bionomics of slender burnished brass (Thysanoplusia orichalcea [Fab- ricius, 1775], Lepidoptera: Noctuidae) on potato (Solanum tuberosum L.) in Kashmir Gousul NISA 1, 2 and Abdul A. BUHROO 1 Received January 11, 2020; accepted December 23, 2020. Delo je prispelo 11. januarja 2020, sprejeto 23. decembra 2020. 1 University of Kashmir, Department of Zoology, Division of Entomology, Hazratbal, Srinagar, India 2 Corresponding author, e-mail: gousulnisa98@gmail.com Bionomics of slender burnished brass (Thysanoplusia orichal- cea [Fabricius, 1775], Lepidoptera: Noctuidae) on potato (So- lanum tuberosum L.) in Kashmir Abstract: A study on biology, morphometrics and geo- metrical progression of Thysanoplusia orichalcea was conduct- ed on Solanum tuberosumunder laboratory conditions. Differ- ent stages viz., egg, larva, pupa and adult of T. orichalcea were observed for their duration and morphometric measurements. The pest depicted five larval instars and moultedfour times dur- ing the entire period. The average pre oviposition and oviposi- tion period was observed to be 3.80 and 2.80 days respectively. Further, fecundity ranges between 381.0-400.0 with an average of 388.0 eggs. The mean incubation period was found to be 4.2 days. The average larval, pre pupal and pupal period was completed in 23.1, 1.4 and 9.5 days respectively. While mean adult longevity on S. tuberosum was 7.45 days. The total life cycle was completed in 41.0-51.0 days with an average of 45.65 days. Moreover, mean head capsule width of first, second, third, fourth and fifth larval instar was found to be 0.33, 0.54, 0.88, 1.46 and 2.24 mm respectivelywith Dyar’s ratio/ growth ratio of 1.63 mm. The expected head capsule width of first, second, third, fourth and fifth larval instar wasobserved as 0.33, 0.53, 0.86, 1.40 and 2.28 mm respectively. Key words: biology; Dyar’s ratio; fecundity; larval instars; morphometry; Solanum tuberosum; Thysasnoplusia orichalcea Bionomija sovke Thysanoplusia orichalcea (Fabricius, 1775) (Lepidoptera: Noctuidae) na krompirju (Solanum tuberosum L.) v Kašmirju Izvleček: Raziskava biologije, morfometrije in geome- trijske progresije sovke Thysanoplusia orichalcea (Fabricius, 1775) je bila izvedena na krompirju (Solanum tuberosum L.) v laboratorijskih razmerah. Opazovano je bilo trajanje raličnih razvojnih stadijev škodljivca, kot so jajčeca, gosenica, buba in odrasli osebki, na njih so bile opravljene morfometrične me- ritve. Škodljivec je imel pet razvojnih stopenj gosenic in se je v celotnem obdobju štirikrat levil. Poprečni obdobji pred in med odleganjem jajčec sta trajali 3,8 in 2,8 dni. Samice so v povpre- čju odložile med 381,0 in 400,0 jajčec, s povprečjem 388,0 jaj- čec. Poprečno inkubacijsko obdobje je trajalo 4,2 dni. Povpreč- na obdobja gosenice, obdobja pred za bubljenjem in bube so trajala 23,1, 1,4 in 9,5 dni. Povprečna življenska doba odraslih osebkov na krompirju je bila 7,45 dni. Celoten življenski krog se je zaključil v 41,0-51,0 dneh, v poprečju v 45,65 dneh. Povpreč- na širina glave je za prvo, drugo, tretjo, četrto in peto stopnjo gosenice znašala 0,33, 0,54, 0,88, 1,46 in 2,24 mm, s količnikom Dyarjevo razmerje/ rastno razmerje 1,63 mm. Pričakovana ši- rina glave je bila za prvo, drugo, tretjo, četrto in peto razvojno stopnjo gosenice 0,33; 0,53; 0,86; 1,40 in 2,28 mm. Ključne besede: bionomija; Dyarjevo razmerje; plodnost; larvalne stopnje; morfometrija; Solanum tuberosum; Thysasno- plusia orichalcea Acta agriculturae Slovenica, 117/1 – 20212 G. NISA and A. A. BUHROO 1 INTRODUCTION Vegetables are indispensable as they make up a major part of the human diet in many parts of the world. Vegetables play an imperative role in human nourishment since they supply chief phyto nutriceu- ticals like vitamins, minerals, dietary fiber and phy- tochemicals (Craig and Beck, 1999; Dias and Ryder, 2011). Amongst various vegetables, Potato (Solanum tuberosum L.) lines as the third vital food crop after wheat and rice. In terms of production, it is leading tu- ber crop all over the globe (Anonymous, 2011a). India generates about 42.34 million tons of potato from 1.86 million hectares of land, thus positions fourth in terms of area and third in terms of potato production in the entire world (Anonymous, 2011a). Around 34.000 t of potatoes are harvested in Kashmir valley from an area of about 1.7 thousand hectares (Anonymous, 2011b). Potato is professed not only as a source of carbohy- drates, but also an exceptional basis of essential amino acids.The starch present in the potato augments satiety, enhances glucose tolerance & insulin sensitivity and diminishes plasma cholesterol & triglyceride concen- trations (Raben et al., 1994; Cummings, 1996). Despite of the numerous health benefits of potato, its produc- tion is still less because of the massive crop damage due to amplified pest menace. Insects also act as vectors for viral diseases, thus further increases the yield losses (Shivalingswami et al., 2002). Due to the lack of resist- ant characters because of rigorous hybrid cultivation, vegetables are more prone to insect pests and diseases (Dhandapani et al., 2003). Among various insect pests, Thysanoplusia orichalcea (Fab.) commonly called as green semiloop- er, slender burnished moth and golden wing moth is one of the serious polyphagous pest infesting potato in Kashmir (Bhagat, 2018). The larval stages of T. orichal- cea are serious defoliators feeding on potato leaves from vegetative to maturity stages. The young larvae puncture holes widely into the lamina of leaves and lat- er instars feed voraciously leaving only the main veins thus plummeting photosynthetic competence. This pest was also found feeding on other crops belonging to familiesCompositae, Cruciferae, Leguminosae, Li- naceae, Cucurbitaceae and Chenopodiaceae (Laute et al., 2015). A little has been done on the biology of T. orichalcea on potatoin Kashmir though its biology has been worked out on soybean (Glycine max (L.) Merr.) and common bean (Phaseolus vulgaris L.) in Akola, Maharashtra and Kishtwar, J&K respectively (Laute et al., 2015 and Kotwal & Bhatia, 2016). Therefore, the ob- jective of the current investigation was to study the life cycle of T. orichalcea on potatoin the laboratory. 2 MATERIAL AND METHODS 2.1 COLLECTION AND REARING OF T. orichalcea For studying the life cycle and morphometrics of T. orichalcea on potato (S. tuberosum L.) variety Kufri Jyoti ??, preliminary culture of different larval instars were gathered from potato fields of Kashmir valley during the year 2018. Each larval instars collected was transferred to separate rearing boxes made of glass at the temperature of 25 ºC and 65 % RH in the Entomology Research Labo- ratory of the Department of Zoology, University of Kash- mir. Fresh potato leaves were provided as food for the larval instars on daily basis till the larvae entered into last stage and the excreta was removed regularly.The rear- ing boxes were monitored regularly for the exuvia. The number and duration of each larval instar was recorded. The last larval instars of T. orichalcea were collected from rearing boxes and transferred to pupation chambers hav- ing one-third part of it filled with soil, which provides fa- vourable conditions for pupation. The pupae were moni- tored regularly until adult eclosion. In order to determine the duration of adult moths, freshly eclosed adults were individually placed in rearing boxes lined with filter paper and were provided with 10  % honey solution as food. Ten adults were used for this experiment and the duration of each adult was monitored and documented.. To determine the fecundity, 10 individual pair of adult moths were placed in oviposition chambers immediately after eclosion lined with fresh potato leaves for egg lay- ing and containing cotton swabs soaked in 10 % honey solution as food for adults at the temperature of 25  ºC and 65  % RH. Each day the number of eggs produced by these moths was monitored. Further, the data on pre oviposition period, oviposition period and incubation period was also recorded. Moreover, the rearing, pupa- tion and oviposition chambers were covered with muslin cloth fastened with rubber bands. 2.2 MORPHOMETRIC AND GROWTH STUDIES Ten individuals of each developmental stage viz., eggs, larval instars, pupae and adults were observed un- der stereozoom binocular microscope and the morpho- metric measurements were documented with the help of vernier calliper as well as simple scale. For determining the duration of different larval instars, the individual lar- vae were observed regularly for exuvia and head capsule. The presence of casted head capsule corroborated the moulting. The numbers of larval instars of T. orichalcea were determined using Dyar’s law (1890). The growth ra- tio (Dyar’s ratio) was obtained by the following formula: Acta agriculturae Slovenica, 117/1 – 2021 3 Bionomics of slender burnished brass (Thysanoplusia orichalcea [Fabricius, ... on potato (Solanum tuberosum L.) in Kashmir 2.3 STATISTICAL ANALYSIS The data associated with different stages of insect viz., egg, larva, pupa and adult were examined for cal- culating mean and standard deviation using SPSS 16.0. 3 RESULTS 3.1 FECUNDITY In the laboratory conditions, average fecundity was observed to be 388.0 (± 8.58) eggs with a range of 381.0 to 400.0 eggs (Table 1). The mean preoviposition period was found to be 3.80 (± 0.83) days with a range of 3.0 to 5.0 days. Moreover, the oviposition period lasted be- tween 2.0 to 4.0 days with an average of 2.8 (± 0.83) days (Table 1). 3.2 EGGS In the laboratory conditions, oviposition occurred during night time at 25 ºC and 65 % RH and the eggs were laid on lower surface of the leaves of potato The freshly laid eggs of T. orichalcea were green, spherical and shiny. Prior to hatching, egg colour changed to dark brown. The average incubation period of eggs was observed to be 4.2 (± 0.83) days with a range of 3.0-5.0 days (Table 7). The length of the egg varied between 0.67 mm to 0.72 mm with an average of 0.70 (± 0.01) mm and width ranges between 0.59 mm to 0.66 mm with an average of 0.63 (± 0.02) mm (Table 2). 3.3 LARVAE The current investigation depicted five larval instars in the life cycle of T. orichalcea. The larvae shed its exui- vae four times during the entire period. The caterpillars flaunted a typical half loop movement and thus attaining a common name as semilooper to its recognition. The ne- onate larvae emerging from the eggs were creamy white in colour with dark black head, which later on changes to green. The first larval period ranged for about 2.0-3.0 days with a mean duration of 2.4 (± 0.51) days (Table 7). The length and breadth of first larval instar ranged between 2.00-3.00 mm and 0.40-0.50 mm with a mean of 2.50 (± 0.52) and 0.44 (± 0.05) respectively (Table 3). Further, the mean width of head capsule was observed to be 0.33 (± 0.02) with a range of 0.29-0.37 mm before moulting (Table 3).The second larval instars were green in colour and their duration lasted for 3.0-4.0 days with an average of 3.40 (± 0.51) days (Table 7). The length and breadth of 2nd larval instar varied from 8.50-10.50 mm and 1.00-1.40 mm with a mean of 9.50 (± 0.88) and 1.27 (± 0.15) respectively (Table 3). Moreover, the width of head capsule ranged between 0.51-0.59 mm with a mean of 0.54 (± 0.03) (Table 3). The first and second larval in- stars were found scraping the epidermis of leaf thereby, causing their skeletonization. The third instar larva displayed two lateral lines running along the plural region. The fourth and fifth larval instars flaunted extra blackish mid dorsal line (Fig 2). From third instar onwards, larvae starts feeding vo- raciously engulfing the entire leaf including mid rib and vein. The third, fourth and fifth larval period ranges be- tween 4.0-5.0, 5.0-6.0 and 7.0-8.0days with an average of 4.3 (± 0.48), 5.6 (± 0.51) and 7.4 (± 0.51) days respectively (Table 7). The mean length and breadth of third larval in- star was 17.35 (± 1.08) and 2.40 (± 0.51) mm with a range of 16.00-18.50 mm and 2.00-3.00 mm respectively (Table 3). In case of fourth larval instar, the average length and S. No. Stage Minimum (days) Maximum (days) Mean ± S. D. 1. Pre oviposition period 3.00 5.00 3.80 ± 0.83 2. Oviposition period 2.00 4.00 2.80 ± 0.83 3. Fecundity 381.00 400.00 388.0 ± 8.58 Table 1: Duration of pre oviposition period, oviposition period (days) and fecundity of T. orichalcea S. No. Variable N Minimum (mm) Maximum (mm) Mean ± S. D. 1. Egg length 10 0.67 0.72 0.70 ± 0.01 2. Egg width 10 0.59 0.66 0.63 ± 0.02 Table 2: Measurements of eggs of T. orichalcea N = Number of observations; SD: Standard deviation Acta agriculturae Slovenica, 117/1 – 20214 G. NISA and A. A. BUHROO breadth was obtained as 24.75 (± 0.67) mm and 3.95 (± 0.43) mm with a range of 24.0-25.50 mm and 3.50- 4.50 mm respectively (Table 3). The length and breadth of fifth larval instar ranged between 32.00-35.00 mm and 4.50-5.50 mm with a mean of 33.30 (± 1.15) and 4.95 (± 0.43) respectively (Table 3). The width of head capsule for third, fourth and fifth larval instar ranged between 0.85- 0.90 mm, 1.39-1.50 mm and 2.19-2.28 mm with a mean of 0.88 (± 0.01) mm, 1.46 (± 0.03) mm and 2.24 (± 0.03) mm respectively (Table 3). Further, the total larval period ranged between 21.00 to 26.00 days with an average of 23.10 (± 2.52) days (Table 7). Growth ratio (Dyar’s ratio) for Thysanoplusia orichalcea was calculated and the expected widths of head capsule of first, second, third, fourth and fifth larval instar were 0.33, 0.53, 0.86, 1.40 and 2.28 mm respective- ly (Table 4). The Dyar’s ratio was computed as 1.63 mm (Table 4) and authenticated that successive larval instars followed a regular geometrical development (Fig 1). Mean observed head capsule width of first larval in- star (N =10) = 0.33 mm Mean observed head capsule width of second larval instar (N =10) = 0.54 mm 3.4 PRE PUPA In this stage, the mature 5th larval instar stops feed S. No. Developmental stage Minimum (mm) Maximum (mm) Mean ± S. D. 1. First larval instar Length 2.00 3.00 2.50 ± 0.52 Breadth 0.40 0.50 0.44 ± 0.05 Width of head capsule 0.29 0.37 0.33 ±0.02 2. Second larval instar Length 8.50 10.50 9.50 ± 0.88 Breadth 1.00 1.40 1.27 ± 0.15 Width of head capsule 0.51 0.59 0.54 ± 0.03 3. Third larval instar Length 16.00 18.50 17.35 ± 1.08 Breadth 2.00 3.00 2.40 ± 0.51 Width of head capsule 0.85 0.90 0.88 ± 0.01 4. Fourth larval instar Length 24.00 25.50 24.75 ± 0.67 Breadth 3.50 4.50 3.95 ± 0.43 Width of head capsule 1.39 1.50 1.46 ± 0.03 5. Fifth larval instar Length 32.00 35.00 33.30 ± 1.15 Breadth 4.50 5.50 4.95 ± 0.43 Width of head capsule 2.19 2.28 ± 0.03 Table 3: Morphometric measurements of larval instars of T. orichalcea *Mean of 10 individuals; SD: Standard deviation S. No. Larval instars Head capsule width (mm) Difference (mm) Observed (Mean ± S. D.) Range Expecteda 1. First larval instar 0.33 ± 0.02 0.29-0.37 0.33 0.00 2. Second larval instar 0.54 ± 0.03 0.51-0.59 0.53 0.01 3. Third larval instar 0.88 ± 0.01 0.85-0.90 0.86 0.02 4. Fourth larval instar 1.46 ± 0.03 1.39-1.50 1.40 0.06 5. Fifth larval instar 2.24 ± 0.03 2.19-2.28 2.28 - 0.04 Table 4: Comparison of observed mean and expected values of head capsule widths (mm) of the larvae of T. orichalcea a Expected width of head capsule determined by Dyar’s ratio. MultiplyingDyar’s ratio (1.63) with the observed width of head capsule of first larval instar provides the expected width of head capsule of second instar larva which when multiplied again with Dyar’s ratio gives expected width of head capsule of third larval instar and so on. Acta agriculturae Slovenica, 117/1 – 2021 5 Bionomics of slender burnished brass (Thysanoplusia orichalcea [Fabricius, ... on potato (Solanum tuberosum L.) in Kashmir ing and becomes sluggish by contracting its body and appendages (Fig 2). This phase lasted for 1.0-2.0 days with an average of 1.4 (± 0.51) days (Table 7). 3.5 PUPA The colour of pupa appeared creamish initially which turns into deep brown towards maturity. The pupa is obtect type. Prior to pupation, a mature full grown larva starts to spin white silken cocoon around the body and protects it externally by leaf fold (Fig 2).The male pupa was found slightly smaller than female pupa. The length and breadth of male pupa varies between 17.50- 19.00 mm and 4.50-5.50 mm with an average of 18.25 (± 0.63) mm and 5.05 (± 0.43) mm respectively (Table 5). Further, the length and breadth of female pupa varies between 19.00-24.00 mm and 5.00-6.00 mm with a mean of 21.70 (± 1.76) mm and 5.50 (± 0.52) mm respectively (Table 5). The pupal period ranged between 9.0 to 10.0 days with a mean of 9.5 (± 0.52) days at 25 ºC and 65 % RH (Table 7). 3.6 ADULT The adult moth comes out of puparium by punctur- ing an escape hole with the aid of some secretions. The forewing of adult moth is olive brown in colour with large conspicuous L shaped metallic golden patch (Fig 2). The male and female moths’ possess pectinate and filliform type of antennae respectively. The average male longevity was found to be 8.4 (± 0.51) days with a range of 8.0-9.0 days (Table 7). The female longevity lasted be Figure 1: Relation between larval growth and head width of T. orichalcea S. No. Variable N Minimum (mm) Maximum (mm) Mean ± S. D. 1. Pupal length (Male) 10 17.50 19.00 18.25 ± 0.63 2. Pupal width (Male) 10 4.50 5.50 5.05 ± 0.43 3. Pupal length (Female) 10 19.00 24.00 21.70 ± 1.76 4. Pupal width (Female) 10 5.00 6.00 5.50 ± 0.52 Table 5: Morphometric measurements of male and female pupae of T. orichalcea N = Number of observations; SD: Standard deviation S. No. Variable N Minimum (mm) Maximum (mm) Mean ± S. D. 1. Adult length (Male) 10 16.50 19.00 17.65 ± 1.08 2. Adult width (Male) 10 28.00 31.00 29.70 ± 1.33 3. Adult length (Female) 10 19.50 22.00 20.60 ± 1.04 4. Adult width (Female) 10 35.00 40.00 38.00 ± 2.00 Table 6: Morphometric measurements of adult male and female of T. orichalcea Acta agriculturae Slovenica, 117/1 – 20216 G. NISA and A. A. BUHROO tween 6.0-7.0days with a mean of 6.5 (± 0.52) days (Table 7). The average adult (male and female) longev- ity was found to be 7.45 (± 0.51) days with a range of 7.0-8.0 days (Table 7). Adults are medium in size with male moths slightly smaller than females. The length of adult male moth varies between 16.50-19.00 mm with an average of 17.65 (± 1.08) mm (Table 6). The length of female moth ranges between 19.50-22.0 mm with a mean of 20.60 (± 1.04) mm (Table 6). Further, the width of male and female moth varies between 28.00-31.0 mm and 35.00-40.00 mm with a mean of 29.70 (± 1.33) mm and 38.00 (± 2.00) respectively (Table 6). The average life S. No. Developmental stage Minimum* (Days) Maximum* (Days) Mean ± S.D. 1. Incubation period 3.00 5.00 4.20 ± 0.83 2. First larval instar 2.00 3.00 2.40 ± 0.51 3. Second larval instar 3.00 4.00 3.40 ± 0.51 4. Third larval instar 4.00 5.00 4.30 ± 0.48 5. Fourth larval instar 5.00 6.00 5.60 ± 0.51 6. Fifth larval instar 7.00 8.00 7.40 ± 0.51 7. Total larval period 21.00 26.00 23.10 ± 2.52 8. Pre pupal period 1.00 2.00 1.40 ± 0.51 9. Pupal period 9.00 10.00 9.50 ± 0.52 10. Male longevity 8.00 9.00 8.40 ± 0.51 11. Female longevity 6.00 7.00 6.50 ± 0.52 12. Average longevity 7.00 8.00 7.45 ± 0.51 13. Total life cycle 41.00 51.00 45.65 ± 4.88 Table 7: Developmental period of various life stages of T. orichalcea *Mean of 10 individuals; SD: Standard deviation Figure 2: Life Cycle of Thysanoplusia orichalcea Acta agriculturae Slovenica, 117/1 – 2021 7 Bionomics of slender burnished brass (Thysanoplusia orichalcea [Fabricius, ... on potato (Solanum tuberosum L.) in Kashmir cycle ranges between 41.0-51.0 days with an average of 45.6 (± 4.88) days (Table 7). 4 DISCUSSION Thysanoplusia orichalcea (Lepidoptera: Noctuidae) is one of the serious defoliator feeding on various vegeta- ble crops. In light of the damage caused by T. orichalcea, the evaluation of its bionomics could be advantageous for the better management of this obnoxious pest. The pre- sent results revealed that the total life cycle lasted from 41.0-51.0 days with an average of 45.6days under tem- perate conditions of Kashmir valley.On the contrary, the total life cycle of T. orichalcea on Phaseolus vulgariswas completed in 34.5-43.7 days under semi-temperate con- ditions of Kishtwar (Kotwal and Bhatia, 2016). Similarly, Laute et al. (2015) found that the total life cycle on soy- bean was completed in 34.3 days under tropical condi- tions in Maharashtra. The variation in the total life cycle of T. orichalcea may be attributed to different climatic conditions and assessment of life cycle on different host crops. These observations revealed that the longevity in the life cycle of green semilooper declines due to higher temperature at warmer regions compared to temperate climatic conditions of Kashmir. The current study depicted that the incubation period of T. orichalcea on potatolasted for 3.0-5.0 days with an average of 4.2 days. However, Laute et al. (2015) reported the average incubation period of 2.8 days on soybean. The discrepancy observed between the current and previous studies might be due to difference in host plant species and varieties. Since there was no disparity in the number of larval instars and the morphometric measurements between the host plants, this specifies that host plant does not affect these parameters. Laute et al. (2015) and Kotwal & Bhatia (2016) also found the simi- lar results. In the present study, it was found that larval development of T. orichalcea on potatowas completed in 21.0-26.0 days with an average of 23.1 days. Conversely, Laute et al. (2015) and Kotwal & Bhatia (2016) revealed total larval period of 16.0-23.0 and 15.0-20.0 days on soy- bean and common bean respectively. These differences might be due to either the variability or diminution of nutritional quality of the host plant species or ecologi- cal distinction with respect to geographical location and time of studies. Perchance of the most realistic implica- tion of the current study is the fact that T. orichalcea lar- val development time differs considerably depending on host plants. Furthermore, the average head capsule width of first, second, third, fourth and fifth larval instar of T. orichalcea in the present finding was viewed as 0.33, 0.54, 0.88, 1.46 and 2.24 mm respectively which coincides with the results of Begum, 1999. Thus it was concluded that the average head capsule widths of each larval instar did not intersect and can be employed as precise indication of each larval instar. Laute et al. (2015) and Kotwal and Bhatia (2016) reported pupal period of 6.0-7.0 and 14.0-15.5 days on soybean and common bean respectively. However, the present investigation showed pre pupal and pupal period ranging between 1.0-2.0 and 9.0-10.0 days with a mean of 1.4 and 9.5 days respectively on potato.The discrepancy monitored between the current and earlier results might be due to the fact that life history parameters are vastly affected by host plant species and test varieties. Generally, the present study revealed that the lon- gevity of adult male and female moth ranged between 8.0-9.0 and 6.0-7.0 days with an average of 8.4 and 6.5 days respectively on potato.The average longevity of adults (males and females) in the present finding was observed to be 7.45 days with a range of 7.00-8.00 days. Plausibly, the male lived longer than the female. Like- wise, other studies also divulged the same results (Goel and Kumar, 1987; Laute et al., 2015 and Kotwal and Bha- tia, 2016). 5 CONCLUSIONS The current investigation on biological descrip- tion of Thysanoplusia orichalcea offers comprehensive information on fecundity, growth and survival, which provides a baseline data for any management practice. Results from these studies can remark the venerable stages of T. orichalcea and also may provide information important for predicting the field population phenology. T. orichalcea commonly called as semilooper is a poly- phagous pest found feeding on many vegetables includ- ing potato. The larval instar of this pest represents the main damaging stage. The larva targets the epidermis of leaves thus causing serious defoliation. This influences the growth of plant and in turn affects the production and yield. The annotations on morphometrics and life cycle parameters will turn out to be advantageous in the identification of pest.. By this approach, we can observe different stages of T. orichalcea and develop integrated management tactics on potato. 6 REFERENCES Anonymous (2011 a). Agriculture statistics at a glance. Directo- rate of Economics and Statistics, Ministry of Agriculture, Govt. of India. Anonymous (2011 b). Statement showing ten year district wise data of area, production and productivity of potato in Kash Acta agriculturae Slovenica, 117/1 – 20218 G. NISA and A. A. BUHROO mir division. Directorate of Agriculture (Kashmir), Govt. of Jammu and Kashmir, pp. 1-3. Begum, M. (1999). Biology of the semilooper, Plusia orichalcea (Fab.) (Lepidoptera: Noctuidae): A pest of soybean in Bang- ladesh. M. S. Thesis. Bhagat, R. C. (2018). Diversity of noctuid caterpillars belong- ing to subfamily Amphipyrinae, Catocalinae, Cuculiinae, Heliothinae, Noctuinae and Plusiinae on host crops/ plants of Jammu & Kashmir state (India). 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World vegetable industry: Pro- duction, Breeding, Trends. Horticultural Reviews, 38, 299- 356. https://doi.org/10.1002/9780470872376.ch8 Dyar, H. G. (1890). The number of moults of lepidopterious lar- vae. Psyche, 5, 420-422. https://doi.org/10.1155/1890/23871 Goel, S. C. & Kumar, V. (1987). Life history of a noctuid, Plu- sia orichalcea Fab. on sunflower. Geobios New Reports, 6(2), 111-114. Kotwal, S. & Bhatia, S. (2016). Biological study of Thysanoplu- sia orichalcea on Phaseolus vulgaris from Kishtwar, J & K, India. International Journal of Current science, 8(3), 27316- 27319. Laute, M. S., Patil, N. V. & Barkhade, U. P. (2015). Biology of green semilooper on soybean. Plant Archives, 15(1), 603- 606. Raben, A. Tagliabue, A., Christensen, N. J., Madsen, J., Holst, J. & Astrup, A. (1994). Resistant starch: The effect on post- prandial glycemia, hormonal response and satiety. The American Journal of Clinical Nutrition, 60(4), 544-551. htt- ps://doi.org/10.1093/ajcn/60.4.544 Shivalingswami, T. M. Satpathy, S. & Banergee, M. K. (2002). Estimation of crop losses due to insect pests in vegetables Ed. By Sarath Babu, B., Varaprasad, K. S., Anitha, K., Pras- ada Rao, R. D. V. J., Chakrabarthy, S. K. and Chandurkar, P. S. Resource management in plant protection, 1, 24-31. Acta agriculturae Slovenica, 117/1, 1–11, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1964 Original research article / izvirni znanstveni članek Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage Ali DOĞRU 1, 2 Received November 12, 2020; accepted December 25, 2020. Delo je prispelo 12. novembra 2020, sprejeto 25. decebra 2020. 1 Sakarya University Faculty of Arts and Science Department of Biology, Esentepe, Sakarya, Turkey 2 Corresponding author, e-mail: adogru@sakarya.edu.tr Effects of salt stress on physiological and biochemical re- sponses of three maize genotypes at the early seedling stage Abstract: Salt stress is one of the major global problems for crop productivity in the arid and semi-arid regions of the world. In this study, variations in some physiological param- eters, water relations, and antioxidant systems under salinity (300 mM NaCl) among three maize (Zea mays L.) genotypes (‘P3167’, ‘32K61’, and ‘Bora’) were investigated. Our result in- dicated that shoot growth is more sensitive to salinity as com- pared to root growth. Salt stress led to physiological drought in all maize genotypes as indicated by the significant decrease in relative water content and increase in water deficit index. Salt stress increased SOD activity in all genotypes showing an ef- ficient formation and detoxification of superoxide radical. The constant level of oxidative markers (MDA and H2O2) and the increased level of the reduced ascorbate and phenolic may in- dicate that non-enzymatic antioxidants are responsible for the elimination of oxidative stress. Changes in ascorbate peroxi- dase and glutathione reductase activities under salinity demon- strated a functional failure in the ascorbate-glutathione cycle, especially in ‘P3167’ and ‘32K61’. Based on the presented results we may conclude that the genotype ‘Bora’ is tolerant to salinity while ‘P3167’ and ‘32K61’ are sensitive. Key words: antioxidant system; oxidative stress; pheno- lics; salinity; soluble sugars Učinek solnega stresa na fiziološki in biokemični odziv treh genotipov koruze v zgodnji razvojni stopnji semenke Izvleček: Solni stres je eden največjih globalnih proble- mov za uspevanje gojenih rastlin v sušnih in polsušnih ob- močjih sveta. V raziskavi so bili preučevani nekateri fiziološki parametri, vodni režim in antioksidacijski system v razmerah slanosti (300 mM NaCl) pri treh genotipih koruze (Zea mays L.; ‘P3167’, ‘32K61’, in ‘Bora’). Izsledki so pokazali, da je rast poganjka bolj občutljiva na slanost v primerjavi z rastjo kore- nin. Solni stres je povzročil fiziološko sušo pri vseh genotipih koruze, ki se je izražala kot značilen upad v relativni vsebnosti vode in povečanju indeksa vodnega deficita. Solni stres je po- večal aktivnost SOD pri vseh genotipih, kar kaže na učinkovito razstrupljanje superoksidnega radikala. Stalna raven vsebnosti označevalcev oksidacije (MDA in H2O2) in povečana vsebnost reduciranega askorbata ter fenolov lahko nakazujejo, da so ne- encimski antioksidanti odgovorni za odpravo oksidacijskega stresa. Spremembe v aktivnosti askorbat peroksidaze in gluta- tion reduktaze v razmerah slanosti so pokazale funkcionalni zlom askorbat-glutationskega cikla, še posebej pri ‘P3167’ in ‘32K61’. Na osnovi predstavljenih izsledkov lahko zaključi- mo, da je genotip ‘Bora’ toleranten na slanost, medtem ko sta ‘P3167’ in ‘32K61’ občutljiva. Ključne besede: antioksidacijski sistem; oksidativni stres; fenoli; slanost; topni sladkorji Acta agriculturae Slovenica, 117/1 – 20212 A. DOĞRU 1 INTRODUCTION Salt stress is one of the most prominent agricultural problems for plant productivity in the arid and semi- arid soils in the world. High salinity in soils is respon- sible for the reduced yield for several crops. Munns and Tester (2008) reported that 45 million hectares of land had been affected by salinity worldwide, and 1.5 mil- lion hectares are taken out of cultivation each year due to over-accumulation of salts in the soil. Salinity leads to reductions in several metabolic processes associated with growth, development, and crop productivity (Fayez and Bazaid, 2014). Seed germination, for example, is inhibit- ed by salt stress in several plant species (Ahammed et al., 2018; Gu et al., 2018; Orlovsky et al., 2016; Wilayasinghe et al., 2019; Khayamim et al., 2014). In black gram and mung bean plants, it was observed that salt stress affected water relations as well (Hasan et al., 2019). It was also reported that salt stress reduced root and shoot growth in barley plants depending on genotypes and salt concen- tration (Doğru and Yılmaz Kaçar, 2019). In plants under optimal growth conditions, the balance between reactive oxygen species (ROS) formation and detoxification is tightly controlled by the antioxidant system (Hameed et al., 2011). However, salt stress may cause the accelerated production of reactive oxygen species (ROS) and oxida- tive stress in the plant cells as a result of higher leakage of electrons toward O2 during photosynthetic and respira- tory electron transport reactions (Asada, 2006). Mittler (2002) has indicated that much of the damage on plants under salt stress is linked to oxidative stress at the cellular level. Plants that have higher antioxidant enzyme activi- ties were considered salt-tolerant (Gapinska et al., 2008). Higher plants have developed several adaptive mecha- nisms to cope with oxidative stress under saline condi- tions such as the increased synthesis of osmoprotectants. Proline and soluble sugars, for example, accumulate in plant tissues and contribute to osmoregulation, struc- tural protection of some biomolecules and membranes, and detoxification of ROS in plants under salt stress (Hare et al., 1998; Ashraf and Foolad, 2007; Abdelkader et al., 2019). Proline may also serve as an organic nitro- gen reserve that could be utilized during stress recovery (Sairam and Tyagi, 2004). Among phenolic compounds anthocyanins have been well known to accumulate under salt stress and play an important role in scavenging ROS in plant tissues as well (Petridis et al., 2012; Chunthabu- ree et al., 2016). Breeding for salt tolerance in crop plants has usu- ally been limited because of the lack of reliable traits for selection (Yıldırım et al., 2008). Salt tolerance is very complex because multiple genes are involved. In this case, it seems that the most effective way to overcome the salinity problem may be the introduction of salt-tolerant crops. Therefore, this experiment was conducted to de- termine some physiological and biochemical responses in three maize genotypes grown under salt stress through some growth parameters (root and shoot length), water relations (relative water content end water deficit index), photosynthetic pigment content (chlorophyll a and b), oxidative stress markers (malondialdehyde and hydro- gen peroxide content) and some endogenous resistance mechanisms (anthocyanin, free proline, total phenolics, total soluble carbohydrate contents and activities of some antioxidant enzymes). 2 MATERIAL AND METHODS 2.1 PLANT MATERIALS, GROWTH CONDITIONS, AND EXPERIMENTAL DESIGN Maize (Zea mays L.) cultivars (‘P3167’, ‘32K61’, and ‘Bora’) were grown in a growth chamber in plastic pots (14 x 14 cm; upper diameter x height) containing Ho- agland nutrient solution. The average temperature for day/night was 25/18 °C respectively, relative humidity was 40-50%, the photoperiod for the day/night cycle was 16/8 h respectively, and the maximum photosyntheti- cally active radiation was about 200 µmol m-2 s-1. After 20 days of growth, plants were divided into two groups. The first group of plants were control (no salt treatment) and watered with Hoagland nutrient solution until the end of the study. In the second group of plants, salt stress was induced by applying 300 mM NaCl. For every individual genotype, we had 20 pots each of which contains 4 plants per treatment. The seedlings were harvested after 5 days of application and leaves are kept at -80 °C until analysis. 2.2 DETERMINATION OF ROOT AND SHOOT LENGTH Measurement of root and shoot length were done with a millimetric ruler. The longest root was taken into consideration for measurement. Root and shoot length were expressed as cm plant-1. 2.3 DETERMINATION OF LEAF RELATIVE WA- TER CONTENT (RWC) AND WATER DEFICIT INDEX Leaf samples were taken and its fresh mass was re- corded immediately. The sample was then incubated in deionized water overnight and the turgid mass of the leaf Acta agriculturae Slovenica, 117/1 – 2021 3 Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage sample was recorded. The leaf sample was oven-dried at 70 °C for 48 h and the dry mass of the sample was esti- mated. The relative water content and water deficit index were calculated according to Sairam et al. (2002). 2.4 PHOTOSYNTHETIC PIGMENT AND ANTHO- CYANIN ANALYSIS Photosynthetic pigments were extracted from leaf segments in 3 ml 100 % acetone. The absorbance of the extracts was measured at 644.8 and 661.6 nm using a Shimadzu mini 1240 UV visible spectrophotometer. The concentrations of chlorophyll a and chlorophyll b were calculated according to Lichtenthaler (1987). Total anthocyanin content was analyzed by the pro- cedure of Mancinelli et al. (1975). Leaf sample (0.1 g) was soaked in 10 ml of a mixture of methanol and 1 N HCL (85/15; v/v) for 72 h at 4 °C. The crude extracts were fil- tered through a 0.45 µm syringe filter before measure- ment of total anthocyanin content at 530 and 657 nm. The content was expressed as mg g-1 fresh mass. 2.5 MALONDIALDEHYDE (MDA) AND HYDRO- GEN PEROXIDE (H2O2) ANALYSIS MDA and H2O2 content were measured by the method of Heath and Packer (1968) and Ohkawa et al. (1979), respectively. Fresh leaf material (0.1 g) was ho- mogenized in 6 ml of 5 % TCA (4 °C) and centrifuged at 10 000 g for 15 min and the supernatant was used in the subsequent determination. To 0.5 ml of the supernatant were added 0.5 ml of 0.1 M Tris-HCl (pH 7.6) and 1 ml of TCA–TBA reagent. The mixture was warmed at 95 °C for 60 min and then quickly cooled in an ice bath. After centrifugation at 10 000 g for 5 min to remove suspended turbidity, the absorbance of the supernatant at 532 nm was recorded. Non-specific absorbance at 600 nm was measured and subtracted from the absorbance recorded at 532 nm. The concentration of MDA was calculated us- ing its extinction coefficient of 155 mM-1 cm-1. For deter- mination of hydrogen peroxide, 0.5 ml of 0.1 M Tris-HCl (pH 7.6) and 1 ml of 1 M KI were added to 0.5 ml of supernatant. After 90 min, the absorbance was measured at 390 nm. A standard curve for hydrogen peroxide was prepared to determine hydrogen peroxide concentration in each sample. 2.6 FREE PROLINE ANALYSIS Approximately 10 mg powdered dry leaf material was extracted with 4 ml distilled water on a hot plate at 100 ºC for 10 min according to Bates et al. (1973). Ex- tracts were filtered and the same procedure was repeated two times. The liquid phase of the homogenate was col- lected and centrifuged at 3500 rpm for 10 min. Two ml of the supernatant was reacted with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid at 100 °C for 1 h. The reac- tion mixture was mixed with 4 ml toluene and vortexed for 20 s. The chromophore containing toluene was sepa- rated and the absorbance of the pink upper phase was re- corded at 520 nm against toluene blank. A standard curve for proline in the range of 0.2-1 µmol ml-1 was prepared to determine free proline concentration in each sample. 2.7 TOTAL PHENOLIC COMPOUND ANALYSIS The total phenolic content of leaves was determined according to Chandler and Dodds (1983). Accordingly, 0.2 g leaf material was powdered in liquid nitrogen and extracted with 80 % methanol. This mixture was placed in a refrigerator at 4 °C for 48 h. homogenates were cen- trifuged at 4,000 rpm for 10 min. An appropriate amount of supernatant was reacted with 50  % Foline Ciocalteu Reagent (FCR) and 5  % sodium carbonate and kept at room temperature at a dark place for 1 h. The mixture was vortexed and absorbance was read at 725 nm. The total phenolic content of the leaves was calculated by us- ing a standard curve prepared with gallic acid. 2.8 TOTAL SOLUBLE CARBOHYDRATE (TSC) ANALYSIS TSC content in leaves was measured by the phenol- sulphuric method according to Dubois et al. (1956). For this purpose, leaf material (50 mg) was oven-dried until the constant dry mass was reached. Dried leaf material was powdered in a mortar and pestle and TSS was ex- tracted by 70 % ethanol. After centrifugation of extract at 3,500 rpm for 20 min, a reaction mixture was prepared. This mixture consisted of 1,000 µl supernatant, 300 µl phenol, and 2,000 µl concentrated sulphuric acid. Ab- sorbances of these mixtures were read at 470 nm and the TSC content of the leaves was calculated by a standard curve using sucrose. 2.9 REDUCED ASCORBATE ANALYSIS The reduced ascorbate content was determined ac- cording to Law et al. (1983). For this purpose, leaf mate- rial (0.2 g) was extracted by 10 % TCA. After centrifu- Acta agriculturae Slovenica, 117/1 – 20214 A. DOĞRU gation of extract at 10,000 rpm for 20 min, a reaction mixture was prepared. This mixture consisted of 400 µl supernatant, 10 % TCA, 5 M NaOH, NaPO4 buffer (150 mM, pH 7.4), 10 mM dithiothreitol, 0.5 % N-ethylma- leimide, 44 % H3PO4 and 3 % FeCl3. The mixture was in- cubated at 37 °C for 60 min and absorbances were read at 525 nm. The reduced ascorbate content was determined by a standard curve. 2.10 ANTIOXIDANT ENZYME ACTIVITIES For determination of enzyme activities, 0.3 g fresh leaves material from non-acclimated and cold-accli- mated leaves were powdered with liquid nitrogen and suspended in a specific buffer with proper pH values for each enzyme. The homogenates were centrifuged at 14,000 rpm for 20 min at 4 °C and resulting supernatants were used for enzyme assay. The protein concentrations of leaf crude extracts were determined according to Bradford (1976), using BSA as a standard. Activity of superoxide dismutase (SOD; EC 1. 15. 1. 1) was determined by the method of Beyer and Fridovich (1987), based on the photoreduction of NBT (nitro blue tetrazolium). Extraction was in 1.5 ml homogenization buffer containing 10 mM K2HPO4 buffer (pH 7.0), 2 % PVP and 1 mM Na2EDTA. The reaction mixture consist- ed of 100 mM K2HPO4 buffer (pH 7.8), containing 9.9 x 10-3 M methionine, 5.7 x 10-5 M NBT, 1 % triton X-100, and enzyme extract. The reaction was started by the ad- dition of 0.9 µM riboflavin and the mixture was exposed to light with an intensity of 375 µmol m-2 s-1. After 15 min, the reaction was stopped by switching off the light, and absorbance was read at 560 nm. SOD activity was determined by a standard graphic and expressed as unit mg-1 protein. Activita of ascorbate peroxidase (APX; EC 1. 11. 1. 11) was determined according to Wang et al. (1991) by estimating the decreasing rate of ascorbate oxidation at 290 nm. APX extraction was performed in 50 mM Tris– HCl (pH 7.2), 2  % PVP, 1 mM Na2EDTA, and 2 mM ascorbate. The reaction mixture consisted of 50 mM KH- 2PO4 buffer (pH 6.6), 2.5 mM ascorbate, 10 mM H2O2, and enzyme, containing 100 µg proteins in a final volume of 1 ml. The enzyme activity was calculated from the ini- tial rate of the reaction using the extinction coefficient of ascorbate (E = 2.8 mM cm-1 at 290 nm). Activity of glutathione reductase (GR; EC 1. 6. 4. 2) was measured with the method of Sgherri et al. (1994). Extraction was in 1.5 ml of suspension solution, contain- ing 100 mM KH2PO4 buffer (pH 7.0), 1 mM Na2EDTA, and 2  % PVP. The reaction mixture (total volume of 1 ml) contained 100 mM KH2PO4 buffer (pH 7.8), 2 mM Na2EDTA, 0.5 mM oxidised glutathione (GSSG), 0.2 mM NADPH and enzyme extract containing 100 µg protein. The decrease in absorbance at 340 nm was recorded. The correction was made for the non-enzymatic oxidation of NADPH by recording the decrease at 340 nm without adding GSSG to the assay mixture. The enzyme activity was calculated from the initial rate of the reaction after Figure 1: Effect of the salt stress on (a) root length and (b) shoot length of maize plants (Different letters mean significant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Acta agriculturae Slovenica, 117/1 – 2021 5 Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage subtracting the non-enzymatic oxidation using the extinc- tion coefficient of NADPH (E = 6.2 mM cm-1 at 340 nm). 2.11 STATISTICAL ANALYSIS Experiments were a randomized complete block de- sign with three independent replicates. Analysis of vari- ance (ANOVA) was using SPSS 20.0 statistical software for Windows. To separate significant differences between means, the Duncan test was used at *p = 0.05. 3 RESULTS 3.1 GROWTH Root growth was adversely affected by salinity in ‘P3167’ and ‘32K61’ (p < 0.05) while it was not changed by salt stress in Bora (P > 0.05) (Fig. 1a). The decrease was around 28 %, 26 %, and 13 % in ‘P3167’, ‘32K61’, and ‘Bora’ under 300 mM salinity, respectively. Shoot growth declined by 27 % in ‘P3167’, 12 % in ‘32K61’, and 22 % in ‘Bora’, and all these changes were significantly different from respective controls (p < 0.05) (Fig. 1b). 3.2 WATER RELATIONS RWC in leaves significantly decreased in all maize genotypes by salt stress (p < 0.05) (Fig. 2a). It was 8 %, 9 %, and 7 % lower than respective controls in ‘P3167’, ‘32K61’, and ‘Bora’, respectively. The water deficit index was also adversely affected in all cultivars by salinity. It was found to be 2.75-2.80- and 2.2-fold higher than con- trols in ‘P3167’, ‘32K61’, and ‘Bora’, respectively (p < 0.05) (Fig. 2b). 3.3 PHOTOSYNTHETIC PIGMENT AND ANTHO- CYANIN According to the results, there was an inverse rela- tionship between salt stress and photosynthetic pigment content. Chlorophyll a content, for example, significantly decreased by 30 %, 42 %, and 22 % in comparison with controls when salt stress was applied in the growth me- dium (p < 0.05) (Fig. 3a). Similarly, chlorophyll b content in the leaves of genotypes ‘P3167’ and ‘32K61’ was 40 % and 48 % lower than respective controls and these chang- es were found to be statistically significant (p < 0.05). In the genotype ‘Bora’, however, chlorophyll b content was decreased by 15 % in comparison with control and this change was not significant (p > 0.05) (Fig. 3b). Salt treatment did not affect total anthocyanin con- tent in the leaves of maize genotypes significantly (p > 0.05) (Fig 3c). In the genotypes ‘P3167’ and ‘32K61’ under salt stress, total anthocyanin content in the leaves were 60 % and 129 % higher than respective controls while salt stress decreased it by 24 % in the leaves of ‘Bora’. Figure 2: Effect of the salt stress on (a) relative water content and (b) water deficit index of maize plants (Different letters mean significant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Acta agriculturae Slovenica, 117/1 – 20216 A. DOĞRU Figure 3: Effect of the salt stress on (a) chlorophyll a, (b) chlorophyll b and (c) anthocyanin content of maize plants (Different letters mean significant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Figure 4: Effect of the salt stress on (a) MDA and (b) H2O2 content of maize plants (Different letters mean significant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Acta agriculturae Slovenica, 117/1 – 2021 7 Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage 3.4 MALONDIALDEHYDE (MDA) AND HYDRO- GEN PEROXIDE (H2O2) The rate of MDA accumulation was 146 % and 113 % higher in the salt-stressed ‘P3167’ and ‘32K61’ leaves while it was found to be 8 % lower in the ‘Bora’ leaves as com- pared to control (Fig. 4a). However, these changes were not statistically significant (p > 0.05). Similarly, H2O2 content in the salt-stressed ‘P3167’ and ‘32K61’ leaves represented insignificant increases, with 19 % and 7 % in comparison with respective controls, respectively. The H2O2 accumu- lation in the leaves of ‘Bora’ under salinity was 12 % and insignificantly lower than control (p > 0.05). 3.5 FREE PROLINE, TOTAL PHENOLIC, AND TOTAL SOLUBLE CARBOHYDRATE The rate of free proline accumulation was 13 % and 22 % higher in the salt-stressed ‘P3167’ and ‘32K61’ leaves while it was found to be 6 % lower in the ‘Bora’ leaves as compared to control (Fig. 5a). However, these changes were not statistically significant (P > 0.05). Phenolic content in the leaves of ‘P3167’ and ‘32K61’ was induced by salinity (Fig. 5b) and it was significantly increased by 19  % and 83  %, respectively (p < 0.05). In ‘Bora’, however, change in the phenolic content was not significant and it was increased by 10 % under salt stress (p > 0.05). Higher but insignificant amount of TSC content (54  % higher than control) was measured in the salt- stressed leaves of ‘P3167’ (p > 0.05) (Fig. 5c). In the salt- stressed leaves of ‘32K61’, however, TSC content was not significantly affected but it was 18 % lower than the re- spective control. Salinity led to the significantly decreased TSC content (75 % of the control) in the leaves of ‘Bora’ (p < 0.05). 3.6 ANTIOXIDANT SYSTEM Salt stress remarkably increased the reduced ascor- Figure 5: Effect of the salt stress on (a) free proline, (b) phenolic and (c) TSC content of maize plants (Different letters mean signifi- cant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Acta agriculturae Slovenica, 117/1 – 20218 A. DOĞRU bate content to 7-, 7- and 5-fold in ‘P3167’, ‘32K61’, and ‘Bora’, respectively (p < 0.05) (Fig. 6a). Similarly, SOD ac- tivity was found to be significantly higher than respective controls in all maize genotypes under salt stress (p < 0.05) (Fig 6b). SOD activity was 506 %, 191 %, and 75 % higher than controls in ‘P3167’, ‘32K61’, and ‘Bora’, respectively. APOD activity was significantly and 42 % lower than the respective control in ‘P3167’ under salinity (p < 0.05) (Fig 6c). In ‘32K61’ and ‘Bora’, however, insignificant changes were determined in APOD activity as a result of salt stress (p > 0.05). It was 31 % and 15 % lower than controls (Fig. 6c). In the case of GR activity, significantly higher val- ues were measured in ‘P3167’ and ‘Bora’ (57 % and 50 %, respectively) as compared to control (p < 0.05) while it was not affected and was only 1 % higher than control in ‘32K61’ under salinity stress (p > 0.05) (Fig 6d). 4 DISCUSSION Salt stress generally causes reduction in the growth rate in plants as result of the decreased ability of plants to take up water from the soil (Munns, 2002). It was re- ported that the growth ability of plants under salt stress maight be a reliable criterion to determine the salt tol- erance degree of plants (Parida and Das, 2005). In the present study, root and shoot growth of all maize geno- types were negatively affected by salt stress (300 mM NaCl). When genotypes were compared to each other, root growth in the genotypes ‘P3167’ and ‘32K61’ was found to be more sensitive to salinity while ‘Bora’ was more tolerant. With regard to organ type, however, shoot growth was more sensitive to salinity in all maize geno- types. These results showed that the roots and shoots of maize genotypes used in this study showed considerable variation with respect to salinity tolerance. These results are also in agreement with the findings of Doğru and Yılmaz Kaçar (2019), who manifested that sensitivity and/or tolerance of different barley genotypes may rep- resent great variation under saline conditions. It was also indicated that salt stress may interfere with mitotic activ- ity in the meristematic cells in plants, depending on the Figure 6: Effect of the salt stress on (a) the reduced ascorbate content, (b) SOD activity, (c) APOD activity and (d) GR activity of maize plants (Different letters mean significant differences between the treatments according to Duncan’s multiple range test (p < 0.05)) Acta agriculturae Slovenica, 117/1 – 2021 9 Effects of salt stress on physiological and biochemical responses of three maize genotypes at the early seedling stage plant species, genotype, organ type, and exposure time (Munns, 2002). Accordingly, we may conclude that the meristematic cells in the shoot apex are more sensitive to salinity as compared to the root meristems, probably due to more efficient transport of salt ions from roots to shoots, as reported by Zaimoğlu and Doğru (2016). Growth retardation in roots and shoots can be related to the inhibited cell elongation in plants under salt stress (Bendeoğlu et al., 2014). In the present study, it was ob- served that salt stress decreased RWC and increased wa- ter deficit index in the leaves of maize genotypes. There- fore, another possible reason for the reduced growth rate in maize genotypes under salt stress may be associated with the reduced RWC and induced water deficit index in the leaves. In addition, closure of the stomata and further disruption of the transpiration stream as result of water deficit-induced stimulation of abscisic acid synthesis may be indirectly responsible for the reduced growth rate in the salt-stressed maize genotypes used in this study (Po- lash et al., 2018). The results of the present study indicated a signifi- cant decrease in chlorophyll a content in the salt-stressed maize genotypes which is in agreement with the previous studies of Turan et al. (2007) on Phaseolus vulgaris L. and Taffouo et al. (2010) on Vigna subterranean L. Chloro- phyll b content was found to decrease only in ‘P3167’ and ‘32K61’. The decreased chlorophyll level can be attributed to the inhibition of chlorophyll biosynthesis and/or to the degradation by chlorophyllase (Santos, 2004). In ad- dition, the results showed that chlorophyll b molecules were better preserved in the genotype ‘Bora’ under salt stress. In salt-stressed plants, the reduced chlorophyll content was considered as a typical symptom of oxidative stress (Smirnoff, 1996). On the other hand, the reduced chlorophyll content had been considered as a protective mechanism against oxidative stress as well (Elsheery and Cao, 2008). The results of the present study showed that salt stress did not cause oxidative stress in maize geno- types used in this study, as demonstrated by lower-level MDA and H2O2. Accordingly, total anthocyanin content did not represent considerable changes in the salt-stressed maize leaves. Anthocyanins are diverse group of second- ary metabolites that could be produced in response to oxidative stress (Chunthaburee et al., 2016). Proline is a water-soluble amino acid and is involved in ROS detoxifi- cation (Ashraf and Foolad, 2007). In this study, salt stress did not induce free proline accumulation in the maize cultivars, confirming the hypothesis that maize geno- types are not under oxidative stress as result of salt ap- plication. An alternative explanation for this may be that oxidative stress on the maize genotypes under salt stress is eliminated by different defence mechanism as well. A possible defence mechanism that could eliminate oxida- tive stress on the salt-stressed maize genotypes may be the accumulation of phenolic compounds in leaf tissues. In this study, we observed that salt stress increased the phenolics in the leaves of ‘P3167’ and ‘32K61’. This result is in agreement with the previous reports on Aloe vera (L.) Burm.f. and radish (Moghbeli et al., 2012; Sakamo- to and Suzuki, 2019). Phenolics are believed to prevent the formation of ROS under drought stress (Mayer and Harel, 1991). Parida et al. (2004) have also reported that increases in phenolic content in plant tissues ameliorate the ionic effects of salt stress. Therefore, the enhanced level of phenolic compounds in the leaves of ‘P3167’ and ‘32K61’ under salt stress may be beneficial to achieve salt tolerance. Phenolic compounds have also been indicated to prevent lipid peroxidation and accumulation of MDA (Potapovich and Kostyuk, 2003), as reported in this study. In the present study, the level of the reduced ascorbate in the leaves of maize genotypes was increased by salinity as reported by several authors previously (Panda and Upad- hyay, 2004; Chen et al., 2005). The reduced ascorbate is known to be a detoxifier or neutralizer of superoxide, H2O2, and singlet oxygen species. This result may explain the constant level of H2O2 content and APOD activity in the leaves of the salt-stressed maize genotypes used in this study. In other words, H2O2 accumulation in the salt-stressed leaves of the maize genotypes are prevented by the direct action of the reduced ascorbate instead of APOD activity. The elevated SOD activity in maize geno- types under salt stress may be an indicator of the accel- erated rate of superoxide formation and detoxification (Doğru and Çakırlar, 2020a). GR activity increased in the salt-stressed leaves of the ‘P3167’ and ‘Bora’ while it did not change in ‘32K61’. These results could be interpreted as the lower activity of the ascorbate-glutathione cycle because of the absence of harmony between APOD and GR activities (Doğru and Çakırlar, 2020b). TSC content was not affected in the salt-stressed leaves of the ‘P3167’ and ‘32K61’ while it was decreased in the leaves of ‘Bora’. It has been reported that soluble sugars play an important role in osmotic adjustment in plant cells under stressful conditions (Doğru and Ecem Bayram, 2016). Accord- ing to the results in this study, it could be concluded that TSC is not involved in the osmotic regulation in the salt- stressed ‘P3167’ and ‘32K61’. However, TSC may serve as carbon reserves in these genotypes. In the salt-stressed leaves of ‘Bora’, a lower level of TSC may show that they are used for growth and development. In conclusion, the present study showed that salt stress reduced the growth rate in the salt-stressed maize genotypes, and shoot growth was more sensitive to sa- linity in comparison with root growth. In addition, salt stress led to the water deficit (physiological drought) in all genotypes, probably resulting in growth retardation. Acta agriculturae Slovenica, 117/1 – 202110 A. DOĞRU In ‘P3167’ and ‘32K61’, salt stress predominantly and adversely affected chlorophyll a content while ‘Bora’ re- tained both chlorophylls a and b. The elevated SOD activ- ity in all maize genotypes under salt stress may indicate an efficient dismutation of superoxide radical. Changes in APOD and GR activities under salinity clearly showed an increased pressure on the ascorbate-glutathione cycle, especially in ‘P3167’ and ‘32K61’. The constant level of H2O2 and MDA in the salt-stressed leaves of maize geno- types may show that the reduced ascorbate and phenolic compounds may be responsible for avoiding the adverse effects of oxidative stress. Finally, the genotype ‘Bora’ could be considered as having tolerance to salinity while ‘P3167’ and ‘32K61’ are sensitive ones. 5 ACKNOWLEDGMENT This study is supported by Sakarya University Sci- entific Research Projects Coordination Unit. 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Acta agriculturae Slovenica, 117/1, 1–9, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1768 Original research article / izvirni znanstveni članek Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new records Samia AIT AMAR 1, 2 and Karima BENOUFELLA-KITOUS 1 Received July 08, 2020; accepted December 14, 2020. Delo je prispelo 08. julija 2020, sprejeto 14. decembra 2020. 1 University of Mouloud Mammeri, Faculty of Biological Sciences and Agronomic Sciences, Animal and Plant Biology Department, Tizi-Ouzou, Algeria, Laboratory of Production, Improvement and Protection of Plants 2 Corresponding author, e-mail: samia.aitamar@ummto.dz Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new re- cords Abstract: Aphids are among the phytophagous pests that cause serious damage to crop plants. In Northern Algeria, we have little information on their diversity. In this context, the study of the diversity of aphids was carried out in three regions of Tizi-Ouzou (North of Algeria) namely Tizi-Ouzou center, Tizi-Rached and Aghribs on the potato crop using yellow traps. The results showed a total richness of 65 aphid species, divided into 36 genera, 9 tribes and 8 sub-families, of which 11 spe- cies were identified for the first time in Algeria. These are Aphis coreopsidis (Thomas, 1878), Capitophorus hippophae (Walker, 1852), Cavariella theobaldi (Gillette & Bragg, 1918), Hyada- phis coriandri (B. Das, 1918), Macrosiphoniella linariae (Koch, 1855), Monelliopsis pecanis Bissell, 1983, Myzus hemerocal- lis Takahashi, 1921, Pseudoregma panicola (Takahashi, 1921), Rhopalosiphoninus staphyleae (Koch, 1854), Schizaphis eastopi Van Harten & Ilharco, 1971 and Ovatus inulae (Walker, 1849). The field located in the center of Tizi-Ouzou is the richest with 55 species, followed by the field of Tizi-Rached with 30 spe- cies, and 24 species have been recorded in Aghribs. During the sampling season, Hyperomyzus lactucae (Linnaeus, 1758) and Brachycaudus helichrysi (Kaltenbach, 1843) are the most abun- dant species with 24.44 % and 21.8 % respectively. Three aphid species have been observed on potato leaves, namely Macro- siphum euphorbiae (Thomas, 1878), Aphis gossypii Glover, 1877 and Myzus persicae (Sulzer, 1776). The latter species was ob- served in all three study regions. Key words: aphids; diversity; potato; new species Pestrost listnih uši (Hemiptera: Aphididae) v nasadih krom- pirja na območju Tizi-Ouzou (Severna Alžirija) z novimi najdbami Izvleček: Listne uši so med rastlinojedimi škodljivci tisti, ki lahko povzročijo občutne poškodbe na gojenih rastlinah. V severni Alžiriji je o njihovi pestrosti malo znanega. Z ozirom na to je bila izvedena raziskava pestrosti listnih uši na treh ob- močjih Tizi-Ouzou Severne Alžirije in sicer v središču območja Tizi-Ouzou, in na območjih Tizi-Rached in Aghribs v nasadih krompirja z uporabo rumenih pasti. Izsledki so pokazali, da je celokupna pestrost obsegala 65 vrst listnih uši, ki so pripada- le 36 rodovom, 9 plemenom in 8 poddružinam, od katerih je bilo 11 vrst novih za Alžirijo. Te vrste so bile Aphis coreopsidis (Thomas, 1878), Capitophorus hippophae (Walker, 1852), Ca- variella theobaldi (Gillette & Bragg, 1918), Hyadaphis coriandri (B. Das, 1918), Macrosiphoniella linariae (Koch, 1855), Mo- nelliopsis pecanis Bissell, 1983, Myzus hemerocallis Takahashi, 1921, Pseudoregma panicola (Takahashi, 1921), Rhopalosipho- ninus staphyleae (Koch, 1854), Schizaphis eastopi Van Harten & Ilharco, 1971 in Ovatus inulae (Walker, 1849).Polje v središču območja Tizi-Ouzou je vrstno najbogatejše s 55 vrstami, sledi mu polje na območju Tizi-Rached s 30 vrstami in območje Ag- hribs s 24 vrstami. Med vzorčenjem sta bili vrsti Hyperomyzus lactucae (Linnaeus, 1758) in Brachycaudus helichrysi (Kalten- bach, 1843) najpogostejši s 24,44 % in 21,8 % deležem. Na listih krompirja so bile opažene tri vrste listnih uši in sicer Macrosip- hum euphorbiae (Thomas, 1878), Aphis gossypii Glover, 1877 in Myzus persicae (Sulzer, 1776), slednja je bila opažena ns vseh treh območjih. Ključne besede: listne uši; pestrost; krompir; nove vrste Acta agriculturae Slovenica, 117/1 – 20212 S. AIT AMAR and K. BENOUFELLA-KITOUS 1 INTRODUCTION The potato (Solanum tuberosum L.), is one of the main food resources in the world. It remains the most consumed vegetable or field crop (Brillaud, 2008; Mar- choux, 2008). This crop is subject to many pests, in- cluding aphids, they are small sap-sucking insects. The direct damage is caused by these pests to the crop by sap sucking, thus reducing the resources available for growth and development of the plant (Blackman and Eastop, 2000; 2006). However, the most significant damage is caused indirectly by plant pathogenic viruses transmitted by aphids. Myzus persicae Sulzer, for ex- ample, is known to be a vector of more than 100 plant viruses (Blackman and Eastop, 2000) including Potato Virus Y (PVY), Potato Leafroll Virus (PLRV) or Cu- cumber Mosaic Virus (CMV) (Lin et al., 2016; Shi et al., 2016). Aphids are very widespread, more than 4700 spe- cies have been recorded in the world (Remaudière and Remaudière, 1997), among them about 450 aphid spe- cies have been identified as pests of cultivated plants (Blackman and Eastop, 2000). At least 220 species have been recorded in sub-Saharan Africa (Autrique and Ntahimpera, 1994). One hundred fifty species have been identified in Morocco (Sekkat, 2015). Aphids in Tunisia are represented by one hundred and three spe- cies (Bodenheimer and Swirsky, 1957; Blackman and Eastop, 1994; 2000, 2006; Ben Halima-Kamel, 1991, 1995; Ben Halima Kamel and Ben Hamouda, 1993, 1998, 2004, 2005; Boukhris-Bouhachem et al. 1996; Boukhris-Bouhachem et al., 2007). Ninety nine species are recorded from Egypt (Theobald, 1922; Habib and El Kady, 1961; Darwish, 2009), seventy-three species are listed from Libya (Trotter, 1912; 1914; Damiano, 1961; 1962; Blackman and Eastop, 1994; Ahmeid Al-Nagar, 2000; Ahmeid Al-Najar and Nieto Nefrya, 1998). The Algerian aphid fauna is known partly (Black- man and Eastop, 1994, 2000, 2006; Laamari and Akkal, 2002; Lamaari et al., 2010, 2013; Hidalgo et al., 2012; Laamari, 2016; Laamari et al., 2016; Benoufella-Kitous et al., 2014a, 2014b, 2016, 2019). In Tizi-Ouzou, we have very little data on aphid diversity (Benoufella-Kitous et al., 2014a, 2014b, 2016, 2019). Hence the objective of this study is to evaluate the diversity and abundance of aphids in three potato fields in Tizi-Ouzou region (Northern Algeria). 2 MATERIALS AND METHODS 2.1 THE STUDY REGION This study was carried out in three localities of Tizi-Ouzou (Fig.1) in open field potato crops: The first, Resita variety, was planted on March 03rd, 2013, over an area of 3000 m2 is located in Tizi-Ouzou Center (36 ° 43ʹ 00" N and 4 ° 03ʹ 00" E) at 200 m altitude. The second, Fabula variety, was planted on December 27th, 2016, on an area of 300 m2 is located in Tizi Rached (36 ° 40ʹ N and 4 ° 11ʹ E) at 412 m altitude and the third, Timate variety, was planted on February 23th, 2017, on an area of 250 m2 is located in Aghribs (36 ° 48ʹ 08" N, 4 ° 19ʹ 22" E) at an altitude of 800 m. The fields were sampled during the period from April 2nd to June 18th, 2013 for the Resita variety, from February 13th to May 8th, 2017 for the Fabula variety and from April 1st to June 3rd, 2017 for the Timate variety. The experimental protocol followed for the trapping of winged aphids and the visual control of aphids is that Figure 1: Map of Tizi-Ouzou region and location of study sites, (A) Map of Algeria showing the study region, (B) the study region Acta agriculturae Slovenica, 117/1 – 2021 3 Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new records described by Atesebeha et al. (2009). Two methods were combined for monitoring aphidofauna. 2.2 TRAPPING OF WINGED APHIDS To do this, the study field was divided into 9 blocks. In the middle of each block, a yellow Von Moerik trap (Ѳ = 27 cm, h = 10 cm) was placed, filled with water and a few drops of detergent (dishwashing liquid). The attrac- tion of aphids to the yellow color has been known for long time. This color tends to cause these insects to land (Yat- tara et al., 2013). The contents of the container are neces- sary for optimum yield (EDES, 2011). Aphids caught in the yellow traps were removed once a week with a brush and stored in test tubes filled with 70 % ethanol. 2.3 VISUAL OBSERVATION OF APHIDS ON PO- TATO LEAVES Once a week, in each block of the field, one plant was chosen and the aphids found on the potato leaves were collected in a tube filled with 70 % ethanol, using a brush. Aphids trapped and taken from the plants are sorted and then identified up to the species level. Accord- ing to Lascaux (2010), the identification of aphids was carried out by observing some morphological characters of the aphid, in particular: the antennae, the frontal tu- bercles, the tarsi, the cauda, the color and the shape of the sphinculi, the pigmentation of the abdomen and ve- nation of the wings. The samples collected were deter- mined by Dr. Benoufella-Kitous at the Plant Production, Improvement and Protection Laboratory of the Faculty of Biological Sciences and Agronomic Sciences at Mou- loud Mammeri University in Tizi-Ouzou. The identifi- cation was carried out based on the identification keys of Stroyan (1961), Jacky and Bouchery (1982), Autrique and Ntahimpera (1994), Remaudière et al. (1985) and Leclant (1999) and Blackman and Eastop, (2000; 2006). 3 RESULTS During the study, 2144 aphids were collected from all three regions (Table 1). More than two thirds of the catches (73.27 %) were made in Tizi-Ouzou. A total of 65 species were caught using yellow traps, divided into 36 genera, 9 tribes and 8 subfamilies: Aphidinae, Anoe- ciinae, Calaphidinae, Chaitophorinae, Eriosomatinae, Hormaphidinae, Myzocallidinae, and Pterocommatinae, 11 species are new to Algeria. The Aphidinae subfamily is predominant with 2 tribes, that of Aphidini and that of Macrosiphini. The latter is the richest with 39 species. Tizi-Ouzou centre is the richest in species with 55 species, divided into 32 genera, 7 tribes and 5 subfamilies, namely the Aphidinae, Anoeciinae, Calaphidinae, Eriosomatinae and Myzocal- lidinae. Followed by Tizi Rached station, with 30 spe- cies belonging to three subfamilies, namely Aphidinae, Chaitophorinae and Eriosomatinae. Besides, 24 species were recorded at Aghribs belonging to 4 subfamilies, those of Aphidinae, Eriosomatinae, Hormaphidinae, and Pterocommatinae. Considering all stations, the most abundant species during the sampling season are: Hy- peromyzus lactucae (24.44  %), Brachycaudus helichrysi (21.8 %), Macrosiphum rosae (19.93 %) and Myzus per- sicae (13.11 %). On the other hand, Macrosiphum euphorbiae, Aphis gossypii and M. persicae, were observed on potato leaves. The last two species develop in all potato fields. M. eu- phorbiae was the most observed in Tizi-Ouzou centre. In Aghribs, M. persicae was the most abundant, while in Tizi Rached, A. gossypii was the most abundant (Table 2). 3.1 DESCRIPTION OF SOME NEW SPECIES 3.1.1 Aphis coreopsidis Apterae are yellow to green with darker legs, anten- nae and siphunculi. The abdomen has marginal scleritis (Blackman and Eastop, 2020). The siphunculi are black, relatively long and thick the cauda is clear and narrow 3.1.2 Capitophorus hippophae In spring the apterae colonies are pale green, slen- der. The alatae are greyish-green with a black head and thorax, dark antennae, legs and siphunculi and a a large quadrate dark green patch on the dorsal abdomen (Black- man and Eastop, 2020). Their primary host are species from the genus Polygonum , and the secondary host are species from the same genus (Remaudière et al., 1985). 3.1.3 Hyadaphis coriandri The alatea are yellow-green in color, dusted with greyish wax. They have short, dusky, slightly swollen, siphunculi. that are about twice as long as wide (Hal- bert, 2003). This species colonizes Hydrocotyle vulgaris L. (Hydrocotylaceae), Anethum sp. Coriandrum sativum L., Daucus sp. Foeniculum vulgare Mill., Peucedanum sp. Acta agriculturae Slovenica, 117/1 – 20214 S. AIT AMAR and K. BENOUFELLA-KITOUS Aphid species Tizi-Ouzou Centre Aghribs Tizi Rached Acyrthosiphon pisum (Harris, 1776) 0.76* - - Anoecia corni (Fabricius, 1775) 0.13 - - Aphis coreopsidis (Thomas,1878)1 - - 0.32 Aphis craccivora Koch, 1854 7.51 2.6 4.2 Aphis fabae Scopoli, 1763 9.70 11.8 8.04 Aphis gossypii Glover, 1877 6.87 16.41 6.8 Aphis idaei van der Goot, 1912 3.63 - 1 Aphis nerii Boyer de Fonscolombe, 1841 2.36 0.7 1.61 Aphis pseudocardui Theobald, 1915 - 0.7 - Aphis spiraecola Patch, 1914 0.95 1.1 1.3 Aphis sp. 0.20 - 0.32 Aulacorthum solani Kaltenbach, 1843 1.78 1.52 2.6 Brachycaudus cardui (Linnaeus, 1758) 0.57 3 1.61 Brachycaudus helichrysi (Kaltenbach, 1843) 1.65 21.8 2.3 Brevicoryne brassicae (Linnaeus, 1758) 2.23 1.14 0.32 Capitophorus hippophae (Walker, 1852)1 0.13 - - Cavariella aegopodii (Scopoli, 1763) 0.25 - 0.32 Cavariella pastinacae (Linnaeus, 1758) 0.20 - - Cavariella theobaldi (Gillette & Bragg, 1918)1 0.13 - - Diuraphis noxia (Kurdjumov, 1913) 0.20 - - Dysaphis plantaginea (Passerini, 1860) 4.39 1.14 0.64 Dysaphis tulipae (Boyer de Fonscolombe, 1841) 0.20 5.58 1 Dysaphis apiifolia (Theobald, 1923) 0.13 - - Dysaphis foeniculus (Theobald, 1923) - 1.5 - Eriosoma lanuginosum (Hartig, 1839) 0.13 - - Hyadaphis coriandri (B. Das, 1918)1 0.95 - - Hyadaphis foeniculi (Passerini, 1860) 0.57 - - Hyalopterus pruni (Geoffroy, 1762) 0.32 - - Hyperomyzus carduellinus (Theobald, 1915) 0.20 - - Hyperomyzus lactucae (Linnaeus, 1758) 6.17 6.4 24.44 Hyperomyzus picridis (Börner & Blunck, 1916) 0.13 1.14 - Lipaphis erysimi (Kaltenbach, 1843) 0.95 0.76 0.64 Macrosiphoniella linariae (Koch, 1855)1 2.74 - - Macrosiphum euphorbiae (Thomas, 1878) 3.69 1.14 4 Macrosiphum funestum (Macchiati, 1885) - - 0.4 Macrosiphum rosae (Linnaeus, 1758) 1.72 8.77 19.93 Megoura viciae Buckton, 1876 1.25 - - Metopolophium dirhodum (Walker, 1849) 0.06 - 0 .4 Metopolophium festucae Theobald, 1917 - 3.1 - Monelliopsis pecanis Bissell, 19831 0.13 - - Myzocallis castanicola Baker, 1917 0.20 - - Table 1:Proportions and relative wealth of aphids trapped in potato fields, the nomenclature of all species is follows that of orders in Fauna Europaea (2020) Acta agriculturae Slovenica, 117/1 – 2021 5 Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new records *The proportions are expressed in percentage. 1Species reported for the first time in Algeria. Continued Myzus ascalonicus Doncaster, 1946 2.04 - - Myzus cerasi (Fabricius, 1775) 0.51 - - Myzus hemerocallis Takahashi, 19211 0.13 - - Myzus persicae Sulzer, 1776 13.11 3.1 9.00 Myzus sp. - - 0.32 Nasonovia ribisnigri (Mosley, 1841) 0.45 - 0.32 Ovatus crataegarius (Walker, 1850) - - 0.32 Ovatus inulae (Walker, 1849)1 0.13 - - Pemphigus sp. 1.85 0.3 1.3 Phorodon humuli (Schrank, 1801) 0.45 - - Pseudoregma panicola (Takahashi, 1921)1 - 0.7 - Rhopalosiphum insertum (Walker, 1849) 0.13 - - Rhopalosiphum maidis (Fitch, 1856) 8.30 - 4.50 Rhopalosiphum padi (Linnaeus, 1758) 7.38 6.10 1.4 Rhopalosiphum rufiabdominale (Sasaki, 1899) 0.64 - - Rhopalosiphoninus staphyleae (Koch, 1854)1 0.06 - - Schizaphis eastopi Van Harten & Ilharco,19711 0.06 - - Schizaphis rotundiventris (Signoret, 1860) 0.20 - - Sipha maydis Passerini, 1860 - - 0.4 Sitobion avenae (Fabricius, 1775) 0.38 - - Sitobion fragariae (Walker, 1848) 0.70 - - Smynthurodes betae Westwood, 1849 0.20 0.7 - Takecallis taiwana (Takahashi, 1926) 0.10 - - Uroleucon sonchi (Linnaeus, 1767) - - 0.64 Number of aphid individuals 1571 262 311 Number of species 55 24 30 Species Tizi-Ouzou Centre Aghribs Tizi Rached Aphidsa Frequency Aphids a Frequency Aphids a Frequency A. gossypii 492 16 % 64 25.4 % 403 67.3 % M. euphorbiae 2438 78 % - - 133 22.2 % M. persicae 196 6 % 188 74.60 % 63 10.5 % Total 3126 100 % 252 100 % 599 100 % Table 2: Proportion and relative abundance of species observed in the three potato fields a: number of aphids. Acta agriculturae Slovenica, 117/1 – 20216 S. AIT AMAR and K. BENOUFELLA-KITOUS Pituranthos sp. Steganotaenia sp. (Umbelliferae) (Remaudière et al., 1985). 3.1.4 Myzus hemerocallis Apterae are pale yellowish green or greenish, they measure between 1.6 and 2.4 mm (Blackman and Eastop, 2020). They at- tack basal parts of Hemerocallis spp. and Agapanthus umbellatus. L’Herit. 3.1.5 Pseudoregma panicola Apterae are brownish black or dark brownish red- violet, secreting columns of dense white ; the adults mea- sure between 1,1 and 1,9 mm (Blackman and Eastop, 2020). This species colonize underside of bambou leaves Arundinaria alpina K. Schum. (Remaudière et al., 1985). 3.1.6 Rhopalosiphoninus staphyleae In spring, the Apterae on Staphylea are yellowish- white or pale yellow with a translucent whitish spot on anterior part of dorsal (Blackman and Eastop, 2020). This species lives on Viola tricolor L. (Violaceae). 3.1.7 Schizaphis eastopi The abdomen is devoid of pigmentation, siphunculi are black, cylindrical with apical constriction. The cauda is clear, small than the siphunculi. This species lives on Typha domingensis Pers., Typha angustifolia L. and Typha capensis (Rohrb.)  N.E. Br. (Typhaceae) (Remaudière et al., 1985). 4 DISCUSSIONS Based on the trapping results of winged aphids caught using yellow traps installed in three potato fields in the Tizi-Ouzou region, 65 species were identified, af- ter comparison of our results and those of Laamari et al. (2010; 2013), 11 species are new for Algeria, 27 species were collected in Tizi-Ouzou in Draâ Ben Khedda on the bean (Benoufella-Kitous et al., 2014a). Benoufella- Kitous et al. (2014b) affirmed the presence of 28 species on citrus fruits in Oued-Aissi (Tizi-Ouzou, Algeria). On the food legumes, 55 species have been recorded in Tala Amara (Tizi-Ouzou) (Benoufella-Kitous et al., 2016) and 43 species in Tizi Rached (Benoufella-Kitous et al., 2019). Most of these aphid species are pests of cultivated plants, including M. persicae, an effective vector, espe- cially of potato viruses. The difference in the specific richness of one region to another can be explained by the difference in the floristic composition which has a direct influence on the aphid richness. Among the plant spe- cies recorded in large numbers in the study stations: Pic- ris echioides L., Sonchus oleraceus L., Solanum nigrum L., Malva sylvestris L. and Aven asterilis L. which can host up to 20 aphidian species (Laamari et al., 2010; Moussadegh et al., 2016). B. helichrysi is the most abundant species in Aghribs with 21.8 %. This abundance can be explained by the presence of its primary host plant: thevine crop. The sec- ondary host of this species is represented by the Astera- ceae (sow-thistle, lettuce), where it is found in colonies of several individuals living inside of the leaves, sometimes in mixed colonies with Nasonovia ribisnigri (Hullé et al., 1999). In Tizi-Ouzou centre, M. persicae is the most dom- inant with 206 individuals with a frequency of 13.11 %. Similar results have been noted in China by Lopes et al. (2012). These authors highlighted the predominance of this aphid over other captured species. Also in Mali, Yattara et al. (2013), report the presence of this species with 21.2 % on potato. This dominance is explained by the presence of its primary host: the peach tree Prunus persica (L.) Batsch,  and also its secondary host which is the potato. The species A. coreopsidis was reported for the first time in Saudi Arabia by Hussain et al. (2015), in spring this species has a several secondary hosts such as Com- positae, Asteraceae, Malvaceae and Lamiaceae fam- ily (Blackman and Eastop, 2020), and several species of these families are found around the field of Tizi Rached. C. hippophae has as secondary host Polygonaceae such as Polygonum persicaria L. (Forbes and Chan, 1985). The presence of R. staphyleae in the Tizi-Ouzou centre is probably due to the presence of flowering plants and also weeds belonging to the Chenopodiaceae family which are secondary hosts of this aphid. Three species of aphids are found on potato leaves, namely M. euphorbiae, A. gossypii and M. persicae. Ac- cording to Hullé et al. (1999), the latter three species are characteristic of this crop. M. persicae is reported by Laa- mari and Akkal (2002) in Setif (Algeria). Laamari (2004), in the Guellal region of Biskra (Algeria), notes the pres- ence of M. euphorbiae and M. persicae on a potato crop. In Kati and Sikasso (Mali), Yattara et al. (2013) highlights the presence of A. gossypii and M. persicae on this crop for three consecutive years. Lopes et al. (2012) report the presence of the latter two species on potato in China. The difference between the abundance of the three species observed on the leaves of potato crops in all fields, could Acta agriculturae Slovenica, 117/1 – 2021 7 Diversity of aphids (Hemiptera: Aphididae) associated with potato crop in Tizi-Ouzou (North of Algeria), with new records be due to the arrival order of the winged adults, and could be to the competition phenomenon, the aphids can respond differently when they are feeding on the same plant. This dietary variation is related to the capacity of aphid species to encourage the plant to produce a richer diet, especially on amino acid (Telang et al., 1999). 5 CONCLUSION This preliminary study, which focuses on the di- versity of aphids associated with potato crop in three localities of the Tizi-Ouzou region (North of Algeria), has shown the existence of 65 species, which 11 species are reported for the first time in Algeria. The presence of aphids and their diversity is favored by the presence of their host plant. In the three study fields several weeds and fruit trees are present, this would explain the abun- dance of these pests within these fields. Therefore, it would be interesting to continue several studies in dif- ferent regions, in order to identify the host plants of each aphid species. 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Phytoprotection, 94(1), 1-7. https://doi. org/10.7202/1024719ar Acta agriculturae Slovenica, 117/1, 1–9, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1523 Original research article / izvirni znanstveni članek Effect of selected Rwandan wheat varieties on the physicochemical char- acteristics of whole wheat flour Bernard RWUBATSE 1, 2, Michael Wandayi OKOTH 1, Angela Adhiambo ANDAGO 1, Sophia NGALA 1, Anastase KIMONYO 3, Clement BITWAYIKI 3 Received February 18, 2020; accepted January 13, 2021. Delo je prispelo 18. februarja 2020, sprejeto 13. januarja 2021. 1 University of Nairobi, Department of Nutrition, Food Science and Technology, Nairobi, Kenya 2 Corresponding author, e-mail: brwubatse@students.uonbi.ac.ke 3 University of Rwanda, Department of Food Science and Technology, Musanze, Rwanda Effect of selected Rwandan wheat varieties on the physico- chemical characteristics of whole wheat flour Abstract: The present study aimed to evaluate the effect of the wheat varieties newly introduced in Rwanda on the physi- cochemical characteristics of their whole wheat grains in order to know their potentials for processing. Gihundo wheat grain variety had the highest values for extraction yield (99.20  %), contents of ash (1.47 %) and total dietary fiber (15.97 %), wa- ter absorption capacity (89.00  %), dough development time (7.62  min) and brightness (84.67 %). For the same physico- chemical characteristics, whole flour from Nyaruka wheat va- riety showed the lowest values for extraction yield (96.20%), water absorption capacity (80.00 %), dough development time (6.33 min) and brightness (80.33), while whole flour from Re- beraho wheat variety exhibited the lowest values for the con- tents of ash (0.98 %) and total dietary fiber (12.44 %). The pro- tein content ranged between 10.00  % and 10.85  % for whole flours from all wheat varieties. The results showed that whole flour from Gihundo wheat grain variety exhibited high values for most of the physicochemical characteristics determined in comparison to the other three varieties. It is important to select grains or flour from these wheat varieties newly introduced in Rwanda based on the individual cultivar because their deriva- tive products could have a more desired quality. Key words: physicochemical characteristics; whole wheat flour; wheat varieties Vpliv izbranih ruandskih sort pšenice na fizikalno-kemijske lastnosti polnozrnate pšenične moke Izvleček: Cilj raziskave je bil proučiti vpliv v Ruandi na novo vpeljanih sort pšenice na fizikalno-kemijske lastnosti pripadajočih celih zrn z namenom prepoznave njihovega po- tenciala za nadaljnjo predelavo. Zrna iz pšenice sorte Gihundo so imela največji izplen moke pri mletju (99,20  %), polnozr- nata moka je vsebovala največ pepela (1,47 %), največ skupne prehranske vlaknine (15,97 %), imela je najboljšo sposobnost vezave vode (89,00 %), najdaljši razvoj testa (7,62 min) in bila je najbolj svetla (84,67). Za iste fizikalno-kemijske lastnosti je polnozrnata moka iz sorte Nyaruka dosegla najmanjši izplen meljave (96,20 %), najslabšo sposobnost vezave vode (80,00 %), najkrajši razvoj testa (6,33 min) in bila je najbolj temna (80,33), medtem, ko je polnozrnata moka iz sorte Reberaho vsebova- la najmanj pepela (0,98  %) in skupne prehranske vlaknine (12,44  %). Vse štiri pšenične polnozrnate moke so vsebovale med 10,00 % in 10,85 % beljakovin. Rezultati so pokazali, da je imela polnozrnata moka iz pšenice sorte Gihundo v primerja- vi z mokami iz zrn ostalih treh sort boljše vrednosti za večino fizikalno-kemijskih parametrov. Pomembno je izbrati pšenična zrna ali moko iz tistih zrn na novo vpeljanih sort v Ruandi, ki imajo najboljše lastnosti, saj lahko to vodi k večji kakovosti iz- delkov. Ključne besede: fizikalno-kemijske lastnosti; polnozr- nata pšenična moka; sorte pšenice Acta agriculturae Slovenica, 117/1 – 20212 B. RWUBATSE et al. 1 INTRODUCTION Whole wheat flours have been gaining an increase in demand and utilization for their nutritive value and health benefits in different food products for human consumption worldwide. AACC (2000) defines whole grain flour as flour which consists of the intact, ground, cracked or flaked cary- opsis, whose principal anatomical components (the starchy endosperm, germ, and bran) are present in the same rela- tive proportions as they exist in the intact caryopsis. Whole grain flour contains more vitamins, minerals, antioxidants, bioactive phytochemicals and dietary fiber than refined flour (Chung et al., 2009; Adom and Sorrells, 2005; Slavin, 2004). Due to health promoting effects of phytochemicals and dietary fiber, an increased consumption of whole grains is recommended (Seal, 2015). While the extraction yield affects the (dough) farin- ograph test properties of whole wheat flours, these phy- tochemical compounds can also affect the quality of the end-use products such as low loaf volume and dense crumb structure, grainy, nutty and bitter flavors (Heinio, 2009; Chang and Chambers, 1992), and darker crumb and crust color (Lebesi and Tzia, 2011). Among the phytochemicals, carotenoids and phenolic compounds such as anthocyanins can act as antioxidants and impact the color and flavor of the food product (Rao and Rao, 2007). On average, the total carotenoid content was 2.57 mg kg-1 for the whole wheat grain, 1.92 mg kg-1 for the endosperm, 9.11mg kg-1 for the germ and 0.74 mg kg-1 for the bran. Those values for the endosperm, the germ and the bran were calculated based on seed fraction mass proportions to whole grain (Ndolo and Beta, 2013). Therefore, depending on the varieties, the physicochemical characteristics of whole wheat grains would significantly differ and influence the processing and quality of end-products. Some physicochemical attributes of the grains and flours of these new wheat varieties released in Rwanda in 2017 have not yet been determined so far in order to provide industries and consumers wheat with pre- ferred specific quality traits and functionality (MINAGRI, 2017; Newtimes, 2017; RAB, 2017). This may often result in promising wheat cultivars that can be rejected by a farm- er, seller, processor or consumer (Battenfield et al., 2016). With regard to this, the present study aimed to determine the physicochemical characteristics of whole wheat grains which can influence the quality of their based products. 2 MATERIALS AND METHODS 2.1 COLLECTION OF THE SAMPLES Four (4) dry wheat grain varieties namely TAI, EN161, Eagle10 and Korongo with their local names Gihundo, Ki- batsi, Nyaruka, and Reberaho, respectively, were collected from the stores of Rwanda Agriculture and Animal Re- sources Development Board (RAB) located at Kinigi, Mu- sanze district, Rwanda. These wheat varieties were grown in RAB farms at the same location and under the same agro ecological conditions in the crop year 2019.The wheat grains were sampled in the same year, packed in high den- sity polyethylene bags and stored at room temperature prior to milling. 2.2 PREPARATION OF THE RAW SAMPLES 2.2.1 Wheat grains milling Before milling, the wheat grains were conditioned to 15.5 % moisture content by the addition of distilled wa- ter and were left for at least 24 h at ambient conditions in a closed plastic container for the absorption of the mois- ture (Mishra, 2016) in order to get a fine particle size whole wheat flour. AACC (2003) method was used to calculate the amount of water to be added for wheat grains tempering: The conditioned wheat grains of each variety were wholly milled for 15 min by using a laboratory hammer mill (CM 1090 Cemotec, 2009, China). All bran and germ were mixed with the flour. The flour was packed in high density polyethylene envelop and stored at -20 oC until the day of analysis. 2.3 DETERMINATION OF PHYSICOCHEMICAL CHARACTERISTICS 2.3.1 Moisture content of whole wheat flour Moisture content of the whole wheat flour was deter- mined using moisture analyser (Model: HE 53/01, Mettler Toledo, China). 2.3.2 Ash content of whole wheat flour Ash content of the whole wheat flour was determined by AOAC (2005) method using a muffle furnace (Lindberg/ Blue 1100°C Box furnace BF 51800 series Ashville, NC). 2.3.3 Total fat content of whole wheat flour Total fat content was determined by AOAC (2005) method using Soxhlet extraction procedure. Acta agriculturae Slovenica, 117/1 – 2021 3 Effect of selected Rwandan wheat varieties on the physicochemical characteristics of whole wheat flour 2.3.4 Total protein content of whole wheat flour Total protein content was determined by AOAC (2005) method using the Kjeldahl procedure. 2.3.5 Total dietary fiber content of whole wheat flour Total dietary fiber content was determined by AOAC (2005) method where the extraction was done using petro- leum ether. 2.3.6 Total carbohydrate content of whole wheat flour Total carbohydrate was determined by AOAC (1990) method. 2.3.7 Bulk density of whole wheat flour The bulk density was determined as described by Khalid et al. (2017). The flour samples were gently filled into 10 ml graduated plastic cylinders. The bottom of the cylin- der was gently tapped on a laboratory bench covered with foam several times until there was no further diminution of the sample level. The mass of the sample was calculated and the bulk density was calculated as mass of sample per unit volume of sample (g ml-1). 2.3.8 Oil absorption capacity of whole wheat flour The oil absorption capacity was determined as de- scribed by Khalid et al. (2017). Each sample of 0.5 g was mixed with 6 ml of mustard oil in pre-weighed centrifuge tubes. The contents were vortexed for 1 min to disperse the sample in the oil. The samples were then kept for 30 min in vertical positions and then subjected to centrifugation of 3000 rpm for 15 min (3-18KS, Sigma Laborzentrifugen GmbH, Germany). The layer of the oil was removed by pi- pette and the tubes were kept in inverted position for 10 min to drain the oil before reweighing. The gain in mass was expressed as grams of oil absorbed per gram of flour. 2.3.9 Wheat grain hardness The wheat hardness was determined as described by Manley (1995).The ground grain samples were obtained by passing the whole wheat grain (15.5 % moisture content) through a Model 3100 hammer mill (Falling Number AB, Huddinge, Sweden) equipped with 1mm screen. The wheat hardness was calculated as the % of ground wheat grain (10 g) forcing through 75 micrometer air jet sieve in 90 s. The mass less than 4.0 g indicated a hard wheat, while 4.0 g or more indicated a soft wheat. 2.3.10 Extraction yield of whole wheat flour The flour extraction yield was determined as a per- centage of all bran and germ mixed with the flour from the mass of grains milled (AACC, 2000): 2.3.11 Farinograph test of whole wheat flour The farinograph parameters were determined using CW Brabender Instruments, inc., Germany following the AACC method (AACC, 2000). The flour sample was placed into a mixing bowl and distilled water was added up to the optimum dough consistency (500 BU). The water absorp- tion, dough development time, dough stability time and mixing tolerance index during mixing were evaluated from the farinograph. 2.3.12 Color of whole wheat flour The color was determined by a color reader (CR-10, Konica Minolta,inc. Japan) on the basis of L*, a* and b* val- ues. 2.4 STATISTICAL ANALYSIS Data in triplicate were subjected to one-way analysis of variance (ANOVA) using SAS System for Windows (ver- sion 9.3, SAS Institute, Cary, NC). Treatment means were separated using the Tukey post hoc test and least significant difference was accepted at p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 EFFECT OF WHEAT VARIETY ON GRAIN HARDNESS, EXTRACTION YIELD AND ON THE PROXIMATE COMPOSITION OF WHOLE WHEAT FLOURS The extraction yield was between 96.20  % and Acta agriculturae Slovenica, 117/1 – 20214 B. RWUBATSE et al. 99.20 % and grain hardness was from 3.03 g to 3.86 g. The results for extraction yield were in the range with those reported by Cauvain (2015). As the product was the whole wheat flour, the wheat milling extraction was supposed to be 100 %. However, the flour yield was not 100 % due to that some quantities of the flour were held, disappeared in parts of the mill and were not collected into the vessel. During brushing the flour off the parts of the hammer mill, some few quantities of it were also transported/conveyed by air. The highest flour yield of Gihundo variety may be due to the low grain hardness (Table 1), producing a lower quantity of damaged starch and tailings during the milling process. The extraction yields of Nyaruka and Reberaho wheat varieties were not significantly different (p > 0.05) and were the lowest. The level of extraction yield indicated the particle size of the wheat flour. This means that when the yield was high, the particle size of the flour was small. The results showed that all wheat grains were possibly hard as their results were below 4 g or 40 % (Manley ,1995). The results for total protein content were between 10.00 % and 10.85 %. There was no significant difference in protein content (p > 0.05) among whole wheat flours from the four wheat varieties, may be because they were grown at the same location and under the same agro eco- logical conditions. Wheat flours from all varieties could possibly fall under all purpose flour category as their pro- tein contents were not below 9 % or above 12 % (Chu, 2004). Moisture content ranged from 11.56 % to 12.09 %. The moisture contents of the wheat flours from Nyaruka and Reberaho varieties were not significantly different (p > 0.05) and were higher than those from the other two wheat varieties. Increase in moisture content could have been associated with increase in fibre (Maneju and Udobi, 2011). Excess moisture content above 12 % may be detrimental as it can lead to mould growth, toxin for- mation, insect infestation, sprouting in storage (Ezeama , 2007) and has the effect of reducing the protein content as a percentage of total mass. On the other hand, grain with excessively low moisture would result in a hard grain with low flour yield (Mishra, 2016). The content for ash was between 0.98 % and 1.47 % and the content for total dietary fiber was from 12.44 % to 15.97  %. The ash content results were in line with 0.42 % found by Khalid (2017) and from 1.46 to 1.56 % reported by Mishra (2016). Khalid et al. (2017) worked on the proximate composition of the native and irradi- ated whole wheat flour from wheat grains var. WH-1021, while Mishra (2016) compared the proximate composi- tion of whole wheat flours and wheat flours made from five wheat cultivars (Advance, Prevail, Select, Brick and Forefront). The results for dietary fiber levels were simi- lar to those found by Bressiani et al. (2016), where they were between 12.45 % and 15.95 % in the study done on the effect of particle size on the damaged starch content, proximate composition, gluten content, phenolic content and free sulfhydryl groups of fine whole wheat flour, me- dium whole wheat flour and coarse whole wheat flour from BRS Guabiju, wheat variety. There was a significant difference (p ≤ 0.05) of the ash and dietary fiber contents among whole wheat flours from all wheat varieties. The ash content may have differed due to mineral content which varied according to wheat cultivar (Bressiani et al.,2016) and the amount of minerals in flour which in- creases with extraction rate (Scade, 1975). The extraction yield may have influenced the levels of ash and dietary fiber of whole wheat flour because generally when ex- traction yield is high, the ash and dietary fiber contents are high as well. It is explained by the fact that the bran portion which is the main source of ash and dietary fiber could be highly concentrated in flour when there was a high extraction yield. Therefore, it could be the reason why the whole wheat flour from Gihundo variety had the highest ash and dietary fiber contents, while the whole wheat flour from Reberaho variety had the lowest ash and dietary fiber contents. The high ash content of the flour indicates that whole grain flour could be an impor- tant source of minerals. Increased dietary fibre content of the flour has several health benefits; it aids in the diges- Wheat variety grains Grain hardness (g) Extraction yield (%)* Moisture content (%) Total protein content (% dmb) Ash content (% dmb) Total fat content (% dmb) Total carbohy- drate content (% dmb) Total dietary fiber content (% dmb) Gihundo 3.03a ± 0.10 99.20c±0.12 11.73b±0.25 10.85a± 0.15 1.47c± 0.31 1.61c± 1.11 74.34a± 1.19 15.97c± 0.99 Kibatsi 3.36b± 0.01 97.70b± 0.04 11.56a± 0.02 10.45a± 1.03 1.31b± 0.98 1.76d±0.23 74.92b±1.56 14.97b± 0.22 Nyaruka 3.86c± 0.02 96.20a± 0.10 12.07c± 0.10 10.00a± 0.61 1.38b± 0.93 1.46b± 0.01 75.09c± 0.99 15.01bc± 0.11 Reberaho 3.82c± 0.21 96.50a± 0.13 12.09c± 0.01 10.30a± 0.95 0.98a± 0.22 1.38a± 2.09 75.28d± 0.19 12.44a± 0.04 Table 1: Effect of wheat variety on grain hardness, extraction yield and on the proximate composition of whole wheat flours Values are means ±SD of 3 replications. Treatment means followed by different letters in the same column are significantly different at p ≤ 0.05. dmb: Dry mass basis. *Percentage of all bran and germ mixed with the flour from the mass of grains milled. Acta agriculturae Slovenica, 117/1 – 2021 5 Effect of selected Rwandan wheat varieties on the physicochemical characteristics of whole wheat flour tion of the bread in the colon and reduces constipation often associated with bread produced from white wheat flour (Jideani, 2009). Dietary fibre plays a significant role in the prevention of several diseases such as cardiovascu- lar diseases, diverticulosis, constipation, irritable colon, cancer and diabetes (Slavin, 2005). The minimum and maximum total fat contents were 1.38 % and 1.76 %, respectively. Fat content differed sig- nificantly (p ≤ 0.05) from one type of whole wheat flour to another one. Flour from Kibatsi had the highest fat content, while flour from Reberaho wheat variety had the lowest fat content. Wheat grains with a high amount of germ could have given whole wheat flours with a high amount of fat (Mishra, 2016). Fat plays a significant role in the shelf life of food products and as such relatively high fat content could be undesirable in food products. This is because fat can promote rancidity in foods, lead- ing to development of unpleasant and odorous com- pounds. Bread loaf volume can increase with free polar fat contents that are naturally present in a particular cul- tivar, mostly when utilized samples are from pure wheat breeding lines (Chung et al.,1982). In the study of inves- tigating the relationships between textural qualities of noodles and flour lipids, Qiyu and Siyuan (2009) found that as the free lipid content of the flour was increased, hardness of noodles linearly increased, reaching a maxi- mum at a level of 1.84 g 100 g-1 flour, thereafter falling to a low value. The same author reported that cohesiveness of noodles was significantly (p ≤ 0.05) decreased due to removal of free lipid while the highest cohesiveness value was obtained at a free lipid content of 1.24 g 100 g-1 flour. A higher free lipid content in flour would reduce cohe- siveness of noodles. According to these findings, whole wheat breads made from the analysed wheat varieties would be harder and less cohesive. Total carbohydrate content ranged between 74.34 % and 75.28 %. There was a significant difference (p ≤ 0.05) in carbohydrate contents among whole wheat flours from all wheat varieties, where whole wheat flours from Re- beraho and Gihundo varieties had the highest and the lowest carbohydrate contents, respectively. B-vitamins and minerals from the wheat bran and germ of the whole wheat breads avail carbohydrate for proper assimilation in humans (Connection, 2017). Whole wheat flour from Reberaho wheat variety could be a good source of me- tabolisable energy and could assist also in fat metabolism because of its high carbohydrate content (Ifie, 2011). 3.2 EFFECT OF WHEAT VARIETY ON THE PHYSICAL PROPERTIES OF WHOLE WHEAT FLOURS The range for oil absorption capacity was from 1.24 g g-1 to 1.45 g g-1. These results were in line with the ones obtained by Khalid et al. (2017). Oil absorption capac- ity changed with the types of whole wheat flour, where Gihundo and Nyaruka varieties gave whole wheat flours with the highest and the lowest oil absorption capacity, respectively. The high amount of dietary fiber in Gihun- do wheat flour (Table 1) might have been responsible for the high oil absorption capacity of the flour (Chou and Huang, 2003). The high oil absorption capacity makes the flour suitable in facilitating enhancement in flavor and mouthfeel when used in food preparations (Kaushal and and Kumar, 2012). The results for bulk density of the whole wheat flours were between 0.32 g ml-1 and 0.56 g ml-1. These results were near to those found by Offia et al. (2015). The bulk density of whole wheat flours from Kibatsi, Nyaruka and Reberaho did not differ significantly (p > 0.05). Flour from Gihundo wheat variety could be dense in comparison to flours from other wheat varieties, and it meant that Gihundo grain endosperm could be filled out better than the endosperm from the other variety grains. The high bulk density of flours suggests their suit- ability such as thickener in food products and for use in food preparations since it helps to reduce paste thickness which is an important factor in convalescent and child feeding (Kaushal and Kumar, 2012). In contrast, low bulk density would be an advantage in the formulation of complementary foods (Akpata and Akubor, 1999). The results for least gelation concentration were in the range of 40.36 % and 70.23 %. Whole wheat flours Wheat variety grains Oil absorption capacity (g g-1) Bulk density (g ml-1) Least gelation concentration (%) Gihundo 1.45d± 0.01 0.56b± 0.13 70.23d± 0.03 Kibatsi 1.31c± 0.10 0.35a± 0.09 56.23c± 0.02 Nyaruka 1.24a± 0.12 0.34a± 0.01 40.36a± 0.15 Reberaho 1.28b± 0.01 0.32a± 0.02 47.42b± 0.21 Table 2: Effect of wheat variety on the physical properties of whole wheat flours Values are means ± SD of 3 replications. Treatment means followed by different letters in the same column are significantly different at p ≤ 0.05. Acta agriculturae Slovenica, 117/1 – 20216 B. RWUBATSE et al. from Gihundo and Nyaruka varieties were the highest and the lowest in least gelation concentration, respec- tively. The lower the least gelation concentration, the better is the gelating ability of the protein ingredient and the swelling ability of the flour is enhanced (Kaush- al and Kumar, 2012). The low least gelation concentra- tion of wheat flour may be an asset as an additive to other gel forming materials in food products. 3.3 EFFECT OF WHEAT VARIETY ON THE FA- RINOGRAPH TEST PROPERTIES OF WHOLE WHEAT FLOURS The range for water absorption capacity was be- tween 80  % and 89  %. The range for those results in- cluded 0.85 g g-1 (85 %) found by Khalid et al. (2017) for wheat grains var. WH-1021 in the study on physi- co-chemical properties of native and irradiated whole wheat flour. Gihundo variety followed by Kibatsi variety gave whole wheat flours with high values of water ab- sorption capacity in comparison to Nyaruka and Reber- aho varieties. The last two were not significantly differ- ent (p > 0.05) in water absorption capacity. Maghirang et al. (2006) reported that an increase or decrease in wa- ter absorption capacity may be explained by differences in flour extraction yield and presumably higher starch damage content as well, after finding that some wheat varieties with differences in grain protein content had similar water absorption requirement. Similarly, to this report, the total protein contents of whole wheat flours from all wheat varieties (Table 1) were not significantly different (p≤0.05), but the flours with high extraction yield (Table 1) showed high water absorption capacity. Sluimer (2005) also showed that water absorption in- creased with increasing extraction rate. It could be the reason why the high extraction yield (Table 1) impacted the whole wheat flours from Gihundo and Kibatsi va- rieties to require a high amount of water to center the farinograph curve on the 500-Brabender Unit (BU) line compared to flours from other remaining wheat varie- ties. The small particle size of bran and high amount of fiber in the flour might have been responsible for high water absorption capacity as reported by Chou and Huang (2003). Water absorption is a key parameter in the purchase of flour for breadmaking (Webb and Ow- ens, 2003).The high water absorption capacity of the flours may assure product cohesiveness (Houson and Ayenor, 2002). The time for dough development was between 6.33 min and 7.62 min and for stability, it was between 10.72 min and 11.65 min. Gihundo variety followed by Kibatsi variety produced whole wheat flours with long development time in comparison to Nyaruka and Re- beraho varieties. The last two were not significantly different (p > 0.05) in development time. As the dough development time is the time taken from when water is added up to when the dough reaches maximum consist- ency, the results indicated that whole wheat flours from Nyaruka and Reberaho varieties took short time for op- timum mixing compared to flours from Gihundo and Kibatsi varieties. The stability time shows the maximum consistency for a dough and is a good indication of its strength. Flours from Nyaruka and Gihundo varieties took the longest and the shortest time, respectively, for dough stability. Thus, the dough obtained from Nyaruka wheat variety lasted long with high consistency, while similar consistency for the dough from Gihundo wheat variety lasted a short time. The results for dough de- velopment time (DDT) and dough stability time ranged in the results found by Bressiani et al. (2016). In the time evaluation of the effect of particle size on mixture properties, extensional properties and paste properties of refined flour and whole grain wheat flour from BRS Guabiju, wheat variety, those authors reported between 7.60 min and 12.93 min for DDT, between 11.6 min and 12.8 min for dough stability for fine whole grain wheat flour, medium whole grain wheat flour and coarse whole grain wheat flour. For the flours which showed low dough development and stability time, they could have been affected by weakened gluten network with fiber-rich bran particles (Bae et al., 2014). Wheat variety Water absorption capacity (%) Dough development time (min) Dough stability time (min) Mixing tolerance index (BU) Gihundo 89.00c± 0.09 7.62c± 0.07 10.72a± 0.03 22.88d± 0.11 Kibatsi 85.00b± 0.03 6.73b± 0.12 12.09c± 0.02 24.73c± 0.01 Nyaruka 80.00a± 0.10 6.33a± 0.01 13.12d± 0.10 20.17a± 0.25 Reberaho 81.00a± 0.15 6.79a± 0.04 11.65b± 0.12 21.02b± 0.02 Table 3: Effect of wheat variety on the farinograph test properties of whole wheat flours Values are means ± SD of 3 replications. Treatment means followed by different letters in the same column are significantly different at p ≤ 0.05. Acta agriculturae Slovenica, 117/1 – 2021 7 Effect of selected Rwandan wheat varieties on the physicochemical characteristics of whole wheat flour Mixing tolerance index (MTI) was from 20.17 BU to 22.88 BU. Mixing tolerance index highly differed (p ≤ 0.05) in whole wheat flours obtained from all wheat varieties. Whole wheat flour from Gihundo and Kibatsi varieties showed the highest and the lowest values of mixing tolerance index, respectively. This indicated that dough from Gihundo variety whole wheat flour was hard and dough from Kibatsi variety whole wheat was soft during mixing. The hardness of dough mixing could contribute to firm product texture. Mishra (2016) who compared the farinograph parameters among ground whole wheat flours and flour incorporated with treated bran, found the results in same range, where MTI was between 18.13 BU for ground whole wheat flours from five wheat cultivars (Advance, Prevail, Select, Brick and Forefront). 3.4 EFFECT OF WHEAT VARIETY ON THE COLOR OF WHOLE WHEAT FLOURS The color ranged between 80.33 and 84.67, 1.4 and 2.30 and 10.14 and 10.67 for L*, a* and b* values, respectively. Gihundo variety followed by Kibatsi va- riety produced whole wheat flours with high L*values in comparison to Nyaruka and Reberaho varieties. The last two were not significantly different (p > 0.05) in L* values. The results indicated that whole wheat flour from Gihundo variety was brighter than the remaining flours. The whole wheat grain with high flour extrac- tion yield produces a fine size flour containing high amounts of bran and germ, which later can make the flour to be less bright (Maghirang et al., 2006). This as- sumption was not the case for whole wheat flours from Gihundo and Kibatsi varieties which demonstrated the highest yield extraction (Table 1). Thereby, the flour brightness was considered to mainly be caused by the wheat cultivar (Maghirang et al., 2006). Redness (a*) and yellowness (b*) values were not significantly differ- ent in whole wheat flours from all wheat varieties (p > 0.05). Anthocyanins and carotenoids, located mainly in the germ and bran, may have contributed to the colors of the whole wheat flour (Schwinn, 2004; Rao and Rao, 2007). Anthocyanins are phenolic compounds (flavo- noids), responsible for most blue to blue-black, and red to purple colors of diverse plant organs (Schwinn, 2004). Meanwhile, carotenoids are responsible for the yellow, orange and red colors in various plants (Rao and Rao, 2007). The beneficial effect of wheat bran against colon cancer is attributed to the presence of a high con- centration of polyphenolic compounds, which are likely released into the colon as a result of bacterial fermenta- tion (Monica and Whole Grain Connection, 2017). The obtained L*, a* and b*values were very close to those found by Mishra (2016). The author obtained 82.08, 1.71 and 10.64 for L*, a* and b* average values, respec- tively, for whole grain flour from five hard red spring wheat cultivars. 4 CONCLUSION The results showed that the wheat varieties affected the extraction yield and other qualities of their whole wheat grain flours. The proximate composition was sig- nificantly different among whole wheat flours from the wheat grain varieties, except protein content. Gihundo and Nyaruka wheat grain varieties have the highest and the lowest extraction yields, respectively. The farino- graph test parameters of the doughs were impacted by wheat varieties. The whole wheat flour brightness was considered to mainly be caused by the wheat cultivar. Beyond the quantity and economic reasons that most of bakeries qualify wheat grains by their extraction yields, it is also important to consider other qualities such fa- rinograph (dough) test characteristics, color and proxi- mate composition to select grains or flour from these wheat varieties newly introduced in Rwanda based on the individual cultivar because their derivative products could have a more desired quality for competitive mar- kets. For their nutritive value and health benefits, whole wheat flours have been gaining an increase in demand and utilization in different food products for human consumption worldwide. Wheat variety grains Extraction yield (%) L* a* b* Gihundo 99.20c±1.40 84.67c±1.04 1.50a±3.01 10.67a± 3.07 Kibatsi 97.70b±0.22 82.67b± 2.08 1.90ab±0.27 10.23a± 0.54 Nyaruka 96.20a±1.01 80.33a±1.04 2.10b± 1.04 10.47a±1.50 Reberaho 96.50a±1.82 80.52a± 3.03 2.30b±0.58 10.14a±1.04 Table 4: Effect of wheat variety on the color of whole wheat flours Values are means ± SD of 3 replications. Treatment means followed by different letters in the same column are significantly different at p ≤ 0.05. % Percentage of all bran and germ mixed with the flour from the weight of grains milled. Acta agriculturae Slovenica, 117/1 – 20218 B. 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Boca Raton, FL. https://doi. org/10.1533/9781855737129.1.200 Acta agriculturae Slovenica, 117/1, 1–9, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1297 Original research article / izvirni znanstveni članek Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria Chris Adegoke FAYOSE 1 and Morakinyo Abiodun Bamidele FAKOREDE 1, 2 Received October 05, 2019; accepted January 18, 2021. Delo je prispelo 05. oktobra 2019, sprejeto 18. januarja 2021. 1 Obafemi Awolowo University,Department of Crop Production and Protection, Ile-Ife 2 Corresponding author, e-mail: dele_fakorede@yahoo.com Planting date effects on maize (Zea mays L.) growth and de- velopment in the rainforest of southwestern Nigeria Abstract: This study was conducted to determine the op- timum planting date for maize (Zea mays L.) to cope with the negative impacts of climate change in the marginal rainforest agro-ecology as typified at Ile-Ife, SW Nigeria. Five maize vari- eties were planted weekly, in 3-replicate randomized complete block design experiments at the Obafemi Awolowo University Teaching and Research Farm, throughout the 2016 and 2017 cropping seasons. The varieties were monitored for seedling and adult plant traits including grain yield with its components. Statistical analysis showed significant effect of planting dates (DOP) on all traits. The first few DOPs in March and April had the highest grain yield which reduced with delayed planting till June and increased again mid July/August before finally drop- ping off thereafter. The higher yield in the earlier dates each year, was due to early flowering and taller plants with higher ear placement. Planting after the first few rains in March/April was the optimum for the first cropping season, and late July to mid-August was best for the second cropping season in this agroclimatic zone. Planting beyond these periods results in poor grain yield and pre-disposes the crop to terminal drought, which could result in complete crop failure. Key words: agronomy; climate change impact and adap- tation; drought; Zea mays L. Vplivi datuma setve na rast in razvoj koruze (Zea mays L.) v območjih deževnega gozda jugozahodne Nigerije Izvleček: Raziskava je je bila izvedena za določitev opti- malnih datumov setve koruze (Zea mays L.) za spoprijemanje z negativnimi posledicami podnebnih sprememb na mejnih območjih deževnega gozda v Ile-Ife, jugozahodna Nigerija. Pet sort koruze je bilo posejanih tedensko v popolnem naključnem bločnem poskusu s tremi ponovitvami na raziskovalnem pose- tvu Obafemi Awolowo University Teaching, v rastnih sezonah 2016 in 2017. Pri vseh sortah so bile spremljane lastnosti rastlin od sejank do odraslih rastlin, vključno s pridelkom in njego- vimi komponentami. Statistična analiza je pokazala značilen vpliv datuma setve (DOP) pri vseh lastnostih. Prvi datumi setve v marcu in aprilu so imeli največji pridelek zrnja, ki se je nato zmanjševal do setve v juniju in spet povečeval do setve v sredi- ni julija in avgusta, nakar je končno upadel. Večji pridelek pri zgodnejših datumih setve je bil vsako leto povezan z zgodnej- šim cvetenjem, višjimi rastlinami in z višjim položajem storžev. Setev po prvih deževjih v marcu in aprilu je bila optimalna za prvi del rastne sezone, setev koncem julija do sredine avgusta pa je bila najboljša za drugi del rastne sezone v teh agroekolo- ških razmerah. Setve izven teh obdobij do dale majhen pridelek zrnja zaradi izpostavljenosti suši, kar bi lahko vodilo k popolni izgubi letine. Ključne besede: agronomija; vpliv podnebnih sprememb in prilagoditve; suša; Zea mays L. Acta agriculturae Slovenica, 117/1 – 20212 C. A. FAYOSE and M. A. B. FAKOREDE 1 INTRODUCTION Maize (Zea mays L.) is a popular crop in sub-Sa- haran Africa (SSA) and its popularity in the region has continued to grow because it provides a cheap source of calories for the relatively poor population, who process it to different food forms in addition to consuming it boiled or roasted. A good number of industries, including the livestock industries also depend on maize for the produc- tion of feed and other products as a result of which the demand for the grains remains high (Talabi et al., 2017). Unfortunately, in Nigeria today, there is a considerable reduction in the area of land available for agricultural production due to increased urbanization. There is also continous decline in soil fertility, resulting from persis- tent cultivation of a piece of land over several or many years. Furthermore, there is the impact of climate change which shows as poor condtions of climate, including drought, and a reduction in the effective growing season due to delayed onset of rainfall followed by early rainfall recession (Fakorede and Akinyemiju, 2003). These are key factors limiting maize production from keeping up with the high demands for its grains. In Nigeria as in many other countries of the world, maize has received relatively large research attention from agronomists, plant breeders and other crop scien- tists over the years. Genetic improvement has been made on many important aspects of the crop, including the de- velopment of high yielding, disease resistant, drought tol- erant and nutritionally fortified varieties (Badu-Apraku and Fakorede, 2017). However, yield could be further en- hanced with improved cultural practices, including op- timum planting dates. Studies have shown that planting maize outside its optimum dates of planting results in re- duced yield (Fakorede, 1985). Investigations had started since the late 1970s at the Obafemi Awolowo University (formerly University of Ife), Ile-Ife to determine the opti- mum planting dates for maize in the marginal rainforest agro-ecology of Ile-Ife in Southwestern Nigeria. Fakore- de (1985) found at Ile-Ife that yield decreased by 30, 38 and 34 kg ha–1 for each day by which sowing was delayed after the first planting in March of 1978, 1980 and 1981 respectively. He suggested early planting of maize with the first few rains in the year to ensure optimum yield. Oluwaranti et al. (2008) evaluated the yield performance of different maturity groups of maize varieties at different planting dates (DOP) at the same location and found that grain yield and yield components decreased with delayed planting in the late season. Planting date studies in the savanna regions of Nigeria have shown similar results. In the Sudan savanna zone, for example, Jibrin et al. (2012) found late June to early July as the optimum planting window for maize; grain yield decreases drastically with delayed planting beyond this period. Rainfall is the primary climatic factor that deter- mines the timing of most agronomic practices, especially planting operations in Nigeria. False start of rainfall and unpredictable frequent occurrence of short duration drought during the growing season, both of which have become more severe over time, are now features of the rainforest agro-climatic zone of SW Nigeria typified by Ile-Ife, a location at 7o 28’ N, 4o 33’ E and 244 m asl in the zone. Such weather anomalies have been attributed to climate change (Fakorede and Akinyemiju, 2003). Re- duced seedling emergence and low vigor, culminating in missing stands, are usually associated with the weather anomalies that characterize early planted maize in the zone. Despite these negative impacts, early planted maize under research as well as farmer practices produce higher yields than plantings delayed till when the environments are seemingly more favorable. Based on this observation, Fakorede (1985) hypothesized that cultivars for early planting would be more productive if they emerged rap- idly, with high seedling and vegetative vigor under rela- tively hot, dry conditions. Furthermore, most of the DOP studies thus far in Nigeria involved open-pollinated vari- eties (OPVs), although Fakorede’s (1985) study included some varietal hybrids, a type of hybrid that has never been commercialized in Nigeria. The varieties were also not screened for drought and or heat tolerance. However, drought and or heat tolerant OPVs belonging to different maturity groups (extra early, early, intermediate, late), as well as conventional types of hybrid (single-cross, dou- ble-cross, 3-way cross hybrids) also of different maturity groups, are now available and are cultivated by farmers in Nigeria. In addition, most DOP studies conducted so far in the rainforest agroclimatic zone of Nigeria involved relatively few dates within each planting season. The study by Oluwaranti et al. (2008) which was conducted during a 4-year period, showed contrasting trends in the early season, a clear indication of the need for more in- vestigations. Studies conducted in advanced countries, such as that by Jong et al. (1982) inter alia suggested that the larger the sampling size of the environment, the more reliable the results obtained for purposes of prediction and recommendation of agronomic practices. The primary objective of the present study, there- fore, was to further investigate the trends in growth, grain yield and yield components of maize planted at weekly interval, starting with the first few rains in March till the recession of rainfall in November for a 2-year pe- riod. A secondary objective was to investigate the pres- ence of genotype x DOP interaction with the goal of rec- ommending varietal types adapted to specific planting periods in the agroclimatic zone. Acta agriculturae Slovenica, 117/1 – 2021 3 Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria 2 MATERIALS AND METHODS The study was carried out at the Teaching and Re- search Farm of Obafemi Awolowo University, Ile-Ife (OAU T&R Farm) in years 2016 and 2017. In each ex- periment, five maize varieties (four OPVs and one sin- gle-cross hybrid), well adapted to the tropical rainforest environments, were planted in 3-replicate randomized complete block design. The OPVs included ‘White DT STR SYN1 – TZL Comp. 1–W’, ‘TZL Comp. 4 DT F2’, ‘TZL Comp. 1 C6/DT – SYN – 1 – W’ all of which were drought tolerant (DT) and of intermediate/late maturity, ‘ACR 94 TZE Comp 5 C3’ (early maturing), and ‘Oba Su- per 1’, an intermediate-late single-cross hybrid obtained from Premier Seeds, Zaria. The four OPVs were obtained from the IITA Maize Improvement Program, and all five varieties are white-grained, high yielding and have been released for commercial production in Nigeria and sev- eral other West and Central African (WCA) countries. The experiments were planted weekly from March to November each year. Each planting date (DOP) repre- sented the individual environment. However, there were some weeks when planting could not be done due to some logistic problems, but 56 plantings (28 each year) were effected, out of which 54 (data unavailable to calculate emergence parameters for the first DOP in March each year due to rainfall anomalies) were analysed for seedling emergence and vigour. Only 42 DOPs (20 in 2016 and 22 in 2017) attained maturity and were analyzed for flower- ing, plant and ear heights, and grain yield. Each plot con- tained six or four rows which were 5 m long and 0.75 m apart; within row spacing was 0.5 m resulting in plot size of about 15 m2 and 22.5 m2 for the four and six-row plots. Prior to planting, the experimental land was ploughed and harrowed and seeds were treated with Apron* which contains thiamethoxam, mefenoxam (metalaxyl-M) and difenoconazole, to control damage by soil-borne dis- eases and insect pests. Three seeds were planted per hill and thinning was done at 9 days after planting (DAP) to two plants per stand giving an estimated plant popula- tion density of 53, 333 plants ha-1. Fertilizer was applied immediately after thinning at the rate of 60 kg ha-1 each for N, P2O5 and K2O. Primextra, which contains atrazine (2-chloro-4- (ethyl amino)-6-isopropylamino-s-triazine) and alachlor (N-(methyl-2-methoxy-ethyl)-2-ethyl- 8-methyl-chloroacetanilide) as active ingredients was ap- plied as post-planting, maize pre-emergence herbicide at the rate of 5 l ha-1. Further weed control was done using paraquat (N,Nʹ-dimethyl-4,4ʹ-bipyridinium dichloride), carefully applied as a post-emergence, non-selective and contact herbicide at the rate of 3.0 l ha-1. Emergence counts were made daily from five to nine days after planting (DAP); from which emergence percentage (E %) and emergence index (EI) were com- puted as follows (Fakorede and Agbana (1983): The dates of the appearance of the first flower (tas- sel) and number of days to 50 % tasselling, anthesis (pol- len shed) and incipient silk extrusion were recorded for each plot in both years. Plant and ear heights (PHT and EHT) were obtained by measuring the distance from the soil surface to the first branch at the base of the tassel, and the node bearing the top ear for 10 random plants in each plot and their means taken as PHT and EHT per plot, respectively. Data were collected on grain yield and yield components (ear number and mass, ear length and ker- nel moisture content). The grain yield data were adjusted to 15 % moisture content. Variance analysis was done on all data using PROC GLM of Statistical Analysis System (SAS, 2000). The linear additive model for the ANOVA was: Yijk = µ + αi + βj(i) + Σk + αΣ(ik) + Σijk, in which Yijk is the observed measurement of the kth genotype grown in the jth rep under the ith environment; µ is the grand mean; αi is the main effect of the i th environment, i = 1,2,….., 42 or 54; βj(i) is the effect of the j th replication nested within the ith environment, j = 1,2,3; Σk is the effect of the k th genotype, k = 1,2,….5; α Σ(ik) is the first order interaction of the ith environment with the kth genotype, and Σijk is the random error (residual) term. Furthermore, correlation and regression analyses were done for all data to deter- mine the trends in response of maize to planting dates. The measured traits (emergence, flowering, PHT, EHT and yield) were regressed on DOP in linear model: Y = a + bX and polynomial model: Y = a + b1X1 +....bnXn, Y and X are the measured traits and DOP, respectively; a and b are the intercept and slope/coefficient of the re- gression, respectively; n is the order of the polynomial (quadratic, cubic, quartic). Pearson correlation analysis was done where the coefficient. 3 RESULTS Results from the variance analysis revealed highly significant DOP effect for all traits (Table 1). Emergence, adult plant traits and grain yield along with its compo- nents were highly significant (p ≤ 0.01) for DOP and other sources of variation.Fairly high emergence for the Acta agriculturae Slovenica, 117/1 – 20214 C. A. FAYOSE and M. A. B. FAKOREDE Ta bl e 1 : M ea n sq ua re s f ro m th e A N O VA o f s ee dl in g vi go ur tr ai ts , y ie ld an d ot he r a du lt pl an t t ra its o f fi ve m ai ze v ar ie tie s e va lu at ed in 5 4 or 4 2 D O P at th e O ba fe m i A w ol ow o U ni - ve rs ity T ea ch in g an d Re se ar ch F ar m , I le -I fe in 2 01 6 an d 20 17 cr op pi ng se as on s. So ur ce D F E% @ 9 D A P EI (d ay s) A du lt pl an t t ra its O th er y ie ld co m po ne nt s D F Si lk in g A SI PH T EH T Yi el d (t/ ha )D F EN D F EL ED K RN D O P† (D ) 53 32 56 .5 8* * 3. 55 ** 41 23 4. 38 ** 47 .1 8* * 51 05 .8 7* * 20 83 .0 48 ** 7. 61 ** 39 41 1. 35 ** 40 23 .0 8* * 50 .7 9* * 3. 26 ** Re p w ith in D 10 8 26 8. 92 ** 0. 57 ** 84 15 .6 4* * 5. 70 ** 37 9. 56 ** 17 1. 18 9* * 0. 49 ** 80 32 .5 2* * 82 1. 98 3. 65 0. 74 Va rie ty (V ) 4 14 33 2. 64 ** 7. 33 ** 4 87 8. 69 ** 83 .0 9* * 66 72 .6 2* * 18 53 .7 02 ** 4. 76 ** 4 57 2. 83 ** 4 23 .4 2* * 4. 31 10 .0 4* * D x V 21 2 60 7. 57 ** 0. 37 ** 16 4 6. 67 * 3. 36 ** 22 7. 88 14 2. 98 ** 0. 45 ** 15 6 65 .6 1* * 16 0 2. 65 ** 2. 39 0. 77 Er ro r 43 3 69 .5 9 0. 21 33 6 5. 01 2. 22 18 7. 99 91 .2 0 0. 22 32 0 21 .4 7 32 8 1. 53 2. 82 0. 64 To ta l 80 9 51 6. 49 0. 56 62 9 27 .3 7 6. 43 58 5. 77 25 6. 42 3 0. 83 59 9 63 .5 1 61 4 3. 43 5. 96 0. 92 C V, % 11 .9 7 6. 90 3. 42 32 .4 5 9. 3 16 .1 1 30 .6 7 23 .0 6 10 .0 8 36 .4 3 6. 01 *,* * F - st at ist ic al ly si gn ifi ca nt at 5  % a nd 1  % le ve l o f p ro ba bi lit y, re sp ec tiv el y. † - D O P = da te o f p la nt in g, E % @ 9 D A P = em er ge nc e pe rc en t a t 9 D A P, E I = e m er ge nc e in de x in d ay s, sil ki ng is in d ay s, A SI = a nt he sis -s ilk in g in te rv al in d ay s, PH T an d EH T ar e pl an t a nd e ar h ei gh ts in cm , E N = n um be r o f e ar s p er p lo t, EL = e ar le ng th (c m ), ED = e ar d ia m et er (c m ), K RN = n um be r o f k er ne l r ow s p er e ar . Acta agriculturae Slovenica, 117/1 – 2021 5 Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria first DOPs in March each year was followed by a period of low emergence in late March/April (Figure 1). There- after emergence increased steadily on the average from May and was fairly uniform mid-season when rainfall is expected to be high, until around October when it de- creased steadily, even though the R2 value (34  %) was quite low despite the high R2 value (81 %) observed in 2016. Emergence was generally higher and faster in 2016 than 2017 with an opposite trend between the years for EI (Figures 1 and 2). There was really no definite pattern for silking with respect to DOP over the two years, except for the earli- ness in silking observed in the first couple of environ- ments in 2016. Similar trends occurred for tasseling and anthesis. The interval between anthesis and silking, ASI, showed a similar trend to the other flowering traits, but for a wider margin where differences occurred between the two years (Figure 3). Plant and ear heights generally showed a trend where they appeared to be fairly uniform mid-seasons, although planting very early or at the latter part of the season produced contrasting results. However, plants were taller and ears placed higher in most dates in 2016 (Figure 4). Grain yield was highest for the first few DOPs in March and April (Figures 5 and 6) even though other en- vironments planted later in March and April each year had some of the poorest yield performances compared to the other months – which, in turn, showed no defi- nite trends with DOPs in 2016 scoring higher yield on the average (1.65 ± 0.78 t ha-1) than DOPs in 2017 (1.39 ± 0.68 t ha-1). Combined analysis of both years confirmed higher average yield in March and a steady decline in Figure 1: Mean values by months for emergence percentage (%) at 9 DAP of five maize varieties evaluated in 54 environments at the OAU T&R Farm in 2016 and 2017 seasons. Figure 2: Mean values for the emergence index (in days) of five maize varieties evaluated in 54 environments at the OAU T&R Farm in 2016 and 2017 seasons. Acta agriculturae Slovenica, 117/1 – 20216 C. A. FAYOSE and M. A. B. FAKOREDE Figure 3: Mean values for the days from anthesis to silking of five maize varieties evaluated in 42 environments at the OAU T&R Farm in 2016 and 2017 seasons. Figure 4: Mean values for ear height (cm) of five maize varieties evaluated in 42 environments at the OAU T & R Farm in 2016 and 2017 seasons. Figure 5: Mean grain yield (t ha-1) of five maize varieties evaluated over 20 DOPs at the OAU T&R Farm in 2016 cropping season. Acta agriculturae Slovenica, 117/1 – 2021 7 Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria subsequent months up until July when yield picked up declining again in September plantings (Figure 7). Figures 5 and 6 indicate a somewhat linear decrease in yield with delays in planting, although the extreme- ly low R2 values (2016 = 2 %, 2017 = 17 %) especially for 2016 indicate that the linear yield response to DOP is almost certainly a decoy. The R2 values increased sig- nificantly (2016 = 72 %, 2017 = 62 %) when cubic and especially quartic polynomials were plotted for the yield response to DOP with the high grain yield of the early DOPs in March of 2016 decreasing to the lowest level in April/May, and later increasing very slightly mid-season, and was somewhat uniform thereafter. In 2017, yield was highest in March/April, lowest mid-season, increased slighlty in late July/August before hitting another low in September. The R2 from the regression analysis of both years combined was at 45  % when a cubic polynomial was plotted for yield in response to DOPs but the linear response of yield to DOPs was very low at R2 = 5 %. There were no correlations of seedling vigour traits with flowering and other adult plant traits including grain yield despite the fact that E %, PHT and EHT were all higher in 2016 than 2017 (Table 2). However, earlier flowering and shorter ASI as well as taller plants and higher ear placement increased yield significantly. 4 DISCUSSION Results of this study decidedly reinforces the posi- tion that maize growth and productivity change in re- sponse to variation in planting date, as indicated by re- sults of earlier studies (Fakorede, 1985; Oluwaranti et al., 2008) as well as common observations in farmers’ fields. The present study, therefore, provides an additional sci- Figure 6: Mean grain yield (t ha-1) of five maize varieties evaluated over 22 DOPs at the OAU T&R Farm in 2017 cropping seasons. Figure 7: Mean grain yield (t ha-1) by DOPs of five maize varieties evaluated over 42 different planting dates at the OAU T&R Farm in 2016 and 2017. Acta agriculturae Slovenica, 117/1 – 20218 C. A. FAYOSE and M. A. B. FAKOREDE entific proof the optimum DOP to maximize maize pro- duction in the rainforest agro-climatic zone of SW Nige- ria is very early in the growing season. Highly significant mean squares were associated with the DOP source of variation for all traits at different growth stages, with the DOP sum of squares accounting for the largest propor- tion of the total sum of squares in each case, ranging from about 41 % for E % and EI to 59 % for grain yield. Envi- ronmental conditions vary widely from day to day and from one environment to the other due to temporal and spatial variations in climatic factors such as temperature, precipitation, solar radiation, evapotranspiration, relative humidity, and edaphic factors relating to soil conditions. In this study, climatic factors varied widely among the environments (Fayose and Fakorede, 2021), probably ag- gravated by the impact of climate change which had been reported as affecting maize growth and production at the location (Fakorede and Akinyemiju, 2003). For instance, the “August break” normally distinct between early and late seasons at Ile-Ife, was not experienced in 2016 and there was a delay in the onset of rainfall in 2017 beyond what had previously been experienced at the location. These probably resulted in the large differences observed among the DOPs for most traits assayed in this study. For that reason, analysis of the data for the individual years separately was justified (Fig. 1). Emergence is expected to be lower early in the sea- son (because of low soil moisture) and later increase to an optimum mid-season before declining. This trend was more noticeable in 2016 as emergence in 2017 did not follow a discernible pattern. Also, flowering, PHT and EHT showed no particular trend from one DOP to an- other. Grain yield on the other hand, was significantly higher with the first few DOPs each year which might seem to support the results of the study by Fakorede (1985) who found that early planting ensured optimum maize grain yield and steady decrease in yield occurred with delays in planting. This makes sense because there is usually low cloud cover during the early DOPs thereby allowing optimum solar radiation, a necessary condition for maize optimum yield, to reach the surface. However, there were dates in March and April that performed very poorly in comparison to later DOPs which might seem to be contradictory to the findings of the aforementioned study. This, could be attributable to the relatively poor edaphic conditions probably resulting in inadvertently inadequate agronomic practices and the attack by army worm, Spodoptera frugiperda (Smith, 1797), which was a problem at a point during the study. Oluwaranti et al. (2008) observed similar results to those of Fakorede (1985) in the late season at the same location. Analysis of the yield in the present study may have indicated no discernible trend in yield performance for both years under consideration. Beyond the early DOPs, however, a polynomial response (cubic) of yield to DOP was observed in which yield was highest very early in the season, dropping to its lowest value around mid-season before rising ever so slightly later and flat- tening out or somewhat decreasing afterwards. Further analysis of DOPs combined for both years showed a clear trend where average yield decreased steadily in each suc- cessive month till June and picked up in July/August be- fore finally dropping off in September in what is clearly a cubic response of yield to DOPs. Year 2016 had higher emergence, earlier flowering, higher ear placement, and taller plants than 2017. This may be due to the higher amount of rainfall received during the growing season at the OAU T&R Farm in 2016 (> 1000 mm) compared to 2017 (675 mm). According to Fakorede and Agbana (1983) and Fayose and Fakorede (2014), varieties with high E  % and low values for EI and ERI tend to accu- mulate dry matter at a higher rate than slowly emerging EI Days to tassel Days to anthesis Days to silk ASI, days PHT, cm EHT, cm YIELD, t/ha E%_9 -0.43** -0.15 -0.22 -0.12 0.12 0.14 0.22 0.26 EI 0.18 0.28 0.11 -0.23 -0.10 -0.23 -0.10 Days to tassel 0.98** 0.90** 0.18 -0.50** -0.58** -0.40** Days to anthesis 0.90** 0.12 -0.58** -0.67** -0.45** Days to silk 0.55** -0.51** -0.56** -0.54** ASI, days -0.06 0.01 -0.37* PHT, cm 0.92** 0.63** EHT, cm 0.62** Table 2: Correlation coefficients among agronomic traits and grain yield of five maize varieties planted over several environments at OAU T&R Farm, Ile-Ife in 2016 and 2017 cropping seasons. *, ** Significantly different from zero at 0.05 and 0.01 level of probability, respectively. Acta agriculturae Slovenica, 117/1 – 2021 9 Planting date effects on maize (Zea mays L.) growth and development in the rainforest of southwestern Nigeria varieties resulting in vigorous plants, thereby increasing yield directly or indirectly. This perhaps, is one of the rea- sons why yield was also higher in 2016 than 2017. Early flowering, short ASI, and taller plants were indeed associ- ated with increased grain yield in this study even though seedling vigour had no significant relationship with yield. Seedling vigour could therefore be assumed to have influ- enced yield indirectly. There was also significant DOP x Variety interac- tion for yield and other traits monitored in this study except PHT, indicating that the different maize varieties responded differently to the different climatic conditions. This suggests the tendency of certain varieties to perform better when planted in specific DOPs. Such varieties when planted in other DOPs outside their favoured DOP would struggle to reproduce the good performance. Each of the varieties indeed performed differently in one environment relative to others as indicated by the ANOVA. The hybrid, ‘Oba Super 1’ had the poorest average performance across DOPs on the one hand, whearas ‘TZL Comp. 4 DT F2’ and ‘TZL Comp. 1 C6/DT – SYN – 1 – W’ had the best aver- age performances on the other hand. It would seem there- fore that drought tolerant late maturing varieties would have better adaptation to this agro-ecology especially in the early season, which provides a window long enough for the partitioning of enough dry matter for the grain filling stage which, in turn, is extended to ensure higher grain return. However, the perfornance of ‘White DT STR SYN1 – TZL Comp. 1 – W’ was unlike the two varieties despite sharing similar characteristics, which casts a cloud on the earlier hypothesis. Nevertheless, ‘TZL Comp. 1 C6/ DT – SYN – 1 – W’ for its consistency across DOPs would seem the best of the varieties evaluated in this study for planting in the marginal rainforest agro-ecology of Ile- Ife in SW Nigeria. The gxe effect could be deeply investi- gated, however, with a larger sample size and that would give a larger degrees of freedom, allowing more room for comparison, with the goal of recommending the varieties that are best adapted to the rainforest agro-ecologies. 5 CONCLUSIONS In conclusion, despite the prevailing climate change scenarios which has no doubt impacted agricultural ac- tivities especially crop production in the past few decades, this study established that planting maize early in the sea- son ensures optimum grain yield. Yield decreased steadily as long as planting is delayed up till June. For optimum yield, planting should be done with the first few rains in March as long as soil moisture could be maintained either with supplementary irriagation or better soil management and other agronomic practices in case of the occurrence of drought of any duration, which is very probable with the present reality of climate change. Planting should not be delayed beyond early to mid April in the early season. Planting in the late season should be done around late July to mid-August. Planting beyond this period reduces yield and predisposes the plants to terminal drought. 6 REFERENCES Badu-Apraku, B. and Fakorede M.A.B. (2017). Morphology and physiology of maize, p 33-53 In Advances in genetic enhance- ment of early and extra-early maize for sub-Saharan Africa, Springer International Publishing Company,Gewerbestrasse 11, 6330, Chan, Switzerland. https://doi.org/10.1007/978-3- 319-64852-1_3 Fakorede, M.A.B. (1985). Response of maize to planting dates in a tropical rainforest location. Experimental Agriculture, 21, 19-30. Fakorede, M.A.B. and Agbana, S.B. (1983). Heterotic effects and association of seedling vigor with mature plant characteris- tics and grain yield in some tropical maize cultivars. Maydica, 28, 327-338. https://doi.org/10.1017/S0014479700012217 Fakorede, M.A.B. and Akinyemiju, O.A. (2003). Climate change: effects on maize production in a tropical rainforest location. P. 272-282. In Badu-Apraku, B., Fakorede, M.A.B., Oue- draogo, M., Carsky, R.J. and Menkir, A. (Eds.). Maize revo- lution in West and Central Afica. Proceedings of a Regional Maize Workshop, IITA-Cotonou, Benin Republic. 14-18 May 2001. WECAMAN/IITA. Fayose, C.A. and Fakorede M.A.B. (2014). Growth analysis of maize varieties at the seedling and vegetative developmental stages. Proceedings of the Genetics Society of Nigeria Confer- ence, Rubber Research Institute, Benin, Edo State, Nigeria, October 20-24. pp 425-429. Fayose, C.A. and Fakorede M.A.B. (2021). 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Genetic variances and relationship among traits of an early maturing maize population under drought-stress and low nitrogen environments. Crop Science, 57, 681-692. https:// doi.org/10.2135/cropsci2016.03.0177 Acta agriculturae Slovenica, 117/1, 1–10, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1829 Original research article / izvirni znanstveni članek Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) Hussein J. SHAREEF 1, 2, Jameel M. AL-KHAYRI 3 Received August 13, 2020; accepted January 20, 2021. Delo je prispelo 13. avgusta 2020, sprejeto 20. januarja 2021. 1 University of Basrah, Date Palm Research Center, Basrah, Iraq 2 Corresponding author, e-mail: husseinshareef@live.com 3 King Faisal University, College of Agriculture and Food Sciences, Department of Agricultural Biotechnology, Al-Ahsa, Saudi Arabia Salt and drought stress exhibits oxidative stress and modulat- ed protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) Abstract: The formation of new proteins under the influ- ence of harsh environmental conditions is a plant adaptation reaction. Two-year-old date palm tissue culture-derived plants from ‘Barhee’ grown in the field were subjected to salt stress (70 g l-1 NaCl) and dehydration-induced by applying 70 g l-1 polyethylene glycol or without irrigation and withholding ir- rigation (0 g l-1) for one month. The soluble carbohydrate con- tent increased in response to salinity and polyethylene glycol treatment in leaves compared to the control and drought treat- ment without irrigation. Proline increased in all treatments. Malondialdehyde and hydrogen peroxide increased under salinity. Salinity treatment increased the activity of ascorbate peroxidase and catalase enzyme. Salinity and polyethylene gly- col treatments increased abscisic acid, whereas the indoleacetic acid level decreased. The protein pattern of roots and leaves in one-dimensional polyacrylamide gel electrophoresis showed that the stress conditions led to new protein bands’ appearance and other proteins’ disappearance. A comparison of protein patterns between the control and stress treatments revealed that the relative intensity of proteins in roots and leaves were more associated with salinity treatment than the drought. The results may be clearing important the molecular mechanism of tolerance under the influence of extreme environmental stress. Key words: abscisic acid; ascorbate peroxidase; lipid per- oxidation; malondialdehyde; polypeptide Solni in sušni stres se izražata kot oksidacijski stres in vpliva- ta na vzorce beljakovin v koreninah in listih dateljeve palme (Phoenix dactylifera L.) Izvleček: Tvorba novih beljakovin je pod vplivom neu- godnih okoljskih razmer prilagoditveni odziv rastlin. Dvoletne dateljeve palme, vzgojene v tkivnih kulturah iz sorte ‘Barhee’, posajene na prostem, so bile izpostavljene solnemu stresu (70 g l-1 NaCl) in dehidraciji z uporabo 70 g l-1 polietilen glikola ali brez zalivanja za en mesec (0 g l-1). Vsebnost topnih ogljiko- vih hidratov se je v listih povečala kot odziv na slanost in obrav- navanje s polietilen glikolom v primerjavi s kontrolo in obrav- navanjem s sušo brez zalivanja. Vsebnost prolina se je povečala v vseh obravnavanjih. Vsebnosti malondialdehida in vodikove- ga peroksida sta se povečali v razmerah slanosti. Obravnavanje s slanostjo je povečalo aktivnosti encimov askorbat peroksidaze in katalaze. Obravnavanji s slanostjo in polietilen glikolom sta povečali vsebnost abscizinske kisline, a zmanšali vsebnost indo- locetne kisline. Vorec beljakovin v koreninah in listih določen z enodimenzionalno poliakrilamidno gelsko elektroforezo je po- kazal, da so stresne razmere vodile k novemu vzorcu beljakovin v stresnih razmerah in izginjanju drugih. Primerjava vzorcev beljakovin med kontrolo in stresnimi obravnavanji je pokazala, da je relativna jakost pojavljanja beljakovin v koreninah in listih bolj povezana z obravnavanjem s slanostjo kot s sušo. Rezultati bi lahko bili pomembni pri pojasnitvi molekularnega mehaniz- ma tolerance pod vplivom ekstremnega okoljskega stresa. Ključne besede: abscizinska kislina; askorbata peroksida- za; peroksidacija lipidov; malondialdehid; polipeptid Acta agriculturae Slovenica, 117/1 – 20212 H. J. SHAREEF and J. M. AL-KHAYRI 1 INTRODUCTION Protein pattern analysis is a helpful instrument for testing plants’ reactions to abiotic stress (Piasecka et al., 2019). Changes in natural conditions influence the quality and amount of plant proteins. Protein groups react signif- icantly to ecological stress, for example, drought (Hellal et al., 2018)a laboratory experiment was conducted in a factorial randomized complete design with four replica- tions. The controlled experiment included ten of Egyp- tian barley cultivars namely; (Giza 123, 124, 125, 126, 127, 129, 130, 134, 135 and 2000 and salt stress (Patankar et al., 2018)the molecular basis of this tolerance is complex and poorly understood. Therefore, this study aimed to identify the genes involved in salinity tolerance using a basic yeast functional bioassay. To achieve this, a date palm cDNA library was overexpressed in Saccharomyces cerevisiae cells. The expression levels of selected genes that make yeast cells tolerant to salt were subsequently validated in the leaf and root tissues of date palm seedlings using a quantitative PCR method. About 6000 yeast transformant cells were replica printed and screened on a synthetic minimal medium containing 1.0 M of NaCl. The screen- ing results showed the presence of 62 salt-tolerant trans- formant colonies. Sequence analysis of the recombinant yeast plasmids revealed the presence of a group of genes with potential salt-tolerance functions, such as aquapo- rins (PIP. Salt stress is a major limiting factor in plant de- velopment and can lead to water stress (Elsheery et al., 2020; Shareef et al., 2020). Salinity reduces the plant’s abil- ity to absorb water (Naeem et al., 2013). Acosta-Motos et al. (2017) explained the mechanism of a direct effect of salinity on plants by triggering specific morphological, physiological, and biochemical changes. Determination of stress resistance involves useful molecules that induce adaptation reactions of the developing plant under stress conditions, such as specific proteins ( Ghatak et al., 2017; Mohamed et al., 2018). The root is the vital organ that first experiences salt stress in the plant (Komatsu & Hossain, 2013). Some pro- teins respond to salt stress induced in the roots more than other plant parts. The main proteins produced by salinity or drought stress are linked to carbohydrates and energy metabolism (Xu et al., 2015). Paul et al. (2015) stated that the main class of specific proteins resulting from stress ul- timately leads to oxidative stress. These defense proteins accumulate in plant roots and play an initial role in plant endurance to drought (Omar et al., 2018). A plant’s tolerance to water stress or its avoidance depends on multiple mechanisms activated by different molecular signals in the roots, stimulated by abscisic acid such as SnRK2-interacting calcium sensor and AREB/ ABFs-ABRE-binding protein/ABRE-binding factor to send the message to the leaf to start the endurance mecha- nisms (Chae et al., 2007)we have isolated a dehydration- inducible gene (designated OSRK1. Abscisic acid repre- sents the long-distance signal that moves from the roots to the leaves, increasing its concentration in the leaves due to expressing genes responsible for ABA biosynthesis, which leads to stomatal closure (Lokhande & Suprasanna, 2012; De Smet & Zhang, 2013). Drought and salinity are essential environmental issues prevailing in date palm (Phoenix dactylifera L.) cultivation regions where they can negatively impact growth and productivity (Al-Khayri & Al-Bahrany, 2004; Al-Bahrany & Al-Khayri, 2012; Shareef, 2019). Several types of genetic markers have been used to characterize date palm varieties under different stress conditions based on phenotypic (Elsafy et al., 2015), biochemical (Yaish et al., 2015)little is known about the underlying molecular mechanisms that contribute to its salt tolerance. Only re- cently, investigators have uncovered microRNA-mediated post-transcriptional gene regulation, which is critical for typical plant development and adaptation to stress con- ditions such as salinity. To identify conserved and novel miRNAs in date palm and to characterize miRNAs that could play a role in salt tolerance, we have generated sRNA libraries from the leaves and roots of NaCl-treated and untreated seedlings of date palm. Deep sequencing of these four sRNA libraries yielded approximately 251 million reads. The bioinformatics analysis has identified 153 homologs of conserved miRNAs, 89 miRNA variants, and 180 putative novel miRNAs in date palm. Expression profiles under salinity revealed differential regulation of some miRNAs in date palm. In leaves, 54 of the identi- fied miRNAs were significantly affected and the majority (70%, and molecular markers (Al Kharusi et al., 2017). Ponnaiah et al. (2019)crude protein (CP has shown that changes in the levels of proteins of pearl millet under salinity and drought conditions have helped to identify the genes involved in controlling tolerance to these stress conditions. In a previous experiment, El Rabey et al. (2015) used date palm seedlings to analyze proteomics for salinity and sensitive protein associated with water stress resulting from gene expression of salinity and dehydra- tion in leaves. The current experiment aimed to analyze the protein pattern change and oxidative stress response in offshoots from tissue culture (true to type) date palms’ leaves and roots exposed to drought and salinity. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL AND STRESS CONDITIONS Two-year-old date palm plants ‘Barhee’ regenerated Acta agriculturae Slovenica, 117/1 – 2021 3 Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) from tissue culture were used in this experiment, which was conducted at Alharthah, Basrah, Iraq (30°38’47.1”N 47°45’08.2”E) during 2019. The plants were grown in silty clay loam soil with an organic fertilizer in September and expanded the offshoot cultivation area. On 1 October, the treatments were conducted. The minimum temperature was 18 °C, the maximum 32 °C, the relative humidity was 30 %, and the light intensity 1350 μmol m-2 s-1. Five repli- cates of each treatment were investigated. The treatments consisted of regular irrigation, (control) withholding irri- gation for one month, induced drought stress with field ca- pacity was 12.5 % (20 ml depth of water), induced drought stress using 70 g l-1 polyethylene glycol (PEG), and salinity stress with 70 g l-1 sodium chloride. Field capacity was 75 % (120 ml depth of water) for PEG and salt treatment. After 30 days, samples were washed with distilled water, dry mat- ter taken, whereas fresh samples frozen in liquid nitrogen. 2.2 ESTIMATION OF TOTAL CARBOHYDRATE CONTENT The amount of total soluble sugar was estimated spec- trophotometrically at 490 nm following the phenol sulfuric acid reagent method of Dubois et al. (1951). The leaves and roots (100 mg) segments were homogenized in 80 % etha- nol and centrifuged at 2000 x g for 25 min. The supernatant (1 ml) was mixed with 0.05 % phenol (2 ml) (pH 6) and 98 % sulfuric acid (2 ml). The blends were hatched at 80 ºC for 20 min in a water bath. Total soluble carbohydrate content was calculated using a standard curve of glucose and expressed as mg g-1 F.M. 2.3 DETERMINATION OF PROLINE Proline was extracted and estimated, according to Bates et al. (1973). Root and leaf (0.5 g dry matter) samples were homogenized separately in cold aqueous sulfosalicyl- ic acid 3 % (10 ml). The homogenate was sieved through Whatman No. 2 paper. In a test tube, 2 ml of the filtrate was blended with acidic ninhydrin (2 ml) and glacial acetic acid (2 ml) and incubated in a 100 ºC water bath for 1 hr. After quick cooling in an ice bath, toluene (4 ml) was in- cluded, and the mixture was shaken by hand. The toluene phase, containing the chromophore, was aspirated, and the absorbance of this phase, using samples of 1 ml, was deter- mined at 520 nm. 2.4 MEASUREMENT OF LIPID PEROXIDATION Lipid peroxidation was determined by evaluating malondialdehyde (MDA). MDA was estimated depen- dent on the strategy of Esterbauer & Cheeseman (1990). A blend of 0.6 g fresh plant tissue and TCA 5 % (5 ml) was centrifuged at 12000 x g for 25 min. The supernatant was blended in thiobarbituric acid 0.67 % (2 ml) and heated for 30 min at 100 °C in a water bath. Sample absorbance was evaluated at 450, 532, and 600 nm using a blank contain- ing all reagents. MDA content of the sample was calculated using the formula: C (μmol g-1) = 6.45 (A532 – A600) – 0.56 A450. 2.5 ESTIMATION OF HYDROGEN PEROXIDE Leaves and roots (500 mg) samples were crushed in trichloroacetic acid (TCA) 5 %, and the mixture was uti- lized for the assurance of H2O2 by the technique of Sagisaka (1976). The response blend with 50 % of TCA, 10 mmol ammonium sulfate ferrous, 2.6 mol potassium thiocyanide, and plant separate, the 480 nm used to read absorbance. 2.6 EXTRACTION OF ANTIOXIDANT ENZYMES Leaves and roots (200 mg) samples were absorbed in fluid nitrogen and crushed in an extraction buffer (2.0 ml): 100 mmol potassium phosphate (pH 7.8), 0.1 mmol ethyl- ene diamine tetraacetic acid (EDTA), and 10 mmol ascor- bic acid. 13000 xg for 15 min at 4 °C used to centrifuged the mixture. The catalase activity (CAT) and ascorbate peroxidase (APX) was measured by collecting the super- natant. All proteins were resolved with a similar support arrangement. Also, the evaluation was made utilizing the Bradford (1976) strategy. CAT activity determined by the technique depicted by Azevedo (1998) was utilized with specific alterations. Its operation was checked by a spec- trophotometer assessing the H2O2 at 240 nm for 2 min in a response medium containing 100 mmol potassium phos- phate buffer (pH 7.0), 12.5 mmol H2O2, and 50 µl of plant separate incubated at 28 °C. As a new solution, a similar response medium free of separate was utilized. The activ- ity of APX was resolved following Nakano & Asada (1981) by observing the ascorbate’s oxidation pace at 290 nm. The response medium was incubated at 28 °C. It was com- prised of 100 mmol potassium phosphate buffer (pH 7.0), 0.5 mmol ascorbic acid, and 0.1 mmol H2O2. The reduction in absorbance was observed for 2 min from the beginning of the response. 2.7 HORMONES ANALYSIS Five grams of a fresh tissue sample, which was homog- Acta agriculturae Slovenica, 117/1 – 20214 H. J. SHAREEF and J. M. AL-KHAYRI enized in 70 % methanol, was stirred overnight at 4 °C. The extract was filtered through Whatman filter paper (No.1) and evaporated under a vacuum. The pH of the aqueous phase was adjusted to 8.5, using 0.1 mol phosphate buf- fer. Later the aqueous phase was partitioned twice using methanol. A rotary evaporator removed the methanol phase. The aqueous phase pH was adjusted to 2.5, using 1 N hydrochloric acid (HCl). The injection of the con- centrate determined phytohormones into a reversed- phase HPLC, C18 column in an isocratic elution mode utilizing a portable stage comprising of acetone: water (26:74) with 30 mmol phosphoric acid as per Tang et al. (2011). The pH was kept up at 4, utilizing 1 N sodium hy- droxide. The temperature was kept at 25 ºC. The flux rate was 0.8 ml min-1, and the elution of the phytohormones was observed at 208 and 265 nm for indoleacetic acid and abscisic acid, respectively. 2.8 EXTRACTION OF PROTEINS AND GEL ELEC- TROPHORESIS Proteins were extracted by homogenizing the 300 mg of solidified dried leaf in 1 ml of extraction buf- fer [0.2 mol, tris-hydroxymethyl aminomethane (Tris) + 0.001 mol ethylenediamine tetra acetic acid + (Na2 + EDTA) + 12 % glycerol + 0.01 M dithiothreitol (DTT) + 0.05 mmol phenylmethylsulfonyl fluoride (PMSF)] and homogenized by a mortar and pestle. At that point, the samples were centrifuged at 15,000 ×g for 15 min; the buffer consisted of 0.125 M Tris HCl (pH 6.8) + 4 % SDS + 20 %, glycerol + 10 % b-mercaptoethanol + 0.01 % bromophenol blue. Protein samples were denaturized by heat in the water bath at 90 °C for 3 min. Protein elec- trophoresis was performed in SDS polyacrylamide gel following procedures described by Laemmli (1970). The protein bands demonstrated clear changes, which were investigated by ImageJ programming. 2.9 STATISTICAL DESIGN AND ANALYSIS The experiment was conducted according to the completely randomized blocks design for four treat- ments: control, drought, polyethylene glycol, salinity, with five replications per treatment. The mean values ± the standard error were displayed. The data were subject- ed to analysis of variance (ANOVA) utilizing IBM (SPSS Statistics V23.0), and Duncan’s multiple range test at p < 0.05 were separated by the means. 3 RESULTS 3.1 ANTIOXIDATIVE AND OXIDATIVE RE- SPONSE TO DROUGHT AND SALINITY STRESS Drought and salinity treatments altered soluble car- bohydrate and proline in roots and leaves (Fig. 1a,b). Af- Figure 1: Oxidation change in response to drought, PEG and salinity stress, (a) Soluble carbohydrate, (b) Proline, (c) Malondialde- hyde (MDA), (d) Hydrogen peroxide. Data are shown as means (of five replicates) ±S.E. Different letters indicate significant differ- ences between treatments of leaves and roots (p < 0.05) Acta agriculturae Slovenica, 117/1 – 2021 5 Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) ter one month of treatment, the soluble carbohydrates’ content increased significantly in response to salinity and PEG treatments in leaves related to the control and drought treatment. In contrast, without irrigation, the drought treatment decreased carbohydrates’ content considerably in the roots (Fig. 1a). Proline in leaves and roots increased significantly in all treatments related to the control (Fig. 1b). Salin- ity treatment increased MDA in leaves and roots related to the control (Fig. 1c). In contrast, drought treatment showed no significant difference in MDA compared to the control in roots. Salinity treatment increases the hy- drogen peroxide in roots and leaves related to other treat- ments. In contrast, drought treatment had no significant difference in H2O2 related to the control in both roots and leaves (Fig. 1d). 3.2 ENZYMES ACTIVITY AND HORMONAL LEVELS IN RESPONSE TO DROUGHT AND SALINITY STRESS Salinity treatment increased APX enzyme activity by 16.46 and 49.19 % higher than the control in roots and leaves, respectively. At the same time, the drought treat- ment reduced APXenzyme activity by 20.81 and 15.65 % compared to the control in roots and leaves, respectively (Fig. 2a). The CAT enzyme activity decreased significant- ly in response to drought in the roots and leaves related to the control. In contrast, the salinity treatment signifi- cantly increased CAT activity in leaves related to the con- trol treatment (Fig. 2b). Salinity and PEG treatments increased ABA’s level in the leaves significantly by 47.86 and 216.02 % com- pared to the control, respectively. Also, both the salinity and PEG treatments increased ABA’s level significantly in the roots by 38.85 and 33.63 % related to the control, re- spectively (Fig. 2 c). In the roots, the IAA level decreased significantly in response to salinity, 32.85 % compared to the control. Whereas, in leaves, the IAA level decreased significantly in response to drought treatment, where 46.60 % reduction was observed compared to the control (Fig. 2d). 3.3 PROTEIN PATTERN CHANGES IN RESPONSE TO DROUGHT AND SALINITY STRESS The protein patterns of roots and leaves in one- dimensional polyacrylamide gel electrophoresis (SDS- PAGE) and subsequent examination by the ImageJ program demonstrated some striking contrasts (Fig. 3). The root protein patterns and relative intensity of pro- tein expression showed that the appearance of protein bands 7 and 8 (molecular mass 59.492 and 37.410 kD, respectively) was up-managed by drought, while the ex- pression of protein bands 4 and 5 (molecular mass 97.400 and 97.391 kD, respectively) was down-controlled under drought. The relative intensity of protein expression in protein bands 4, 5, and 7 was up-managed, while protein Figure 2: Enzymes activity and hormonal levels in response to drought, PEG and salinity stress, (a) Ascorbate peroxidase activity (APX), (b) Catalase activity (CAT), (c) Abscisic acid (ABA), (d) Indoleacetic acid (IAA). Data are shown as means (of five replicates) ± S.E. Different letters indicate significant differences between treatments of leaves and roots (p < 0.05) Acta agriculturae Slovenica, 117/1 – 20216 H. J. SHAREEF and J. M. AL-KHAYRI bands 1, 3, 8, and 9 (molecular mass 185.674, 114.794, 37.410, and 16.658 kD, respectively) were down-con- trolled by PEG. The relative intensity of protein expres- sion in protein bands 4, 5, and 7 was up-managed, while protein bands 1, 3, 8, and 9 (molecular mass 185.674, 114.794, 37.410, and 16.658 kD, respectively) were down- controlled by PEG and salt stress except for protein band 3 which was not present in the salt stress treatment. The appearance of novel protein bands (including band 1 and 7 with molecular mass 147.222 and 59.492 kD, re- spectively) by drought, EPG, and salt stress related to the control, whereas band 6 (molecular mass 87.059 kD) was detected at PEG treatment and band 4 (molecular mass 97.391 kD) was found in the presence of salt stress treat- ment. The leaf protein patterns and relative intensity of protein expression showed that the appearance of pro- tein band 2 (molecular mass of 106.588 kD) was up-man- aged by drought. In contrast, the expression of protein bands 3, 4, 5, 6, and 7 (molecular mass 97.758, 75.875, 61.825, 46.900, and 27.626 kD, respectively) was down- controlled under drought. The relative intensity of pro- tein expression in protein band 2 was up-managed, while protein bands 3, 4, and 5 were down-controlled by PEG. The relative intensity of protein expression in protein band 3 was up-managed, while protein bands 2, 5, and 6 were down-controlled by salt stress. The appearance of new protein bands, including band 1 with a molecular mass of 177.221 kD by drought, EPG, and salt stress com- pared to the control.Whereas band 1 (molecular mass 136.349 kD) was not present in all treatments. The ap- pearance of novel protein bands, including band 8 with a molecular mass of 16.658 kD, responded to drought. The protein bands 6 and 7 with a molecular mass of 43.888 Figure 3: Analysis of protein patterns by one-D SDS-PAGE extracted from root and leaf, showing protein pattern changes in re- sponse to drought, PEG, and salinity stress Figure 4: Dendrogram of hierarchical clustering to root (R) and the leaf (L) of date palm offshoot under drought, polyethylene glycol (PEG), and salinity stress by using protein patterns Acta agriculturae Slovenica, 117/1 – 2021 7 Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.) and 23.789 kD responded to EPG. Also, bands 4 and 7 (with molecular mass 87.059 and 31.799 kD, respec- tively) appearance by salt stress (Fig. 3). Comparing the protein pattern in the control and all stress treatments revealed that the relative intensity in roots and leaves was associated more with salinity treatment than other tested factors. 3.5 CLUSTER ANALYSIS OF PROTEIN PATTERN TO ROOT AND LEAF IN RESPONSE TO DROUGHT AND SALINITY STRESS Hierarchical cluster analysis of the protein pattern to root and leaf in response to drought and salinity stress (Fig. 4) showed two distinct clusters. The first group in- cluded drought, PEG, salinity, and control treatments to leaf (L-drought, L-PEG, L-Salinity, L-Control) and root control (R-Control). A significant similarity was ob- served between the drought treatments (L-drought) and PEG (L-PEG) in the leaf ’s protein pattern. At the same time, the second group included drought, PEG, and sa- linity treatments (R-Drought, R-PEG, R-Salinity), which showed the similarity between the treatments drought and PEG in the protein pattern of the root. 4 DISCUSSION Salt and water stress are among the most severe en- vironmental stress factors determining plant growth and development (Elsheery & Cao, 2008). The relationship between new proteins and tolerance to extreme environ- mental conditions is essential for environmental stress research. Drought and salinity treatments alter soluble carbohydrates’ and proline concentration in roots and leaves (Fig. 1). Soluble carbohydrates contribute to os- motic adjustment during stress and protect giant mole- cules and membranes' structure during extreme environ- mental conditions (De Lacerda et al., 2005; Helaly et al., 2018). Carbohydrates play an essential role as an antioxi- dant resulting from plant tissue damage (Al Hassan et al., 2015). Soluble carbohydrates contribute to IAA build- ing, especially glucose, which increases the IAA primers, such as (ANT and TRP) (Sairanen et al., 2013). The root is known to be a sink organ of carbohydrates. The carbo- hydrates are reduced under drought treatment (Fig. 1a). Decreased polysaccharides’ content in the root under the influence of dehydration results from sugars' consump- tion in the process of respiration (Jaleel et al. 2009). Proline concentration in leaves and roots increased significantly in all treatments than the control (Fig. 1b). Proline increases in plants that are more tolerant to harsh environmental conditions. One of the most important adaptations to the surrounding conditions is the accu- mulation of proline under the influence of drought or salt stress (Amini & Ehsanpour, 2005). Proline has a vital role in protecting the plant, promoting growth, and effective antioxidants in water stress conditions on the date palm (Dhawi & Al-Khayri 2008; Butt et al. 2020). Also, proline helps protein inversion and a regular signal that activates multiple planting adaptation responses to extreme en- vironmental conditions (Khedr et al., 2003). Increasing the proline concentration provides for protein building because it is an amino acid and improves endurance by maintaining cell water potential, osmotic balance, and membrane stability by preventing leaching. Salinity treatment resulted in a significant increase in MDA and H2O2 in leaves and roots (Fig. 1 c,d). The high MDA and H2O2 content shows increased oxidative stress and reflect tissue damage due to environmental stress (Ben Abdallah et al., 2017). The accumulation of sodium and chloride causes plant tissue damage, thus activating oxidative stress (Baghalian et al., 2008). MDA modifies many proteins into the photosynthetic center II (PSII) to stimulate the plant to adapt to environmental stress (Chen et al., 2018). Proteins are significant focuses for radicals and two-electron oxidants in biological sys- tems because of their plenitude and high rate constants for the response (Davies, 2016). Hydrogen peroxide's most essential property is the capacity to cross cell mem- branes freely, which superoxide generally cannot do (Kurutas, 2016). High-level H2O2 can stimulate new enzymatic proteins such as APX and CAT enzymes that scavenge free radicals (Sofo et al., 2015). Salinity treatment increased APX and CAT activity in leaves and roots (Fig. 2 a,b). APX activity increased under saline stress conditions due to gene expression that contributes to the maintenance of enzyme activity and physiological processes ( Omar et al., 2012; Khan et al., 2020b). Gene expression of the enzyme is regulated dur- ing plant development responds to environmental stress (Huseynova et al., 2013). The effectiveness of APX direct- ly contributes to the protection of plants against extreme environmental conditions (Khan et al., 2020a). Also, the APX enzyme's high activity can reduce the damage of reactive oxygen species (ROS) in the roots and thus in- crease the conversion of ROS to H2O2 and the use of the enzyme to maintain root cells (Huang et al., 2019). Dehydration leads to reduced growth in general and thus affects gene expression resulting in reduced synthe- sis of enzymes. Different enzyme activity under various stress factors confirms the validity of the antioxidant system (Khan et al., 2019). The high activity of the CAT enzyme in the leaves is attributed to the stability of the tolerance of environmental conditions in plants (Kawa- Acta agriculturae Slovenica, 117/1 – 20218 H. J. SHAREEF and J. M. AL-KHAYRI mura & Muraoka, 2018). Reduced enzyme activity un- der drought stress compared to the standard conditions showed the role of H2O2 as a signal transmitted from the root to the leaf and thus reduced the concentration of H2O2 in the roots (Fig. 1d). Salinity and PEG treatments increased ABA lev- els in the leaves and roots (Fig. 2c). The ABA signaling pathway receives and transmits hormonal stimulation to activate several events towards the plant's ability to adapt to environmental conditions (Vishwakarma et al., 2017). ABA signaling pathway affects building many proteins that lead to plant adaptation to extreme environmental conditions (Mittler & Blumwald, 2015). The accumula- tion of ROS contributes to collecting the ABA required to activate the gene expression process (Vishwakarma et al., 2017). Salinity and drought treatments decreased the level of IAA in the leaves and roots (Fig. 2d). IAA is involved in stimulating the construction of proteins such as H+- ATP (Salehin et al., 2019). Environmental stress modi- fies IAA transmission and its placement in the roots, so the auxin concentrations in the roots decrease when the plant is exposed to stress (Tanimoto, 2005). The reduc- tion of IAA in the roots and leaves due to the imbalance in the growth regulators and the enzymes' inhibition is responsible for building auxin. Comparing protein patterns in the control and stress treatments revealed that the relative intensity in roots and leaves was more significantly associated with salinity treatment than other factors (Figs. 3, 4). Cluster analysis showed the convergence of the effect of PEG and drought stress on protein patterns, whether in leaf or root, while distinguishing the impact on drought and salinity. Plant growth and adaptation to environmental conditions are strongly influenced by protein metabolism (Ghatak et al., 2017). Various investigations have indicated that the construction of new proteins is associated with changes in plant environmental conditions such as salinity (Yaish et al., 2015) drought (El Rabey et al., 2015), which causes an increase or decrease in polypeptides. The results in- dicate that the environmental conditions of stress led to the emergence of new proteins and other proteins' dis- appearance, accompanied by unmatched stress proteins' appearance. The formation of new proteins under dehy- dration's influence refers to creating these proteins ear- lier on cell death programming as we observed shrinkage and stiffness in plants prone to dehydration. 5 CONCLUSION One of the most challenging difficulties in recent literature related to plant endurance is to explain the mo- lecular basis of adaptation to environmental stress fac- tors. A change in gene expression by drought stress leads to a cascade of changes, including the protein patterns. Exposure of date palm offshoots to dehydration creates specialized proteins whose primary function is to close the conveying channels in roots, close stomata, shrink cells, reduce vital processes, and maintain water in the tissues critical to maintaining plant survival. Exposure the plant to salinity builds multiple specialized proteins that improve the tolerance mechanism by modifying the roots’ selective channels, closing the stomata, and acti- vating the defense mechanism against oxidative stress. 6 ACKNOWLEDGMENT The authors are grateful to Prof. Dennis V. Johnson of Cincinnati, Ohio, USA, for this manuscript’s critical review and editorial enhancement. 7 REFERENCES Acosta-Motos, J.R., Ortuño, M.F., & Bernal-Vicente, A. (2017). Plant responses to salt stress: adaptive mechanisms. Agron- omy, (7),18. https://doi.org/10.3390/agronomy7010018 Al-Bahrany, A.M., & Al-Khayri, J.M. (2012). 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Frontiers in Plant Science, 6(NO- VEMBER), 946. https://doi.org/10.3389/fpls.2015.00946 Acta agriculturae Slovenica, 117/1, 1–6, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1935 Original research article / izvirni znanstveni članek Analysis of gliadin patterns and diversity in Triticum polonicum L. acces- sions from Ethiopia Eleni SHIFERAW 1, 2 Received October 19, 2020; accepted January 24, 2021. Delo je prispelo 19. oktobra 2020, sprejeto 24. januarja 2021. 1 Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia 2 Corresponding author, e-mail: teleni08@gmail.com Analysis of gliadin patterns and diversity in Triticum poloni- cum L. accessions from Ethiopia Abstract: Gliadins from 25 accessions represented by 350 individual seed samples were analysed by acid-polyacrylamide gel electrophoresis (A-PAGE) with the objective of identify- ing gliadin band patterns and examine the extent of diversity in Triticum polonicum L. collections from Ethiopia. Seventy polymorphic bands and 68 different patterns were identified. Eighteen different mobility bands and 16 patterns were identi- fied in ω-gliadin region, 22 bands and 20 patterns in γ-gliadin region, 12 bands and 22 patterns in β-gliadin region and 18 bands and 10 patterns in α-gliadin region. The average genetic diversity calculated from the data of the four gliadin zones of the analysed samples was 0.15. The γ region have the highest diversity (H = 0.193), followed by ω regions (H = 0.177) and β region (H = 0.168) and the lowest diversity was observed in α region (H = 0.127). Cluster analysis based on genetic distances resulted in grouping of the analysed accessions in to seven main groups. Though the level of diversity was relatively lower than other tetraploid wheat species from Ethiopia, the findings are indicative of the existence of variation in the collections which can be exploited for wheat improvement. Key words: A-PAGE; genetic diversity; gliadin; Triticum polonicum Analiza vzorcev gliadinov in raznolikosti akcesij poljske pše- nice (Triticum polonicum L.) v Etiopiji Izvleček: V raziskavi so bili s kislo poliakrilamidno gelsko elektroforezo (A-PAGE) analizirani gliadini 25 akcesij poljske pšenice v 350 vzorcih semen z namenom določiti vzorce glia- dinskih prog in ugotoviti obseg raznolikosti te pšenice v Etiopi- ji. Določeno je bilo 70 polimorfnih prog in 68 različnih vzorcev. Osemnajst različnih mobilnih prog in 16 vzorcev je bilo dolo- čenih v območju ω-gliadinov, 22 prog in 20 vzorcev v območju γ-gliadinov, 12 prog in 22 vzorcev v območju β-gliadinov in 18 prog in 10 vzorcev v območju α-gliadinov. Poprečna genetska raznolikost, izračunana na osnovi podatkov analiziranih vzor- cev za ta štiri območja gliadinov je bila 0,15. Območje γ je ime- lo največjo raznolikost (H = 0,193), sledili sta mu območji ω (H = 0,177) in β (H = 0,168), najmanjša raznolikost je bila ugo- tovljena v območju α (H = 0,127). Analiza grozdov na osnovi genetske oddaljenosti je združila analizirane akcesije v sedem glavnih skupin. Čeprav je raven raznolikosti nekoliko manjša kot pri drugih tetraploidnih vrstah pšenice v Etiopiji, so od- kritja pokazala obstoj spremenljivosti v zbirkah, ki bi jih lahko uporabili pri izboljšanju pšenice. Ključne besede: A-PAGE; genetska raznolikost; gliadin; Triticum polonicum Acta agriculturae Slovenica, 117/1 – 20212 E. SHIFERAW 1 INTRODUCTION Focus given mainly to increasing grain yield and crop productivity has resulted in an increase in micro- nutrient deficiency in food grains (Garg et al., 2018). Agronomic and genetic biofortification are being used as a means to improve the deficiency of micronutrients in modern varieties. Exploitation of underutilized species is one option to meet the demand for enhanced nutri- tion requirements. Evaluation of minor wheat species may provide breeders with important sources of genes suitable for biotic and abiotic stress tolerance and nu- trition improvement. Among the many underutilized crops Triticum polonicum L., commonly known as Polish wheat, offers valuable agronomical and nutritional ben- efits that can be incorporated in to wheat varieties cur- rently in use. Triticum polonicum L., 2n = 28, AABB), is a species which is grown sporadically in Africa (Ethiopia and Algeria), Europe (Spain, Italy, Ukraine and Russia) and some countries in the Asia temperate (USDA, 2020). Lately, T. polonicum is gaining attention due to its desira- ble traits. In comparison with durum and bread wheat, it has abundant protein and ash content and low fat content (Bieńkowska et al., 2019a) and it has good content of iron phosphorus, zinc, sulphur, magnesium, and boron, in addition to a lower content of aluminum (Bieńkowska et al., 2019b). Starch with up to 43.2 % amylase content was also reported in the grain of T. polonicum (Rodrıguez- Quijano et al., 2003). Foods with high amylase content take longer to digest hence to improve diet, cereals with high amylase content are needed (Vaziri et al., 2014). It is also shown to have potential and useful attributes in durum wheat breeding for drought tolerance (Hakimi et al., 1998). Since most of the new varieties of wheat are nota- bly poorer in mineral content breeders have specified T. polonicum as a genetic resource with good potential to enhance wheat’s nutrition, specifically for genetic bio- fortification of durum and bread wheat (Bieńkowska et al., 2019b). The species is also reported to show satisfac- tory resistance to Fusarium head blight (Wiwart, 2013), and could be a potential source of semi-dwarfing genes for the development of durum wheat cultivars (Watan- abe, 2004). T. polonicum can be used in wheat breeding programs since it can be crossed and produce fertile and genetically stable hybrids with T. aestivum L. and T. du- rum Desf. (Akond et al., 2006; Hakimi et al., 1997). Gliadins are mainly monomeric proteins that are not affected by environment (Lookhart & Finney, 1984) and coast effective hence they make good tool for identi- fying genetic differences and determine quality (Bushuk & Zillman, 1978; Payne, 1987). In prior studies high and low molecular weight glutenin subunits composition of Ethiopian tetraploid wheat (Hailu et al., 2006) and al- lelic variation of gliadin coding loci of improved durum wheat varieties from Ethiopia (Hailegiorgis et al., 2017) have been reported. T. polonicum is one of the minor tetraploid wheat species cultivated in Ethiopia with lim- ited importance, usually grown mixed with T. durum and T. aestivum (Demissie & Habtemariam, 1991) with ear- lier reports of cultivation in pure stands as well (Engels & Hawkes, 1991). It is a neglected crop in terms of con- servation, research and utilization. The diversity of glia- din patterns in Ethiopian tetraploid wheat has not been reported so far. Genetic diversity study in conserved ge- netic resources of the species is important to better utilize and manage the resource. This study examined gliadin pattern and diversity of some T. polonicum collections from Ethiopia. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL AND GLIADIN EXTRAC- TION Twenty five T. polonicum accessions from Ethiopia obtained from the Ethiopian Biodiversity Institute were used for this study. Fourteen seeds from each accession (a total of 350 individual seed samples) were used for gliadin profiling and diversity assessment. Each wheat grain was crushed using mortar and pestle and gliadin was extracted with 70 % (v/v) ethanol allowing the mix- ture to stand at 40 oC for 45 minutes and at +4 oC until electrophoresis. Aliquot of the supernatant was mixed with sample buffer (18  % sucrose containing methyl green tracking dye) before loading 25 μl of the extract on the gel. 2.2 A-PAGE Glidins were fractionated using acid -polyacryla- mide gel electrophoresis (A-PAGE), containing acryla- mide (7.5 %) and bis-acrylamide (0.375 %) with ferrous sulfate (0.009 %) and ascorbic acid (0.00018 %) polymer- ized by the addition of hydrogen peroxide (15 %). Elec- trophoresis was performed at 50 V, 150 V and 250 V for 10 min each, at 350 V for 60 min and at 550 V for 80 minutes in 0.05 M aluminum lactate (pH 3.1) buffer. The cultivar Icaro was used as a standard in each gel run to compare electrophoretic bands and patterns. At the end of the run, gels were fixed using 12 % trichloroacetic acid for 15 minutes and stained overnight in staining solution containing 0. 04 % (w/v) Coomassie brilliant blue R-250 in 100 ml ethanol and 80 ml 12 % trichloroacetic acid. Acta agriculturae Slovenica, 117/1 – 2021 3 Analysis of gliadin patterns and diversity in Triticum polonicum L. accessions from Ethiopia After destaining using tap water, gels were photographed on a white illumination source for documentation. Each sample was run twice to confirm the gliadin profile. 2.3 DATA ANALYSIS Banding pattern of each individual seed was com- pared within and among accessions and the standard cul- tivar to distinguish the different gliadin bands and pat- terns. Each different band of individual seeds displayed by gliadin loci was scored for its presence (1) or absence (0). The data obtained were entered as a binary data matrix and used to perform genetic diversity measures using GenAlEx 6.5 (Peakall & Smouse, 2012). Gene di- versity was computed as the expected heterozygosity (H) based on band frequencies of gliadins. Genetic distance matrix was used to perform cluster analysis using the un- weighted pair-group method for the arithmetic average (UPGMA) (Sneath & Soakal, 1973) clustering method by TFPGA version 1.3 (Miller, 1997). 3 RESULTS AND DISCUSSION 3.1 GLIADIN ELECTROPHORETIC PATTERNS Twenty five different accessions of T. polonicum rep- resented by 350 samples were analyzed by A-PAGE. Elec- trophoretic profile for representative accessions is shown in Fig.1. Assuming that the bands with the same relative mobility represent the same subunit, a total of 70 differ- ent bands and 68 gliadin patterns were detected. The av- erage number of gliadin bands per accession was 24 and it ranged from 14 to 37. These bands were grouped into patterns at each of the four gliadin zones (α, β, γ and ω gliadins). The patterns within each gliadin group of α, β, γ and ω were identified by comparing banding patterns of each sample with each other and the standard cultivar. Gliadin patterns in the four gliadin regions observed in the analysed samples is depicted in Fig. 2. Based on the observed gliadin profiles, 10 different gliadin patterns and 18 different mobility bands were identified in the α-gliadin region which showed limited variation com- pared to the other zones. Each α-gliadin pattern showed two to five bands. The most frequent pattern was pattern 1, observed in 84 % of the analysed accessions followed by pattern 2, 3 and 5 each observed in 16 % of the acces- sions. Twenty two different gliadin patterns were identi- fied in the β-gliadin zone each pattern showing three to six bands. The most frequent pattern was pattern 1 de- tected in 30 % of the accessions followed by pattern 20 observed in 20 % of the accessions. In γ-gliadin zone, 20 different patterns having one to five bands in each pat- tern were detected. Pattern 1 was the most frequent one appearing in 68 % of the accessions. In ω-gliadin zone, 16 different patterns having three to seven bands were ob- served. Pattern 1 was the most frequent pattern encoun- tered in 44 % of the accessions followed by pattern 11 ap- pearing in 20 % of the accessions. The number of gliadin bands and patterns, 70 and 68 respectively, detected in the present study is higher than that of Pan et al. (2007) who reported 48 gliadin bands and 65 patterns in T. po- lonicum accessions from different countries. In other studies, Zaefizadeh et al. (2010) reported 66 polymorphic bands and 81 patterns in durum wheat landraces and Ojaghi & Akhundova, (2010) reported 48 bands and 47 patterns in double haploid T. aestivum. When comparing the different gliadin regions, α-gliadin region showed the lowest number of band patterns which is similar to find- Figure 1: Gliadin electrophoretic patterns on A-PAGE for representative T. polonicum accessions showing within accession variation (A) and no variation within accession (B). Acta agriculturae Slovenica, 117/1 – 20214 E. SHIFERAW ings in studies on other wheat species (Ram et al., 2005; Aliyeva et al., 2012). 3.2 GENETIC DIVERSITY A total of 70 bands were scored among the studied samples. The overall frequencies of these bands ranged from 0.007 to 0.871 among all the populations. The band with the highest frequency was detected in γ region while bands with the lowest frequency were detected in γ and α gliadin regions. The majority of the bands (74.28  %) showed frequency of < 25 %. The number of bands with- in each accession ranged from 14 to 37. Within acces- sions, the percentage of polymorphic bands ranged from 0.00 to 47.14 and the gene diversity (H) ranged from 0.00 to 0.153. The highest value was recorded in accession 23 (accession no. 226725) while five accessions (209774, 222726, 222632, 222242 and 226441) showed no within accession variation. The overall gene diversity in the samples based on the patterns observed for each of the four gliadin regions showed that the γ region has the highest diversity (H = 0.193), followed by ω regions (H = 0.177) and β region (H = 0.168) and the lowest diversity was observed in α region (H = 0.14). In all the T. polonicum samples (350 entries) all bands were polymorphic and the mean gene diversity estimate was 0.167. The genetic diversity is rela- tively lower than other tetraploid wheat from Ethiopia examined using seed storage protein (Hailu et al., 2006) and microsatellite markers (Teklu et al., 2006; Yifru et al., 2006). Assessment of agromorphological character- istics of T. polonicum from Ethiopia had also indicated that phenotypic variation is relatively small except for the apical tooth and awn length (Demissie & Habtemariam, 1991). Limited cultivation of the species might have caused genetic erosion resulting in low genetic variation. Hailu et al (2006) reported 100 % genetic erosion of T. polonicum in Tigray and Gojam regions of Ethiopia and 84.4 % genetic erosion throughout the country. Yifru et al. (2006) also reported a much localized use of T. poloni- cum which might result in its loss. Tetraploid wheat used to occupy 60 % of the total wheat area (Tessema and Be- lay 1991). However, because of broader adaptability and yield advantage, bread wheat has been expanding in area of production substituting traditional durum and emmer wheat (Tsegaye & Berg, 2007). Recent reports estimate that tetraploid wheat accounts for around 40 % of the to- tal wheat production (Brasesco et al., 2019), however, de- tail data on production of the different tetraploid wheat species is lacking. 3.3 ANALYSIS OF RELATIONSHIP Nei’s genetic distance (1972) among the accessions ranged from 0.00 to 0.538. The lowest genetic distance was observed among accessions 18, 19 and 21 (0.00) fol- lowed by accession 2 and 3 (0.0002). The highest distance was observed between accessions 22 and 24 (0.538) and accession 22 and 15 (0.529). The UPGMA clustering, based on Nei’s genetic distance, for the 25 accessions re- vealed seven major groups (Fig. 3) containing one to 10 accessions per group. Group A comprises of accessions that showed no within accession diversity while group C contains accessions with the relatively high values of gene diversity, percentage of polymorphic bands and number of bands per accession. Figure 2: Electrophoregram of different gliadin patterns in α, β, γ, and ω gliadin regions observed in the analysed samples. Acta agriculturae Slovenica, 117/1 – 2021 5 Analysis of gliadin patterns and diversity in Triticum polonicum L. accessions from Ethiopia 4 CONCLUSION Despite lower diversity revealed in the analysed accessions, the findings reported here have important implications for T. polonicum conservation and utiliza- tion in the country. In the Ethiopian genebank there are very few T. polonicum accessions. However farmers culti- vate T. polonicum mixed with other wheat species hence wheat accessions conserved as Triticum spp also include T. polonicum as shown by activities that splits these mixed accessions in to their respective species. Splitting mixed species and additional collection of the species from potential growing areas need to be conducted so that the species can be used in improving wheat varieties in nutrient composition and disease resistance. 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Zaefizadeh, M., Somarin, S.J., Ojaghi, J., Seyedi, S.M., Mahmoodabad, R.Z., Ochi, M. (2010). Genetic diver- sity for gliadin patterns of durum wheat landraces in the Northwest of Iran and Azerbaijan. Pesquisa Agropecuária Brasileira, 45, 1425–1432. https://doi.org/10.1590/S0100- 204X2010001200013. Acta agriculturae Slovenica, 117/1, 1–11, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1252 Original research article / izvirni znanstveni članek The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) Ehiokhilen Kevin EIFEDIYI 1, 2, Grace Adejoke ILORI 1, Henry Emeka AHAMEFULE 1, Abduquadir Yusuf IMAM 1 Received August 28, 2019; accepted Janjuary 28, 2021. Delo je prispelo 28. avgusta 2019, sprejeto 28. januarja 2021. 1 University of Ilorin, Department of Agronomy, Ilorin – Nigeria 2 Corresponding author, e-mail: eifediyi.ek@unilorin.edu.ng The effects of zinc biofortification of seeds and NPK fertil- izer application on the growth and yield of sesame (Sesamum indicum L.) Abstract: Soils of the southern Guinea savanna zone of Nigeria are inherently infertile due to shortened fallow period and the continuous use of inorganic fertilizer which depletes the soil of micronutrients such as zinc over time. A field trial was carried out at the Teaching and Research Farm of the Uni- versity of Ilorin, Nigeria during the 2016 and 2017 cropping seasons to evaluate the effect of zinc and NPK fertilizer on the growth, yield and zinc concentration of seeds of sesame. The experiment was laid out as a factorial fitted into a randomized complete block design (RCBD), replicated thrice. The treat- ment consisted of four levels of ZnSO4 (0, 5, 10 and 15 kg ha -1) and four levels of NPK 15:15:15 (0, 100, 200 and 300 kg ha-1). Data collected were subjected to analysis of variance (ANOVA) and means were separated using new Duncan multiple range test at 5 % level of probability. Results obtained showed signifi- cant effects of Zn and NPK rates on plant height, number of leaves, yield per plot and yield per hectare. The application of 15 kg ha-1 Zn and 300 kg ha-1 (15:15:15) NPK resulted in high yield and high zinc content of seeds. Key words: bio-fortification; sesame; growth; yield Učinki biofortifikacije semen s cinkom in uporaba NPK gno- jil na rast in pridelek sezama (Sesamum indicum L.) Izvleček: Tla v južno gvinejski savanski coni Nigerije so nerodovitna zaradi prekratkih obdobij prahe in stalne up- orabe mineralnih gnojil, kar s časom povzroča obubožanje tal na mikrohranilih kot je zink. Poljski poskus je bil izveden na učnem in raziskovalnem posestvu Univerze v Ilorinu, Nigerija, v rastnih sezonah 2016 in 2017 za ovrednotenje učinka cinka in NPK gnojil na rast in pridelek ter vsebnost cinka v semenih sezama. Poskus je bil faktorski, popolni bločni poskus (RCBD) s tremi ponovitvami. Obravnavanja so obsegala štiri ravni cinka kot ZnSO4 (0, 5, 10 in 15 kg ha -1) in štiri ravni NPK 15:15:15 (0, 100, 200 in 300 kg ha-1). Variabilnost zbranih podatkov je bila analizirana z ANOVA, poprečja so bila ločena z multiplim Duncanovim testom rangov pri 5 % verjetnosti. Izsledki so pokazali značilne učinke Zn in NPK na višino rastlin, število listov, pridelek na ploskev in pridelek na hektar. Uporaba 15 kg ha-1 Zn in 300 kg ha-1 (15:15:15) NPK je dala največji pridelek in največjo vsebnost cinka v semenih. Ključne biofortifikacija; sezam; rast; pridelek Acta agriculturae Slovenica, 117/1 – 20212 E. K. EIFEDIYI et. al 1 INTRODUCTION Low input agriculture is no longer sustainable due to increasing population, thus the use of inorganic fer- tilizer for the improvement of growth and yield to pre- vent hunger and starvation has become imperative (Gra- ham et al., 2001), hence, farmers in the Guinea savanna zone of Nigeria cultivate their crops intensively relying on nitrogenous and phosphate fertilizers to increase crop growth and yield. However, the continuous use of inorganic fertilizer can deplete the soil of valuable mi- cronutrients and sometimes can cause zinc antagonism (Haas et al., 2016) and these valuable micro nutrients are vital for crop growth and human health. Subsequently, soils low in mineral composition produce seeds and veg- etative parts with even lower mineral contents. Roughly one third of the world’s population suffers from hidden hunger which results from deficiencies of vitamins (par- ticularly A and C) and minerals (such as zinc, iodine and iron), which leads to illness that is more severe in chil- dren than adults (GNR, 2003; Kennedy et al., 2004). Zinc is involved in many enzymatic reactions in crops such as carbohydrate metabolism and protein synthesis and its shortfall results in discoloration of leaves, impaired growth and delayed sexual development in human (Haas et al., 2016; Vootman & Bindraban, 2015; Haas et al., 2012). Cakmak (2008) reported that over thirty percent of the diet of the world`s population is deficient in zinc. This is attributed to crop production in areas with low soil mineral availability and consumption of crops with inherently low mineral content, low fish intake and other animal products in their diet due to poverty (Graham et al., 2001). Majority of rural dwellers in the southern Guinea savannah zone of Nigeria depends on cereals as staple food, have no access to fruits, vegetables and can- not afford fish and animal products because of poverty. Black et al. (2013) had reported severe zinc deficiency in children under five years old in some parts of Africa including Nigeria due to low zinc in their soil. In addi- tion, this low zinc concentration in soil solution is due to its limited mobility in the soil (Cakmak, 2014); hence difficulty in plant roots to absorb it for its growth, devel- opment and its availability in plant tissues. One of the ways of overcoming this pitfall is through biofortification using zinc. Biofortification is a process of making essential nu- trients bio-available to crops during crop growth using agronomic and genetic methods (Bouis et al., 2011). Sev- eral methods have been used for the biofortification of crops, namely; breeding and agronomic approach using inorganic fertilizer (Cakmak, 2008). Rice has been bio- fortified with zinc in Bangladesh using breeding method (Chowdhury, 2014) whereas Zou et al. (2012) reported an increase in the concentration of zinc by up to 20 parts per million in wheat seeds in India and Pakistan by foliar spray, but in maize and rice, foliar spray of zinc resulted in only a small effect (Phattarakul et al., 2012). Suresh et al. (2013) reported a significant improvement in growth and yield of sesame using 25 kg ha-1 ZnSO4 but no report of its concentration on the seeds. However, biofortified staple foods cannot provide high level of minerals and vitamin supplements compared to industrially fortified foods, but they can help by increasing the daily adequacy of micronutrient intake in humans (Bouis et al., 2011). The biofortification of local staples will help to bridge this micronutrients and vitamins gaps (Hotz & McClafferty, 2007) thereby reducing disease prevalence among the rural populace. The soils of the Guinea savannah zone of Nigeria are deficient in zinc (Aduloju, 2000) and farmers hardly apply zinc fertilizer. Developing biofortified crops can improve the efficiency of growth in these soils with depleted or very low mineral composition (Borg et al., 2009). Cakmak (2008) reported the daily requirement of zinc for different age groups (10 mg for children, 12 mg for women and 15 mg for men) whereas Pavlotska (2007) noted that nursing mothers require 25 mg per day of zinc. The objective of this study was to determine the ef- fect of zinc and NPK fertilizer on the growth and yield of sesame. 2 MATERIALS AND METHODS 2. 1 THE EXPERIMENTAL SITE Field experiment was conducted at the Teaching and Research Farm of the University of Ilorin, Ilorin, Nigeria during the 2016 and 2017 cropping seasons. The study area is situated in the southern Guinea savannah on (Latitude 04°351N and Longitude 08°291E, 307 m above sea level). The average rainfall of the area is be- tween 1000–1540 mm which is bimodal in occurrence. 2. 2 EXPERIMENTAL LAYOUT AND SOWING OF THE SEEDS The land was ploughed and harrowed before mark- ing out into plots. The size of each plot was 3 m × 3 m with a 0.50 m avenue between the plots. The seeds of ses- ame (Ex-Sudan cultivar) which were obtained from the National Cereal Research Institute of Nigeria, Badeggi, Niger State, Nigeria were sown on the 15th of July, 2016 and repeated on July 15; 2017 using the drilling method at a depth of 2.5 cm and the seeds were covered lightly with soil to prevent desiccation by sunlight. The seed- Acta agriculturae Slovenica, 117/1 – 2021 3 The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) lings were later thinned three weeks after sowing to two plants per stand at a spacing of 30 × 30 cm between and within rows. 2. 3 EXPERIMENTAL DESIGN The experiment was laid out in a factorial ar- rangement fitted into a randomized complete block de- sign (RCBD) replicated thrice. The factors were NPK (15:15:15) fertilizer rates (0, 100, 200, 300 kg ha-1) and ZnSO4 levels (0, 5, 10, 15 kg ha -1). The NPK fertilizer was applied 10 cm away from the plant using the side band placement method while the zinc sulphate was applied mid-morning by foliar method using a knap sack sprayer at three weeks after sowing. Zinc is commonly applied to crops as ZnSO4 (Cakmak, 2008), as this will help to in- crease the micronutrient content in edible parts (Prasad et al., 2015). 2. 4 SOIL AND SEED ANALYSES Soil samples from the experimental plot were col- lected from the top soil at a depth of 0–30 cm from a 2.5 x 2.5 m grid, bulked; then a composite was taken for physical and chemical analyses before and at the end of the cropping seasons. The soil samples collected were air dried ground and passed through a 2 mm sieve. The sieved soil samples were taken to the laboratory for chemical analysis, as described by Carter & Gregorich (2007). Soil organic carbon was determined by the proce- dure of Walkley & Black using the dichromate wet oxida- tion method (Nelson & Sommers, 1996). Organic matter was estimated by multiplying carbon (C) by 1.724. Total nitrogen was determined by Micro-Kjeldahl digestion and distillation techniques (Bremner, 1996), and avail- able phosphorus was determined following Bray No 1 (1N NH4F + 0.5N) extractant by vanado-molybdo-phos- phoric acid method (Kuo, 1996), Soil pH was measured (soil: water ratio, 1:2) using a glass electrode; particle-size analysis was done using the hydrometer method (Gee & Or, 2002). Textural class was determined using a textural triangle (Brady & Weil, 1999; Hunt & Gilkes, 1992) and extraction of exchangeable bases was done by using IN ammonium acetate, exchangeable K and Na were de- termined by using flame photometry while calcium and magnesium were analyzed by atomic absorption spectro- photometry. The total zinc concentrations in the seed samples were determined by wet digestion method as described by Minaleshewa et al. (2010) and concentrations in the extracts were determined by flame atomic absorption Spectrometry while its concentration in the soil was de- termined by the method of (Maclean & Langille, 1976). 2. 5 DATA COLLECTION AND ANALYSIS Data were collected on growth parameters (plant height, number of leaves, and leaf area index), dry matter accumulation, yield parameters (number of capsules per plant, length of capsule, yield per net plot and yield per hectare). The plant height was assessed by measuring the plant from the ground level to the terminal point using a measuring tape, while the number of leaves was assessed visually by counting the green leaves of the five tagged plants in the net plot (60 plants) whereas the leaf area was calculated based on the work of Silva et al. (2002) using: S = 0.3552 x C2 where S = leaf area in cm2 and C = leaf longitudinal length x breadth while the leaf area index was estimated as; The data on yield components were collected on number of capsules per plant, length of capsule, seed mass per net plot and seed mass per hectare. Data on number of capsules per plant was estimated by counting, the length of capsule was calculated by the use of digital Vernier caliper, while the yield per net plot (60 plants) was carried out by weighing the seeds by using a sensitive balance whereas the yield per hectare was extrapolated from the yield per net plot and the data collected were subjected to general analysis of variance (ANOVA) using Genstat statistical software (17th edition). Treatment dif- ferences were compared using the new Duncan Multiple range test at 5 % level of probability. 3 RESULTS AND DISCUSSION 3.1 PHYSICAL AND CHEMICAL PROPERTIES OF SOIL The data on physical and chemical properties of soil of the experimental site is presented in Table 1. The result showed that the soil pH was slightly acidic to moderately acidic before and after the experiment. The organic mat- ter content was low throughout the experimental peri- od. The chemical soil properties such as total nitrogen content, available phosphorus and exchangeable cations such as potassium, calcium and magnesium were very Acta agriculturae Slovenica, 117/1 – 20214 E. K. EIFEDIYI et. al low; and available micronutrients such as zinc was very low before and at the end of the cropping seasons. These low soil nutrients of the study site are below the critical levels for optimum crop growth and yield using soil data rating as suggested by Linsday & Norvell (1978); FDALR (1990) which is as follows; organic carbon, 1–1.4 %, to- tal nitrogen, 0.151-0.200 %; available phosphorus, 7.0– 20 mg kg-1; exchangeable potassium, 0.3–0.6 cmol kg-1; exchangeable calcium, 5-10 cmol kg-1; exchangeable mag- nesium 1-3 cmol kg-1 and available zinc 0.6 –1.0 cmol kg-1. The low nutrient status of the experimental site, which is typical of Alfisols could be attributed to burning of crop residues, short fallow period and seasonal bush fire which deprives the soil of valuable organic matter. This is the reason why the cultivation of crops is done solely with the application of external inputs such as nitrogenous ferti- lizer which has led to the depletion of valuable micronu- trients such as zinc over time. Eifediyi et al. (2016) had reported that soils of the study area are deficient in macro and micro nutrients which are essential for crop growth and development. 3.2 METEOROLOGICAL DATA The rainfall data of the study site for 2016 and 2017 is presented in Figure 1. The data on rainfall indicated that no precipitation was experienced in the month of February during the two cropping seasons. But in 2016, September recorded the highest rainfall of 332 mm while the highest rainfall in 2017 was recorded in the month of August (302.7 mm). The rainfall in the study area has changed drastically in the past few decades and thus in sharp contrast to the rainfall pattern in the past which is bimodal in nature which starts in late march with the first peak in July, an August break and the second peak in Sep- tember. This variation in rainfall could be attributed to the effect of climate change (Odjugo, 2011). The data on tem- perature is shown in Figure 2. The highest temperature in 2016 was experienced in the month of February while in 2017; the highest temperature was recorded in the month of March. The temperature of the study area has been on the increase in the past few years which can also be attrib- uted to the effect of climate change (Ajibade, 2002). The relative humidity is shown in Figure 3. The high- est relative humidity was recorded in the month of Sep- tember in 2016 while in 2017, it was in August. 3.3 GROWTH PARAMETERS The effect of Zn and NPK fertilizer application rate Year Soil pH Org C (%) Org. Matter (%) Total N mg g-1 P mg g-1 K cmol kg-1 Ca cmol kg-1 Mg cmol kg-1 Zn mg kg-1 2016a 6.1 0.82 1.41 0.07 6.67 0.31 5.05 0.28 0.56 2016b 5.9 0.54 0.93 0.04 5.42 0.32 4.39 0.21 0.54 2017 5.8 0.50 0.86 0.03 3.28 0.30 4.23 0.19 0.53 Table 1: Physical and chemical properties of soil of the experimental site before and after cropping in 2016 and 2017 a Before initial cropping, b. End of first year cropping. Figure 1: Rainfall pattern of the study area in 2016 and 2017. Acta agriculturae Slovenica, 117/1 – 2021 5 The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) on the plant height and number of leaves of sesame at 4, 6, 8 and 10 weeks after sowing (WAS) are presented in Tables 2 and 3. The data revealed that the zinc applied at the rate of 15 kg ha-1 produced the highest plant height and number of leaves at the four sampling periods. There was no significant difference observed in the first season (2016) but in the second season (2017), significant dif- ferences (p < 0.05) were observed between the different rates. A trend was observed when the different rates of application of NPK 15:15:15 fertilizer was applied at the different sampling dates of 4, 6, 8 and 10 WAS with the 300 kg ha-1 producing the tallest plants and the highest number of leaves. No significant difference was observed in the first year (2016), but in the second season (2017), significant differences (p < 0.05) were observed at the four sampling periods. The positive response of crop to the application of fertilizer most especially zinc in the increase in growth attributes could be attributed to the important role it plays in enzymatic reactions, regula- tion of auxin synthesis (Marschner,1995) metabolic pro- cesses, and oxidation-reduction reactions and for many enzymes which are needed for nitrogen metabolism (Ha- feez et al., 2012; Omidian et al., 2012). The effect of Zn and NPK fertilizer application rate on the leaf area index of sesame at 4, 6,8 and 10 weeks after sowing (WAS) is presented in Table 4. The data re- vealed that the zinc applied at the rate of 15 kg ha-1 pro- duced the highest leaf area index at the four sampling periods, significant differences (p < 0.05) were observed between the different rates in the two seasons at 4 WAS. There was no significant difference observed in the first and second seasons at the 6, 8 and 10 WAS. A similar trend was observed when NPK fertilizer was applied, with the 300 kg ha-1 producing the highest leaf area in- dex throughout the period of assessment in both seasons. Significant differences (p < 0.05) were observed between the different rates in the two seasons at 4 WAS. Increase in leaf area index with increase in rate of zinc fertilizer Figure 2: Temperature pattern of the study site in 2016 and 2017. Figure 3: Relative humidity pattern of the study site in 2016 and 2017. Acta agriculturae Slovenica, 117/1 – 20216 E. K. EIFEDIYI et. al application may be due to the fact that zinc exerts a great influence in basic plant life processes, such as nitrogen metabolism, uptake of nitrogen and protein quality, photo synthase-chlorophyll synthesis, carbon anhydrase activity, resistance to abiotic and biotic stresses, protec- tion against oxidative damage (Alloway, 2004; Potarzy- cki & Grzebkisz., 2009;Tekale et al., 2009). The increase in the leaf area index as a result of zinc application is in agreement with the report of Badshah et al. (2013) who reported significant increase in leaf area with foliar ap- plication of zinc. The effect of Zn and NPK fertilizer application Treatments 4 WAS 6 WAS 8 WAS 10WAS Season 1 2 1 2 1 2 1 2 Zn (kg ha-1) 0 34.0 28.17 76.2 73.1 98.2 90.6 107.1 100.0 5 39.4 30.75 81.4 75.2 102.0 93.3 110.9 102.8 10 40.9 32.08 81.6 85.5 103.4 110.8 114.9 120.8 15 46.8 34.67 87.4 80.2 108.3 102.3 117.6 114.4 Sed (p < 0.05) ns 0.740 Ns 5.21 ns 6.27 ns 5.19 NPK (kg ha-1) 0 29.9 18.25 69.3 50.8 86.3 69.3 96.7 80.8 100 40.0 25.00 79.4 79.8 101.1 104.3 111.9 112.7 200 40.4 36.50 84.2 87.4 107.9 109.6 116.7 117.5 300 50.8 45.92 93.8 95.8 116.7 113.7 125.2 124.0 Sed (p < 0.05) 6.48 0.938 8.56 6.28 7.68 6.40 7.01 6.15 Interaction ns 1.785 Ns 12.06 ns 12.73 ns 11.85 Table 2: Effects of Zn and NPK fertilizer on the plant height of sesame at 4, 6, 8 and 10 weeks after sowing Season 1 = data on 2016 cropping season; season 2 = data on 2017 cropping season Treatments 4 WAS 6 WAS 8 WAS 10WAS Season 1 2 1 2 1 2 1 2 Zn (kg ha-1) 0 17.58 24.0 41.3 41.3 55.0 53.3 62.7 60.8 5 18.92 24.0 41.4 41.4 57.7 58.1 66.7 64.5 10 21.92 28.42 52.1 52.1 66.2 65.8 76.3 74.2 15 22.83 30.42 57.1 57.1 68.8 78.1 77.6 75.6 Sed (p < 0.05) ns 1.28 3.63 3.63 3.77 3.13 3.89 3.89 NPK (kg ha-1) 0 12.42 13.00 28.1 28.1 45.1 49.3 54.9 53.1 100 20.00 22.33 53.2 53.2 66.2 65.2 72.3 70.3 200 20.25 30.33 54.1 54.1 64.0 68.2 74.6 72.2 300 28.58 41.17 56.4 56.5 71.6 72.5 81.4 79.4 Sed (p < 0.05) 2.11 1.28 3.63 3.63 3.77 3.13 3.89 3.89 Interaction ns 2.57 Ns 7.26 7.78 6.27 ns ns Table 3: Effects of Zn and NPK fertilizer on the number of leaves of sesame at 4, 6, 8 and 10 weeks after sowing Season 1 = data on 2016 cropping season; season 2 = data on 2017 cropping season Acta agriculturae Slovenica, 117/1 – 2021 7 The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) rate on the dry matter accumulation of sesame at 4, 6, 8 and 10 weeks after sowing (WAS) is presented in Table 5. The data revealed that the zinc applied at the rate of 15 kg ha-1 accumulated the highest mass at the four sam- pling periods. Significant differences (p < 0.05) were ob- served between the different rates at 4, 6 and 10 WAS, but there was no significant difference observed in the first and second seasons at the 8 WAS. A trend was observed when the different rates of application of NPK 15:15:15 fertilizer was applied at the different sampling dates of 4, Treatments 4 WAS 6 WAS 8 WAS 10WAS Season 1 2 1 2 1 2 1 2 Zn (kg ha-1) 0 2.75 2.64 3.75 3.64 4.47 4.58 4.62 4.73 5 2.76 2.65 3.90 3.75 4.50 4.61 4.66 4.77 10 2.94 2.83 3.98 3.86 4.52 4.63 4.70 4.81 15 3.32 3.22 4.49 4.38 6.01 6.12 5.10 5.21 Sed (p < 0.05) 0.386 0.386 ns ns ns ns ns ns NPK (kg ha-1) 0 2.17 2.06 3.59 3.86 4.71 4.82 4.51 4.62 100 2.70 2.59 3.63 3.52 4.79 4.90 4.56 4..67 200 2.82 2.72 4.01 3.90 4.96 5.07 4.82 4.93 300 4.08 3.97 4.88 4.77 5.05 5.16 5.19 5.30 Sed (p < 0.05) 0.386 0.386 ns ns ns ns ns ns Interaction 0.771 0.771 ns ns ns ns ns ns Table 4: Effects of Zn and NPK fertilizer on the leaf area index of sesame at 4, 6, 8 and 10 weeks after sowing Season 1 = data on 2016 cropping season; season 2 = data on 2017 cropping season Treatments 4 WAS 6 WAS 8 WAS 10 WAS Season 1 2 1 2 1 2 1 2 Zn (kg ha-1) 0 0.56 0.58 1.61 1.60 2.04 1.79 2.62 2.50 5 0.58 0.63 1.68 1.68 2.87 2.36 3.84 3.72 10 0.71 0.68 2.04 1.89 2.61 2.62 3.91 3.79 15 0.71 0.73 1.90 2.03 2.89 2.64 3.97 3.85 Sed (p < 0.05) 0.060 0.014 0.187 0.187 ns ns 0.355 0.355 NPK (kg ha-1) 0 0.45 0.41 1.63 1.63 2.29 2.04 3.08 2.96 100 0.58 0.56 1.69 1.69 2.65 2.40 3.42 3.30 200 0.61 0.71 1.84 1.84 2.73 2.48 3.59 3.47 300 0.91 0.95 2.05 2.05 2.75 2.50 4.25 4.13 Sed (p < 0.05) 0.060 0.014 0.187 0.187 0.320 0.320 0.355 0.355 Interaction 0.119 0.029 0.374 0.374 ns ns 0.710 0.710 Table 5: Effects of Zn and NPK fertilizer on the dry matter accumulation (g) of sesame at 4, 6, 8 and 10 weeks after sowing in the 2016 and 2017 cropping seasons Season 1 = data on 2016 cropping season; season 2 = data on 2017 cropping season Acta agriculturae Slovenica, 117/1 – 20218 E. K. EIFEDIYI et. al 6, 8 and 10 WAS with the 300 kg ha-1 producing the high- est dry matter mass. However, significant differences (p < 0.05) were observed at the four sampling periods. There was also an increase in the dry matter accumulation and dry matter is an important parameter in the study of crop canopies. Increase in dry matter as the rate of application of Zn and NPK fertilizer increased could be attributed to the role performed by nitrogen and potassium in im- proving plant metabolism, enhancing plant meristematic activity and increasing the rate of photosynthesis (Allo- way, 2004). 3.4 YIELD AND YIELD COMPONENTS OF SESA- ME The effects of zinc and NPK fertilizer on the yield components of sesame is presented in Table 6. The data showed that the zinc applied at the rate of 15 kg ha-1 produced the highest number of capsules per plant and length of capsules which were significantly (p < 0.05) dif- ferent from the other rates in the two years of study (2016 and 2017). The application of 300 kg ha-1 also produced the highest number of capsules and length of capsules in the two years of study which were significantly different (p < 0.05) from the other rates. There was also a signifi- cant zinc and NPK fertilizer interaction. The data on the yield of net plot and yield per hectare showed a similar trend as what was observed in the num- ber of capsules per plant. The highest net yield of (452 g and 454 g) and yield per hectare (502.2 kg and 505.1 kg) were produced by applying zinc at the rate of 15 kg ha-1 which were significantly different from the other rates in 2016 and 2017 cropping seasons respectively. Similarly, the application of NPK fertilizer at 300 kg ha-1 gave the highest yield (net plot, 555.5 g and 554.3 g in 2016 and 2017 respectively) while the yield per hectare (617.1 kg and 615.8 kg in 2016 and 2017 cropping seasons respec- tively) which were significantly different from the other fertilizer rates. There was also a significant interaction between the zinc and NPK fertilizer application. Increasing the rate of zinc from 5 to 10 and 15 kg ha_1, significantly increased yield and yield components. This is in agreement with Cakmak, (2000) and Omidian et al. (2012) who reported that zinc application increased the yield components of sesame as a result of the role it plays in cell division, cell enlargement and synthe- sis of protein. Zinc also regulates the membrane func- tion and provides resistance to environmental stress in crop plant. The foliar spray of Zn increased the number of capsules and number of seeds per capsule (Bakry et al., 2012). This may be due to the involvement of zinc in photosynthesis, chlorophyll production, pollen function and fertilization (Pandey et al., 2006). Saedi (2002) stated that the foliar application of zinc promoted the vegetative and flowering stages of crops due to increase in the plant metabolism and photosynthesis. This increase in sesame yield may also be due to the abundant efficacy of en- Treatments Number of capsules Length of capsules (cm) Yield of net plot (g) Yield (kg ha-1) Zinc concentration (mg kg-1) Season 1 2 1 2 1 2 1 2 1 2 Zn (kg ha-1) 0 61 62 3.23 3.27 353.6 354.1 392.9 393.4 9.48 8.93 5 62 64 3.44 3.33 378.2 380.4 420.2 422.6 14.20 16.53 10 75 76 3.33 3.44 411.4 412.2 457.1 458.0 16.73 20.45 15 100 101 3.41 3.52 452.0 454.6 502.2 505.1 20.55 21.95 Sed (p < 0.05) 8.55 8.63 0.904 0.094 10.16 11.20 11.29 12.45 0.979 0.637 NPK ((kg ha-1) 0 54 56 3.13 3.22 238.7 242.6 265.2 269.6 11.30 9.45 100 74 74 3.35 3.38 368.2 370.0 409.1 411.1 16.33 17.59 200 75 76 3.42 3.45 432.9 434.4 481.0 482.7 16.60 20.08 300 95 96 3.50 3.50 555.4 554.3 617.1 615.8 17.75 20.78 s.e.d (p < 0.05) 8.55 8.63 0.904 0.094 10.16 11.20 11.29 12.45 0.979 0.638 Interaction 17.09 17.27 0.181 0.189 20.32 22.40 22.57 24.89 1.957 1.275 Table 6: Effects of Zn and NPK fertilizer on the yield components of sesame in the 2016 and 2017 cropping seasons Season 1 = data on 2016 cropping season; season 2 = data on 2017 cropping season Acta agriculturae Slovenica, 117/1 – 2021 9 The effects of zinc biofortification of seeds and NPK fertilizer application on the growth and yield of sesame (Sesamum indicum L.) zyme activities which influence plant pigments because zinc is an important component of all classes of enzymes (CIMMYT, 2006; Malakouti, 2007). Mohsen et al. (2009) also suggested that foliar spray of zinc increased the seed yield of safflower (Carthamus tinctorius L.). Similarly, foliar application of zinc sulfate significantly improved the seed yield of canola (Brassica napus L.) (Omidbeigi, 2005), safflower (Carthamus tinctorius L.) (Movahedy et al., 2009), lentil (Lens culinaris Medik.) (Nakhzari et al., 2011) and soybean (Glycine max (L.) Merr.) (Jamson et al., 2009). Zinc concentration in sesame seed also increased as the rate of application of Zn and NPK fertilizer increased. This increase in the zinc concentration may be attributed to the fact that zinc has favorable effects on the metabo- lism of plant which might be responsible for greater me- tabolite accumulation in the reproductive organs (Babae- ian et al.2011). 4. CONCLUSION The study revealed that the application of zinc at 15 kg ha-1 and NPK 15:15:15 at the rate of 300 kg ha-1 improved the growth, yield attributes and zinc concen- tration of sesame seeds. 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Effect of peanut mixed cropping with graminaceaous species on micronutrient concentra- tions and iron chlorosis of peanut plants grown in a calcar- eous soil. Journal of Plant and Soil, 306, 23-36. https://doi. org/10.1007/s11104-007-9484-1 Acta agriculturae Slovenica, 117/1, 1–6, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1622 Original research article / izvirni znanstveni članek Induction of defence related enzymes and biocontrol efficacy of Tricho- derma harzianum in tomato plants infected with Fusarium oxysporum and Fusarium solani Tavga Sulaiman RASHID 1, Sirwa Anwar QADIR 1, Hayman Kakakhan AWLA 2, 3 Received April 18, 2020; accepted January 27, 2021. Delo je prispelo 18. aprila 2020, sprejeto 27. januarja 2021. 1 Salahaddin University, College of Agricultural Engineering Sciences, Department of Plant Protection, Erbil, Iraq 2 Erbil Polytechnic University, Khabat Technical Institute, Department of Plant Protection, Iraq 3 Corresponding author, e-mail: hayman.awla@epu.edu.iq Induction of defence related enzymes and biocontrol efficacy of Trichoderma harzianum in tomato plants infected with Fu- sarium oxysporum and Fusarium solani Abstract: Fusarium wilt of tomato plants caused by Fu- sarium oxysporum Schlecht. emend. Snyder & Hansen and Fu- sarium solani (Mart.) Sacc. are serious problem limiting tomato production worldwide. Biological control has emerged as one of the most promising alternatives to chemical fungicides. The biological control capability of a T. harzianum isolate against F. solani and F. oxysporum has been investigated. It inhibited col- ony growth of two Fusarium species by more than 80 % in dual culture tests. Results of greenhouse experiments revealed that disease severity in the tomato plants co-inoculated with T. har- zianum was significantly lower than plants only infected with the Fusarium pathogens. Tomato plants inoculated with the an- tagonistic T. harzianum isolate, showed enhanced peroxidase and polyphenol oxidase activities in greenhouse experiments and increased resistance to F. solani and F. oxysporum. The T. harzianum isolate indirectly affected the Fusarium pathogens by enhancing plant defence. Key words: fungal pathogens; antagonistic fungus; perox- idase; polyphenol oxidase; plant growth and biological control Vzpodbuditev aktivnosti encimov povezanih z obrambo in učinkovitost biokontrole z glivo Trichoderma harzianum Rifai v paradižniku okuženem z glivama Fusarium oxyspo- rum Schlecht. emend. Snyder & Hansen in Fusarium solani (Mart.) Sacc. Izvleček: Fuzarijska venenja paradižnika, ki jih povzroča- ta glivi Fusarium oxysporum Schlecht. emend. Snyder & Hansen in Fusarium solani (Mart.) Sacc. so resen problem, ki omejuje svetovno pridelavo paradižnika. Biološka kontrola se je poka- zala kot najbolj obetajoča alternativa kemičnim fungicidom. V raziskavi je bila preučevana sposobnost biološkega uravnavanja gliv F. solani in F. oxysporum z izolati glive Trichoderma harzia- num. Izolati so zavrli rast kolonij obeh vrst iz rodu Fusarium za več kot 80 % v preiskusih dvojnih kultur. Izsledki iz poskusov v rastlinjaku so pokazali, da je bila obolelost pradižnika značil- no manjša, če je bil ta inokuliran hkrati z obema patogenima glivama iz rodu Fusarium in z glivo T. harzianum. Ratline pa- radižnika, ki so bile inokulirane z izolati antagonistične glive T. harzianum so imele v poskusih v rastlinjaku povečani aktivno- sti peroksidaze in polifenol oksidaze ter povečano odpornost proti patogenima glivama F. solani in F. oxysporum. Izolati iz glive T. harzianum so neposredno vplivali na patogena iz rodu Fusarium s povečanjem obrambe rastlin. Ključne besede: paradižnik; patogene glive; antagonistič- ne glive; rast in biološka kontrola; peroksidaza; polifenol oksi- daza Acta agriculturae Slovenica, 117/1 – 20212 T. S. RASHID et al. 1 INTRODUCTION Tomato plants can be attacked by different soil- borne fungi, which cause severe diseases such as wilt and root rot (Morsy et al., 2009). Fusarium species are the most common pathogens, which can live in organic materials and nearly all soil types (Boyaci et al., 2010). They can attack the vascular system in infected plants, block water transport through xylem by inducing vessel plugging, leading to foliage wilt and finally plant death (Portal et al., 2018; Malandrakis et al., 2018). The attempts to minimise the use of fungicides make it necessary to develop economical and effective methods to obtain biopesticides that are based on endog- enous microorganisms (de los Santos-Villalobos et al., 2012). Trichoderma (Hypocreales) accommodates vari- ous soil-borne and pathogen-antagonistic species (Ver- ma et al., 2007). Trichoderma harzianum is well known for its antagonistic effect on pathogenic fungi. It is the most potent biocontrol agent that inhibits the growth of Fusarium species. Mechanisms of biocontrol agent are based on antifungal metabolites, mycoparasitism, com- petition for nutrients and induced resistance (Perello et al., 2003; Silva et al., 2019). Moreover, an increased activity of polyphenol oxi- dase and peroxidase in response to the infection by the pathogen is considered to play an active role in contrib- uting to disease resistance in certain plant hosts (Vid- hyasekaran, 2004). Pradeep and Jambhale (2002) and Vidhyasekaran (2004) observed that plants show in- creased activities of polyphenol oxidases and peroxidases in response to pathogen infections and the ability of a plant to resist disease. The present study investigated the effectiveness of T. harzianum to induce systemic resist- ance against Fusarium solani and Fusarium oxysporum when infecting tomato plants. 2 MATERIALS AND METHODS 2.1 ANTAGONISTIC ACTIVITY Trichoderma harzianum isolate UPM40 was ob- tained from the plant protection department, faculty of agriculture, University Putra Malaysia and was evaluated in vitro for its antagonistic activity against two Fusarium isolates in dual culture method (Rahman et al., 2009). DNA barcoding strategies based on ITS sequences were used to identify one of the Fusarium as a member of the F. solani (KM039055) and the other as a member of the F. oxysporum species complex (KM039054) (from previous study Rashid et al., 2016). Isolate UPM40 was identified as a member of Trichoderma harzianum species complex on the basis of morphological characters. Discs of 6 mm diameter of the antagonist and pathogens were cut from the edge of actively growing colonies on potato dextrose agar (PDA DifcoTM) and placed at opposite sides (4.5 cm from each other) on PDA plates. Each antagonist/patho- gen combination was set up in triplicates. The plates were incubated at 24 ± 2 °C. The antagonistic effect of the test fungus was estimated by measuring their radial growth in comparison to control plates using the following for- mula: I = [(C-T) / C] X 100 where: I = % of inhibition in the mycelia growth, C = the growth of pathogens in control plates and T = the growth of pathogens in the dual culture plates. 2.2 SOIL PREPARATION AND EXPERIMENTAL DESIGN Soil comprising clay, sand and organic matter (1:1:1 v:v) from Erbil Governorate (pH 5.5-6) was air dried and sieved (5 mm), sterilised twice in an autoclave at 121 °C for one hour and allowed to cool for 24 hours before use. The tomato (‘Baccarat 322’) seeds were surface dis- infected by soaking them in 95 % ethanol for 10 seconds, followed by 3 % sodium hypochlorite for 1 minute and then washed 6 times with sterile distilled water. Two tomato seeds were planted per plastic cell in seedling growing trays. The seedlings were trimmed down to one seedling per cell after two weeks. The one-month-old to- mato plants were then moved to clean pots (two plants/ pot). On a daily basis, these plants were watered with tap water. The independently repeated experiment was con- ducted in a completely randomised design. 2.3 IN VIVO TEST A Neubauer haemocytometer was used to adjust the T. harzianum spore concentration to 2 × 105 spore/ml (working solution). The soil with one-month-old tomato plants was drenched with 100 ml of working solution (Mahato et al., 2018). After 24 hours, the same amount of 2 × 105 spore/ml pathogen suspension was added to the soil per pot. 2.3.1 Chlorophyll content measurement The chlorophyll content was measured in ten repeti- tions per leaf (5 plants per treatment) with a CL-01 Chlo- rophyll Content Metre after 30 days of inoculation. This device measured the relative chlorophyll content via dual wavelength optical observance (620 and 940 nm). Acta agriculturae Slovenica, 117/1 – 2021 3 Induction of defence related enzymes and biocontrol efficacy ... tomato plants infected with Fusarium oxysporum and Fusarium solani 2.3.2 Sample collection for the biochemical analysis The roots of the treated and untreated tomato plants were collected 10, 20 and 30 days after the treatment ap- plication; they were washed in running tap water and stored in a deep freezer (-80 °C) until they were used for the biochemical analysis. 2.3.3 Total protein content measurement Protein extracts were generated from roots and pro- tein concentrations were determined using the method described by Arora and Wisniewski (1994), of which the latter was based on Bradford assays. 2.3.4 Quantification of polyphenol oxidase (PPO) ac- tivity The reaction mixture consisted of 0.5 ml of the en- zyme extract and 2.3 ml of 0.1M phosphate buffer (pH = 6.1). Both were mixed in a cuvette and adjusted to the zero absorbance of a spectrophotometer (Mahadevan and Sridhar, 1982). Catechol solution (0.1 M, 0.2 ml) was added and the reactants were quickly mixed. The enzyme activity, measured as the change in absorbance per min- ute (ÄA/min) at 400 çm, was measured immediately after the addition of the catechol solution. 2.3.5 Quantification of peroxidase (PO) activity Peroxidase activity was determined according to Sreedevi et al. (2011). The peroxidase enzyme activity was determined from the roots of the un-inoculated and inoculated tomato plants. About 0.5 g of freshly har- vested material was ground in a pre-chilled mortar with 20 ml of 0.1 M cold ice phosphate buffer (pH 7.1) and centrifuged at 2000 rpm for 10 minutes. The supernatant was made up to 25 ml and used for the assay. Freshly pre- pared pyrogallol (0.2 M) reagent (0.1 ml), 1.0 ml of the enzyme extract, and 1.4 ml of the 0.1M phosphate buffer (pH, 7.1) were mixed in a cuvette, and the mixture was immediately adjusted to the zero absorbance of a spec- trophotometer. The H2O2 solution (0.5 ml of 0.01M) was added to it, and the content was mixed by inverting the cuvette. The enzyme activity was recorded according to the changes in absorbance per minute (ÄA /min/ä) at 430 cm. 3 RESULTS AND DISCUSSION 3.1 ANTAGONISTIC ACTIVITY The results indicated that Trichoderma harzianum (T. h) reduced the radial growth of F. solani (F. s) colony by 82.31 % and F. oxysporum (F. o) by 80.25 %. 3.2 IN VIVO EXPERIMENT Trichoderma treated plants showed increased plant height, fresh shoot mass and root dry mass when co- inoculated with F. o (20.6 cm, 0.79 g and 0.15 g) or F. s (27 cm, 0.66 g and 0.17 g). Plants treated only with F. o had 10.17 cm and 14.00 cm height (0.31 g) shoot mass and (0.06 g) root mass. In addition, plants inoculated only with T. h showed the highest mean values for plant height, shoot dry mass and root dry mass (32.33  cm, 1.02  g and 0.18  g) compared to other treatments and control plants (Table 1). The increased growth response caused by Trichoderma isolate may be due to the modifi- cation of the rooting system (Chao et al., 1986). The re- sults further indicate that T. harzianum had a significant role in reducing disease incidence. Therefore, the results of the study are similar to the findings of Abd- El-Khair et al. (2011), Otadoh et al. (2011) and Alwathnani et al. (2012). Treatments Shoot height (cm) Shoot dry mass (g) Root dry mass (g) Control 22.67a 0.76a 0.14a T. h 32.33b 1.02c 0.18b F. o 10.17c 0.17d 0.06c F. s 14.00d 0.31e 0.06c T. h + F. o 20.67e 0.79a 0.15d T. h + F. s 27.00f 0.66b 0.17be Table 1: Effect of T. harzianum, F. oxysporum and F. solani on shoot height (cm), shoot and root dry mass (g) on tomato plants C: control; F. o: F. oxysporum; F. s: F. solani and T. h: T. harzianum. Acta agriculturae Slovenica, 117/1 – 20214 T. S. RASHID et al. One of the important physiological indicators is the photosynthesis rate, which is related to the chlorophyll content in plant leaves in normal conditions. In the pre- sent study, the chlorophyll content was found to increase significantly in all plants treated with T. h compared to those inoculated with F. s or F. o. The chlorophyll content also significantly went up in plants treated with T. h + F.s (26.5) or T. h + F. o (23.5) in comparison to the F. o and F. s only (14.86 and 20.5) inoculated plants (Figure 1). Previous studies have claimed that applying biocontrol agents to infected plants increases mineral levels [(nitro- gen (N), phosphorous (P), potassium (K) and magnesi- um (Mg)], chlorophyll biosynthesis and photosynthetic activity (Henry et al., 2009; Morsy et al., 2009; Alwath- nani et al., 2012). The total soluble protein content significantly de- creased in tomato plants: F. s (0.082, 0.076 and 0.075) and F. o (0.084, 0.074 and 0.072) after 10, 20 and 30 days, respectively (Figure 2). The significant decrease in the protein content in the tomato tissues as a result of the pathogen infection may be due to certain activities re- lated to a hypersensitive response (Chandra and Bhatt, 1998). Trichoderma harzianum also increased the total protein content in the infected tomato plants (T. h + F. s and T. h + F. o). The defence reaction occurs due to an accumulation of pathogenesis-related (PR) proteins such as chitinase, phenylalanine ammonia lyase and peroxi- dase (Kloepper et al., 1992). This has also been previously reported by Houssien et al. (2010). Tables 2 and 3, show that the activity of peroxidase and polyphenol oxidase,  increased significantly com- pared to the controls. The activity increased during the treatment period from day 10 to day 30. Also Fusarium species are able to produce metabolites, which play a vital role in tissue browning due to their ability to oxidise phe- nols to quinones (Ramadoss, 1991). Except the tomato plant inoculated only with the antagonistic T. harzianum, there was an increase in the activity of the polyphenol ox- Figure 1: Effect of T. harzianum on chlorophyll content of the tomato plants inoculated with F. oxysporum and F. solani. Figure 2: Effect of T. harzianum on the total protein content (mg g-1) of the tomato plants inoculated with F. oxysporum and F. solani.. Acta agriculturae Slovenica, 117/1 – 2021 5 Induction of defence related enzymes and biocontrol efficacy ... tomato plants infected with Fusarium oxysporum and Fusarium solani idase and peroxidase up to the 20th day. After this time, the activity decreased or remained stable. At the initial stage of infection, there is probability of the host plant to secrete more defence enzymes (Ojha et al., 2012). The activity in- creased gradually from the 20th day up to the 30th day in the plants infected with T. h. + F. o. and T. h. + F. s.. The highest activities for both treatments 1.87 and 1.95 min g-1 for fresh mass of peroxidase and 2.71 and 2.55 min g-1 for fresh mass of polyphenol oxidase were recorded after the 30th day of inoculation. This is the most likely related to the fact that in tomato plants, when inoculated with a pathogen and an antagonist, the host plant secretes more of the phenol oxidase enzyme for defence, but at the later stages of infection, the antagonist blocks the activity of the pathogen. Various antioxidant enzymes, such as peroxi- dases and polyphenol oxidases can participate in the re- active oxygen metabolism of the species during infection (Morkunas and Gmerek, 2007). Other researchers have observed increased activity in these enzymes in the host tissues in response to pathogenic infections (Abo-Elyousr et al., 2008, Christopher et al., 2010; Ojha et al., 2012). 4 CONCLUSION The experiments demonstrated that the T. harzianum is effective and can induce systemic and localized resist- ance against Fusarium infection in terms of changes of the plants’ polyphenol oxidase activity, peroxidase activ- ity and the total phenolic content. These mechanisms help to develop resistance in tomato plants. 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CRC Press. https://doi.org/10.1201/9781482277951 Acta agriculturae Slovenica, 117/1, 1–9, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1378 Original research article / izvirni znanstveni članek The role of exogenous silicon to mitigate Al2O3 nanoparticle-induced toxicity in barley (Hordeum vulgare L.) Ghader HABIBI 1, 2 and Maryam SHAHINFAR 1 Received October 12, 2019; accepted January 28, 2021. Delo je prispelo 12. oktobra 2021, sprejeto 28. januarja 2021. 1 Payame Noor University (PNU), Department of Biology, Iran 2 Corresponding author, e-mail: gader.habibi@gmail.com The role of exogenous silicon to mitigate Al2O3 nanoparticle- induced toxicity in barley (Hordeum vulgare L.) Abstract: In this study, we used silicon (Si, in the form of K2SiO3, 2 mM) to alleviate the toxicity of aluminum oxide (Al2O3) nanoparticles (NPs) in barley (Hordeum vulgare L.). Using Inductively Coupled Plasma-Mass Spectroscopy (ICP- MS) analyses, we showed that the Al2O3 NPs were taken up by barley plants. Barley growth was negatively affected by the ad- dition of 3 g l-1 nano-Al2O3, whereas the diminishing effect of NPs on barley growth was not obvious when 1 g l-1 nano-Al2O3 was applied, indicating that the nano-Al2O3 action is dependent on nano-Al2O3 dose. Si pretreatment ameliorated toxic effects of high nano-Al2O3 on root growth. Si pretreatment did not decrease nano-Al2O3 entry into roots but reduced nano-Al2O3 accumulation in the shoot. The restriction of the root-to-shoot translocation of nano-Al2O3 was one of the important mecha- nisms for Si to mitigate high nano-Al2O3 toxicity. The occur- rence of oxidative stress was found under 3 g l1 nano-Al2O3 treatment, as evaluated by the accumulation of malondialde- hyde (MDA). Exogenous addition of Si could alleviate toxicity symptoms induced by Al2O3 nanoparticles by reducing lipid peroxidation via enhancing antioxidant activity of catalase as well as by limiting the root-to-shoot translocation of nano- Al2O3. These data provide the first direct evidence that the Si pretreatment ameliorates nano Al2O3 phytotoxicity in plants. Key words: Hordeum vulgare L.; malondialdehyde; nano- Al2O3; nanotoxicity; silicon Vloga dodajanja silicija za preprečevanje strupenosti nano delcev Al2O3 pri ječmenu (Hordeum vulgare L.) Izvleček: V raziskavi je bil uporabljen silicij (Si), v obliki 2 mM K2SiO3, za preprečevanje strupenosti nano delcev alumi- nijeva oksida (Al2O3;NPs) pri ječmeni (Hordeum vulgare L.). Analiza z ICP-MS je pokazala, da so bili nano delci Al2O3 privze- ti v rastline. Na rast ječmena je negativno vplival dodatek 3 g l-1 nano delcev Al2O3, medtem, ko rast ječmena ni bila občutno zmanjšana pri dodatku 1 g l-1 nanodelcev Al2O3, kar kaže, da je učinek nano delcev Al2O3 odvisen od doze. Predhodno ob- ravnavanje rastlin s silicijem je oblažilo toksičen učinek velikih koncentracij nano delcev Al2O3 na rast korenin. Predhodno obravnavanje s Si ni zmanjšalo privzema nano delcev Al2O3 v korenine ampak zmanjšalo njihovo kopičenje v poganjkih. Omejitev translokacije nano delcev Al2O3 iz korenin v poganjke se je izkazala kot pomemben mehanizem preprečevanja njihove toksičnosti s silicijem. Pojav oksidacijskega stresa pri obravna- vanju z 3 g l-1 nano delci Al2O3 je bil ovrednoten s kopičenjem malondialdehida (MDA). Dodajanje silicija lahko prepreči nastanek toksičnih znakov, ki jih povzročajo nano delci Al2O3 preko zmanjšanja peroksidacije lipidov s povečevanjem ak- tivnost katalaze kot tudi z omejevanjem njihove translokacije iz korenin v poganjke. Ti izsledki so prvi neposreden dokaz, da predobravnavanje s silicijem zmanjšuje strupenost nano delcev Al2O3 pri rastlinah. Ključne besede: Hordeum vulgare L.; malondialdehid; nano-Al2O3; nanotoksičnost; silicij Acta agriculturae Slovenica, 117/1 – 20212 G. HABIBI and M. SHAHINFAR 1 INTRODUCTION Aluminum (Al) toxicity is one of the main stress factors limiting plant growth and crop yields in acid soils (Wang et al., 2004), which now account for ~40 % of the earth’s arable land (Ma et al., 2001; Rahman et al., 2018). To alleviate damaging effect of Al toxicity as well as to prevent root growth inhibition, some plant species used diverse mechanisms such as releasing or- ganic acids that chelate Al, transporting of organic acid anions out of the root cells and forming complexes with organic acids in their leaves, that enable them to grow on acid soils (Ma et al., 2001). Barley is considered to be most sensitive to Al toxicity among cereal species (Wang et al., 2006). Because of the fact that the yield of barley was reduced in acid soils (Fujii et al., 2012), the understanding of the physiological and biochemical mechanisms improving Al tolerance of this species is very important. Nowadays aluminum oxide (Al2O3) nanoparticles (NPs) are one of the most used NPs and developed for applications in cosmetic fillers producing, materials packaging, tools cutting, glass and metal production, etc (Hanemann and Szabó, 2010). Thus these NPs can enter to the waste water streams and may predomi- nantly be applied to agricultural fields (Colvin 2003; Navarro et al. 2008). Regarding to nanotoxicology, many studies have been published concerning the dif- ferent cytotoxic effects of such nanoparticles on mam- malian, animals and bacteria (Wiesner et al., 2006; Lin and Xing, 2007), and only a few studies have focused on the toxicity of NPs to plants (Lee et al. 2008, 2010). Re- cently, root growth inhibition by 2 g l-1 nano-Al2O3 was reported for soybean, cabbage, and carrot (Yang and Watts, 2005), tobacco (Burklew et al., 2012) and wheat (Yanik and Vardar, 2015). Since quantitative methods for determining nanoparticles in plant tissues have not been considered, in this study, uptake and accumula- tion of nano-Al2O3 nanoparticles were quantified by Inductively Coupled Plasma-Mass Spectroscopy (ICP- MS) analyses. In this study, we used exogenous Silicon (Si) to mitigate toxicity symptoms induced by Al2O3 NPs in barley plants. Silicon is a beneficial mineral element for plants. A number of studies have been demonstrated that Si is beneficial for the growth of plants, especially those be- longing to the family Poaceae (Broadley et al., 2012). Si can mitigate the effects of various environmental stress- es such as salinity, drought, chilling, UV radiation (Col- lin et al., 2014; Zhu and Gong, 2014; Habibi, 2016) and Al and Mn toxicity (Zargar et al., 2019). Exogenous ap- plication of Si exhibits the capacity to enhance the plant growth and yield as well as stress tolerance under metal toxicity by reducing the metal uptake and transport in plants (Adrees et al., 2015), formation of silicon bodies in the cell wall (Prabagar et al., 2011) and enhancing the activities of antioxidant enzymes (Habibi, 2014; Shen et al., 2014; Dorneles et al., 2019). This research was conducted to study the effects of Si application on the amelioration of nano-Al2O3 tox- icity. According to the best of our knowledge, there is no information in literature regarding the ameliorating effect of Si on nano-Al2O3 toxicity in plants. To address this issue, we examined in some detail the biochemi- cal mechanisms by which nano-Al2O3 influences the growth, photosynthetic pigments and antioxidant ca- pacity in barley plants. Since the Si alleviates elemental aluminum-induced damages resulting in better plant growth under aluminum toxicity, we hypothesize that Si can also mitigate nano-Al2O3 toxicity damages. 2 MATERIALS AND METHODS 2.1 CHARACTERIZATION AND PREPARATION OF NANOPARTICLE SUSPENSION FOR TREATMENT Aluminum oxide nanoparticles (Al2O3 Nanopo- wder, alpha, 99 %, 80 nm, Hydrophilic) were obtained from US Research Nanomaterials, Inc. The morphol- ogy and diameter of Al2O3 NPs were also evidenced by scanning electron microscope (SEM, Seron Technol- ogy, AIS2100 model) as shown in the Figure 1. After dispersing NPs in distilled water, the solution was soni- cated through ultrasonication (230 V/50–60 Hz) for 30 min in order to obtain a homogeneous mixture. 2.2 GROWTH CONDITIONS AND EXPOSURE OF NANOPARTICLES TO PLANT Seeds of barley (Hordeum vulgare ‘Bahman’) were germinated on filter paper moistened with distilled wa- ter. Ten-day-old seedlings were transported to modified Hoagland nutrient solution (Johnson et al., 1957) con- taining 6 mM KNO3, 4 mM Ca(NO3)2, 2 mM NH4H- 2PO4, 1 mM MgSO4, 50 µM H3BO3, 2 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, 0.5 µM H2MoO4 and 0.02 mM FeSO4-EDTA for 25 days prior to the treatment proce- dure. At 25 days after germination, Al2O3 nanoparticles (0, 1 and 3 g l-1) and Si (K2SiO3, 2 mM) were applied together with the nutrient solution described above. Plants were grown under a temperature regime of 22- 25/17-19 ºC, relative humidity of 60-65 % and daily pho- Acta agriculturae Slovenica, 117/1 – 2021 3 The role of exogenous silicon to mitigate Al2O3 nanoparticle-induced toxicity in barley (Hordeum vulgare L.) ton flux density of about 300-350 μmol m-2 s-1 throughout the experimental period. 2.3 HARVEST PLANTS Plants were harvested 14 days after applying the na- noparticles. Fully expanded and mature leaves were uti- lized for measurement of enzymatic analysis. Shoots and roots were washed with distilled water, blotted dry on fil- ter paper and after determination of fresh mass (FM) they were dried for 48 h at 70 °C for determination of dry mass (DM). Plants height and tap root length were measured using a ruler. 2.4 DETERMINATION OF ALUMINUM According to Yanik and Vardar (2015), shoot and root samples were oven-dried at 80 °C for 24 h, and mixed with 8 ml 65 % HNO3 at 175 °C. To quantify Al2O3 NPs concentration in shoot and roots, we used Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) analyses; and the measured concentration of elemental Se in shoot and root samples was normalized by the dried mass of the shoot and root. 2.5 DETERMINATION OF TOTAL CAROTENOIDS AND CHLOROPHYLLS a AND b Leaf concentration of chlorophyll and carotenoids was measured according to Lichtenthaler and Wellburn (1985). After extraction of fresh pigments in the cold ac- etone, the samples stand for 24 h in the dark at 4 ºC. 2.6 ASSAY OF ANTIOXIDATIVE ENZYMES AND MALONDIALDEHYDE (MDA) CONTENT The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) were determined according to methods described elsewhere (Habibi and Hajiboland, 2012). Lipid peroxidation was estimated from the amount of malondialdehyde (MDA) formed in a re- action mixture containing thiobarbituric acid according to methods described elsewhere (Habibi and Hajiboland, 2012). 2.7 STATISTICAL ANALYSES Experiment was done in complete randomized block design with 4 independent replications. Statistical analysis was carried out using Sigma Stat (3.5) with Tukey test. Re- sults were given as mean ± standard deviation (SD). Dif- ferences between treatments were considered to be signifi- cant, when a p value was less than 0.05 (p < 0.05). 3 RESULTS AND DISCUSSION 3.1 EFFECT OF DIFFERENT NANO-AL2O3 CON- CENTRATIONS ON ITS UPTAKE AND ACCU- MULATION USING ICP-MS Exogenous Al2O3 NPs application increased en- Figure 1: Scanning electron microscopy image of Al2O3 nanoparticles when nano-Al2O3 was mixed with hydroponic solutions. Acta agriculturae Slovenica, 117/1 – 20214 G. HABIBI and M. SHAHINFAR dogenous Al2O3 NPs contents in shoot and roots of bar- ley plants (Figure 2). Al2O3 NPs concentration in plants grown without NP addition was under the analyzing limits. The highest content of Al was found in roots of plants. The most effective uptake and transport of Al was observed for 3 g l-1 Al2O3 NPs. These results agreed with Asztemborska et al. (2015) who reported that Al2O3 NPs was taken up by Zea mays L. plants. Indeed, most research have mainly focused on assessing the nature of the safety and toxicity of these nanoparticles (Yanik and Vardar, 2015), but the uptake and entry of nanoparticles into plant systems is still poorly comprehended (Li et al., 2015). In this study, the relatively high concentrations of Al found in shoots grown in the presence of Al2O3 NPs strongly indicated the transport of intact particles of Al2O3 from the root to the shoot in barley plants. 3.2 Si PRETREATMENT REDUCED NANO-AL2O3 ACCUMULATION IN THE SHOOT Si pretreatment did not reduce nano-Al2O3 entry into roots but decreased nano-Al2O3 accumulation in the shoot (Figure 3). Recently, authors proposed the forma- tion of aluminosilicate complexes in the cell wall (Pra- bagar et al., 2011; Adrees et al., 2015). Similarly, possible retention of aluminum in the cell wall has been studied in relation to ameliorating effect of Si on aluminum toxic- ity in maize (Wang et al., 2004). These authors reported that Si causes higher aluminum tolerance in plants via binding of aluminum to the cell wall. Our results indi- cated significant effect of supplemental Si on nano-Al2O3 concentration in shoots. They are in agreement with the findings of Dorneles et al. (2016) who reported a de- crease in aluminum concentration by Si application in the shoots of potato plants. However, there is no infor- mation in literature regarding the ameliorating effect of Si on nano-Al2O3 toxicity in plants. 3.3 NANO-AL2O3 ACTION IS DEPENDENT ON NANO-AL2O3 DOSE Barley growth was negatively affected by nano- Al2O3 levels up to 1 g l -1. Although no change was ob- served in 1 g l-1, NP treatment at 3 g l-1 decreased the shoot and root fresh mass, and root elongation with re- gard to controls (Figure 3). Moreover, shoot and root dry mass was affected negatively by high concentration of na- no-Al2O3 during the experiment (Figure 4). In this study, the highest applied concentration of Al2O3 was about 1.5 times higher than that reported to be toxic (2 g l-1) for Figure 2: Al2O3 nanoparticles accumulation in barley that was recovered by ICP-MS in shoot and root sections of treated plants. Error bars indicate the standard deviation. Bars indicated with the same letter are not significantly different (p < 0.05). Acta agriculturae Slovenica, 117/1 – 2021 5 The role of exogenous silicon to mitigate Al2O3 nanoparticle-induced toxicity in barley (Hordeum vulgare L.) Figure 3: Effects of different concentration of Al2O3 NPs on the shoot and root fresh mass, and root elongation of barley seedlings exposed to 2 mM Si for 14 days. Error bars indicate the standard deviation. Bars indicated with the same letter are not significantly different (p < 0.05). Figure 4: Effects of different concentration of Al2O3 NPs on the shoot and root dry mass of barley seedlings exposed to 2 mM Si for 14 days. Error bars indicate the standard deviation. Bars indicated with the same letter are not significantly different (p < 0.05). Acta agriculturae Slovenica, 117/1 – 20216 G. HABIBI and M. SHAHINFAR corn, cucumber, soybean, cabbage, and carrot (Lee et al., 2010). Additionally, our results are in agreement with the findings of Burklew et al. (2012) who reported that root growth and development decreased as the concentration of aluminum oxide nanoparticles increased. Probably, higher concentrations of nano-Al2O3 adsorb on the root surface, and interrupt the root functions (Asztemborska et al., 2015). Our results clearly indicated that the di- minishing effect of exogenously applied nano-Al2O3 on growth parameters of barley plants was dependent on doses of nano-Al2O3 used. 3.4 Si PRETREATMENT AMELIORATED TOX- IC EFFECTS OF NANO-AL2O3 ON ROOT GROWTH In current study, root growth was reduced by 3 g l-1 nano-Al2O3, whereas this reduction was alleviated by application of exogenous Si (Figure 4). Exogenous ad- dition of Si can mitigate toxicity symptoms induced by aluminum stress in many plant species (Hammond et al., 1995; Singh et al., 2011; Shen et al., 2014) by enhanc- ing antioxidant protection via modifying the activities of antioxidant enzymes (Shen et al., 2014; Dorneles et al., 2019), and by apoplastic binding of aluminum via forma- tion of aluminosilicate complexes in the cell wall (Wang et al., 2004; Adrees et al., 2015). However, the mecha- nisms of Si-mediated alleviation of nano-Al2O3 stress are still unknown. 3.5 Si PRETREATMENT DID NOT CHANGE THE CONCENTRATION OF PHOTOSYNTHETIC PIGMENTS Leaf photosynthetic parameters including chloro- phyll b and carotenoid contents were not significantly influenced under nano-Al2O3 stress with or without Si treatment (Figure 5). However, a consistent tendency of chlorophyll a to decrease in response to high levels of nano-Al2O3 was observed. It has been reported that Si markedly mitigates Al-induced reduction in photosyn- thetic parameters in peanut plants (Shen et al., 2014). In contrary, our results indicated that the photosynthetic Figure 5: Effects of different concentration of Al2O3 NPs on the chlorophyll (Chl) a, b and carotenoid contents in leaves of barley seedlings exposed to 2 mM Si for 14 days. Error bars indicate the standard deviation. Bars indicated with the same letter are not significantly different (p < 0.05). Acta agriculturae Slovenica, 117/1 – 2021 7 The role of exogenous silicon to mitigate Al2O3 nanoparticle-induced toxicity in barley (Hordeum vulgare L.) pigment concentration was not influenced by Si under nano-Al2O3 stress. 3.6 Si PRETREATMENT MITIGATED AL2O3 NAN- OPARTICLE-INDUCED OXIDATIVE STRESS IN BARLEY PLANTS The activity of SOD was not influenced even under the highest nano-Al2O3 levels applied (Figure 6). How- ever, a consistent tendency of CAT activity to decrease in response to nano-Al2O3 was observed. We observed that CAT activity was enhanced by Si application in the na- no-Al2O3-stressed seedlings, which was consistent with the ability of Si to decrease the content of MDA in this plant. We found that the application of 3 g l-1 nano-Al2O3 was toxic, because it caused the accumulation of MDA, a marker for the ROS-mediated cell membrane damage. However, the MDA content was reduced with Si under nano-Al2O3 stress. Similarly, increasing the activity of an- tioxidant enzymes and mitigating the Al-induced dam- age to membrane lipids was reported in potato genotypes grown with silicon (Dorneles et al., 2019). Indeed, ex- ogenous addition of Si can mitigate toxicity symptoms induced by aluminum stress via modifying the activities of antioxidant enzymes (Shen et al., 2014; Dorneles et al., 2019; Zargar et al., 2019); however, the mechanisms of Si-mediated inhibition of membrane lipids peroxidation and enhancing the activities of antioxidant enzymes un- der nano-Al2O3 toxicity are still unknown and must be further explored. Furthermore, we showed that Si ame- liorated the negative effect of high nano-Al2O3 on pro- ductivity in barley plants by reducing lipid peroxidation and enhancing antioxidant activity of CAT (Figure 6). 4 CONCLUSION In summary, we showed that the toxic effect of na- no-Al2O3 was in a dose-dependent manner. Nano-Al2O3 treatment at 3 g l-1 decreased the shoot and root mass, and root elongation with regard to controls; however, no changes were observed in 1 g l-1. Si pretreatment amelio- rated toxic effects of 3 g l-1 nano-Al2O3 on root growth. This Si-mediated alleviation of nano-Al2O3 toxicity was in parallel with the enhanced antioxidant protection via modifying the activities of antioxidant enzymes and the Figure 6: Effects of different concentration of Al2O3 NPs on the activity of superoxide dismutase (SOD) and catalase (CAT), and malondialdehyde (MDA) content in leaves of barley seedlings exposed to 2 mM Si for 14 days. Error bars indicate the standard deviation. Bars indicated with the same letter are not significantly different (p < 0.05). Acta agriculturae Slovenica, 117/1 – 20218 G. HABIBI and M. SHAHINFAR restriction of the root-to-shoot translocation of nano- Al2O3, as well as lower MDA production. 5 ACKNOWLEDGEMENT We thank Ali Maleki, University of Kharazmi for providing Scanning Electron Microscopy image of Al2O3 nanoparticles. 6 REFERENCES Adrees, M., Ali, S., Rizwan, M., Zia-ur-Rehman, M., Ibrahim, M., Abbas, F., Irshad, M.K. (2015). 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Delo je prispelo 20. avgusta 2020, sprejeto 10. februarja 2021. 1 Institute of Horticulture of National Academy of Agrarian Sciences of Ukraine, Novosilky, Kyiv region, 03027, Ukraine 2 Corresponding author, e-mail: pavliukl.92@ukr.net Variability of some isolates of Prunus necrotic ringspot virus and Prune dwarf virus infecting sour and sweet cherry in Ukraine Abstract: Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV) are the most common pathogens in stone crop orchards. These diseases are easily transmitted with pollen and hence rapidly spread in orchards leading to stunting of trees, their increased susceptibility to abiotic stress factors and, eventually, to significant yield losses. In Ukraine, only monitoring studies on the spread of these viruses were conducted until now. However, phylogenetic comparison of Ukrainian isolates was lacking. In this work, total RNA was iso- lated from plant samples tested positive for PNRSV and PDV by ELISA. The part of viral CP gene sequences were amplified and sequenced with their subsequent phylogenetic analysis. It was determined that PNRSV isolates from Ukraine analyzed in this study belong to different groups – PV-96 (MT828889) and PV- 32 (MT892676) with a maximum identity level of 100 % with known isolates from NCBI GenBank. PDV isolates (MT828888 and MT828887) showed high identity with each other (99.6 %), and Slovakian isolate from sweet cherry was shown as the most related to them with identity of 95.3 %. Key words: PNRV; PDV; ELISA; RT-PCR; sweet cherry virus; phylogenetic analysis Variabilnost izolatov virusa nekrotične obročkaste pegavosti breskve (PNRSV) in virusa pritlikavosti slive (PDW) na višnji in češnji v Ukrajini Izvleček: Virus nekrotične obročkaste pegavosti breskve (PNRSV) in virus pritlikavosti slive (PDW) sta najbolj pogosta patogena v sadovnjakih koščičastega sadja. Bolezni se z lahkoto prenašata s cvetnim prahom in se tako hitro širita v sadovnja- kih, kar vodi k slabitvi dreves, povečuje njihovo občitljivost na abiotske stresorje in včasih k znatni izgubi pridelka. V Ukrajini so bile do sedaj izvedene le raziskave o razširjenosti teh virusov, ni pa bilo njihovih filogentskih raziskav.V tej raziskavi so bili z ELISA testom analizirani izolati iz vzorčenih rastlin, pozitivnih na PNRSV in PDV. Del zaporedij virusovega CP gena je bilo namnoženih in sekvenciranih za njihovo kasnejšo filogenetsko analizo. Ugotovljeno je bilo, da izolati PNRSV virusov iz Ukra- jine, analizirani v tej raziskavi, pripadajo različnim skupinam in sicer skupinama PV-96 (MT828889) in PV-32 (MT892676) z največjo stopnjo istovetnosti 100 % z znanimi izolati iz NCBI GenBank. PDV izolata (MT828888 in MT828887) sta pokazala veliko medsebojno istovetnost (99.6 %), in s slovaškim izola- tom iz češnje, ki se je izkazal najbolj soreden z njima z istove- tnostjo 95.3 %. Ključne besede: PNRV; PDV; ELISA; RT-PCR; češnjev virus; filogenetska analiza Acta agriculturae Slovenica, 117/1 – 20212 L. PAVLIUK et al. 1 INTRODUCTION Sour cherry (Prunus cerasus L.) and sweet cherry (Prunus avium L.) are traditional fruit crops in Ukraine. They have an important role in the structure of fruit and berry crop production. According to the State Statistics Service of Ukraine (http://www.ukrstat.gov.ua/), the vol- ume of sour and sweet cherry production in Ukraine in 2019 amounted to 236.13 thousand tons. However, virus diseases are one of the limiting factors for increasing yields. The most common sour and sweet cherry viruses in the world are Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV) (Caglayan et al., 2011). According to the preliminary data, prevalence of these viruses in mother plant orchard of cherries in Ukraine is 11.8 % for PNRSV and 8.7 % for PDV (Pavliuk et al., 2018). Both viruses belong to Ilarvirus genus, Bromoviri- dae family (Pallas et al., 2012). In fruit trees, infection by these viruses leads to a variety of symptoms, the manifes- tation of which depends on climatic conditions and the type of host plant (Kamenova et al., 2019). PDV causes chlorotic rings and spotting, deforma- tion (thinning and curling) of leaf blades, and shorten- ing of internodes (Kamenova et al., 2019). PNRV induce similar symptoms to PDV, and is also manifested by necrotic and chlorotic spots, mosaic and deformation of leaves, flowers and fruits, but often remains latent (Sokhandan-Bashir et al., 2017). PNRSV symptoms may vary depending on the time passed since the initial infec- tion. Immediately after the virus enters the plant (“shock phase”), the most acute necrotic symptoms are typically observed. In the following chronic course of the disease, the symptoms may be partially masked or slightly al- tered. Also, in the initial infection, the virus delays the flowering on individual branches or of the whole tree, and young leaves are often smaller than those of healthy plants. The spotting pattern may vary depending on the cultivar (Verderevskaya & Marinesku, 1985; Nyland et al., 1976; Wells & Kirkpatrick, 1986). Mixed infection of plants with PDV and PNRSV leads to the development of more severe symptoms (Caglayan et al., 2011). These two viruses are often found in combination as both are readily transmitted by pollen. These pathogens are particularly dangerous for sour and sweet cherry, peach, and almond (Umer et al., 2019). Crop yield is reduced due to the decrease in the number of fruit buds caused by these two viruses (Kamenova et al., 2019). Under the influence of these pathogens, peach yield can be reduced by 60 % (Umer et al., 2019). Infec- tion of Prunus spp. plants by PNRSV reduces tree growth by 30 %, and reduces yield by 20-56 %, also making the trees very susceptible to low temperatures (Amari et al., 2007). In susceptible crops, PDV monoinfection can cause yield losses of up to 99 %, also significantly reducing fruit quality (Çağlayan et al., 2011). In addition to transmission by grafting and other agronomic measures, spreading by pollen is a typical way of transmission (Çağlayan et al., 2011; Pallas et al., 2012). Due to their ability to transmit by pollen, PDV and PNRSV can spread rapidly in stone crop orchards and cause significant economic damage (Amari et al., 2007; Çağlayan et al., 2011; Kamenova et al., 2019; Nemeth, 1986; Umer et al., 2019). As other ilarviruses, these pathogens have segment- ed genome consisting of three fragments of single-strand- ed positive sense RNA (Pallas et al., 2012; Roosinck et al., 2005). PNRSV has isometric virions (Jakab-Ilyefalvi et al., 2011) while PDV can form two types simultaneously – icosahedral and bacilli forms (Kozieł et al., 2017). RNA1 and RNA2 encode replicase subunits, while RNA3 encodes coat protein (CP) and movement pro- tein (MP). The CP is synthesized from subgenomic RNA (RNA4) derived from RNA3 (Pallas et al., 2012). CP of ilarviruses forms a shell for packaging of genome segments and participates in its activation (Pallas et al., 2013). RNA3 length is 1683  nt for PDV and 1943 or 1951  nt for PNRSV. Ilarvirus MP is required for virus cell-to-cell trafficking (Pallas et al., 2012). For phyloge- netic analysis of ilarviruses, CP or MP gene sequences are typically used (Codoñer & Elena, 2006), or partial sequences of RdRp (Maliogka et al., 2007). Viral genome variability contributes to the emer- gence of new highly virulent isolates, so there is a need for phylogenetic analysis, which allows to determine the variability of the genome and to establish its genetic iden- tity with already known isolates. The aim of the research was to analyse part of CP gene of Ukrainian PNRSV and PDV isolates sampled from sour and sweet cherry trees, and to establish their phylogenetic relationships with known isolates by comparing the nucleotide sequences. 2 MATERIALS AND METHODS In order to identify the symptoms of viral infection, a visual examination of the mother plantations was per- formed during the period of the most pronounced mani- festation of symptoms – May-June of 2019 year. In order to confirm the presence of viral infection, leaf samples were taken from four spots in tree canopy. Samples were taken (Table 1) from plants with symptoms typical for to PDV and PNRSV infections, as well as from asymptomatic plants. The latter were select- Acta agriculturae Slovenica, 117/1 – 2021 3 Variability of some isolates of Prunus necrotic ringspot virus and Prune dwarf virus infecting sour and sweet cherry in Ukraine ed from trees near to those having the most expressed symptoms. Positive samples previously tested by ELISA were confirmed by RT-PCR. One sample from each in- fected cultivar was taken for sequencing. These samples included those from sour cherry cultivars Kseniia (PN- RSV), Boguslavka (PDV), and a sample of sweet cherry Nizhnist cultivar (PDV and PNRSV). Extraction of total RNA was performed using RNe- asy Plant Mini kit (Qiagen, UK) according to the manu- facturer’s recommendations. 75  mg of fresh tissue was used for extraction. Extracted RNA was stored at -20 °С. Extraction quality was checked by DeNovix DS-11 spec- trophotometer (DeNovix, USA), at wavelength of 260, 260/230, 260/280 nm. PNRSV and PDV identification was carried out by RT-PCR using primer pairs amplifying a part of CP gene: PNRSV–10F (5’-TTC TTG AAG GAC CAA CCG AG AGG-3’)/PNRSV–10R (5’-GCT AAC GCA GGT AAG ATT TCC AAG C-3’) with expected fragment size of 348 bp, and PDV–17F (5’-CGA AGT CTA TTT CCG AGT GGA TGC-3’)/PDV–12R (5’-CAC TGG CTT GTT TCG CTG TGA AC-3’) with the expected fragment size of 303 bp (Massart et al., 2008.) In order to control RNA amplification, internal control with nad5-f/nad5-r primers with expected fragment size of 181 bp was used (Menzel et al., 2002). In order to conduct RT-PCR, commercial kit Verso 1-Step RT-PCR Kit ReddyMix (Thermo Scientific, USA) was used according to the manufacturer’s recom- mendations. The following components were tak- en per reaction mixture with a volume of 20  μl: 2X 1 Step PCR ReddyMix – 10 μl, Verso Enzyme Mix – 0,4 μl, RT Enhancer – 1 μl, Primer forward – 0,4 μl (10  mmol), Primer reverse – 0,4  μl (10  mmol), H2O – 6,6 μl, RNA – 50 ng for one reaction. The same reaction was performed to check RNA quality with nad5-f/nad5- r primers. Amplification was performed in «Epрendorf Mastercycler Рersonal» programming thermostat (Epрendorf AG, Germany) with the following param- eters: 50 оС – 15 minutes, 95 оС – 2 minutes, 40 amplifica- tion cycles (95 оС – 20 s, 55 оС – 30 s, 72 оС – 15 s), and 72 оС – 5 minutes. The presence of amplification fragments was checked by separating the PCR products in 2 % agarose gel, with TBE buffer with the addition of ethidium bro- mide 0.5 μl -1. Obtained amplicons of domestic isolates of the Ilarvirus genus were sequenced by Sanger sequenc- ing method. The sequences of PNRSV and PDV capsid protein gene were compared with the known sequences in GenBank (www.ncbi.nlm.nih.gov) using function BLAST  2.10.0 software. For phylogenetic analysis, 25 PNRSV and 30 PDV isolates were selected from the Gen- Bank (www.ncbi.nlm.nih.gov), that were extracted from different crops and geographic origin. Multiple sequence alignments were performed by Clustal  W algorithm of MEGA 10 (Kumar et al., 2018). Sequences were cut cor- responding to the length of our fragment. Construction of the phylogenetic tree was carried out using MEGA 10 software by the methods Neighbour-Joining (NJ) (Saitou & Nei, 1987) using Kimura two parameter. The statisti- cal significance was calculated using 500 bootstrap repli- cates (Felsenstein, 1985). Standard error of the pairwise identities of groups was calculated using STATISTICA software. 3 RESULTS AND DISCUSSION Careful examination of the trees revealed leaf de- Figure 1: Chlorosis and deformation of the leaf. A – ‘Boguslavka’ (PDV infected), B – ‘Nizhnist’ (PDV and PNRSV infected) (virus isolates recovered from these samples were used for phylogenetic analysis) Acta agriculturae Slovenica, 117/1 – 20214 L. PAVLIUK et al. After preliminary ELISA testing, in order to study the molecular characteristics samples were selected: ‘Kse- niia’ infected with PNRSV, ‘Bohuslavka’ – with PDV and ‘Nizhnist’ with mixed infection of these viruses (Table 1). Isolates were named based on the position of in- fected trees in the orchard. RT-PCR method confirmed the presence of viruses in the tested samples (Fig. 2). As a result, the sequences of isolates PNRSV 4-1 (828889), PNRSV 16-15 (MT892676), PDV 1-67 (MT828888), PDV 16-15 (MT828887) were obtained. Sequenced fragments of the PNRSV coat protein gene after its alignment had 295 nucleotides correspond- ing to nts 1601-1891 in RNA3. This part contains a 177 nt part of the coding region (nts 1601- 1777) and a part of UTR. The sequenced fragment of the genome of the Ukrainian isolates PNRSV 4-1 and PNRSV 16-15, which is the C-terminal end of the CP gene, is extremely con- served as it includes the dimerization region at position of 1694-1744 nt (198-215 amino acids) (Aparicio et al., 2006). It should be noted that the presence of a non-cod- ing sequence does not affect the phylogenetic grouping of isolates. formations along the veins (Fig. 1A), dark brown spots with small holes and chlorotic areas. It should be noted that chlorosis was the most common symptom among all surveyed trees. These symptoms are characteristic of PDV infection and have been repeatedly described by a number of authors (Massart et al., 2008; Smith et al., 1988; Sanchez et al., 2015; Kamenova et al., 2019). Chlorotic spots and small necrotic holes were de- tected on sweet cherry of ‘Nizhnist’ cultivar (Fig. 1B). The presence of both viruses in the plant was confirmed by ELISA and RT-PCR. The course of viral diseases is often latent and dis- plays no symptoms of infection. This is especially true when the disease becomes chronic. Therefore, the exami- nation of ‘Kseniia’ sour cherry trees did not reveal any symptoms of virus infection, although ELISA and RT- PCR diagnostics confirmed virus presence. Despite many trees showing symptoms similar to virus infection, the presence of pathogens was not con- firmed in all samples. This is because the symptoms of virus infection can be very similar to the effects of abiotic factors, lack of nutrients, insect damage etc. Cultivar Number of tested samples (n) ELISA test Location PDV PNRV Mixed infection Nizhnist (sweet cherry) 6 - - 1 Zaporizhzhya Vidrodzhennia (sour cherry) 7 - - - Zaporizhzhya Boguslavka (sour cherry) 11 4 - - Kyiv Kseniia (sour cherry) 7 - 3 - Kyiv Malva (sour cherry) 4 - - - Kyiv Total 35 4 3 1 - Table 1: Sour and sweet cherry samples tested for PDV and PNRSV by ELISA method Figure 2: Fragments amplified from extract of infected sour and sweet cherry samples, M – SibEnzyme 100bp molecular weight marker Acta agriculturae Slovenica, 117/1 – 2021 5 Variability of some isolates of Prunus necrotic ringspot virus and Prune dwarf virus infecting sour and sweet cherry in Ukraine Based on the molecular characteristics of the nucle- otide sequences of the CP and MP genes, PNRSV isolates are divided into three groups: PV32, PE5 and PV96 (Pal- las et al., 2012). According to the results of the analysis, Ukrainian isolates extracted from cherry cultivars were attributed to two different groups. PNRSV 4-1 isolate fell into PV96 group while PNRSV 16-15 – into PV32 group. Isolates belonging to the PV96 group are charac- terized by asymptomatic course of infection, while PV32 can cause severe disease symptoms, and PE-5 can cause both mild and severe symptoms. However, information on pathogenicity of isolates is limited, so virus group af- filiation and respective symptoms do not necessarily cor- relate (Hammond, 2003). In our case, trees of ‘Kseniia’ infected by the isolate of PV96 group did not show infec- tion symptoms, while the plant of ‘Nizhnist’ the symp- toms were apparent. In the latter case, we cannot say for sure that such manifestation of symptoms depends on the aggressiveness of the isolate, as this sample was also infected with PDV. The identity between the nucleotide sequences of the whole investigated fragment of PNRSV 4-1 and PNRV 16-15 isolates was 97.9 %, but was 100 % at the amino acid level. The non-coding section turned out to be the most variable 95.5 % identity between the isolates. The identity between the coding fragments was 98.7 %. The difference of 1.3 % between domestic isolates is caused by two nucleotide T/C substitutions that occurred at posi- tions 1642 and 1714 nt. During the construction of the phylodendrogram, three main clusters were formed. The first cluster (Fig. 3) consisted of 12 isolates representing the PV-96 group, including PNRSV 4-1 isolate. The identity of this iso- late with other from the same group ranged from 100 to 99.3 % at the nucleotide level. The most similar isolates were sAJ133205.1 (peach, Italy), AJ133208.1 (nectarine, Spain), JQ005044.1 and JQ005057.1 (sweet cherry and peach, Canada), MH730938.1 (peach, China). The range of pairwise identities within this group was 99.7-99.3 %. This indicates nucleotide substitutions that occur in the middle of the group. PNRSV 16-15 isolate from Ukraine belongs to PV- 32 group in its molecular characteristics. In addition to the Ukrainian isolate, the group includes 10 isolates from the NCBI GenBank. Of this group, the most simi- lar isolates were AJ133213.1 (plum, Italy), MF069040.1 (sweet cherry, Czech Republic), AY948440.1 (rose, In- Figure 3: Phylogenetic tree of PNRV isolates, constructed on the basis of sequences of fragments of the CP gene and the UTR using the Neighbour-Joining algorithm option of MEGA 10. Bootstrap analysis of 500 replicates was performed. Sequences obtained in our study are highlighted in bold. The scale bar represents the number of substitutions per nucleotide. Acta agriculturae Slovenica, 117/1 – 20216 L. PAVLIUK et al. dia), KX650619.1 (sweet cherry, China). Other members of this group were 99.6-98.6% identical to PNRSV 16-15. Comparison of the nucleotide sequences of the iso- lates detected nucleotide substitutions, both within the group and between groups. These substitutions were syn- onymous and did not affect the amino acid sequence. All isolates included in PV 96 and PV32 groups were 100 % identical at the amino acid level. When comparing this section of the representatives of the two groups, a total of 13 nucleotide substitutions were found which did not affect the amino acid sequence. The PE-5 group stood out separately. Isolates of this group were characterized by low identity with PV groups. The identity of Ukrainian isolates with representatives of PE-5 was 90.3-91.5 % with PNRSV 4-1 and 89.8-90.7 % with PNRSV 16-15 The analysis showed that the grouping of isolates does not depend on the geographical origin and the host plant. The same conclusions were made in previous stud- ies (Aparicio et al., 1999; Aparicio & Pallás, 2002; Scott et al., 1998), as CP is highly conservative and does not depend on the host plant or geographical area (Sala-Re- jczak & Paduch-Cichal, 2013). Sequenced fragment of the coat protein of PDV with a size of 262 nt, which corresponds to the position of 1340-1601 nt in RNA3. This section encodes 87 amino acids at the position 43-129. The studied fragment is part of ORF3b (Kozieł et al., 2017; Bahman et al., 1994). When comparing the fragment of the nucleotide sequence of PDV 1-67 and PDV 16-15 isolates, the iden- tity was at the level of 99.6 %, while for the amino acids – 98.8 %. A single nucleotide substitution in PDV 1-67 isolate – A/T at position 1455 resulted in the replace- ment of the amino acid F (phenylalanine) / Y (tyrosine) at position 81. It should be noted that these substitutions were absent in all other isolates. Also, Ukrainian isolates were characterized by the presence of C nucleotide at po- sition 1519, while all other isolates had T in this position. However, this replacement was synonymous and did not change the protein composition. The range of pairwise identities of PDV 1-67 and PDV 16-15 to other isolates was quite wide and ranged from 95.3 to 85.9 % at the nu- cleotide level, which indicates a fairly high variability of the genome of isolates. Amino acid sequence identity was slightly higher at 97.7-86.5 %, indicating that some ami- no acid substitutions were still synonymous. In total, in the analysis of all studied isolates, 24 positions in which amino acid substitutions took place were calculated. Figure 4: Phylogenetic tree of PDV isolates, constructed on the basis of sequences of fragments of the CP gene using the Neighbour- Joining algorithm option of MEGA 10. Bootstrap analysis of 500 replicates was performed. Sequences obtained in our study are highlighted in bold. The scale bar represents the number of substitutions per nucleotide. Acta agriculturae Slovenica, 117/1 – 2021 7 Variability of some isolates of Prunus necrotic ringspot virus and Prune dwarf virus infecting sour and sweet cherry in Ukraine When constructing a phylogenetic tree, all isolates were divided into two main groups (Fig. 4). The first group included two subgroups. Subgroup Ia consisted of 17 isolates, which differed in geographical origin and host plant. The Ia subgroup also included Ukrainian iso- lates. KU949346 isolate (sweet cherry, Slovakia) was the closest in amino acid and nucleotide sequences. The level of identity by nucleotide sequences was 95.3 % with PDV 16-15 and 94.9 % with PDV 1-67 and 98.8 and 97.7 % by amino acid sequences, respectively. Identity of this subgroup with other isolates varied between 94.4-88.3 %, while the mean pairwise identity within this subgroup was 93.81 %. Subgroup Ib included 11 isolates. The range of pairwise identities with PDV 1-67 and PDV 16-15 was 93.6-91.4 % by nucleotide sequences. Isolates of this group were more similar to each other – 95.97  %. The overall mean identity of all isolates of the first group was 93.41 % (se = 0.3). The second group was formed only by isolates of sweet cherry as the host plant while the geographical distribution of these isolates was different. In most pre- vious studies, no grouping based on the host plant or geographical origin was observed (Vaskova et al., 2000; Pallas et al., 2012, Öztürk & Çevik, 2015, Predajňa et al., 2017). While study of PDV isolates from Turkey revealed four groups. The first and second groups contained only isolates of cherry and almond, respectively, while the third and fourth contained isolates extracted from dif- ferent host plants (Ulubaş Serçe et al., 2009). Identity of this group with PDV 1-67 and PDV 16-15 isolates ranged from 90.9 to 85.9 % by nucleotide sequences. In general, this group was the most distant in nucleotide sequences with the mean identity of the isolates 93.67 % (se = 0.6). Construction of the phylodendrogram based on amino acid sequences showed similar results of isolate grouping into two groups. The same results were ob- tained in a previous study, which analyzed the amino acid sequences of the CP gene of isolates contained in the GenBank (Pallas et al., 2012). Thus, PDV isolates that differ in molecular char- acteristics may circulate on the territory of Ukraine, al- though they are extracted from host plants of the same species and have a common geographical origin. 4 CONCLUSIONS Based on of nucleotide sequence analysis of PNRSV, it was determined that isolates belonging to PV-96 and PV-32 groups circulate in Zaporizhzhya and Kyiv re- gions, respectively. The manifestation of symptoms may differ based on the group explaining the fact that some infected PNRSV trees show no signs of viral infection. 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Delo je prispelo 11. maja 2020, sprejeto 28. februarja 2021. 1 isfahan University of Technology, Department of Natural resources, isfahan, iran 2 isfahan University of Technology, research institute for biotechnology and bioengineering, isfahan, iran 3 Corresponding author, e-mail: poorangolkar@gmail.com; golkar@cc.iut.ac.ir 4 isfahan University of Technology, College of agriculture, Department of agronomy and Plant breeding, isfahan, iran Discrimination of drought tolerance in a worldwide collec- tion of safflower (Carthamus tinctorius L.) genotypes based on selection indices Abstract: improvement of elite safflower genotypes for drought-tolerance is hampered by a deficiency of effective se- lection criteria. The present study evaluated 100 genotypes of safflower in terms of their drought tolerance over a period of three years (2016–2018) under both non-stress and drought- stress conditions. The eight drought-tolerance indices of toler- ance index (TOL), mean productivity (mP), geometric mean productivity (GmP), stress susceptibility index (SSi), stress tolerance index (STi), yield stability index (ySi), drought re- sistance index (Di), and harmonic mean (Harm) were calcu- lated based on seed yield under drought (ys) and non-drought (yp) conditions. a high genetic variation was found in drought tolerance among the genotypes studied. The mP, GmP, and STi indices were able to discriminate between tolerant and drought-sensitive genotypes. Plots of the first and second prin- cipal components identified drought-tolerant genotypes aver- aged over the three study years. Cluster analysis divided the genotypes into three distinct groups using the drought toler- ance indices. Ultimately, eight genotypes (namely, G3, G11, G13, G24, G33, G47, G58, and G61) from different origins were detected as more tolerant to drought stress suitable for use in safflower breeding programs in drought-affected areas. The most tolerant and susceptible genotypes could be exploited to produce map- ping populations for drought tolerance breeding programs in safflower. Key words: cluster analysis; drought stress; principal component analysis; selection index; yield; safflower Abbreviations: TOL: Tolerance; mP: mean productiv- ity; SSi: drought susceptibility index GmP: geometric mean productivity; ySi: yield stability index; Di: drought resistance index. Odkrivanje tolerance na sušo v mednarodni zbirki genotipov žafranike (Carthamus tinctorius L.) na osnovi izbranih inde- ksov Izvleček: izboljšanje elitnih genotipov žafranike na prena- šanje suše ovira pomanjkanje učinkovitih selekcijskih kriterijev. V raziskavi je bilo ovrednoteno 100 genotipov žafranike glede na njihovo prenašanje suše v obdobju treh let (2016–2018) v razmerah brez stresa in razmerah sušnega stresa. izračunanih je bilo osem indeksov tolerance na sušni stres kot so tolernca na stres (TOL), poprečna produktivnost (mP), geometrijska poprečna produktivnost (GmP), indeks stresne občutljivosti (SSi), indeks stresne tolerance (STi), indeks stabilnosti pridelka (ySi), indeks odpornosti na sušo (Di), in harmonično poprečje na osnovi pridelka semena v sušnih (ys) in nesušnih (yp) raz- merah. med preučevanimi genotipi je bila ugotovljena velika genetska variabilnost v toleranci na sušo. z indeksi mP, GmP, in STi je bilo mogoče razlikovati na sušo tolerantne in občutljive genotipe. Polja prve in druge glavne komponente so določila na sušo tolerantne genotipe v vseh treh letih raziskave. klasterska analiza je z uporabo indeksov tolerance na sušo razdelila geno- tipe v tri jasno ločene skupine. Na koncu je bilo ugotovljenih osem genotipov (G3, G11, G13, G24, G33, G47, G58, in G61) različ- nega izvora, ki so bili bolj tolerantni na sušo in so primerni za uporabo v žlahtniteljskih programih žafranike na od suše ogro- ženih območjih. Na sušo najbolj prilagojene genotipe žafranike bi lahko uporabili za odkrivanje populacij, ki bi bile primerne pri žlatnjenju žafranike na sušo. Ključne besede: klasterska analiza; sušni stres; analiza glavnih komponent; selekcijski indeks; pridelek; žafranika Okrajšave: TOL: toleranca; MP: poprečna produktivnost; SSI: indeks občutljivosti na sušo; GMP: geometrijska poprečna produktivnost; YSI: indeks stabilnosti pridelka; DI: indeks odpornosti na sušo. Acta agriculturae Slovenica, 117/1 – 20212 P. Golkar et al. 1 INTRODUCTION Droughts due to alterations in rainfall patterns and climate change form a most devastating factor in food pro- duction on a global scale (blum, 2018; anjum et al., 2017). This warrants a blue revolution in agriculture concentrat- ed on increasing productivity per unit of water to produce more crops per drop of water. recently, an important target in crop breeding programs is the development of drought- tolerant genotypes that possess a high capability for adap- tion to arid and semi- arid climates (kirigwi et al., 2004; basu et al., 2016). The challenges in understanding the mechanisms in- volved in plant behavior under water scarcity include: 1) mutagenic control of drought tolerance, 2) genetic variabil- ity and differences among species in responding to changes in water availability, and 3) interactions with other factors such as drought stress duration and intensity (Varshney et al., 2018). moreover, breeding programs are adversely af- fected by the high interaction of genotype × environment, low heritability of drought tolerance traits, lack of efficient selection particularly under field conditions, and the dif- ficulties associated with simultaneous selection, sharp climate changes, and unpredictable rainfall in different re- gions (ashraf, 2010; rauf et al., 2016; blum, 2018). Since the genotypes with a high yield under optimum conditions may not be drought tolerant (blum, 2018), many studies preferred selection under both stress and non-stress condi- tions (Fernandez, 1992). To have a high and durable yield in a drought-prone environment drought-tolerant genotypes are needed (abdolshahi et al., 2012). The capacity of genotypes to per- form reasonably well in drought-stressed environments is the paramount reason for their stable production (raman et al., 2012). To decrease the impacts of abiotic stress with- out any substantial yield loss, researchers tend to develop drought-tolerant genotypes based on prior evaluation and identification of drought-tolerant germplasm. The high cost of drought soil amelioration has encouraged breed- ers to use selection indices as an economic and efficient method for resolving the problems associated with drought stress breeding (Vieira et al., 2016). in this regard, a variety of selection indices to identify stress-tolerant cultivars have been proposed that some of the important and most applicable of them include: Toler- ance (TOL) (rosielle $ Hamblin, 1981) (Table 1) , mean productivity (mP) (rosielle & Hamblin, 1981) (See Table 1), stress susceptibility index (SSi) (Fischer and maurer, 1978) (See Table 1), geometric mean productivity (GmP) (kristin et al., 1997) (See Table 1), stress tolerance index (STi) (Fernandez, 1992) (See Table 1), yield stability index (ySi) (Gavuzzi et al., 1997) (See Table 1), and drought re- sistance index (Di) (Lan, 1998) (See Table 1). Our literature review have reported on the efficiency of different selection indices for selecting drought-tolerant genotypes in different crops as like as rice (raman et al., 2012); canola (khalili et al., 2012);sunflower (Gholinezhad et al., 2014); maize (Hao et al., 2011) and bread wheat (abdolshahi et al., 2012). Safflower (Carthamus tinctorius L.) is an annual oil Table 1: Different drought tolerance indices used for screening safflower genotypes Acta agriculturae Slovenica, 117/1 – 2021 3 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices seed crop with diverse industrial and pharmaceutical ap- plication that is grown commercially in iran (Golkar & ka- rimi, 2019). The deep roots of safflower make it a drought- tolerant plant viable under the drought stress conditions in arid climates (mirzahashemi et al., 2014; Hussain et al., 2016). Drought stress is one of the most devastating abiotic stresses that poses a serious threat to worldwide safflower production (Hussain et al., 2016). Given the declining water resources in the arid and semi- arid regions of the world due to consecutive droughts, increased safflower cultivation can be an economic and valuable alternative to other drought- tolerant genotypes. in this regards, some studies is known about drought tolerance of local iranian cultivars (Omidi et al., 2012; bahrami et al., 2014; mirzahashemi et al., 2014). Despite of current efforts intended for assessing tolerance criteria based on tolerance indices in safflower, little has been reported at maturity (bahrami et al., 2014). Further- more, this tolerance undoubtedly appears to be stage-de- pendent and must be evaluated at the yielding phase. Variations in drought patterns such as differences in location, year, and drought intensity as well as genotypic differences call for safflower genotypes with different lev- els of drought tolerance to be cultivated in different areas. However, the differences in the genotypes recommended might have stemmed from the variability in the drought tolerance potential of safflower genotypes. moreover, cli- mate changes increase drought frequency in some regions but drought is a global issue. Given the broad distribution of safflower around the world, it is the objective of the pre- sent study to identify drought-tolerant genotypes from a new worldwide collection based on drought selection indi- ces. The new identified genotypes could be used for cultiva- tion in arid regions of the world. 2 MATERIALS AND METHODS 2.1 PLaNT maTEriaL One hundred safflower genotypes originating from different geographical regions of the world were selected for screening drought tolerance (Table S1). The exotic genotypes were obtained from Leibniz institute of Plant Genetics and Crop Plant research (iPk), Germany. iranian genotypes were taken from the genotype inventory at the agricultural research Center, isfahan, iran. 2.2 FiELD EXPErimENT aND irriGaTiON rE- GimES an experiment was carried out in three consecutive years from early march 2016 to the end of 2018 at Lavark research Farm, affiliated to isfahan University of Tech- nology, 40 km southwest of isfahan (32° 32΄N, 51° 23΄ E, 1630 m above sea level), iran. mean annual precipitation and temperature at this site are 149 mm and 15.4°C, re- spectively. The soil was silty clay loam with a bulk density of 1.3 g cm‒3 in the top 50 cm and a pH level of 7.4-7.9. The field experimental design was a square lattice design (10 by 10) with two replications for each different irrigation (drought stress and non-drought) regimes in each year. The seeds were planted in rows of 3 m long and spaced 25 cm from each other to yield a plant density of 40 plants m‒2 in the plots. all the plants received the first irrigation before planting. after this period, irrigation was applied every week until the budding stage. From budding stage to full maturity stage, the non-stress treatment involved irrigation when 50 % of the total available water was depleted from the root zone, but in the drought stress conditions, irriga- tion was applied when 85 % of the total available water was depleted from the root zone. The irrigation interval (number of days between two irrigations) during the growing season (budding to full ma- turity) was variable because of the variation in evapotran- spiration (ET). Soil samples were taken from a depth of 0 to 60 cm of the soil from both drought and non- drought plots to determine the soil water content and calculate the irriga- tion water content on the basis of a 60 cm rooting depth. Soil samples were taken before each irrigation when evap- oration from a Class a pan indicated 70 and 140 mm of evaporation under normal and drought-stress conditions, respectively. Then, irrigation depth was determined using the for- mulae: i = [(θFC - θi)/100] D × b); where, i represents irriga- tion depth (cm), θFC (–0.03 mPa) is soil gravimetric mois- ture percentage at field capacity (22 %), θi (–1.5 mPa) is soil gravimetric moisture percentage at irrigation time (10 %), D is root-zone depth (50 cm), and b is soil bulk density at the root zone (1.3 g cm–3) (Clarke et al., 2008). The volume of irrigation water applied was monitored at each irriga- tion by calculating the depth of water over a Parshall flume which was calculated as: id = i ×p, where p is the fraction of i that can be depleted from the root zone. Then ig = (id/ Ea) × 100, which Ea is irrigation efficiency (%), assumed to be 65 % on the average. The differences in available water related to different mean for temperature in growing sea- sons across three years of study. No growth regulators or fungicides were applied. Surface application of 130 (kg ha‒1) N and 25 (kg ha‒1) P was carried out in both treatments with an additional 55 kg ha‒1 of N during the rosette stage. Plants were harvested in the middle row at matu- rity and seed yield was recorded in each plot. Ten differ- ent selection indices were calculated using the equations reported in Table 1. in these equations, YS represents the Acta agriculturae Slovenica, 117/1 – 20214 P. Golkar et al. yield of genotypes under stress; YP, the yield of genotypes under normal conditions (kg ha‒1); and denote mean yields of all the genotypes under stress and non-stress conditions, respectively. 2.3 STaTiSTiCaL aNaLySiS a combined analysis of variance (aNOVa) was per- formed using SaS software (SaS. Ver. 9.3.1), for seed yield and selection indices using GLm procedure. Principal Component analysis (PCa) and 3D biplot diagrams were exploited to identify tolerant and susceptible genotypes us- ing r software (Ver. 3.6.1). Correlations between seed yield in the non-stress and drought-stress treatment as well as the relevant drought tolerance indices were determined us- ing SaS PrOC COrr and Heat map Graph (r software ver 3.6.1). The safflower genotypes were classified using the seed yields obtained from each of the water treatments and drought tolerance indices data using the Ward algorithm based on the squared Euclidean distances in the r Software (Ver. 3.6.1). 3 RESULTS analysis of variance indicated the non-significant effect of year on all the studied traits (Table 2). a highly significant (p < 0.01) variation in seed yield was observed among the studied genotypes under both (stress and non- stress) conditions and for all the tolerance indices exam- ined (Table 2). The genotype × year interaction effect was not significant for any of the indices, except for Di and Harm (Table 2). The significant genotype × environment interaction for both Di and Harm, indicating consider- able variability among the genotypes across different years and different irrigation treatments for these selection indi- ces. Table 3 reports the ten highest and the lowest seed yields, yp and ys, for the studied genotypes. Clearly, the highest yp values were obtained for G13 (5680 kg ha -1) (from iran) and G61 (5310 kg ha ‒1) (from morocco), but the least yp value was obtained for G79 (900 kg ha ‒1). Under stress conditions, the highest seed yield (ys) was obtained in gen- otypes 47 (3038 kg ha‒1) and 24 (2670 kg ha‒1), but the low- est (590 kg ha‒1) was observed in G79 (Table 3). G13 recorded the highest values of TOL (4130), SSi (1.61), and Harm (1.14) indices, whereas G86 (from Tajikistan) recorded the least values for TOL (260), SSi (0.24), and Harm (0.13). The highest (0.87) and the lowest (0.27) values of ySi were obtained for G86 and G13, respectively. Finally, the geno- types 47 and 25 had the highest (1.55) and lowest (0.20), respectively, mean values of the Di index (Table 3). The correlation coefficients among yp, ys, and other drought tolerance selection indices were calculated to determine the most desirable drought tolerance criteria (Table 4). it was found that seed yield and ySi exhibited negative (- 0.5**) and positive (0.34**) correlations under the non-stress and stress conditions, respectively. Seed yield under the non-stress treatment showed positive and signif- icant correlations with all the selection indices, except for ySi (‒0.50**) and Di (Table 4). Seed yield under the stress treatment showed positive and significant correlations with mP, GmP, STi, and Di but negative and significant ones with SSi and Harm (Table 4). Principal Component analysis (PCa) as a representa- tive for distinguish the relationships among the indices re- vealed that the first component (PC1) explained 54 % of the total seed yield variation and exhibited positive correlations with yp, mP, GmP, and STi (Figure 1). PC2 explained 44 % of the total yield variation and had higher positive correla- tions with Di, ySi, and ys but higher negative correlations with SSi and Harm (Figure 1). S.O.V DF yP ys SSi ySi TOL mP GmP STi Di Harm year (y) 2 3947374.2 77841 0.00019 0.05 133849.6 29145.2 303800.5 0.047 0.34 0.2 block/ year 3 282227.2 117545.25 0.36 0.06 696640.8 25726.0 14486.5 0.003 0.13 0.18 Genotype (G) 99 3305183.4** 844416.53** 0.40** 0.08** 2177268.3** 1530482.9** 1260048.7** 0.53** 0.34 0.23** G × y 198 89064.9 80535.49 0.071 0.01 143568.0 48908.2 55476.1 0.028 0.05** 0.54** residual 297 89064.9 154372.9 0.12 0.025 350774.0 122390.6 125950.5 0.06 0.09 0.07 Table 2: Combined analysis of variance for seed yield under non-stress (yp) and stress (ys) conditions and different susceptibility indices in safflower genotypes growing under drought stress and normal conditions evaluated in 2016 and 2018 * and **, significant at p < 0.05 and p < 0.01, respectively. abbreviations: DF: degree of freedom; yp: seed yield under non-stress; ys: seed yield under stress; SSi: stress susceptibility index, ySi: yield stability index; TOL: stress tolerance; mP: mean productivity; GmP: geometric mean productivity; STi: stress tolerance index; Di: Drought resistance index; and Harm: Harmonic mean Acta agriculturae Slovenica, 117/1 – 2021 5 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices Ten highest indices yP¥ (kg ha-1) yS (kg ha-1) SSi ySi TOL mP GmP STi Di Harm 5680(G13) 3080(G47) 1.613(G13) 0.8724(G86) 4130(G13) 3985(G47) 3820.2(G47) 2.2172(G47) 1.5516(G47) 1.1488(G13) 5310(G61) 2670(G24) 1.58(G61) 0.8552(G50) 3770(G61) 3615(G13) 3233.2(G24) 1.6356(G11) 1.3097(G24) 1.1104(G61) 4910(G58) 2390(G11) 1.5697(G25) 0.84(G99) 3140(G58) 3425(G61) 3159.8(G11) 1.5709(G24) 1.1934(G94) 1.1069(G76) 4890(G47) 2385(G3) 1.5629(G76) 0.8148(G38) 2800(G76) 3380(G11) 3046.4(G3) 1.4443(G3) 1.1672(G97) 1.1029(G25) 4520(G33) 2350(G97) 1.5285(G18) 0.8042(G81) 2760(G33) 3340(G58) 2957.2(G13) 1.33(G13) 1.15(G69) 1.0677(G18) 4370(G11) 2167.5(G48) 1.5057(G59) 0.7948(G94) 2750(G18) 3305(G24) 2931(G58) 1.2916(G58) 1.1144(G48) 1.03(G59) 3980(G27) 2160(G2) 1.4701(G27) 0.7841(G41) 2690(G27) 3142.5(G3) 2846.3(G61) 1.2356(G61) 1.1077(G99) 1.0095(G27) 3980(G18) 2130(G63) 1.4483(G66) 0.7749(G12) 2450(G59) 3140(G33) 2824.3(G97) 1.215(G97) 1.0974(G63) 0.9724(G66) 3940(G24) 2118.3(G94) 1.4284(G58) 0.7713(G69) 2440(G25) 2880(G97) 2817.8(G33) 1.2063(G33) 1.0941(G86) 0.97(G21) 3900(G3) 2100(G69) 1.4258(G21) 0.7668(G37) 2300(G66) 2845(G2) 2759(G2) 1.1386(G2) 1.0793(G11) 0.9508(G58) Ten lowest indices yP (kg ha-1) yS (kg ha-1) SSi ySi TOL mP GmP STi Di Harm 1 2660(G8) 930(G78) 1.3358(G95) 0.7411(G10) 1915(G39) 2660(G57) 2568.3(G48) 1.0026(G48) 1.0463(G96) 0.8926(G95) 2 1510(G81) 920(G95) 0.5084(G69) 0.3546(G21) 410(G71) 1210(G67) 1167.2(G64) 0.209(G31) 0.3026(G79) 0.2604(G69) 3 1460(G64) 920(G87) 0.4939(G12) 0.3461(G66) 380(G99) 1200(G64) 1161.3(G31) 0.2038(G64) 0.3007(G95) 0.2553(G12) 4 1450(G67) 910(G37) 0.4824(G41) 0.3334(G27) 340(G38) 1175(G71) 1155.7(G71) 0.2032(G71) 0.3003(G66) 0.2536(G41) 5 1380(G89) 890(G21) 0.4545(G94) 0.3204(G59) 339(G5) 1160(G89) 1138.2(G89) 0.1942(G89) 0.2884(G18) 0.2367(G94) 6 1380(G71) 860(G31) 0.4392(G81) 0.3093(G18) 320(G81) 1150(G9) 1071.8(G9) 0.173(G9) 0.2834(G87) 0.2299(G81) 7 1350(G82) 801(G5) 0.4147(G38) 0.2939(G76) 310(G79) 1070(G82) 1046.6(G37) 0.1647(G37) 0.2647(G59) 0.208(G38) 8 1210(G37) 790(G82) 0.3429(G99) 0.2904(G25) 300(G50) 1060(G37) 1026(G82) 0.1582(G82) 0.2497(G21) 0.1803(G99) 9 1140(G5) 780(G9) 0.3284(G50) 0.2884(G61) 300(G37) 970.5(G5) 952.4(G5) 0.1363(G5) 0.2346(G76) 0.1587(G50) 10 900(G79) 590(G79) 0.2846(G86) 0.2719(G13) 260(G86) 745(G79) 718.9(G79) 0.0788(G79) 0.2024(G25) 0.1392(G86) Table 3: Ten highest and lowest values for seed yield under non-stress conditions (yp), Seed yield under stress conditions (ys), and different selection indices among the 100 different safflower genotypes investigated yp ys SSi ySi TOL mP GmP STi Harm Di yp ¥ 1 ys 0.59 ** 1 SSi 0.50** -0.34** 1 ySi -0.50** 0.34** -0.99** 1 TOL 0.86** 0.10 0.83** -0.83** 1 mP 0.95** 0.80** 0.24* -0.24* 0.67** 1 GmP 0.88** 0.89** 0.08 -0.08 0.52** 0.98** 1 STi 0.86** 0.88** 0.06 -0.06 0.50** 0.96** 0.98** 1 Harm 0.53** -0.32** 0.99** -0.99** 0.86** 0.27** 0.10 0.08 1 Di 0.17 0.89** -0.70** 0.70** -0.31** 0.47** 0.61** 0.61** -0.67** 1 Table 4: Correlation coefficients between seed yield (kg ha–1) of safflower genotypes under non-stress (yp) and stress (ys) conditions and each of the stress susceptibility indices averaged over three years * and ** Significant at p < 0.05 and p < 0.01; respectively; ns, not significant. abbreviations ¥: yp: Seed yield under non- stress condition; ys: Seed yield under stress condition; SSi: stress susceptibility index, ySi: yield stability index, TOL: stress tolerance, mP: mean productivity, GmP: geometric mean productivity, STi: stress tolerance index, Harm: harmonic mean, Di: drought resistance index. Acta agriculturae Slovenica, 117/1 – 20216 P. Golkar et al. 3.1 CLUSTEr aNaLySiS a dendrogram was drawn based on the cluster anal- ysis using seed yield under drought and non-drought conditions along with the selection indices TOL, mP, GmP, STi, SSi, ySi, Di, and Harm (Figure 3). The clus- ter analysis performed classified the 100 genotypes of saf- flower investigated into three distinct groups consisting of 7, 44, and 49 genotypes. The genotypes in the smallest group (1) including G13, G61, G33, G58, G47, G11, G24, and G3 showed the highest seed yield under both non-stress and drought stress conditions (Figure 3). The genotypes clustered in Group 2 (i.e., G51, G93, G39, G60, G2, G57, G14, G16, G97, G48, G63, G99, G50, G86, G10, G65, and G81) recorded medium levels of seed yield under drought stress. The because of the positive and significant correlation of STi with seed yield under both conditions, a three- dimensional graphs based on the STi index were drawn (Figure 2). These graphs split the genotypes into four groups, each of which represents one combination of the genotypes. The genotypes 47, 24, 97, 3, and 11 (Group a) are those with high yields under drought and non- stress environments. Those in Group b (e.g., G13, G59, and G61) consisted of genotypes with high yields in a normal environment but low seed yields under drought condi- tions. No genotype was, however, detected as one with a high yield in a stressful environment (Group C). The genotypes with low yields under both environmental conditions were assigned to Group D (e.g., G3, G11, G79, and G5). Figure 1: biplot drawn based on the first and second components obtained from principal component analysis using seed yield of safflower genotypes under non stress (yp) and stress (ys) conditions. abbreviations: stress susceptibility index (SSi), yield stability index (ySi), stress tolerance (TOL), mean productivity (mP), geometric mean productivity (GmP), stress tolerance index (STi), drought resistance index (Di); harmonic mean (Harm), and conditions in 100 safflower genotypes. Figure 2: Three-dimensional diagram for identifying drought-tolerant genotypes based on seed yield under non-stress (yp) and stress (ys) conditions as well as the stress tolerance index (STi). Acta agriculturae Slovenica, 117/1 – 2021 7 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices third group consisted of genotypes with a low productiv- ity under either environmental conditions (i.e., G79, G37, G89, G55, G21, and G25). 4 DISCUSSION This study evaluated drought tolerance in a world collection of safflower accessions under the effects of year and genotype. The analysis of variance showed a large genetic variation in drought tolerance among the accessions studied as an unpredictable factor affecting seed yield in the genotypes from different geographical regions. year factor was not found to have any significant effect on seed yield or selection indices; hence, the indi- ces selected for this germplasm can be effectively used if seed yield is adequately heritable. Considering the fact that traits involved in drought tolerance mechanisms are polygenic ones, the requirement to screen tolerant geno- types has encouraged plant breeders to seek a reliable in- dex. in response to this need, the present study evaluated eight different selection indices (i.e., mP, GmP, TOL, SSi, STi, ySi, Di, and Harm) for use in the estimation of seed yield under drought stress. based on the correlation analysis performed, the positive and significant correla- tion between TOL and yp (0.86 **) implies that the geno- types superior in terms of seed yield (such as G79 and G9) showed greater reductions in seed yield under drought conditions. also, the non-significant correlation between TOL and ys (0.10) revealed the failure of the TOL index Figure 3: Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices. Acta agriculturae Slovenica, 117/1 – 20218 P. Golkar et al. to identify the most tolerant genotypes, confirming the results reported by rizza et al. (2004). The greater TOL values indicated the higher sensitivity of the genotypes investigated to drought stress; thus, smaller values of this index is favored. The positive and significant corre- lation between yp and SSi (0.50 **) and that between yp and Harm (0.53**) demonstrated that the genotypes with higher values for yp or the SSi index exhibited a higher sensitivity to drought stress (Table 2). On the other hand, the negative and significant correlation be- tween SSi and ys (-0.34**) or that between Harm and ys (-0.32 **) implied that the superior genotypes under drought stress recorded lower values for SSi and Harm. Hence, the SSi and Harm indices are able to discrimi- nate superior safflower genotypes (the ones with lower values of SSi or Harm indices) in drought prone areas. Studying spring wheat, Guttieri et al. (2001) maintained that SSi values >1 and <1 might indicate above-average and below-average susceptibility to drought stress, re- spectively. The most suitable index for selecting stress- tolerant genotypes is an index that establishes a positive and strong correlation with seed yield under both stress and non-stress conditions (Fernandez, 1992). To select drought-tolerant genotypes, based on the most desirable indices, use is made of the correlation coefficient of each index with yp and ys (Golabadi et al., 2006; Ebrahymian et al., 2012; abdolshahi et al., 2012; Naghavi et al., 2013). Seed yield was found to have a highly significant positive correlation with GmP, mP, STi, and Harm indices un- der both the environmental (drought and non- drought conditions) conditions examined (Table 2). based on the correlation analysis conducted in this study, GmP, STi, and mP were found to favor genotypes with a high-yield potential under stress conditions (Table 2), which agrees with the findings reported Sio-Se mardeh et al. (2006), Hao et al. (2011), and Ebrahimiyan et al. (2012). Given the fact that G47 recorded the highest values for mP and STi, this genotype was identified as the most productive and stable safflower ones from among the ones investi- gated under both stress and non-stress conditions. The results of the present study indicating the capability of the selection indices GmP, mP, and STi to identify geno- types satisfactorily under both conditions are consist- ent with those reported for GmP and mP in mungbean (Fernandez, 1992); STi and GmP in rice (raman et al., 2012); safflower (bahrami et al., 2014) Brassica napus L. (khalili et al., 2012) and durum wheat (ilker et al., 2011); as well as GmP, STi, and mP in tall fescue (Ebrahymian et al., 2012) and maize (Hao et al., 2011). GmP is often used by plant breeders interested in calculating relative performance since drought stress might vary in sever- ity both under field conditions and over different years (Fernandez, 1992). in the present study, GmP established significant and positive correlations with TOL, yp, and ys (Table 3). Di, which is commonly accepted as an index to identify genotypes with high yields under both stress and non-stress conditions (Lan, 1998), showed only a highly significant and positive correlation with ys (0.89 **) (Table 4), demonstrating that selection of safflower genotypes with high Di values might be useful for severe drought- stricken regions but that the genotypes selected based on this index do not have very high yields or yields equiva- lent to those of genotypes currently cultivated under nor- mal irrigation. Seed yield under non-drought conditions (yp) was positively correlated with yS, confirming previ- ous reports on safflower (bahrami et al., 2014) other crop species such as bread wheat (El-rawy and Hassan, 2014), corn (Naghavi et al., 2013) and bread wheat (abdolshahi et al., 2012). it may also be noted that the satisfactory responses shown by some genotypes under stress condi- tions could be ascribed to the good adaptation mecha- nisms in these genotypes (Naghavi et al., 2013). The impacts of the different indices in each PC indicate that PC1 and PC2 could be identified as yield potential and stress susceptibility groups, respectively. The genotypes (such as G47, G11, G3, and G24) recording high values for both PC1 and PC2 may be considered as superior ones for seed yield under both experimental conditions; hence, they are designated as stable genotypes (Figure 1a). The genotypes recording low PC1 but high PC2 values includ- ed those also with high values of Di, ySi, and seed yield under drought stress, but low values of SSi and Harm values (Figure 1b). The genotypes (such as G13 and G61) recording high PC1 but low PC2 values included geno- types with high values for GmP, STi, mP, and seed yields under non-stress conditions (Figure 1C). On the other hand, the majority of the genotypes with low PC1 and PC2 values were identified as susceptible genotypes; these included G79, as the most tolerant one, and the genotypes G9, G82, and G89 (Figure 1D), that were recognized as un- stable genotypes. The majority of the genotypes investi- gated (more than 60 %) were classified in Groups b and D (Figure 1). This biplot may also be used for identifying contrasting genotypes (genotypes in group a versus D) for planning fine mapping populations for safflower ge- nome studies of drought tolerance. based on our cluster analysis, the genotypes assigned to Group 3 were recog- nized as the most tolerant ones to be used as parents for improving drought tolerance in safflower breeding pro- grams. Thus, the genotypes in Group 1 and Group 3 were identified as drought tolerant and drought susceptible, respectively. Cluster analysis has been widely used not only to discriminate high distance genotypes but also to determine genetic diversity based on similar traits under drought stress conditions (Golabadi et al., 2006; moham- madi et al., 2011; Naghavi et al., 2013).The results of the Acta agriculturae Slovenica, 117/1 – 2021 9 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices present cluster analysis of the genotypes investigated were consistent with the PCa results obtained. Thus, drought- tolerant genotypes recording high PC1 and PC2 values as well as those assigned to Groups of 1 and 3 in the cluster analysis can be used as extreme parental genotypes with the highest genetic distance to develop new hybrid varie- ties in safflower aimed at production of drought-tolerant cultivars. However, further evaluation of genotypes using drought tolerance indices across multiple locations is re- quired to confirm their stability for developing improved safflower genotypes. 5 CONCLUSION From the results obtained, it may be concluded that it is preferable to use simultaneously different drought tolerance indices for screening drought-tolerant safflow- er genotypes. The results of different multivariate analy- ses revealed that STi, mP, and GmP, in this descending order, were not only capable of efficient selection of high seed-yield genotypes under both the environmental con- ditions examined but also of discrete identification of drought-tolerant from drought-sensitive safflower geno- types. The G47 genotype (Spanish origin) was identified as the most drought-tolerant one with the highest seed yield under both drought and non-stress conditions. based on the results obtained in this study, the elite genotypes (i.e., G24, G13, G3, G11, G33, G58, and G61) may be recommended as promising cultivars for cultivation in drought-affected areas or as appropriate donor parents in safflower hybrid- ization programs. These genotypes may also be exploited for improving seed yield and stability in safflower for cul- tivation in drought prone regions through appropriate selection methods. 6 ACKNOWLEDGMENT The authors would like to thank research institute for biotechnology and bioengineering, isfahan Universi- ty of Technology, isfahan, iran. research Center, isfahan, iran, is also acknowledged for their financial support as a research project number # 97/ 70649. 7 REFERENCES abdolshahi, r., Safarian, a., Nazari, m., Pourseyedi, S., mo- hamadi-Nejad, G. (2013). 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Genetics and Molecular Research. 15, 1-10. https://doi.org/10.4238/ gmr.15027719 Acta agriculturae Slovenica, 117/1 – 2021 11 Discrimination of drought tolerance in a worldwide collection of safflower (Carthamus tinctorius L.) genotypes based on selection indices Genotype code Genotype name Geographical origin Genotype code Genotype name Geographical origin Genotype code Genotype name Geographical origin G1 a2 iran (azerbayejan) G34 Car159 Germany G67 Car64 Slovakia G2 ac- Stirling Canada G35 Car160 russia G68 Car67 Germany G3 aC-sunset Canada G36 Car161 russia G69 Car68 Germany G4 arak 2811 iran (markazi) G37 Car169 Hungary G70 Car70 Lybyan G5 C111 iran(isfahan) G38 Car175 india (kusum) G71 Car72 North korea G6 Car118 india G39 Car181 india G72 Car74 North korea G7 Car 116 india G40 Car188 Poland G73 Car75 North korea G8 Car 9 Slovaki G41 Car19 Poland G74 Car76 North korea G9 Car100 italy G42 Car190 iran (isfahan) G75 Car77 North korea G10 Car106 Spain G43 Car198 azerbaijan G76 Car78 Hungary G11 Car114 india G44 Car199 korean republic G77 Car79 Japan G12 Car117 Sudan (tozi) G45 Car200 unknown G78 Car80 North korea G13 k21 iran (kordestan) G46 Car201 Sudan G79 Car83 Tajikistan G14 Car124 Pakistan G47 Car210 Spain G80 Car86 Tunisia G15 Car125 russia G48 Car211 Germany G81 Car87 romania G16 Car126 belgium G49 Car214 Poland G82 Car89 Tunisia G17 Car127 Germany G50 Car215 Germany G83 Car94 Spain G18 Car129 Germany G51 Car216 Germany G84 GE62918 Germany G19 Car130 morocco G52 Car217 Germany G85 Gila USa G20 Car131 Paraguay G53 Car218 Germany G86 Hartman USa G21 Car132 Germany G54 Car219 Germany G87 iL111 iran (aur- oumieh) G22 Car135 Portugal G55 Car221 Germany G88 isf-14 iran (isfahan) G23 Car137 Pakistan G56 Car224 Germany G89 isf28 iran(isfahan) G24 Car138 Poland G57 Car226 Germany G90 k21 iran (kord- estan) G25 Car146 Egypt G58 Car227 Germany G91 kmS 36 iran (karaj) G26 Car147 Pakistan G59 Car228 Germany G92 mex.17-45 mexico G27 Car148 Pakistan G60 Car230 Germany G93 mex.7-147 mexico G28 Car151 india G61 Car24 morocco G94 mex.7-38 mexico G29 Car152 iraq G62 Car37 Sudan G95 mex-13-216 mexico G30 Car155 russia G63 Car42 Sudan G96 mex2-138 mexico G31 Car156 Pakistan G64 Car49 Spain G97 mex22-191 mexico G32 Car157 morocco G65 Car55 Poland G98 mex6-97 mexico G33 Car158 Paraguay G66 Car56 Nebraska 8 (USa) G99 Pi 301055 Turkey G100 Saffire Canada Table supplementary 1: Characteristics of the 100 different genotypes of safflower used in this study Acta agriculturae Slovenica, 117/1, 1–13, Ljubljana 2021 doi:10.14720/aas.2021.117.1.1716 Original research article / izvirni znanstveni članek Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria Chioma Patricia ADEKUNLE 1, Augustine Adebayo KUTU2, 3, David Alaba ALORI 4 Received June 06, 2020; accepted March 03, 2021. Delo je prispelo 06. junija 2020, sprejeto 03. marca 2021. 1 Federal University of Agriculture, Department of Agricultural Economics and Farm management, Abeokuta, Nigeria 2 Federal University of Technology, Department of Agricultural Economics & Extension, Minna, Nigeria 3 Corresponding author, e-mail: kutuA@ukzn.ac.za 4 University of KwaZulu-Natal, School of Accounting, Economics & Finance, South Africa Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria Abstract: This study examines whether greater women’s household bargaining power is associated with the improve- ment in children’s welfare in Ogun State, Nigeria. Using data from 320 farm households with a Logit regression model, the study revealed that 31.86 % of children under-five years of age were stunted, 32 % were underweight and 16.2 % were wasted. Children growing up healthy were 62 %, implying that one – third of under-five children in the study area still experience nutrition deficiency. About 3.33 % and 1.05 % children simul- taneously experienced stunting and wasting together, which perhaps suggests a harsh deprivation environment. In addition, 63.33 % of women in the study area had low bargaining power implying that they lack control over important decisions in their households. Women who enjoy decision-making power in their households, particularly with large purchasing power, are associated with having children with better height-for-age, mass-for-age, and mass-for-height ratios. Women’s inequality as relates to intra-household bargaining power negatively af- fects children’s welfare and leads to chronic malnutrition. As a policy recommendation, it is therefore, important to enhance women’s status, which, with time will lead to more investment in their children’s education, health, and overall welfare. Key words: bargaining power; child welfare; mass index; Ogun state Ali ima enakopravnost žensk v gospodinstvih vpliv na dobro- bit otrok? Primeri iz kmečkih gospodinjstev iz države Ogun, Nigerija Izvleček: V raziskavi je bilo preučevano, kako je enako- pravnost žensk v gospodinjstvih povezana z izbolšanjem dob- robiti otrok, v državi Ogun, Nigerija. Z regresijskim logit mo- delom so bili obdelani podatki iz 320 kmečkih gospodinjstev. Raziskava je odkrila, da je bilo 31,86 % otrok , v starosti pod pet let, zaostalih v razvoju, 32 % jih je imelo premajhno maso in 16,2 % jih je bilo podhranjenih in bolnih. Otrok, ki so do- raščali zdravo je bilo 62 %, kar kaže na to, da so trije od petih otrok še vedno podhranjeni. Okrog 3,33 % in 1,05 % otrok se hkrati sooča z zaostankom v razvoju in podhranjenostjo, kar kaže na zelo nevzpodbudno okolje za razvoj otrok. Dodatno ima na preučevanem območju 63,33 % žena majhno enako- pravnost, kar kaže na pomankljivost njihovega odločanja o pomembnih zadevah v gospodinjstvih. Žene, ki imajo večjo moč odločanja v gospodinjstvih, še posebej tiste z večjo ku- pno močjo, imajo otroke, ki so višji glede na njihovo starost, močnejši in z večjim razmerjem med maso in višino. Ženska neenakopravnost je povezana z njihovo neenakopravnostjo v gospodinjstvu, kar vpliva negativno na dobrobit otrok in vodi h kronični podhranjenosti. Priporočilo bi torej bilo, da je po- membno izboljšati položaj žena, kar bi s časom vodilo v več vlaganja v izobraževanje njihovih otrok, njihovega zdravja in v splošno izboljšanje stanja. Ključne besede: enakopravnost; dobrobit otrok; masni indeks; država Ogun Acta agriculturae Slovenica, 117/1 – 20212 C. P. DEKUNLE et al. 1. INTRODUCTION Globally, the significance of women’s empowerment in intra-household decision-making on household wel- fare has been a topical issue and debate among scholars. The debate centres on the need to enhance women’s bar- gaining power and increase their participation in intra- household decision-making (Grown et al., 2005; Mal- hotra and Schuler, 2005). This subject is also captured in the Millennium Development Goals (MDGs) and the Sustainable Development Goals (SDGs) in order to achieve international development. The need to enhance women’s empowerment and achieve economic growth also led to the formation of the World Bank’s Gender Ac- tion Plan in 2006 (Zuckerman, 2007). Policymakers and development practitioners are of the opinion that wom- en’s empowerment is a necessity for societal development (Pratley, P. (2016). Based on this, an empirical research study in this nature is essential in order to be able to suggest to policymakers an effective policy to empower women both in the rural and urban areas for the purpose of achieving desired outcome of empowering women and promote their bargaining power in all economic fronts. It is important to note that lack of women’s empowerment manifests in various ways, including in the labour market (increase unemployment), increase poverty and limited economic opportunities (Guvuriro and Booysen, 2019). In line with the need for women’s empowerment, Yusof and Duasa (2010) and Doepke and Tertt (2014) revealed that women’s empowerment can influence the allocation of resources including those that concern children and the one that relates to national development. This study finds that women remain economically disadvantaged in Nigeria when compared to their men counterparts. They are also disadvantaged in the intra-household decision- making power and are sometimes prevented from ac- tively participate in cash-based work or labour market. This development accounts for the significance and mo- tivation of this study, especially on the need to examine whether greater women’s household bargaining power or empowerment will lead to the improvement in children’s welfare in Nigeria. According to Duflo (2000), there is a correlation between women’s greater involvement in household decision-making and the allocation of spending in the family and among farm households, which benefit chil- dren. Duflo revealed that an increase in the women’s decision-making power in the family would positively affect the expenditure allocation and overall children’s wellbeing. This means that as the share of household in- come controlled by women increases, the welfare of the children is enhanced (World Bank, 2011). This is because an improvement in women’s intra-household bargaining power and resources allocation lead to improved welfare outcomes of children (Wang, 2014). According to the data released by UNICEF/WHO/ World Bank Group (2017), about 151 million of children under-five years suffer from malnutrition and 67 million were wasted globally in 2017 due to lack of women’s em- powerment. Studies have revealed that among the pub- lic health problems that resulted from lack of women’s empowerment, malnutrition remains the highest among children in developing countries including Nigeria. Mal- nutrition affects the mental reasoning and physical de- velopment of children, increases the danger of infections and contributes to the child’s illness and death (Pelletier and Frongillo, 2003). According to De Onis and Blössner (2003), stunting, wasting and underweight are the three major symptoms of a child’s malnutrition. Stunting and wasting are an indication of chronic and acute malnutri- tion while underweight is a combination of both acute and chronic malnutrition (De Onis and Blössner 2003). All these problems and symptoms are because of lack of women’s empowerment. Various studies have shown that malnutrition con- tributes to the global burden of diseases. According to Liu et al. (2015), malnutrition accounts for 50  % of all the global death rate among children under the age of five. The WHO (2016) also revealed that about 99 million of under-five year’s children were either stunted, wasted or underweight globally while approximately 6 million children were stunting and wasting at the same time (Khara et al., 2018). Based on these figures, 55 % of all stunted children were from Asia and 39 % from Africa, while 69  % of the stunting and wasting children were from Asia and 27 % from Africa (UNICEF/WHO/World Bank Group, 2017) since women are lagging behind in terms of empowerment and involvement in households’ decision-making. In Nigeria, and especially among farm households, malnutrition is one of the most important factors that contribute to child mortality among under-five year’s children. This occurs most among the farm households. According to UNICEF/WHO/World Bank (2017), mor- tality rates of about 128 deaths per 1000 births were recorded in 2017 while 37 % of under-five children are stunted. Because of deficiency in women’s empowerment and poor intra-households decision-making power, there is also the problem of maternal nutrition in which 11 % and 25 % prevalence of undernutrition and overweight/ obesity were recorded among women (National Popu- lation Commission and ICF International, 2014). This problem is more prevalent among farm households and as a result, children in rural areas are more likely to be stunted than those in urban areas (Herrador et al., 2014). It is against this view and background that this study Acta agriculturae Slovenica, 117/1 – 2021 3 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria seeks to examine whether greater women’s household bargaining power is associated with the improvement in children’s welfare using farm households in Ogun State as a case study. This study has meaningful implications for policy and contributes to the growing literature, which finds evidence supporting increased female bargaining power as a method to promote equality and improve child health outcomes. The rest of the article is as follows. The next section presents the empirical review of the lit- erature. Section 3 presents the methodology employed in analyzing the research data. Section 4 contains the women’s bargaining power and the questions that were contained in the questionnaire. Section 5 presents the results and discussion and section 6 concludes the study. Many studies have established strong evidence of women’s empowerment and higher intra-households decision-making power to translate into improved child wellbeing and societal development. For example, Gokhale et al. (2004) and Chen and Li (2009) show a strong link between women’s education, decision-mak- ing and child survival or better living condition. Angel- Urdinola and Wodon (2010) also revealed that women’s earning and income generation contribute to women’s empowerment and intra-households’ decision-making. According to Urdinola and Wodon, wage gap is a sign of discrimination against women and this can hinder chil- dren’s access to education, healthcare and other social- economic wellbeing. Richards et al. (2013) carried out a study on gen- dered intra-household bargaining as a social determi- nant of child health and nutrition in the low and middle- income countries. They revealed that “intra-household bargaining power operates through inter-linked mecha- nisms that shape how resources are channelled to chil- dren in terms of nutrition and health inputs which lead to better child health and nutrition outcomes”. Tolhurst et al. (2008) advocated a gender transformatory ap- proach, which aims to promote women’s empowerment and intra-household decision-making power needed in order to achieve gender relationships and quality wellbe- ing for the children. Behrman and Skoufias (2006) highlighted the sig- nificance of income in achieving women’s empowerment and greater intra-households decision-making. Based on their view, income in the hands of women will lead to a complete income pooling in the households and height- en the development of the entire family. Gummerson and Schneider (2013) supported this view and revealed that women are the more responsible managers of financial resources in the family. This is also in line with Behrman and Skoufias (2006) who stated that “policies targeted to- wards women can generate immediate consequences by either improving a women’s voice in the household and/ or contributing to an improvement in human capital in- vestments in children”. Expanding on the link between improved women’s bargaining power and resource allocation, Doss (2013) revealed that there is a sufficient evidence to support the view that women’s bargaining power does affect resource allocation and developmental outcomes. This means that there is a link between women’s empowerment or bargaining power and the development of the society. Invariably, there is a link between the relative decision- making power at the household level and greater expan- sion of the development of the society. Martínez (2013) shared similar view and indicated that increased women’s bargaining power would promote equality and improve child welfare. While carrying out a study of Women’s intra-household bargaining power and child welfare out- comes, Saaka (2018) also revealed that women’s empow- erment is associated with benefits for women and their children. However, despite the numerous studies on the wom- en’s intra-household bargaining power and children’s welfare outcomes around the world, there is scanty re- search on the subject in Nigeria. Realizing this empirical gap in the literature, this study is undertaken to contrib- ute to the debate on whether greater women’s household bargaining power is associated with the improvement in children’s welfare in Nigeria. It is expected that the findings of the study will help enlighten policymak- ers on the wisdom of formulating policies that enhance women’s empowerment and increase their bargaining power within the household as well as increase spending on long-term durable goods (education and nutrition of children). 2. METHODOLOGY 2.1 STUDY AREA The study area is Ogun State in South West Nigeria. The state was created in February, 1976 with Abeokuta as the capital. It has a land of approximately 1.7 million hectares and occupies about 1.9 % of Nigeria’s total land and about 2.5  % of the country’s population. It has 20 local government areas spread across the four main agri- cultural zones of the state- Egba, Ijebu, Remo, and Yewa/ Awori. The state shares an international boundary with Benin Republic to the west and Oyo state to the north, Lagos state to the south, and Ondo state to the east. In addition, the state has a population of 3.7 million based on 2006 national population census. There are two dis- tinct seasons in the state namely, the rainy season and the Acta agriculturae Slovenica, 117/1 – 20214 C. P. DEKUNLE et al. dry season and in terms of farming activities, the state is well known for livestock and cocoa production. Fasina et al. (2020) revealed that the death of new- born babies resulting from malnutrition and neonatal mortality is very high in the state. There is poor access to quality education and health care facilities especially among the rural farm households. Poor care-seeking be- haviour of families due to unemployment and poor sala- ries are high in the area. Ogunlesi and Ogunlesi (2012) also revealed that low health care education and the use of home remedies are the common living status of farm households in the study area. 2.2 STUDY DATA AND METHODS OF DATA COL- LECTION The data for the study were collected through a well-structured questionnaire and interview from the ru- ral farmers. Data were collected on the socio-economic characteristics such as age, age at marriage, number of years in marriage, gender, marital status, educational status, and other variables of mother and child (such as employment status, monthly income, land ownership, livestock ownership and access to credit). The mass and height of children were collected through the anthropo- metric measurement procedures and counter-checked using their vaccination cards. 2.3 SAMPLE AND SAMPLING TECHNIQUE This study employed the multistage sampling tech- nique; at stage one, two agricultural zones of Ijebu and Ilaro were selected out of the four zones in the state. At stage two, 50  % of the blocks were selected from each zone. This gives three blocks for Ijebu and two blocks for Ilaro and translates to five blocks. At stage three, four cells each per block were randomly selected to give twenty cells. At stage four, sixteen (16) farm households were selected from each cell, which gave three hundred and twenty (320) households. Women in the productive age (15-49 years) and under-five children were targeted by the study. In total, 141 boys and 281 girls, aged 0-59 months from 320 households were considered for the study. 2.4 ANALYTICAL TECHNIQUES AND RESEARCH DESIGN Both descriptive and quantitative techniques were used to analyse the data. The choice of both approaches was to ensure that the results are valid and reliable. It is also based on Vetter (2017) that a descriptive research design can answer the questions of who, what, where, when and how. These were the concepts used in the ques- tionnaire in framing the research questions of this study. The descriptive analysis using percentages and frequency analysis were used to describe the sociodemographic characteristics of the variables employed while logit re- gression model was used to analyze the data and deter- mine the factors related to child malnutrition. 2.4.1 Welfare Estimates of Children under the Age of Five: Analysis of Anthropometric Measure- ments The welfare status of children under the age of five is usually measured using three indices: mass-for-height (wasting) which reflects acute growth disturbances, height-for-age (stunting) which reflects long-term growth faltering and mass-for-age (underweight) which is a combination of both long and short-term effects. Anthropometric data were analyzed to generate welfare indicators of children under the age of five. The study ad- opted the method of Abera et al. (2017) to convert mass, height, and age of children into height-for-age (HAZ), mass-for-age (MAZ), and mass-for-height (MHZ) in or- der to assess malnutrition. At the end, the variables for stunting, underweight, and wasting were classified as 1 for stunted and 0 for not stunted, 1 for underweight and 0 for not underweight, and 1 for wasted and 0 for not wasted. On the other hand, the WHO (2006) child- growth standard was employed for the anthropometric measurements of children. In line with WHO (2008) and Yalew et al. (2014), the minimum dietary diversity and breastfeeding were used in the study. Seven food groups containing grains, roots and tubers, legumes and nuts, dairy products (milk, yogurt, and cheese), flesh foods (meat, fish, poultry, and liver/organ meats), eggs (vitamin-A rich fruits and vege- tables) and other fruits and vegetables were used as good meal frequency. 2.4.2 Women’s bargaining power This study made use of the women’s decision-mak- ing processes within the household as proxy for women’s bargaining power. Their ability to make decision about food preparation and consumption (Patel et al., 2007), decision about family asset or investment (Reggio, 2011), decision about gender roles, children education, number of children and health care decision (Allendorf, 2007). This study assumed that women who participate in the Acta agriculturae Slovenica, 117/1 – 2021 5 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria intra-households decision-making have relative bargain- ing power. This means that women who partake in family decision-making can be said to have greater bargaining strength than those ones that are excluded. This study was based on farm households and spe- cifically on married female households. In line with An- derson et al. (2017), this study restricts sample to female’s spouses who are living together as husband and wife. The reason for this selection was that when there is no spouse, only the one parent makes decisions. Therefore, female spouses were interviewed taking into consideration that the wife’s present husband is the father of the children. This helps to ascertain how they reach important socio- economic decisions in the family. In order to assess the allocation of household decision-making power, twenty- two (22) questions about household and farm activities were contained in the questionnaire. Using a likert scale ranging from 1 to 10, respondents were asked to choose the best option that align with their views over a given decision. Questions included in the questionnaire include the following: This study adopts questions from Doss (2013), An- derson et al. (2017) and Mengesha and Merkeb (2020). In a scale of 1 to 10, how is decision-making in the family shared between yourself and your spouse? The decision- making power is in the following regards: (a) Asset ownership • Land ownership, by plot or parcel, at the indi- vidual level? • Rights associated with the land, by plot/parcel and individual owner? • Documentation of land ownership, including names on documents? • Ownership of dwelling? • Ownership of livestock? (b) Crop and livestock • What types of crop to be cultivated on the farm? • Where to sell crops? • What types of livestock to be raised on the farm? • When to sell off the livestock? • How to spend money raised from the sale of crops? • How to spend money raised from the sale of livestock? (c) Decisions about children • What number of children to have? • What foods to feed the children? • Whether to send children to school? • Type of health practices? (d) Advance decisions • Whether to buy a new high-yield seed or use the ordinary seeds? • Whether to buy new farm equipment or use the old tools? • What types of information or training the household needs? • Who to attend farm training? (e) Broad decision-making authority: livelihood versus overall • Overall decision-making for the household? • Can you travel to visit your family and friends? • Can you go to the market alone? • Can you go to a health clinic for your own health needs? • Can you take your children to a health clinic alone? The report of decision-making authority is mea- sured on a scale (0–10) that is assigned to the choice that each spouse choose for themselves and their spouse. The statistics for each of the variables used are summarized in Table 1. Decision-Making Index (DMI) was used to mea- sure women bargaining power. The whole responses for each respondent (husband and wife) were calculated. However, this study focuses on farm households women bargaining power. The result gotten is the respondent’s score on the women decision-making index. The closer the value of the index is to 1, the higher the women bar- gaining power. This is as stated below: 2.4.1 Estimation model In this study, the welfare outcome of children under the age of five is determined by the mother’s bargaining power in the family, the child’s and women’s socioeco- nomic characteristics, and other household factors. This study focuses on three main welfare outcomes: under- weight, stunting and wasting of children under the age of five. The corresponding econometric model is specified as follows: Where Yi is a measure of the children welfare out- comes, Pi is the women’s bargaining power, Xi is the variables of children under the age of five and women’s characteristics which are variables that contribute to im- provements in child welfare outcomes. Acta agriculturae Slovenica, 117/1 – 20216 C. P. DEKUNLE et al. 3. RESULTS AND DISCUSSION 3.1 SOCIOECONOMIC CHARACTERISTICS OF WOMEN The results of the socio-demographic characteristics Characteristics Frequency Percent Mean Age ≤ 30 176 55.00 31-40 104 32.50 41 - 49 40 12.50 Mean age 29 Age at marriage ≤ 20 201 62.81 21-25 86 26.88 26-30 18 5.63 > 30 15 4.68 Mean 8 Number of marriages Once 277 86.56 More than once 43 13.44 Number of years in marriage 1-5 67 20.94 6-10 188 58.75 11 and above 65 20.31 9 Education No formal education 171 53.44 Primary 94 29.38 Secondary 44 11.75 Tertiary 11 3.44 Body Mass Index (kg m-2) Underweight 73 22.81 Healthy weight 148 46.25 Overweight/grade I obesity 64 20.00 Obese/grade II obesity 35 10.94 Land ownership 38 11.88 Livestock ownership 102 31.88 Access to credit 77 24.06 Employment status Working 288 90.00 Full housewife 32 10.00 Monthly income Less than N 50,000 231 72.19 N 51,000- N 100,000 76 23.75 N 101,000 and above 13 4.06 Mean N41,540 Table 1: Women’s socioeconomic characteristics Source: Author’s field survey, 2019 Acta agriculturae Slovenica, 117/1 – 2021 7 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria of the respondents, as presented in Table 1, show that the dominant age group for women in the study area was less than 30 years, which comprises 55 % of women respon- dents with a mean age of 29 years. Of 320 respondents, 62.81 % were women under 20 years of age when married and the mean age at marriage was 18 years. This clearly shows that the study concentrated on women in the re- productive age group (15-49 years) and evidence of early marriage is common in rural farm households in Nige- ria. About 43 women (13  %) have married more than once and 67 (21 %) women have been married for less than 5 years. Besides, 53.44 % have no formal education. It shows a lower literacy rate among rural women farm households. Overall, nutritional status of 46.25 % of the mothers is normal, that is, they have normal Body Mass Index (BMI) while 28.1  % mothers suffer from lack of energy or thinness and 20 % of women are overweight. In addition, 12 % of the female respondents own land while 32 % own livestock. However, 24 % have access to credit. 3.2 INFANT AND YOUNG CHILD FEEDING PRACTICES AMONG FARM HOUSEHOLD’S CHILDREN UNDER FIVE YEARS OF AGE Table 2 gives the results of the feeding practices among the farm households. While breastfeeding was popular among farm households children (0-23 months), young children’s feeding like the minimum dietary diver- sity was still inadequate and lacking. For example, about 87 % of the children (6-24 months) were inadequately fed with the minimum recommended meal frequency and 77.08 % lack the minimum dietary diversity. 86.12 % of newly born babies do not timely received milk and colos- trum. The findings also show that exclusive breastfeeding is done for 0-5 months (33.535) which is less than the six months recommended by WHO/UNICEF. Children were given other complementary foods, which exposed them to unhealthy feeding and sicknesses. 3.3 ANTHROPOMETRY RESULTS OF THE WEL- FARE OF FARM HOUSEHOLD’S CHILDREN UNDER FIVE YEARS OF AGE The WHO (2006) growth standard was employed for anthropometric measurements. As stated under sec- tion 3.4.1, the variables for stunting, underweight, and wasting were classified and calculated for farm house- hold’s children of 0 to 59 month. As shown in Table 3, the incidence of underweight among children was 32 %, and boys (38.15  %) are more likely to be underweight than girls (26.43 %). On the other hand, the incidence of stunting was recorded in which about 32 % of the chil- dren have stunted growth. The result shows that stunting rises with age, climaxing at slightly above 35 % among children in their second and third year of life. Similar re- sults were found in the previous studies of Yimer (2000); Asfaw and Giotom (2000) in Ethiopia. Severe stunt- ing among farm households with children of age 12-23 months (38.71 %) was also recorded with boys (38.48 %) more likely to be stunted than girls (30.12 %). This means that male children are more likely to be stunted and un- derweight than their female counterparts. This finding is consistent with a meta-analysis in sub-Saharan Africa (Wamani et al., 2007), a study in the Northern Ethio- pia (Alemayehu et al., 2015), and research in Myanmar (Mya et al., 2019). With underweight and stunting as a chronic malnutrition problem, Omilola (2010) revealed that “stunted children may never regain height lost due to stunting, and most children will never gain the cor- responding body mass”. Finally, wasting was found to be the least preva- lent malnutrition among farm households’ children in the study. It was half (16.2 %) the rate of prevalence in underweight and stunting. However, this value is higher than the prevalence rate of 11 % recorded in Nigeria in 2003 but lower than the figure of 18 % recorded in 2013 (National Population Commission, 2014). In line with Dabale and Sharma (2014), wasting was higher among boys (16.5 %) than in girls (15.2 %). The highest incidence of wasting was seen among chil- dren aged 0–23 months as compared to lowest figure seen among children aged 36–47 months. This is in line with Feeding practices across categories Frequency Percent Ever breastfed (0-23 months) 414 98.10 Breastfed exclusively (0-5 months) 141 33.53 Meal frequency (6-24 months) 369 86.61 Not timely receive milk and colostrum within one hour of birth 363 86.12 Not meet the minimum dietary diversity 326 77.08 Table 2: Feeding practices of farm household’s under-five year’s old children Source: Author’s field survey, 2019 Acta agriculturae Slovenica, 117/1 – 20218 C. P. DEKUNLE et al. Prevalence of overall, moderate and severe underweight status (Mass-for-Age Z-score) in children 0 to 59 months of age Characteristics % Prevalence of underweight % Prevalence of moderate underweight % Prevalence of severe under- weight Overall 32.00 27.65 4.35 Sex Boy 38.15 31.74 6.41 Girl 26.43 21.01 5.42 Age 0-5 Months 28.17 18.62 9.55 6-11 Months 39.26 32.96 6.30 12-23 Months 36.83 29.27 7.56 24-35 Months 30.11 24.50 5.61 36-47 Months 32.54 26.81 5.73 48-59 Months 29.62 25.18 4.44 Prevalence of overall, moderate and severe stunting status (Height-for-Age) in children 0 to 59 months of age Characteristics % Prevalence of stunting % Prevalence of moderate stunting % Prevalence of severe stunting Overall 31.82 23.76 8.06 Sex Boy 38.48 29.78 8.70 Girl 30.12 25.88 4.24 Age 0-5 Months 30.11 24.87 5.24 6-11 Months 32.65 28.82 3.83 12-23 Months 38.71 29.85 8.86 24-35 Months 35.27 30.82 4.45 36-47 Months 33.54 29.09 4.54 48-59 Months 32.03 24.09 7.94 Prevalence of wasting (Mass-for –Height) in children 0 to 59 months of age Characteristics % Prevalence of wasting % Prevalence of moderate wasting % Prevalence of severe wasting Overall 16.20 10.30 5.72 Sex Boy 16.50 10.30 5.72 Girl 15.20 10.00 5.20 Age 0-5 Months 16.60 11.40 5.20 6-11 Months 16.70 11.60 5.10 12-23 Months 16.40 10.10 6.30 24-35 Months 15.80 11.20 4.60 36-47 Months 15.10 10.90 4.20 48-59 Months 15.30 10.25 5.05 Table 3: Prevalence of underweight, stunting and wasting in children 0 to 59 months of age Source: Author’s field survey, 2019. Akombi et al. (2017), which posited that the incidence of wasting and severe wasting was higher in the age group of 0-5 months and 6-23 months. Acta agriculturae Slovenica, 117/1 – 2021 9 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria 3.4 EXTENT OF (DUAL MALNUTRITION DEFI- CITS) WASTING AND STUNTING IN CHIL- DREN UNDER FIVE YEARS OF AGE Table 4 shows the combined burden of stunting and wasting of children less than five year of age. Chil- dren growing up healthy are 62.44 %, implying that one – third of farm household’s children less than five years of age in the study area still experiences nutrition defi- ciency. Children suffering from the simultaneous occur- rence of stunting and wasting are 3.33  % while 1.05  % are undergoing severe stunting and wasting together. This might not be unconnected with the environmental condition and harsh deprivation of the farm household’s children, especially in the rural area. As a policy recom- mendation, it is therefore, advisable that good nutrition be introduced to the children and treatment for malnu- trition illnesses be carried out among the farm house- hold’s children in the study area. This recommendation is in line with Babatunde et al. (2011) that suggested that government should enact food policy that will increase daily per capita household calorie supply especially in the rural area. 3.5. THE PATTERN OF WOMEN BARGAINING POWER Table 5 shows that 63.33 % of women in the study area have low bargaining power, while 26.25  % have moderate bargaining power and 10.42 % have high bar- gaining power. This implies that the men were adequate in more indicators than the women; the women were ad- equate in more indicators than the man in 10 % of house- holds; and the man and the women are equally adequate in 26 % of households. On average, the male respondents are adequate in 63  % more indicators (approximately two indicators) than the female respondent in the same household. Therefore, women lack control over impor- tant decisions in their households. 3.5.1 Effects of women’s bargaining power on farm household’s welfare of the children less than five years of age Table 6 presents the results of the logit regression model. Women farm households’ involvement in pro- ductive activities have a positive effect on their child’s welfare outcomes. When they are employed or involved in other income generating activities, there is a lower likelihood of their being underweight, stunted and wast- ed. Belch and Willis (2002) revealed that women (farm households) with more financial resources wield more power in household decision-making and have stronger bargaining power in the family. In addition, Getahun and Villanger (2017) also revealed that wives that are employed have high bargaining power, as they are able to contribute to household expenditures and this improves their participation in the intra-household decision-mak- ing. Women’s incomes provide them with the bargaining power and control over major decision like schooling and consumption expenditures (Doss, 2013). It explains how they can live on during family challenges or mar- riage break-up, and reflects how the children can be well taken care of without the man. This means that higher No stunting Moderate stunting Severe stunting Total No wasting 62.44 17.38 4.16 83.98 Moderate wasting 4.12 3.33 2.85 10.30 Severe wasting 1.62 3.05 1.05 5.72 Total 68.18 23.76 8.06 100.00 Table 4: Extent of wasting and stunting in under five year old children Source: Author’s field survey, 2019 Level of bargaining power Frequency Percentage Low bargaining power (the decision is taken mainly by men) (DM1 =0.33) 203 63.33 Moderate bargaining power (the decision is shared) (DMI = 0.34 - 0.66) 84 26.25 High bargaining power (the decision is taken mainly by women) (DMI0.67) 33 10.42 Total 320 100 Table 5: Pattern of women bargaining power Source: Field survey, 2019. Acta agriculturae Slovenica, 117/1 – 202110 C. P. DEKUNLE et al. incomes or earnings increase women’s bargaining power and ability to participate in intra-households decision- making especially among the rural farmers. Age also plays a significant role in women’s em- powerment. McElroy (1990) contends that “spousal age measures how well each family member can do in the marriage or remarriage market”. Therefore, newer wives may have more bargaining power during negotiations. Equally, older wives may have greater bargaining power and influence on decision-making that positively con- tribute to the welfare of their children (Chari et al., 2017) and increase the nutritional status of their children. This is because older wives would have more influence on their children’s welfare, leading to a higher bargaining power. The coefficient on the variable for bargaining power that captures mother participation in household’s de- cision-making was significant at the 1 % and positively related to child’s health outcomes. This means that the higher the women’s bargaining power, the lower the like- lihood of the under-five children in the farm household to be underweight, stunted and wasted. Therefore, wom- en with greater decision-making power in the household, especially concerning large purchases, are more empow- ered and associated with having children with better nutrition, which will translate to better height-for-age, mass-for-age and mass-for-height ratios. 4. CONCLUSION This paper examines whether greater women’s household bargaining power is associated with the im- provement in children’s welfare in Ogun State, Nigeria. It examines the relationship between farm households women’s intra-households decision-making power and child’s welfare outcomes. Using cross-sectional farm household data from Abeokuta-Ogun State, the findings revealed that an increase in mother’s bargaining power can benefit the child and increase his or her welfare out- Underweight Stunting Wasting Variables Coefficient t-value Coefficient t-value Coefficient t-value Children’s characteristics Sex (male dummy) 1.09* 1.95 0.73** 2.28 0.73 1.28 Age (years) 2.52** 2.04 1.98** 2.33 -1.98** -2.33 Birth order 11.01*** 3.43 5.82** 2.19 5.82** 2.19 Birth mass (kg) -0.33*** -3.82 -0.33*** -4.45 -0.12*** -5.12 Multiple birth 1.21* 1.89 -0.42 -1.48 -0.42 -1.42 Mother’s characteristics Mother’s BMI (overweight dummy) -0.22*** -2.62 -0.06** -2.13 -0.06** -2.13 Maternal stature (short dummy) 2.35** 2.28 1.13*** 2.94 -1.13 -1.02 Age at first marriage -3.50*** 6.12 -2.14** 2.31 1.11 0.98 Educational attainment (number of years of schooling) -0.18*** -5.11 -0.23** -2.02 -0.34*** 4.19 Employment status (working dummy) -0.72* -1.91 -0.35** -2.31 -0.34*** -3.32 mother’s first child (male dummy) 0.09 0.83 0.09 0.92 0.09 0.92 Income 0.16 1.38 0.13 1.44 0.13 1.46 Bargaining power -7.68*** -3.77 -5.23*** -3.25 -5.21*** -3.25 Frequency of antenatal visit 0.02 0.14 -0.17** -2.14 -0.17 -1.14 Constant 0.04** 2.02 0.03*** 5.12 0.01* 1.99 LR Chi2 = 412.04 Prob > Chi2 = 0.0000 Pseudo R2 = 0.2514 Log likelihood = -1664.24 Table 6: Logit regression results of the effects of farm households women’s bargaining power on welfare of under-five years old children Note: ***, **, & * implies significant at p < 0.001, p < 0.05, & p < 0.01 respectively. Acta agriculturae Slovenica, 117/1 – 2021 11 Does women’s intra-household bargaining power have effect on child welfare? Evidence from farm households in Ogun state, Nigeria comes. The study shows that women with greater intra- household decision-making power are associated with having children with better height-for-age, weight-for- age, and weight-for-height ratios. Women farm households’ involvement in produc- tive activities have a positive effect on their child’s wel- fare outcomes. The broader social economic implication is that when women are employed or involved in other income generating activities, there is a lower likelihood of their children being underweight, stunted and wasted. This means that wives that are employed have high bar- gaining power, as they are able to contribute to house- hold expenditures and this improves their participation in the intra-household decision-making (Getahun and Villanger, 2017). Women’s incomes provide them with the bargaining power and control over major decision like schooling and consumption expenditures. This study is however, limited in scope as it was car- ried out in Southwest part of the country. The research- ers believe that further studies (future researchers) could scale-up the scope and focus on other part of the coun- try particularly those that fall outside of the Southwest region, as the finding is very relevant and germane for developing countries and particularly in Nigeria where gender inequality persists when it comes to intra-house- hold decision-making in the family. The paper deals with relevant social issue and contributes to existing literature on women’s intra-households bargaining power using the farm households as a case study. There was an established relationship between women’s empowerment and child welfare. 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Delo je prispelo 05. oktobra 2020, sprejeto 26. decembra 2020. 1 Kmetijski inštitut Slovenije, Hacquetova, Ljubljana 2 Korespondenčni avtor, e-naslov: eva.praprotnik@kis.si 3 Univerza v Ljubljani, Biotehniška fakulteta, Odd. za agronomijo, Jamnikarjeva, Ljubljana Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae) Izvleček: Strune so ličinke hroščev pokalic (Coleoptera: Elateridae) in so znane kot gospodarsko pomembni škodljivci. Poznavanje vrstne diverzitete strun na določenem območju je pomembno, saj se ekološke zahteve posameznih vrst razlikuje- jo, to pa vpliva na izbor ustreznih načinov zatiranja. Morfolo- ško določanje strun je zahtevno in pogosto je razlikovanje med posameznimi vrstami skoraj nemogoče. Molekularne metode zato ustrezno dopolnjujejo morfološke metode, in sicer se za molekularno določanje strun najpogosteje uporablja mitohon- drijski gen za citokrom oksidazo I. V članku navajamo moleku- larne, morfološke in vedenjske metode za določanje strun, prav tako izpostavljamo tudi prednosti in slabosti naštetih metod. Na koncu članka je priložen poenostavljen morfološki dolo- čevalni ključ za določevanje gospodarsko pomembnejših vrst strun rodu Agriotes v slovenskem jeziku. Ključne besede: strune; morfološko določanje; moleku- larno določanje; vedenjski vzorci Comparison of traditional and modern methods for identifi- cation of economically important wireworm species (Coleop- tera: Elateridae) Abstract: Wireworms are larvae of click beetles (Co- leoptera: Elateridae) and are well known pests of economic importance. Knowing the species diversity in a particular area is important, as the ecological requirements and consequent management strategies of individual species vary. Morphologi- cal identification of wireworms is challenging; separating be- tween individual species is often almost impossible. Molecular methods therefore complement morphological methods. Mi- tochondrial gene for cytochrome oxidase I is most commonly used for molecular identification of wireworms. In this study we list molecular, morphological and behavioural methods for wireworm identification and also highlight the advantages and disadvantages of these methods. At the end of the article, a sim- plified morphological identification key for determining eco- nomically important wireworm species of the genus Agriotes in Slovenian language is attached. Key words:wireworms; morphological identification; molecular identification; behavioural patterns Acta agriculturae Slovenica, 117/1 – 20212 e. PRAPROTNIK et al. 1 UVOD Strune so polifagne ličinke hroščev pokalic (Cole- optera: Elateridae) in so na svetovni ravni znane kot eni najpomembnejših talnih herbivorov na kmetijskih ze- mljiščih. Družina Elateridae je ena izmed vrstno najšte- vilčnejših družin hroščev, vendar njeni filogenetski od- nosi še vedno niso dorečeni (Gur’yeva, 1974; Kundrata in Bocak, 2011; Lawrence in Newton, 1995). Večina naj- pomembnejših kmetijskih škodljivcev v Evropi pripada rodu Agriotes (Furlan, 2014; Tóth in sod., 2003), v Sever- ni Ameriki rodovom Hypnodius, Agriotes, Limonius, Cte- nicera, Aeolus in Melanotus (Morales-Rodriguez in sod., 2014; Saguez in sod., 2017), v Aziji rodovoma Agriotes in Melanotus (Oba in sod., 2015), v Avstraliji pa rodovom Conoderus, Agrypnus, Heteroderes, Arachnodima in Ha- patesus (Calder, 1996). Strune lahko uvrstimo v kriptične vrste, saj je pred- stavnike določenih vrst med seboj težko oziroma prak- tično nemogoče ločiti na podlagi morfoloških znakov. Zaradi zapletene sistematike in življenjskega kroga v tleh so bile strune do nedavnega obravnavane kot en sam ško- dljivec. Temu primerni so bili tudi varstveni ukrepi, kjer so smernice narekovale enake načine zatiranja, ne glede na vrstno sestavo strun na določeni lokaciji. Vendar je potrebno upoštevati, da so strune talne žuželke z raznoli- kimi prehranjevalnimi navadami in njihova vloga v eko- sistemu ni vedno jasna. Na primer vrsta Glyphonyx bi- marginatus Schaeffer, 1916 je ena izmed pomembnejših škodljivcev sladkornega trsa na Floridi (Cherry, 2007), vendar ne velja za pomembnega škodljivca krompirja (Langdon in Abney, 2017). Vrsti Conoderus exsul (Sharp, 1877) in Agrypnus variabilis (Candeze, 1857) sta na Ha- vajih in v Avstraliji pomembna škodljivca sladkornega trsa, na Novi Zelandiji pa sta vrsti pomembna plenil- ca ličink bojevniške muhe Inopus rubriceps (Macquart, 1847), ki je škodljivec trav na pašnikih (Robertson in Pottinger, 1979; Williams in Galbreath, 1987). Danes, ko družba zahteva okolju prijaznejše načine zatiranja ško- dljivcev in je uporaba biotičnih agensov v porastu (Ansa- ri in sod., 2009; Kabaluk in sod., 2007; Kleespies in sod., 2013), v ospredje prihaja potreba po znanju o posebnih ekoloških zahtevah posameznih vrst (Hermann in sod., 2013) in njim ustreznih metodah za učinkovito integri- rano varstvo pridelkov. Obstoječi morfološki določevalni ključi ter mole- kularne analize so pogosto omejeni na posamezno geo- grafsko območje oziroma se osredotočajo le na izbrane taksonomske skupine. Vendar je potrebno upoštevati, da se zaradi spremenjenih kmetijskih praks in vnosa tuje- rodnih vrst spreminja sestava (kompleks) vrst na posa- meznih območjih. Primer je vnos evropskih vrst Agriotes obscurus (Linnaeus, 1758) in Agriotes lineatus (Linnaeus, 1767) v Britansko Kolumbijo v začetku 20. stoletja (King, 1950). Vrsti sta tako do leta 2000 že veljali za pomembna škodljivca vrtnin ter okrasnih in krmnih rastlin v Severni Ameriki (Vernon in sod., 2001). Ena od metod za določanje strun je njihovo gojenje do stadija imaga, saj je morfološko določanje odraslih osebkov enostavnejše in zanesljivejše (Leseigneur, 1972; Lohse, 1979; Jiang, 1999; Laibner, 2000; Cate, 2007; Pro- svirov, 2013). Ta metoda pri strunah ni najbolj praktična, saj le-te potrebujejo več let, da zaključijo svoj življenjski krog v tleh, hkrati pa se dolžina larvalnega stadija med vrstami močno razlikuje, na primer pri vrstah Agriotes spp. štiri do sedem let (Furlan, 1998; Miles, 1942), pri vr- sti Limonius californicus (Mannerheim) štiri do enajst let (EPPO, 2005) ter eno leto pri vrsti Aelous mellilus (Say, 1836) (Jewett, 1940). Prav tako identifikacija le odraslih predstavnikov ne odslikava realne vrstne sestave strun v specifičnem času in prostoru (Benefer in sod., 2012). Vrste iz rodu Agriotes spadajo med najpomembnejše škodljivce v Srednji Evropi, kjer je bilo do sedaj zabele- ženo dvajset vrst iz omenjenega rodu (Cate, 2007; Fur- lan in Tóth, 2007), kar devet od teh pa predstavlja po- membne talne škodljivce na kmetijskih zemljiščih: A. lineatus, Agriotes brevis Candeze, 1863, Agriotes ustulatus (Schaller, 1783), Agriotes sputator (Linnaeus, 1758), in A. obscurus, ki so hkrati tudi najpogostejše vrste v Slove- niji (Milevoj in sod., 2005), ter Agriotes litigiosus (Rossi, 1792), Agriotes proximus Schwarz, 1891, Agriotes rufipal- pis Brullé, 1832 in Agriotes sordidus (Illiger, 1807). Mor- fološki ključi (Schaerffenberg, 1940; Cocquempot in sod., 1999; Klausnitzer, 2013; Heimbach in sod., 2020) omogo- čajo identifikacijo le devet od vsega dvajsetih vrst iz rodu Agriotes. 2 MORFOLOŠKA IDENTIFIKACIJA Čeprav so ličinke znotraj družine Elateridae precej raznolike, jih od ličink drugih družin razlikujemo po na- slednjih znakih: (1) trije pari pravih nog na oprsju, se- stavljeni iz 5 členov (peti segment predstavlja terminalni krempeljc); (2) hitinizirano telo; (3) zgornja ustna (ang. labrum) odsotna, oziroma zlita z oglavnim ščitom (ang. clypeus) in glavno kapsulo; (4) maksila (ang. maxilla) in spodnja ustna (ang. labium) podaljšani in zliti v samo- stojno enoto; (5) frontalni šiv v obliki lire; (6) tipalke iz treh členov; (7) dihalnice biforalne oblike (tj. z dvema vhodoma); (8) parni izrastki na konici abdomna – analne vilice (ang. urogomphi); (9) telo ravno, z devetimi abdo- minalnimi segmenti; (10) deseti abdominalni segment slabše viden, saj leži ventralno glede na deveti segment. Na njem je lociran anus (Hyslop, 1917; Glen in sod., 1943; Klausnitzer, 2013). Vizualno so strunam podobne Acta agriculturae Slovenica, 117/1 – 2021 3 Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae) ličinke hroščev iz družine črnivcev (Tenebrionidae), še posebno iz rodu Eleodes. Družini zanesljivo med seboj ločimo po tem, da imajo ličinke črnivcev frontalni šiv v obliki črke Y, imajo dobro razvito zgornjo ustno, bolj iz- razite tipalke, prvi par nog na oprsju je pogosto daljši od drugih parov ter na devetem abdominalnem segmentu so pogoste ščetine in manjši trni (Rogers in sod., 1978; Glen in sod., 1943). Določanje strun je težavno zaradi velike morfološke podobnosti med vrstami, hkrati pa grejo ličinke med ra- zvojem skozi večje število larvalnih stopenj (8-16), med katerimi prihaja do morfoloških sprememb znotraj iste vrste (Pic in sod., 2008; Klausnitzer, 2013). Te spremembe so rezultat prehranjevanja in premikanja v tleh ter posle- dičnih poškodb zunanjega skeleta oz. hitinjače. Strukture, ki so podvržene takšnim spremembam je najbolje opazo- vati takoj po levitvi, saj se njihova izrazitost proti koncu larvalnega stadija zmanjšuje (Glen in sod., 1943). Glen in sod. (1943) za najpomembnejše strukture za zanesljivo identifikacijo rodov in vrst strun navajajo (Slika 1, Slika 2): (1) deveti abdominalni segment, in sicer prisotnost/ odsotnost kavdalne zareze, prisotnost/odsotnost »očesc« (ang. muscular impressions, eye-spots), oblika analnih vilic, prisotnost/odsotnost ščetin, oblika stranskih robov; (2) struktura glavne kapsule, nosnica (ang. nasale) je vi- dna pod mikroskopom, pomembna pa je prisotnost/od- sotnost strukture ter oblika zobcev. Nosnica je podvrže- na fizičnim poškodbam in je zato pogosto obrabljena; (3) mandibule so pomemben morfološki znak, in sicer pri- sotnost/odsotnost retinakuluma in pomožnega zobca na notranji strani mandibule ter kót, ki ga pomožni zobec tvori z osjo mandibule. Tudi mandibule so podvržene fi- zičnim poškodbam, hkrati pa lahko pri merjenju kotov tako majhnih objektov pride do napak; (4) prisotnost/ odsotnost izrazitih telesnih zarez in vdolbin. Te struktu- re so manj izrazite na mladih ličinkah in ličinkah, ki so se nedavno levile; (5) oblika ventralnega dela predprsja (ang. presternum), in sicer je lahko sestavljen iz več delov oziroma zlit v en sklerit; (6) prisotnost/odsotnost ščetin; (7) prisotnost/odsotnost oči. Za zanesljivo določitev je potrebno opazovati dobro razvite ličinke, saj se pri mladih ličinkah obarvanost, šte- vilo ščetin, značilnosti nosnice, kavdalne zareze in anal- nih vilic lahko močno razlikujejo; pri vrstah A. sordidus, A. sputator, A. lineatus in A. obscurus morajo ličinke za določitev doseči dolžino vsaj 1 cm (Pic in sod., 2008). Mnogi morfološki določevalni ključi (Tabela 1) datirajo v prvo polovico 20. stoletja in so zaradi tega taksonomsko zastareli. Pogosto so napisani v različnih jezikih, na primer v nemščini (Beling, 1883, 1884; Scha- erffenberg, 1940; Korschefsky, 1941; Klausnitzer, 2013), danščini (Schiødte, 1870), francoščini (Pic in sod., 2008), angleščini (Glen in sod., 1943; Becker, 1956; Eidt, 1954; Etzler, 2013; Glen, 1950; Lanchester, 1946; Riley in Ke- aster, 1979; Wilkinson, 1963; Heimbach in sod., 2020). Prav tako so klasični dihotomni ključi nemalokrat zah- tevni za uporabo za nestrokovnjake, posebno če ključi te- meljijo na besednem opisovanju lastnosti in morfoloških struktur. Uporabnikom prijaznejši so slikovni določeval- ni ključi, ki prikažejo sliko za vsak iskan morfološki znak. Takšni ključi za določevanje strun so redki; Etzler (2013) je ustvaril kombinacijo slikovnega in dihotomnega dolo- Slika 1: Struna rodu Agriotes sp. a: glava, dorzalno. b: glava, ventralno. Dobro vidne mandibule in nosnica. c: deveti abdominalni segment, dorzalno. Dobro vidna »očesca«. d: deveti abdominalni segment, ventralno. Dobro viden anus. e: struna, lateralno. Slike: Eva Praprotnik. Figure 1: Wireworm of the genus Agriotes sp. a: head, dorsal. b: head, ventral. Mandibles and nasale clearly distinguishable. c: ninth abdominal segment, dorsal. Muscular impressions (“eye-spots”) clearly distinguishable. d: ninth abdominal segment, ven- tral. Anus clearly distinguishable. e: wireworm, lateral. Photo: Eva Praprotnik. Acta agriculturae Slovenica, 117/1 – 20214 e. PRAPROTNIK et al. čevalnega ključa za gospodarsko pomembne vrste strun, ki se pojavljajo v ameriški zvezni državi Montana in na pacifiškem severozahodu. Strokovnjaki v Evropi se z izzivom določevanja strun in iskanjem zanesljivih morfoloških znakov ukvar- jajo že dolgo časa. Beling (1883, 1884) in Schiødte (1870) sta opisala ličinke družine Elateridae, dihotomni dolo- čevalni ključ Schaerffenberga (1940) je namenjen do- ločanju 15 najpomembnejših vrst strun v Nemčiji, delo Korschefskyja (1941) povzema biologijo družine Elateri- dae in sistematiko njihovih ličink, določevalni ključi Co- cquempota in sod. (1999), Heimbacha in sod. (2020) in Pica in sod. (2008) se osredotočajo na rod Agriotes, naj- bolj celovit določevalni ključ pa je Klausnitzerjev (2013), saj v njem dobimo opise in skice za celotno družino Ela- teridae Evrope. Literatura o strunah Severne Amerike je prav tako obširna, saj nam Glen in sod. (1943) podajo dihotomni določevalni ključ za več kot 30 vrst strun Ka- nade, 7 let pozneje pa detajlni določevalni ključ plemena Lepturoidini Severne Amerike (Glen, 1950). Wilkinson (1963) nadgradi in spiše določevalni ključ za 25 vrst strun, ki se pojavljajo na kmetijskih zemljiščih Britanske Kolumbije. Določevalna ključa Eidta (1954) in Becker- ja (1956) se osredotočata na rod Agriotes, Lanchester (1946) se osredotoča na rod Limonius, Riley in Keaster (1979) pa na rod Melanotus. Stibick je napisal pregled skupine Hypnoidinae, ki jo filogenetsko uvršča v samo- stojno poddružino in je eden redkih, ki je naredil pregled na svetovni ravni, in sicer Severne in Južne Amerike (Sti- bick, 1976; Stibick, 1978), Evrazije (Stibick, 1979), Indije (Stibick, 1980b) in Nove Zelandije (Stibick, 1980a). Morfološko določanje strun je pogosto nezanesljiva metoda in zahteva veliko znanja in izkušenj, pa vendar so prve filogenetske študije družine Elateridae v veliki meri temeljile prav na morfoloških značilnostih ličink ter fo- silnih ostankih (Becker, 1956). Takšno filogenetsko drevo temelji na izvornih (pleziomorfnih) in izpeljanih (apo- morfnih) znakih, ki so se pri vrstah, ki jih med seboj pri- merjamo, pojavili v različnih kombinacijah v različnem časovnem obdobju (Rieppel, 2020). Hyslop (1917) je na podlagi morfoloških lastnosti odraslih hroščev in ličink izrisal filogenetsko shemo v geološki časovni lestvici, ki je do razvoja molekularnih metod veljala za najpopolnejši prikaz evolucijske zgodovine družine Elateridae. Evolu- cija družine je v biološkem smislu šla v smeri krajšanja življenjske dobe odraslih hroščev in posledično daljšanja obdobja, ki ga preživijo v stadiju ličink. Larvalni stadiji so tako tekom filogeneze doživeli znatnejše morfološke spremembe kot pa stadiji imaga (Dolin, 1978). 3 MOLEKULARNO DOLOČANJE V zadnjem desetletju se za določanje morfološko težko ločljivih vrst uveljavlja vse več molekularnih me- tod (Tabela 1), ki v veliki meri temeljijo na izolaciji DNK in pomnoževanju specifičnih odsekov z uporabo metode verižne reakcije s polimerazo (ang. polymerase chain re- action - PCR). Veliko le-teh temelji na identifikaciji od- raslih hroščev (Han in sod., 2016; Kundrata in sod., 2016; Oba in sod., 2015), čeprav so ličinke tiste, ki povzročajo znatno škodo v kmetijskih in gozdnih ekosistemih. DNK črtno kodiranje je metoda, pri kateri namno- žimo DNK fragmente, ki so specifični za določen ta- kson. Pri živalih se za določanje najpogosteje uporablja zaporedje v genu za mitohondrijsko citokrom c oksida- zo (COI) (Hebert in sod., 2003). DNK črtno kodiranje je pomembno orodje za določanje kriptičnih vrst, torej Slika 2: Levo: glava, ventralno. pz – pomožni zobec, ret – retinakulum, n – nosnica (nasale). Desno: dihalnica biforalne oblike. Slike: Eva Praprotnik. Figure 2: Left: head, ventral. pz – preapical tooth , ret – retinaculum , n – nasale. Right: biforous spiracle. Photo: Eva Praprotnik. Acta agriculturae Slovenica, 117/1 – 2021 5 Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae) Ta be la 1 : M et od e za d ol oč an je st ru n, v kl ju čn o z n jih ov im i p re dn os tm i i n sla bo st m i Ta bl e 1: M et ho ds o f w ire w or m id en tifi ca tio n, in cl ud in g th ei r a dv an ta ge s a nd d isa dv an ta ge s M et od e Pr eu če va ni o rg an iz em Pr ed no st i Sl ab os ti Re fe re nc a M or fo lo šk o do lo ča nj e 15 n aj šk od lji ve jši h vr st st ru n v N em či ji pr iso tn e sk ic e in fo to gr afi je m or fo lo šk ih st ru kt ur kl ju č v n em šk em je zi ku , u po ra ba za ht ev na za ne st ro ko vn ja ke , p ou da re k le n a ek on om sk o po m em bn ih v rs ta h Sc ha er ffe nb er g, 19 40 ve č k ot 3 0 vr st st ru n K an ad e kl ju č v a ng le šk em je zi ku , n at an čn e sk ic e m or fo lo - šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e, po ud ar ek le na ek on om sk o po m em bn ih v rs ta h G le n, in so d. 1 94 3 vr st e pl em en a Le pt ur oi di ni S ev er ne A m er ik e kl ju č v a ng le šk em je zi ku , n at an čn e sk ic e m or fo lo - šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e G le n, 1 95 0 45 v rs t, ki se p oj av lja jo v a m er išk i z ve zn i d rž av i M on ta na in p ac ifi šk em se ve ro za ho du po dr ob en sl ik ov ni k lju č v k om bi na ci ji z d ih ot om - ni m k lju če m po ud ar ek le n a ek on om sk o po m em bn ih v rs ta h Et zl er , 2 01 3 pr eg le d sk up in e H yp no id in ae pr eg le d po dd ru ži ne n a sv et ov ni ra vn i za ht ev en d ih ot om ni k lju č St ib ic k, 1 97 6, 1 97 8, 19 79 , 1 98 0a , 1 98 0b 25 v rs t s tr un , k i s e po ja vl ja jo n a km et ijs ki h po vr ši- na h Br ita ns ke K ol um bi je kl ju č v a ng le šk em je zi ku , p ris ot ne sk ic e m or fo lo šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e, po ud ar ek le na ek on om sk o po m em bn ih v rs ta h W ilk in so n, 1 96 3 6 ek on om sk o po m em bn ih v rs t s tr un ro du L im o- ni us kl ju č v a ng le šk em je zi ku , p ris ot ne sk ic e m or fo lo - šk ih st ru kt ur , p od ro be n op is m or fo lo šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e, po ud ar ek le na ek on om sk o po m em bn ih v rs ta h La nc he st er , 1 94 6 ne ar kt ič ne v rs te ro du A gr io te s kl ju č v a ng le šk em je zi ku , o br av na va li či nk e in od ra sle p rim er ke , p rik az ra zš irj en os ti vr st n a ze m lje vi du up or ab a za ht ev na za n es tr ok ov nj ak e Be ck er , 1 95 6 dr už in a El at er id ae v E vr op i ce lo vi t k lju č z a id en tifi ka ci jo ce lo tn e dr už in e, po dr ob ne sk ic e m or fo lo šk ih st ru kt ur kl ju č v n em šk em je zi ku , u po ra ba za ht ev na za ne st ro ko vn ja ke K la us ni tz er , 2 01 3 10 v rs t r od u M ela no tu s S ev er ne A m er ik e kl ju č v a ng le šk em je zi ku , p ris ot ne sk ic e m or fo lo - šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e Ri le y in K ea st er , 19 79 ; R ile y, 19 83 4 vr st e ro du A gr io te s i z N ov e Šk ot sk e kl ju č v a ng le šk em je zi ku , p od ro bn e sk ic e in o pi si m or fo lo šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e, po ud ar ek le na ek on om sk o po m em bn ih v rs ta h Ei dt , 1 95 4 4 vr st e ro du A gr io te s po en os ta vl je n in k ra te k do lo če va ln i k lju č kl ju č v fr an co sk em je zi ku , p ou da re k le n a ek on om sk o po m em bn ih v rs ta h Pi c, in so d. 2 00 8 ve č k ot 1 5 ro do v dr už in e El at er id ae po dr ob na si st em at ik a dr už in e El at er id ae , p ris ot ne sk ic e m or fo lo šk ih st ru kt ur kl ju č v n em šk em je zi ku , u po ra ba za ht ev na za ne st ro ko vn ja ke Ko rs ch ef sk y, 19 41 4 vr st e ro du A gr io te s v F ra nc iji pr iso tn e sk ic e m or fo lo šk ih st ru kt ur , p rik az ra zš ir- je no st i v rs t n a ze m lje vi du kl ju č v fr an co sk em je zi ku , p ou da re k le n a ek o- no m sk o po m em bn ih v rs ta h C oc qu em po t,  in so d. 1 99 9 kl ju č z a id en tifi ka ci jo 1 1 ro do v st ru n, s po ud ar - ko m n a vr st ah iz ro du A gr io te s kl ju č v a ng le šk em je zi ku , p ris ot ne fo to gr afi je m or fo lo šk ih st ru kt ur up or ab a za ht ev na za n es tr ok ov nj ak e H ei m ba ch , i n so d. 20 20 Acta agriculturae Slovenica, 117/1 – 20216 e. PRAPROTNIK et al. na da lje va nj e ta be le 1 M ol ek ul ar no d ol oč an je an al iz a m ito ho nd rij sk eg a ge na C O I v k om bi na ci ji z m ul tip le ks P C R 17 v rs t r od u Ag rio te s i z S re dn je E vr op e po ce ni in za ne slj iv a m et od a, p ri ka te ri je v er je t- no st la žn o po zi tiv ni h re zu lta to v m aj hn a om ej en o št ev ilo za če tn ih o lig on uk le ot id ov zn ot ra j en e PC R re ak ci je St au da ch er , i n so d. 20 11 an al iz a m ito ho nd rij sk eg a ge na C O I i n je dr ne ga g en a EF 1- α ro d M ela no tu s i z b am bu so vi h go zd ov Ju žn e K ita jsk e an al iz a ve čj eg a št ev ila lo ku so v po da za ne slj iv ej še re zu lta te an al iz a le m ito ho nd rij sk ih g en ov n e po da v ed no za ne slj iv ih fi lo ge ne ts ki h re zu lta to v Zh an g, in so d. 2 01 9 an al iz a m ito ho nd rij sk eg a ge na C O I 30 ek on om sk o po m em bn ih v rs t v M on ta ni , Z D A po ce ni in za ne slj iv a m et od a, št ev iln i o pt im iz ira ni pr ot ok ol i an al iz a le m ito ho nd rij sk ih g en ov n e po da v ed no za ne slj iv ih fi lo ge ne ts ki h re zu lta to v Et zl er , i n so d. 2 01 4 an al iz a m ito ho nd rij sk eg a ge na C O I 10 v rs t r od u M ela no tu s, 1 vr st a ro du C on od er us po ce ni in za ne slj iv a m et od a, št ev iln i o pt im iz ira ni pr ot ok ol i an al iz a le m ito ho nd rij sk ih g en ov n e po da v ed no za ne slj iv ih fi lo ge ne ts ki h re zu lta to v Li nd ro th in C la rk 20 09 an al iz a m ito ho nd rij sk eg a ge na C O I 7 vr st ro du A gr io te s i z F ra nc ije po ce ni in za ne slj iv a m et od a, št ev iln i o pt im iz ira ni pr ot ok ol i an al iz a le m ito ho nd rij sk ih g en ov n e po da v ed no za ne slj iv ih fi lo ge ne ts ki h re zu lta to v Pi c, in so d. 2 00 8 an al iz a m ito ho nd rij sk eg a ge na C O I 3 vr st e ro du A gr io te s po ce ni in za ne slj iv a m et od a, št ev iln i o pt im iz ira ni pr ot ok ol i an al iz a le m ito ho nd rij sk ih g en ov n e po da v ed no za ne slj iv ih fi lo ge ne ts ki h re zu lta to v Le hm us in N ie po ld , 20 15 T- RF LP 3 vr st e ro du A gr io te s po ce ni , e no st av na in za ne slj iv a m et od a m et od a je o bč ut lji va n a va ria bi ln os t i zo lir an e D N K m ed p os am ez ni m i v zo rc i. U po ra ba ra zl ič ni h ba rv il vp liv a na m ob iln os t D N K fr ag m en to v v ka pi la rn i e le kt ro fo re zi El lis , i n so d. 2 00 9 an al iz a 16 S rR N K 15 ek on om sk o po m em bn ih v rs t v K an ad i po ce ni in h itr a m et od a, u st re zn a m et od a za fi lo ge - ne ts ke št ud ije m et od a se v n aj ve čj i m er i u po ra bl ja za št ud ije pr ok ar io nt sk ih o rg an iz m ov Be ne fe r, in so d. 20 13 Ve de nj sk e la st no st i in te nz ite ta to ni čn e ne gi b- no st i A . l in ea tu s i n A . o bs cu ru s v ko m bi na ci ji z m or fo lo šk o id en tifi ka ci jo la hk o zm an jša m o m ož no st n ap ak e pr i i de nt ifi ka ci ji te m pe ra tu ra im a ve lik v pl iv n a ve de nj e st ru n, za id en tifi ka ci jo p ot re bu je m o ži ve p rim er ke Ri tte r, in so d. 2 01 6 Acta agriculturae Slovenica, 117/1 – 2021 7 Primerjava tradicionalnih in sodobnih metod za določanje gospodarsko pomembnih vrst strun (Coleoptera: Elateridae) dveh ali več vrst, ki so med seboj težko ločljive le na pod- lagi morfoloških lastnosti. Hkrati nam metoda omogoči prepoznavo novih povezav med odraslimi hrošči in prej vrstno nedoločljivimi ličinkami (Etzler in sod., 2014). Pic in sod. (2008) so prvi uporabili molekularno metodo za določanje strun. Pomnoževali so COI regijo mitohondrijske DNK za določitev 7 vrst rodu Agriotes, ki velja za najpogostejši rod pokalic v Franciji. Metoda kombinacije molekularnega in morfološkega določanja se je izkazala za uspešno pri štirih najškodljivejših vrstah A. sordidus, A. sputator, A. lineatus in A. obscurus, vendar z eno izjemo. Tri ličinke, ki so jih raziskovalci morfološko opredelili kot A. sputator, so se na kladogramu razvrstile ob zaporedju odraslega predstavnika vrste Agriotes galli- cus Lacordaire, 1835. Ker je larvalni stadij A. gallicus sko- rajda neznan, predstavlja molekularno določanje edini način za razlikovanje ličink A. gallicus in A. sputator. Lindroth in Clark (2009) sta prav tako pomnože- vala COI regijo mitohondrijske DNK za prepoznavo in filogenetski pregled gospodarsko pomembnih vrst strun srednjega zahoda ZDA iz rodu Melanotus in Conoderus. Dobljeni filogenetski odnosi se v veliki meri skladajo z morfološkimi podatki določevalnega ključa po Riley in Keaster (1981). Podan je zgled vrste Melanotus depres- sus (Melsheimer, 1844), ki se od ostalih vrst omenjenega rodu znatno razlikuje, saj je edina brez parnih prečnih prog na posteriornih segmentih. S tem se skladajo tudi rezultati filogenetske analize, saj vrsta zavzema izhodišč- ni položaj za vse ostale preučevane vrste znotraj rodu Melanotus. Zhang in sod. (2019) so za molekularno določanje treh vrst strun iz rodu Melanotus iz Južne Kitajske, ki se prehranjujejo s poganjki bambusa, uporabili podatke mitohondrijskega gena COI in jedrnega gena EF1-α. Na- mreč analiza filogenetskih odnosov in določitev vrst, ki temelji le na mitohondrijskem genu COI včasih ne zado- stuje in poda zavajajoče rezultate zlasti pri ozko sorodnih vrstah (Rubinoff in Holland, 2005; Shaw, 2002). Tudi Etzler in sod. (2014) so za določanje gospodar- sko najpomembnejših vrst strun na območju ameriške zvezne države Montana uporabili analizo COI gena. Fi- logenetska analiza vrste Hypnoidus bicolor (Eschscholtz, 1829) je pokazala veliko raznolikost med posameznimi kladi (več kot 3%), kar lahko nakazuje na prisotnost dveh ali celo treh kriptičnih vrst, ki jih na podlagi morfolo- ških znakov uvrščamo v eno samo vrsto. Benefer in sod. (2013) so z analizo 16S rRNK gena prav tako pokazali veliko genetsko raznolikost omenjene vrste, v korelaciji z geografsko razdaljo, in sicer do 4,6 %. Za primerjavo, med vrstami A. lineatus, A. sputator in A. obscurus ge- netska raznolikost znaša 3,7  % (Benefer in sod., 2012), oziroma 4,9  % (Pic in sod., 2008). Status H. bicolor je pomemben zaradi vse večjega negativnega vpliva vrste v agroekosistemih, saj se njena številčnost po prepovedi nekaterih kemičnih insekticidov povečuje (Parker in Ho- ward, 2001). Staudacher in sod. (2011) so razvili preprost protokol za določanje 17 vrst iz rodu Agriotes z metodo DNK črtnega kodiranja, v kombinaciji s PCR multiple- ksom pa so določili 9 najpomembnejših in hkrati najpo- gostejših vrst. Princip delovanja PCR multipleksa je enak klasični PCR reakciji, s tem da lahko naenkrat pomno- žujemo več različnih DNK segmentov v vzorcu, in sicer z uporabo večjega števila začetnih oligonukleotidov v re- akcijski mešanici. Zaradi prihranjenega časa in denarja uporaba metode PCR multipleksa narašča, še posebej pri raziskavah z velikim številom vzorcev (Gauthier, 2010; Lagisz in sod., 2010). Potrebno pa je upoštevati, da je šte- vilo začetnih oligonukleotidov, ki jih lahko uporabimo znotraj ene mulitipleks PCR reakcije omejeno in morajo biti optimizirani na delovanje pri enaki temperaturi po- daljševanja novo sintetiziranih nukleotidnih zaporedij, saj lahko v nasprotnem primeru pride do neenakovre- dnega pomnoževanja posameznih fragmentov (Sint in sod., 2012). Filogenetska analiza celotnega COI gena je pokazala veliko podobnost med vrstama A. lineatus in A. proximus. Ti dve vrsti prav tako privablja enak fero- mon (Subchev in sod., 2005), morfološke razlike odraslih osebkov so minimalne, ličinke vrste A. proximus pa tudi še niso morfološko opisane. Ta dejstva sprožijo vprašanje ali je smiselno obravnavati A. lineatus in A. proximus kot dve ločeni vrsti (Staudacher in sod. 2011). Lehmhus in Niepold (2015) sta pomnoževala mi- tohondrijski gen COI za identifikacijo vrst A. lineatus, A. sputator in A. obscurus, Ellis in sod. (2009) pa so za identifikacijo teh treh vrst pomnoževali 16S regijo mito- hondrijske DNK in uporabili tehniko polimorfizma dol- žine končnih restrikcijskih fragmentov (ang. Terminal restriction fragment length polymorphism - T-RFLP). Osnova metode je pomnoževanje specifičnega odseka DNK, pri čemer sta oba, lahko tudi le en začetni oligo- nukleotid, označena s fluorescentnim barvilom (Walker in sod., 2017). Pomnoženim odsekom dodamo encim restrikcijska endonukleaza, ki razreže odseke na specifič- nih mestih, sama dolžina končnih odsekov pa je vrstno specifična. Metoda je poceni, ponovljiva in zanesljiva za določitev nekaterih najpomembnejših vrst strun, vendar je hkrati občutljiva na variabilnost med vzorci (količina izolirane DNK), uporaba različnih barvil pa vpliva na mobilnost DNK fragmentov v kapilarni elektroforezi (Prakash in sod., 2014). 4 DOLOČANJE NA PODLAGI VEDENJSKIH LASTNOSTI Za določanje žuželk lahko v kombinaciji z morfo- Acta agriculturae Slovenica, 117/1 – 20218 e. PRAPROTNIK et al. loškimi značilnostmi uporabimo tudi vedenjske lastnosti (Beaudoin-Ollivier in sod., 2000). Ritter in sod. (2016)so spremljali protipredatorsko vedenje pri različnih vrstah strun iz rodu Agriotes. Strune se v primeru odvzema iz substrata lahko odzovejo s tremi vedenjskimi oblikami, in sicer: 1) tonična negibnost (hlinjenje smrti); 2) faza ori- entacije (gibanje na površju); 3) faza kopanja v substrat. Ugotovili so, da je pri 20 °C tonična negibnost statistično značilno različna med vrstama A. lineatus in A. obscurus, in sicer ličinke, ki ostanejo negibne več kot 4,35 s pripa- dajo vrsti A. lineatus, tiste, ki so negibne manj kot 4,35 s pa vrsti A. obscurus. Pri morfološki določitvi, ki temelji na številu ščetin nad dihalnico (A. lineatus 1 ščetina in A. obscurus 2 ščetini (Lehmhus in Niepold, 2015)), je mo- žnost napačne določitve 19,1  %. Če hkrati spremljamo še čas tonične negibnosti, se ta napaka zmanjša na 2,2 %. Slabost izbrane metode je, da za določanje potrebujemo žive primerke. Hkrati je prisoten velik vpliv temperatur- nih sprememb, saj te različno vplivajo na protipredatorsko vedenje posameznih vrst strun (Staudacher in sod., 2013), zato so za razrešitev teh vprašanj pomembne dodatne eto- loške študije. 5 ZAKLJUČEK Prepoznavanje raznolikosti kriptičnih vrst na do- ločenem območju je pomembno za uporabo ustreznih smernic za varstvo kmetijskih pridelkov. Nezmožnost rutinskega in zanesljivega razlikovanja med posameznimi vrstami strun predstavlja veliko omejitev pri raziskova- nju in upravljanju kmetijskih ekosistemov. Pri določanju strun je pomembna kombinacija različnih metod, še pose- bej pri vrstah, ki jih na podlagi morfoloških lastnosti težko ločimo med seboj. Dihotomni ključi so za nestrokovnjake lahko zahtevni za uporabo, zato lahko s kombinacijo mor- foloških in molekularnih metod izboljšamo in razširimo obstoječe identifikacijske ključe. Analiza mitohondrijske- ga gena COI je dovolj zanesljiva za vrstno določitev strun, za boljše razumevanje med- in znotrajvrstnih odnosov pa so potrebne dodatne filogenetske študije, tudi na globalni ravni. Hkrati metoda ni vedno vsem dostopna in ni upo- rabna za rutinske preiskave kmetijskih svetovalcev. Za lažje razumevanje in določanje smo pripravili morfološki določevalni ključ za ekonomsko pomembnejše vrste strun. Pri uporabi morfoloških določevalnih ključev se je potrebno zavedati, da obstaja velika verjetnost napa- ke pri določitvah, zlasti pri opazovanju živih primerkov. 6 VIRI Ansari, M. A., Evans, M., & Butt, T. M. (2009). Identification of pathogenic strains of entomopathogenic nematodes and fungi for wireworm control. Crop Protection, 28(3), 269– 272. https://doi.org/10.1016/j.cropro.2008.11.003 Beaudoin-Ollivier, L., Prior, R. N. B., & Laup, S. (2000). 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Journal of Asia-Pacific Entomology, 22(1), 140–150. https://doi.org/10.1016/j.aspen.2018.12.017 Za ličinke iz rodu Agriotes je značilno cilindrično, močno hitinizirano telo, deveti abdominalni segment je nedeljen, stožčasto zašiljen, na bazi tega segmenta pa so prisotna »očesca«. 1 Tergiti so vzdolžno valovito nagubani. Deveti abdominalni segment se konča s topo, bolj ali manj izrazito bradavico. Med vrhom mandibule in retinakulumom ni dodatnih zobcev. Srednji zobec pri nosnici je daljši od zunanjih dveh. Ličinka vitka in intenzivno rumene barve, maksimalna dolžina 9 mm, širina 0,6 mm…………………………………………………………... Agriotes pallidulus 1* Tergiti nagubani, gladki ali z vdolbinicami. Konica devetega analnega segmenta koničasta in hitinizirana. Mandibule pred vrhom razširjene ali s prisotnim pomožnim zobcem med vrhom mandibule in retinakulumom. Zobci nosnice enako dolgi (Slika 2)..…... ....................................................................................................................................................................................……………...……..…..…2 2 Prisotne granulacije na začetku segmentov in med kolčkom (ang. coxa). Pomožni zobec z osjo mandibule tvori pravi kot 90 °. Od 1.-8. segmenta: nad dihalnico je ob večji ščetini prisotna še manjša ščetina. Maksimalna dolžina 18 mm, širina 1,4 mm ………… …….....................................................................................................................................................................…………….. Agriotes sputator 2* Odsotne granulacije na začetku segmentov in med kolčkom………………………………...…………………...……………………...3 3 Mandibule pred vrhom razširjene, vendar brez pomožnega zobca. Pore, iz katerih izraščajo ščetine 9. abdominalnega segmenta, so rahlo izbočene. Maksimalna dolžina 25 mm, širina 1,9 mm………………………………………………………… Agriotes ustulatus 3* Med vrhom mandibule in retinakulumom prisoten pomožni zobec…………………………...……………………...…………….4 4 Od 1.-8. segmenta: nad dihalnico je manjša ščetina odsotna. Pomožni zobec z osjo mandibule tvori kot 60 °. Maksimalna dolžina 27 mm, širina 2 mm………………………………………………………...…………………...…………………....… Agriotes lineatus 4* Od 1.-8. segmenta: nad dihalnico je ob večji ščetini prisotna še manjša ščetina. Pomožni zobec z osjo mandibule tvori kot 120 °. Maksimalna dolžina 28 mm, širina 2 mm…………………………………………………………………………...… Agriotes obscurus Priloga 1: Morfološki identifikacijski ključ za najpogostejše vrste iz rodu Agriotes v Sloveniji (Povzeto po: Schaerffenberg, 1940; Klausnitzer, 2013, Heimbach, in sod. 2020) Appendix 1: Morphological identification key for the most common species of the genus Agriotes in Slovenia (See: Schaerffenberg, 1940; Klausnitzer, 2013, Heimbach, et al. 2020) Acta agriculturae Slovenica, 117/1, 1–11, Ljubljana 2021 doi:10.14720/aas.2021.117.1.2106 Review article / pregledni znanstveni članek Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) Žiga LAZNIK 1, 2, Stanislav TRDAN 1 Received Februay 27, 2021; accepted March 03, 2021. Delo je prispelo 27. februarja 2021, sprejeto 03.03. 2021. 1 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo, Jamnikarjeva, Ljubljana, Slovenija 2 Korespondenčni avtor, e-naslov: ziga.laznik@bf.uni-lj.si Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) Izvleček: Marmorirana smrdljivka (Halyomorpha halys [Stål, 1855]; [Hemiptera, Pentatomidae]) je predstavnik družine ščitastih stenic. Gre za invazivno, tujerodno in polifagno vrsto, ki izvira iz Vzhodne Azije. Sredi 90-ih je bila vnesena v ZDA ter leta 2004 v Evropo. Danes se pojavlja v večini evropskih držav. V novih okoljih se hitro prilagaja in uspešno razmnožuje, pri čemer razvije najmanj en popoln rod letno. Z naraščanjem po- pulacij postaja moteč dejavnik v urbanem okolju ter v zadnjem času eden od najpomembnejših škodljivcev v kmetijski pride- lavi. V Sloveniji smo stenico prvič našli leta 2017 v Šempetru pri Gorici. V slabih dveh letih se je razširila po celotni Sloveniji ter začela povzročati škodo v kmetijski pridelavi. V preglednem članku smo zbrali raziskave tujih in domačih raziskovalcev, ve- zanih na preučevanje različnih načinov zatiranja marmorirane smrdljivke. V članku so predstavljeni tako kemični načini za- tiranja, uporaba insekticidnih mrež, atraktantov in repelentov, privabilnih posevkov kot tudi načini biotičnega varstva z upo- rabo plenilcev in parazitoidov. Ključne besede: Halyomorpha halys; kemično zatiranje; atraktanti; repelenti; privabilni posevki; insekticidne mreže; bi- otično varstvo rastlin; Slovenija Management methods for marmorated stink bug (Halyo- morpha halys [Stål, 1855], Hemiptera, Pentatomidae) Abstract: The brown marmorated stink bug (Halyomor- pha halys (Stål, 1855); [Hemiptera, Pentatomidae]) is an inva- sive, alien and polyphagous insect species native to East Asia. It was introduced to the United States in the mid-1990s and to Europe in 2004. Today it is present in most European coun- tries. In new environments, it adapts quickly and reproduces successfully, developing at least one complete generation per year. With the growth of populations, it is becoming a disturb- ing factor in the urban areas and recently one of the most dan- gerous pests in agricultural production. It was first discovered in Slovenia in 2017 in Šempeter near Gorica. In less than two years, it spread to the entire territory od Slovenia and began to cause damage to agricultural production. In a review paper, we have collected research by foreign and domestic researchers related to the study of different ways of controlling the brown marmorated stink bug. The article presents chemical methods of control, the use of insecticide nets, the use of attractants and repellents, trap crops, as well as methods of biotic protection using predators and parasitoids. Key words: Halyomorpha halys; chemical control; attrac- tants; repellents; trap crops; insecticidal nets; biological control; Slovenia Acta agriculturae Slovenica, 117/1 – 20212 Ž. LAZNIK in S. TrdAN 1 UVOD Na področju omejevanja številčnosti marmorirane smrdljivke (Halyomorpha halys) in preprečevanje škode v kmetijski pridelavi v tem trenutku nimamo na razpo- lago prav veliko ukrepov, kot tudi ne učinkovitih rešitev. Opravka imamo z izjemno »robustnim« škodljivcem, ki se ga ne da enostavno obvladovati z insekticidi. Mar- morirana smrdljivka je predstavnik družine Pentatomi- dae. Gre za invazivno, tujerodno in polifagno vrsto, ki izvira iz Vzhodne Azije (Panizzi in sod., 2000; Lee in sod., 2013a). Vrsta je bila sredi 90-ih vnesena v ZDA (Hoebeke in Carter, 2003; Morrison in sod., 2017) ter leta 2004 v Evropo (Leskey in sod., 2012a; Rice in sod., 2014). Danes se pojavlja v večini evropskih držav. V novih okoljih se hitro prilagaja in uspešno razmnožuje, pri čemer razvije najmanj en popoln rod letno (Leskey in sod., 2012a). Z naraščanjem populacij postaja mo- teč dejavnik v urbanem okolju ter v zadnjem času eden najbolj nevarnih škodljivcev v kmetijski pridelavi (In- kley, 2012). V Sloveniji smo jo prvič odkrili leta 2017, in sicer v Šempetru pri Gorici. V slabih dveh letih se je razširila na območju celotne Slovenije ter začela pov- zročati škodo v kmetijski pridelavi (Rot in sod., 2018). Vrsta je polifag. Prehranjuje se s preko 150 različ- nimi rastlinskimi vrstami iz številnih družin, med ka- terimi prevladujejo metuljnice in rožnice (Morrison in sod., 2017). Med sadnimi vrstami so njeni najpomemb- nejši gostitelji hruška, jablana, breskev, leska, kaki in aktinidija (Leskey in sod., 2012a). Škodo povzroča tudi na vinski trti, na plodovkah (paradižnik, paprika, jajče- vec, kumare) in stročnicah (fižol). V pridelavi poljščin so najbolj ogroženi posevki soje ter koruze. Prehranjuje se tudi na številnih okrasnih rastlinah in grmovnicah ter prosto rastočih drevesnih vrstah, kot so: veliki jesen (Fraxinus excelsior L.), visoki pajesen (Ailanthus altis- sima [Mill.] Swingle), pavlovnija (Paulownia tomentosa [Thunb.] Steud.), jerebika (Sorbus aucuparia L.), lovo- rikovec (Prunus laurocerasus L.), oslez (Hibiscus spp.), vrtnica (Rosa sp.), navadna bodika (Ilex aquifolium L.) in številnih druge (Nielsen in Hamilton, 2009; Leskey in sod., 2012a; Morrison in sod., 2017). V ZDA, kjer je marmorirana smrdljivka zastopana najdlje, je škoda, ki jo je povzročila v kmetijski pride- lavi ocenjena na nekaj milijard dolarjev. V letu 2010 je pridelovalcem jabolk na območju Srednjega Atlantika povzročila škodo v višini 34 mio. $, sočasno so pride- lovalci breskev beležili 50 % izgube pridelka (Leskey in sod., 2012a; Morrison in sod., 2017). V Evropi je do se- daj povzročila največ škode v Italiji, v pridelavi hrušk in jabolk (Cesari in sod., 2015). V deželi Emiglia Roma- gna, v provinci Modena je bilo v letu 2015 zaradi moč- nega napada marmorirane smrdljivke poškodovanih od 30 do 50 % hrušk (Cesari in sod., 2015). Z Madžarske poročajo o veliki škodi v pridelavi fižola in paprike, v Gruziji in Abhaziji je zaradi marmorirane smrdljivke ogrožena pridelava lešnikov (Morrison in sod., 2017). Poleg škode v kmetijski pridelavi je po vsej Evropi po- stala moteč dejavnik v urbanem okolju. Zaradi speci- fičnega načina prezimovanja, v jesenskem času stenice množično priletajo v bližino človeških bivališč. V is- kanju skupnega zimskega zavetja izločajo agregacijske feromone ter se množično zbirajo na fasadah hiš, kar povzroča prave invazije žuželk (Inkley, 2012). V nasadih se škodljivec pojavlja od cvetenja do zo- renja plodov gostiteljskih rastlin. Obdobje, v katerem povzroča škodo, je izjemno dolgo. Kemično zatiranje marmorirane smrdljivke, vezano na pragove škodlji- vosti, zahteva veliko število škropljenj, kar negativno vpliva na agroekosistem, obremenjuje okolje in je v popolnem nasprotju z vzpostavljenim sistemom in- tegriranega varstva (Morrison in sod., 2017). Dolgo- ročno gre pričakovati, da se bodo domorodni koristi organizmi sčasoma prilagodili na novega tujerodnega škodljivca ter ga začeli omejevati na dopustno raven. Izkušnje iz tujine kažejo, da je obvladovanje marmo- rirane smrdljivke izjemno zahteven proces, ki mora združevati številne ukrepe varstva rastlin (Morrison in sod., 2017). Temeljiti morajo na zanesljivih metodah spremljanja populacije škodljivih organizmov, pred- videvanju nastanka škode ter pravočasni napovedi in izvedbi ukrepov. 2 SPREMLJANJE POPULACIJSKE DINAMI- KE MARMORIRANE SMRDLJIVKE Za spremljanje populacijske dinamike različnih vrst domorodnih stenic se v praksi navadno upora- bljajo različne detekcijske metode, kot so uporaba me- tuljnice, otresanje rastlin, feromonske vabe in UV oz. črne svetilke (svetilke, ki oddajajo vijolično in modro kratkovalovno sevanje) (Krupke in sod., 2001; Leskey in Hogmire, 2005; Kamminga in sod., 2009; Borges in sod., 2011). Za spremljanje populacijske dinamike marmorirane smrdljivke so se v praksi sprva upora- bljale piramidalne pasti z uporabo agregacijskega fero- mona (metil [2E, 4E, 6Z]-dekatrienoat) stenice Plautia stali Scott (Nielsen in sod., 2011; Laskey in sod., 2012b; Joseph in sod., 2013). Raziskovalci so ugotovili, da omenjeni feromon deluje na marmorirano smrdljivko kot kairomon (Aldrich in sod., 2007; Khrimian in sod., 2008). Nadaljnje raziskave so pokazale, da feromon (metil [2E, 4E, 6Z]-dekatrienoat) ne vpliva na gibanje marmorirane smrdljivke spomladi in zgodaj poleti in je zato uporaben le pri pozno poletnem in jesenskem lo- Acta agriculturae Slovenica, 117/1 – 2021 3 Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) vljenju stenic (Leskey in sod., 2012b). Khrimian in sod. (2014) so ugotovili, da dvokomponentno privabilo, ki ga sestavljata agregacijski feromon (murgantiol) ter sinergistično sredstvo metil-dekatrienoat (MDT) vpli- vata tako na ličinke kot tudi na odrasle osebke marmo- rirane smrdljivke skozi celotno rastno dobo. Njihovo raziskavo so potrdili tudi rezultati nekaterih sorodnih raziskav (Weber in sod., 2014; Leskey in sod., 2015; Morrison in sod., 2017; Rice in sod., 2018), v katerih so se predvsem ukvarjali z ugotavljanjem razmerij različ- nih vrst agregacijskih feromonov in njihovim učinkom na lovljenje populacij marmorirane smrdljivke. Pozna- vanje agregacijskih feromonov marmorirane smrdljiv- ke predstavlja podlago za strategijo »privabi in ubij (angl. “attract and kill”), kjer s feromoni privabimo na izbrano rastlinsko vrsto škodljivca in ga nato z uporabo drugih tehnik (kemično, biotično varstvo, mehansko z uporabo insekticidnih sesalcev) zatremo (Morrison in sod, 2016). Iz ZDA poročajo tudi o spremljanju popu- lacijske dinamike z uporabo svetlobnih vab (bela, črna in modra dolgovalovna svetloba), vendar so potrdili uspešnost uporabe zgolj pri lovljenju odraslih osebkov (Nielsen in sod., 2013). 3 UPORABA ETERIČNIH OLJ Rastlinska eterična olja predstavljajo alternativni način zatiranja škodljivih žuželk v kmetijstvu (Isman, 2006). Eterična olja so vir aktivnih, močnih presnov- kov, ki lahko vplivajo na bionomijo, vedenje in fiziolo- gijo žuželk. Poleg tega imajo kratko obstojnost v okolju in majhno toksičnost za sesalce in so zaradi široke upo- rabe kot dišave in arome navadno na voljo v velikih ko- ličinah po razumnih cenah (Isman, 2006). Dosedanje raziskave so pokazale, da eterična olja navadno delujejo kot repelenti za različne skupine žuželk (Moore in sod., 2007; Zhang in sod., 2013). Zhang in sod. (2014) so preučevali vpliv različnih eteričnih olj (nageljnove žbice, limonina trava, popro- va meta) in ugotovili, da prav vsa vplivajo odvračalno na ličinke kot tudi odrasle osebke marmorirane smr- dljivke. V nadaljevanju so kitajski raziskovalci naredi- li kemično analizo eteričnih olj in določili več kot 20 aktivnih snovi. Nekatere med njimi (evgenol, karvon, menton, pulegon, metil salicilat, trans/cis-citral, metil benzoat in β-kariofilen) so v primerjavi z atraktanti (MDT) zmanjšali ulov marmorirane smrdljivke v lovil- nih posodah za 72 do 99 %. Raziskovalci zaključujejo, da lahko kombinacija repelentov in atraktantov, razpo- rejenih na kmetijskih in urbanih zemljiščih, predstavlja t.i. »odvrni in privabi (angl. “push and pull”) strategijo zatiranja marmorirane smrdljivke. 4 KEMIČNO ZATIRANJE MARMORIRANE SMRDLJIVKE Marmorirana smrdljivka postaja vedno večji pro- blem v kmetijstvu, zato raziskovalci širom sveta iščejo učinkovite ukrepe z željo po omejitvi njene številčnosti in posledično zmanjšanju gospodarske škode na goje- nih rastlinah. Leskey in sod. (2012c) poročajo o neu- činkovitosti insekticidov, ki se navadno uporabljajo za zatiranje nekaterih drugih vrst stenic v ZDA. Lee in sod. (2013a) so pripravili pregled literature, vezane na učinkovitost različnih vrst insekticidov, ki jih v Aziji uporabljajo za zatiranje marmorirane smrdljivke. Ugo- tovljeno je bilo, da so nekateri insekticidi iz skupine kloriranih ogljikovodikov, organskih fosforjevih estrov, karbamatov, piretroidov in neonikotinoidov pokazali zelo veliko učinkovitost pri kontaktnem zatiranju mar- morirane smrdljivke (Bae in sod., 2008). Žal večina teh pripravkov ni registriranih v ZDA in Evropi za zatiranje škodljivca oz. so bile nekatere aktivne snovi celo za- konsko umaknjene iz trgovskih polic. Zato raziskovalci iščejo ustrezne novejše rešitve na področju kemičnega zatiranja marmorirane smrdljivke. V raziskavah, ki so jih opravili v ZDA, so tako v obdobju med leti 2008 in 2017 različne skupine razi- skovalcev opravile serije laboratorijskih preizkušanj učinkovitosti različnih vrst insekticidov pri zatiranju marmorirane smrdljivke (Nielsen in sod., 2008; Kuhar in sod., 2012; Leskey in sod., 2012c; Lee in sod., 2013b; Kuhar in Kamminga, 2017). Nielsen in sod. (2008) so potrdili veliko toksičnost piretroidov (bifentrin, beta- -ciflutrin, lambda cihalotrin, ciflutrin in fenpropatrin) pri zatiranju marmorirane smrdljivke. Zadovoljivo učinkovitost so pokazali tudi nekateri neonikotinoidi (dinetofuran in tiametoksam). Nielsen in sod. (2008) so tudi potrdili, da je zatiranje ličink marmorirane smr- dljivke bolj učinkovito kot zatiranje odraslih osebkov. V sorodni raziskavi so Leskey in sod. (2012c) potrdi- li učinkovitost piretroidov (bifentrin, fenpropatrin, permetrin), organskih fosforjevih estrov (dimetoat, malation, metidation, klorpirifos, acefat), karbamatov (metomil) in kloriranih ogljikovodikov (endosulfan). Krawczyk in sod. (2011) so potrdili veliko učinkovitost aktivnih snovi bifentrin, metomil, endosulfan in veči- ne neonikotinoidov (razen a.s. tiakloprid) pri zatiranju vseh razvojnih stadijev marmorirane smrdljivke. Kuhar in sod. (2012) so potrdili učinkovitost acefata, oksami- la, zeta-cipermetrina, fenpropatrina, lambda-cihalo- trina in acetamiprida pri zatiranju ličink marmorirane smrdljivke. Njihovi rezultati so potrdili tezo Nielsen in sod. (2008), da so ličinke bolj občutljive na delovanje insekticidov kot odrasli osebki marmorirane smrdljiv- ke. Lee in sod. (2013b) poročajo o 100 % učinkovitosti Acta agriculturae Slovenica, 117/1 – 20214 Ž. LAZNIK in S. TrdAN aktivnih snovi metomil, acefat, klorpirifos, dimetoat, malation, metidation, bifentrin, fenpropatrin, perme- trin, dinotefuran in endosulfan pri zatiranju odraslih osebkov marmorirane smrdljivke. Pregled učinkovitosti posameznih aktivnih snovi pri zatiranju marmorirane smrdljivke v laboratorijskih razmerah je predstavljen v preglednici 1. Pridobljeni laboratorijski rezultati so skupine raz- iskovalcev vodili tudi na preizkušanje učinkovitosti in- sekticidov pri zatiranju marmorirane smrdljivke na pro- stem (Bergmann in Raupp, 2014; Leskey in sod., 2014; Monneyham in sod., 2016; Kuhar in Kamminga, 2017). Učinkovitost insekticidov v laboratorijskih preizkusih je dostikrat večja od tiste, ki jo dosežemo v poskusih na prostem zaradi številnih omejujočih dejavnikov, ki jih v laboratoriju lahko reguliramo. Ker se marmorirana smr- dljivka lahko premika na daljše razdalje (Lee in Leskey, 2015) kot tudi med različnimi gostiteljskimi rastlinami Insekticid Učinkovitost Insekticid Učinkovitost Organski fosforjevi estri Neonikotinoidi Dimetoat 4 Dinotefuran 3,3 Malation 4 Klotianidin 2,8 Metidation 4 Tiametoksam 2,8 Klorpirifos 3 Acetamiprid 2,3 Metal paration 3 Imidakloprid 1,5 Acefat 2,5 Tiakloprid 1,5 Formetanat 2 Azinfos-metil 1 Klorirani ogljikovodiki Diazinon 1 Endosulfan 4 Fosmet 1 Druge skupine insekticidov Karbamati Spinetoram 1 Metomil 3,3 Abamektin 1 Oksamil 2 Piriproksifen 1 Karbaril 1,5 Priflukuinazon 1 Novaluron 1 Piretroidi Tlfenpirad 1 Etofenproks 4 Indoksakarb 1 Bifentrin 3,8 Spirotetramat 1 Permetrin 3 Klorantranilprol 1 Fenpropatrin 2,8 Ciantranilprol 1 Lambda-cihalotrin (LC) 2,8 Flonikamid 1 Ciflutrin 2,7 Beta-ciflutrin (BC) 2,5 Kombinirana uporaba Cipermetrin 2,5 Tiametoksam + klorantranilprol 4 Gama-cihalotrin 2 Beta ciflutrin + imidakloprid 3,7 Zeta-cipermetrin (ZC) 2 LC + tiametoksam 3,7 Esfenvalerat 1 Bifentrin + imidacloprid 3 ZC + bifentrin 3 LC + klorantranilprol 1 Preglednica 1: Učinkovitost insketicidov pri zatiranju marmorirane smrdljivke (Halyomorpha halys) v laboratorijskih razmerah. Table 1: Performance of insecticides against the brown marmorated stink bug (Halyomorpha halys) in laboratory bioassays. Legenda: učinkovitost pripravka 1 (< 50%), 2 (50-69 %), 3 (70-89 %), 4 (90-100 %) (Nielsen in sod., 2008; Krawczyk in sod., 2011; Leskey in sod., 2012c; Kuhar in sod., 2012; Lee in sod., 2013b; Kuhar in Kamminga, 2017). Acta agriculturae Slovenica, 117/1 – 2021 5 Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) (Zobel in sod., 2016), je ključnega pomena, da pride v stik z letalno dozo aktivne snovi na tretirani rastlini (Morrison in sod., 2016). Le tako je možno doseči zado- voljivo raven učinkovitosti izbranih insekticidov pri zati- ranju marmorirane smrdljivke. Leskey in sod. (2014) so ugotovili, da je sveži nanos insekticida na rastlino znatno bolj letalen za marmorirano smrdljivko kot nekaj dni star insekticidni ostanek na rastlini. Omenjeni raziskovalci so to ugotovitev potrdili na primeru aktivnih snovi fenpro- patrin ter dinotefuran. Obe aktivni snovi sta se izkazali kot zelo učinkoviti, če je marmorirana smrdljivka prišla v stik z aktivno snovjo takoj, ko je bila le-ta nanesena. Ob poznejšem stiku (po 24h) z aktivno snovjo je bil uči- nek le-te zanemarljiv. Leskey in sod. (2014) so ugotovili, da insekticidni ostanki na rastlini delujejo na marmo- rirano smrdljivko odvračalno (vplivajo na zmanjšano hranjenje), ne povzročijo pa njene smrtnosti. Bargmann in Raupp (2014) sta v sorodni raziskavi potrdila pomen takojšnjega stika marmorirane smrdljivke z insekticidom na primeru aktivnih snovi karbaril, acetamiprid in per- metrin. Ostanki aktivnih snovi permetrin in karbaril so v njuni raziskavi vplivali na več kot 80 % smrtnost ličink marmorirane smrdljivke 48 h po nanosu, medtem ko aktivna snov acetamiprid ni imela učinka na smrtnost le-teh. Mooneyham in sod. (2016) so preučevali učinek devetih registriranih insekticidov, ki se uporabljajo za zatiranje škodljivcev na stavbah. Ugotovili so, da aktivne snovi lambda-cihalotrin (LC), LC + tiametoksam, beta- -ciflutrin (BC), BC + imidacloprid učinkujejo (več kot 80 % smrtnost odraslih osebkov marmorirane smrdljiv- ke) tudi po desetih dneh po nanosu na okenske okvirje. Skupine različnih raziskovalcev so v obdobju med leti 2011 do danes izvedle številne raziskave vezane na preiz- kušanje učinkovitosti insekticidov pri zatiranju marmo- rirane smrdljivke na prostem (Krawczyk in sod., 2011; Kuhar in sod., 2012; Herbert in sod., 2013; Nielsen in Rucker, 2013; Frank, 2014; Walgenbach in Schoof, 2015; Morrison in sod., 2016; Kuhar in Kamminga, 2017). Na splošno so se številni insekticidi, ki so bili učinkoviti v laboratorijskih preizkusih, dobro izkazali tudi na pro- stem. Aktivne snovi, ki so bile najučinkovitejše, so bile iz skupine piretroidov (beta-ciflutrin, bifentrin, permetrin, fenpropatrin, lambda-cihalotrin, zeta-cipermetrin), neo- nikotinoidov (dinotefuran, klotianidin in tiametoksam), karbamatov (metomil) in kloriranih ogljikovodikov (en- dosulfan) (Preglednica 2). Skupina insekticidov Organofosfati Neonikotinoidi Kombinirana uporaba Acefat Dinotefuran Tiametoksam + klorantranilprol Fosmet Klotianidin BC + imidakloprid Tiametoksam LC + tiametoksam Karbamati Bifentrin + imidacloprid Metomil Klorirani ogljikovodiki ZC + bifentrin Oksamil Endosulfan LC + klorantranilprol BC + spinetoram Piretroidi Druge skupine insekticidov Bifentrin + abamectin Etofenproks Flupiridafuron Binfetrin + klorantranilprol Bifentrin Diflubenzuron Metomil + fosmet Permetrin Indoksakarb Metomil + ZC Fenpropatrin Klorantranilprol Lambda-cihalotrin (LC) Ciklaniprol Beta-ciflutrin (BC) Flonikamid Gama-cihalotrin Zeta-cipermetrin (ZC) Esfenvalerat Preglednica 2: Insekticidi, ki so pokazali visoko učinkovitost pri zatiranju marmorirane smrdljivke (Halyomorpha halys) na pros- tem. Table 2: Insecticides that provided a significant reduction of the brown marmorated stink bug (Halyomorpha halys) in field efficacy experiments. (Krawczyk in sod., 2011; Kuhar in sod., 2012; Herbert in sod., 2013; Nielsen in Rucker, 2013; Frank, 2014; Walgenbach in Schoof, 2015; Morrison in sod., 2016; Kuhar in Kamminga, 2017). Acta agriculturae Slovenica, 117/1 – 20216 Ž. LAZNIK in S. TrdAN 5 UPORABA INSEKTICIDNIH MREŽ Uporaba mrež za zaščito ljudi pred škodljivimi žu- želkami je dobro znana in razširjena praksa, katerih upo- raba sega v konec 17. stoletja (Da San Gallo, 1679). V kmetijstvu se zaščitne mreže v zadnjem času uporabljajo tako v rastlinjakih kot tudi na prostem z namenom, da zaščitimo rastline pred škodljivimi organizmi (Saupha- nor in sod., 2012). Novost v pristopu so t.i. insekticidne mreže, kjer je na mrežo nanesen izbrani insekticid, ki poveča učinkovitost zatiranja škodljivca oz. omejevanja poškodb in posledične škode na gojenih rastlinah (Hill in sod., 2007). Zaenkrat je najbolj učinkovit način preprečevanja škode, ki jo povzroča marmorirana smrdljivka, ravno uporaba insekticidnih mrež (Kuhar in sod., 2017; Sab- batini Peverieri in sod., 2018). V pridelavi sadja se za ta namen uporabljajo protitočne mreže, nadgrajene s stran- skimi mrežami, ki popolnoma preprečijo dostop steni- cam do rastlin. V določenih primerih se na mreže lahko nanese tudi insekticid (Kuhar in sod., 2017). Popolno zamreženje nasadov je potrebno izvesti takoj po cvetenju sadnega drevja (Leskey in Nielsen, 2018). V raziskavi, ki so jo opravili v Italiji (Sabbatini Peverieri in sod., 2018), raziskovalci poročajo o veliki učinkovitosti insekticidne mreže, ki je bila tretirana z a.s. alfa-cipermetrinom, pri zatiranju oz. omejevanju škode, ki jo povzroči marmo- rirana smrdljivka. Potrdili so več kot 70 % smrtnost od- raslih osebkov marmorirane smrdljivke, ki so prišli v stik z insekticidno mrežo. Poskus je bil sicer opravljen v laboratorijskih razmerah. Kuhar in sod. (2017) poročajo o 80 % smrtnosti odraslih osebkov marmorirane smr- dljivke, ki so prišli v stik z insekticidno mrežo, ki je bila tretirana z a.s. deltametrin. V poskusu na prostem, so Sabbatini Peverieri in sod. (2018) v nasadu breskev pre- učevali insekticidno mrežo prepojeno z alfa-cipermetri- nom in deltametrinom. Mrežo so dodatno prepojili tudi z atraktantom (murgantiol + MDT). Raziskava je poka- zala veliko umrljivost vseh razvojnih stadijev marmori- rane smrdljivke in posledično občutno zmanjšanje škode na rastlinah. Gre za zgled uporabe strategije »privabi in ubij«, ko z atraktantom privabimo škodljivca, ki nato pri- de v stik z mrežo, ki je prepojena z insekticidom. Prav tako so italijanski raziskovalci preučevali tudi vpliv zašči- tnih mrež brez dodatka insekticida na pojav škode, ki jo naredi marmorirana smrdljivka (Candian in sod., 2018). Rezultati raziskave so pokazali 45 % zmanjšanje škode v breskovih nasadih v primerjavi z netretiranimi nasadi, ter za 20 % zmanjšanje škode v primerjavi z nasadi, ki so bili tretirani z insekticidom deltametrin (Candian in sod., 2018). V nobeni od omenjenih raziskav raziskovalci niso izpostavili vpliva insekticidnih mrež na neciljne or- ganizme kot tudi ne na koristne vrste žuželk. 6 METODA PRIVABILNIH POSEVKOV Metoda privabilnih posevkov je pogosto upora- bljena v primerih, ko za zatiranje škodljivega organiz- ma ni registriranega fitofarmacevtskega sredstva, ko je pripravek predrag oziroma tedaj, ko glavni posevek ni odporen na napad ali okužbo škodljivih organizmov. Pri metodi privabilnih posevkov izkoriščamo lastnosti za škodljivce dovzetnih rastlin (Trdan in sod., 2005; Cook in sod., 2006). Privabilne posevke posadimo oziroma posejemo med rastline glavnega posevka ali v njegovo bližino, z namenom, da bi na dovzetne rastline privabili škodljivce in obenem zmanjšali njihovo številčnost na glavnem posevku (Cook in sod., 2006). Marmorirana smrdljivka je polifag, saj se hrani z različnimi rastlinskimi vrstami. Soergel in sod. (2015) so preučevali vpliv privabilnih posevkov pri pridelavi paprike. Ugotovili so, da je bil ulov marmorirane smr- dljivke na sončnicah (Helianthus annuus L.) v primerja- vi s papriko občutno večji. V sklopu raziskave sicer niso ugotovili zmanjšanja poškodb na papriki, kar avtorji pripisujejo predvsem dejstvu, da marmorirane smrdljiv- ke niso zatirali na privabilnih posevkih. Znano je, da je marmorirana smrdljivka zelo mobilna vrsta (Lee in Le- skey, 2015), zato predvidevajo, da so se osebki, ki so se v večjem številu pojavili na sončnicah pozneje vrnili na papriko in na plodovih povzročili gospodarsko škodo. Do podobnih rezultatov so prišli tudi Mathews in sod. (2017), ki so preučevali vpliv mešanih privabilnih po- sevkov (sončnica in navadni sirek) na zmanjšanje škode, ki jo povzroči marmorirana smrdljivka na papriki. Ulov marmorirane smrdljivke je bil na privabilnih posevkih za 4 x večji kot na glavni rastlini, vendar do zmanjša- nja poškodb na plodovih ni prišlo. V sorodni raziskavi so Nielsen in sod. (2016) preučevali različne rastlinske vrste, kot potencialne privabilne posevke. Poleg sončni- ce so v raziskavo vključili tudi navadni sirek (Sorghum bicolor L.), biserno proso (Pennisetum glaucum [L.]), je- dilni oslez (Abelmoschus esculentus Moench) in njivski grah (Pisum sativum var. arvense [L.]). Ulov marmori- rane smrdljivke je bil na navadnem sirku za več kot 4 x večji v primerjavi z ostalimi preučevanimi rastlinami. V nadaljevanju so preizkusili različne metode zatiranja škodljivca na privabilnih posevkih – kemično (azadi- rahtin), uporaba sesalcev za mehansko odstranjevanje žuželk z rastlin in ožiganje rastlin. Ožiganje rastlin se je izkazalo kot najbolj učinkovito (Nielsen in sod., 2016). Iz dosedanjih rezultatov raziskav je razvidno, da samo- stojna uporaba metode privabilnih posevkov ni zado- stno učinkovit ukrep pri zmanjšanju škode, ki jo pov- zroči marmorirana smrdljivka na glavni rastlini, temveč je potrebno metodo kombinirati z uporabo drugih var- stvenih ukrepov. Acta agriculturae Slovenica, 117/1 – 2021 7 Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) 7 BIOTIČNO VARSTVO Biotično zatiranje marmorirane smrdljivke trenu- tno temelji predvsem na ugotavljanju ustreznih agensov, ki bi se v prihodnje lahko uporabljali v programih biotič- nega varstva rastlin. Parazitske osice iz reda kožekrilcev (Hymenoptera), so znane kot najbolj učinkovit naravni sovražnik marmorirane smrdljivke. Svoja jajčeca odla- Vrsta naravnega sovražnika Tip Razvojni stadij marmorirane smrdljivke Anastatus parazitoid jajčeca Anastatus bifastiatus parazitoid jajčeca Anastatus mirabilis parazitoid jajčeca Anastatus pearsalli parazitoid jajčeca Anastatus reduvii parazitoid jajčeca Arilus cristatus plenilec ličinke, imago Arma chinensis plenilec Astata bicolor plenilec ličinke Astata unicolor plenilec ličinke, imago Astochia virgatipes plenilec Bicyrtes quadrifasciatus plenilec ličinke Bogosia parazitoid imago Geocoris plenilec jajčeca, ličinke Gryon japonicum parazitoid jajčeca Gryon obesum parazitoid jajčeca Harmonia axyridis plenilec jajčeca Isyndus obscurus plenilec Misumenops tricuspidatus plenilec Ooencytrus parazitoid jajčeca Ophiocordyceps nutans patogen Orius plenilec jajčeca Telenomus chlorupus parazitoid jajčeca Telenomus podi parazitoid jajčeca Trichopoda pennipes parazitoid ličinke, imago Trissolcus brochymenae parazitoid jajčeca Trissolcus cultratus parazitoid jajčeca Trissolcus edessae parazitoid jajčeca Trissolcus euschisti parazitoid jajčeca Trissolcus flavipes parazitoid jajčeca Trissolcus itoi parazitoid jajčeca Trissolcus japonicus parazitoid jajčeca Trissolcus mitsukurii parazitoid jajčeca Trissolcus scutellaris parazitoid jajčeca Trissolcus semistriatus parazitoid jajčeca Trissolcus thyantae parazitoid jajčeca Trissolcus utahensis parazitoid jajčeca Preglednica 3: Naravni sovražniki marmorirane smrdljivke (Halyomorpha halys). Table 3: Natural enemies of the brown marmorated stink bug (Halyomorpha halys). (Arakawa in Nakamura, 2003; Li in Liu, 2004; Yang, 2009; Haye in sod., 2015; Abram in sod., 2017, Dieckhoff in sod., 2017; Costi in sod., 2019). Acta agriculturae Slovenica, 117/1 – 20218 Ž. LAZNIK in S. TrdAN gajo v jajčeca gostitelja in povzročajo njihov propad. V Aziji, v izvornem okolju marmorirane smrdljivke, je raz- širjena vrsta Trissolcus japonicus (Ashmead, 1904), ki je znana kot njen najbolj učinkovit parazitoid (Yang, 2009). V Severni Ameriki in Evropi je bilo ugotovljenih več raz- ličnih rodov jajčnih parazitoidov, potencialnih naravnih sovražnikov H. halys, ki pripadajo trem družinam: Sceli- onidae (Telenomus, Trissolcus, Gryon spp.), Eupelmidae (Anastatus spp.) in Encyrtidae (Ooencyrtus spp.). Osice iz rodov Telenomus in Trissolcus (Scelionidae) so znane kot specializirani jajčni parazitoidi ščitastih stenic (He- miptera: Pentatomidae), medtem ko so parazitoidi iz rodov Anastatus in Ooencyrtus bolj generalisti in napa- dajo žuželke iz različnih družin (Abram in sod., 2017, Dieckhoff in sod., 2017). Raziskave, opravljene v Švici, so potrdile zastopanost vrst Anastatus bifasciatus (Geof- froy), Trissolcus cultratus (Mayr), Trissolcus semistriatus (Nees), Trissolcus scutellaris (Thomson), Trissolcus cul- tratus Mayr in Telenomus chloropus (Thomson) (Haye in sod., 2015). Rezultati triletnega spremljanja zastopanosti domorodnih jajčnih parazitoidov v Emigli Romagni v Italiji, so potrdili zastopanost vrste A. bifasciatus. Stopnja parazitizma je znašala od 1 do 3 % (Costi in sod., 2019). V preglednici 3 navajamo naravne sovražnike marmori- rane smrdljivke. V Evropi je v teku več raziskav z name- nom preizkušanja učinkovitosti domorodnih naravnih sovražnikov marmorirane smrdljivke. Kot obetavna sta se pokazala jajčna parazitoida Ooencyrtus telenomicida (Vassiliev 1904) [Hymenoptera] in Anastatus bifasciatus [Hymenoptera] (Costi in sod., 2019). Prvi je učinkovitost izkazal zlasti v laboratorijskih poskusih, medtem ko je v poljskih poskusih pokazal omejeno delovanje. Nadalju- jejo se tudi raziskave vrste A. bifasciatus, ki je v labora- torijskih poskusih pokazala manjšo učinkovitost pri pa- razitiranju H. halys v primerjavi z vrsto O. telenomicida, vendar pa je zaradi bioloških značilnosti najresnejši kan- didat za klasično biotično varstvo marmorirane smrdljiv- ke (Dieckhoff in sod., 2017). A. bifasciatus se razvija in razmnožuje v temperaturnem območju 15-32 °C, letno razvije do 3 rodove v obdobju od junija do oktobra, kar se ujema s periodo ovipozicije pri marmorirani smrdljiv- ki. Samice se hranijo na jajčecih gostitelja, hkrati vanje odlagajo lastna jajčeca, kar povzroči smrt gostitelja. Na podlagi obetavnih rezultatov začetnih raziskav, namera- vajo raziskovalci Univerze iz Modene in Mednarodnega centra za kmetijstvo in bioznanosti (CABI), ki deluje v Švici, razviti protokole za masovno gojenje parazitoida A. bifasciatus, ki bodo podlaga za morebitno komercialno rabo (Costi in sod., 2019). Eno od možnih alternativ predstavlja tudi uporaba entomopatogenih gliv in entomopatogenih ogorčic pri zatiranju marmorirane smrdljivke. Gouli in sod. (2012) so v laboratorijskih razmerah preučevali učinkovitost različnih sevov entomopatogene glive Beauveria bassia- na (Bals.-Criv.) Vuill. ter Metarhizium anisopliae (Met- chnikoff) Sorokin pri zatiranju odraslih osebkov marmo- rirane smrdljivke. Vsi sevi B. bassiana so se v poskusu izkazali za bolj učinkovite od glive M. anisopliae. S sevom B. basssiana GHA (Botanigard®, Mycotech Europe Limi- ted; Metrob d.o.o.) so dosegli med 85 in 100 % smrtnost odraslih osebkov marmorirane smrdljivke. Burjanadze in sod. (2020) so v laboratorijskih razmerah preučeva- li učinkovitost različnih vrst entomopatogenih ogorčic (Heterorhabditis bacteriophora Poinar, Steinernema bor- jomiense n.sp. in Steinernema apuliae sp.n.). Z ogorčico H. bacteriophora so dosegli okoli 80 % smrtnost odraslih osebkov marmorirane smrdljivke. 8 ZAKLJUČEK Na področju omejevanja številčnosti marmorirane smrdljivke in preprečevanje škode v kmetijski pridelavi v tem trenutku nimamo na razpolago prav veliko ukrepov, kot tudi ne učinkovitih rešitev. Kemično obvladovanje marmorirane smrdljivke vezano na pragove škodljivosti, zahteva veliko število škropljenj, kar negativno vpliva na agroekosistem, obremenjuje okolje in je v popolnem na- sprotju z vzpostavljenim sistemom integriranega varstva. Proti škodljivcu imamo trenutno v Sloveniji registrirana zgolj dva kemična pripravka; Karate Zeon 5 CS in Mospi- lan 20 SG, ki imata kot aktivno snov lambda-cihalotrin in acetamiprid, kar ju uvršča pod širokospektralne in- sekticide. Uporaba le teh je samo kratkoročna rešitev, saj lahko stenica s časom razvije odpornost. Ne-ciljno delo- vanje teh sredstev prizadene tudi koristne organizme, kot so naravni sovražniki. Slednjim pripisujemo velik pomen pri obvladovanju marmorirane smrdljivke v Sloveniji. Dolgoročno gre pričakovati, da se bodo domorodni ko- risti organizmi sčasoma prilagodili na novega tujerodne- ga škodljivca ter ga začeli omejevati na dopustno raven. Trenutno potekajo raziskave, kjer raziskovalci iz KGZS – KGZ Nova Gorica preučujejo zastopanost jajčnih pa- razitoidov marmorirane smrdljivke v Sloveniji. Potrdili so zastopanost treh vrst; Anastatus bifasciatus, Trissol- cus mitsukurii ter Ooencyrtus telenomicida (Rot in sod., neobjavljeno). Raziskovalna skupina sočasno preučuje tudi učinkovitost parazitiranja omenjenih vrst. Rezulta- ti raziskav domorodnih jajčnih parazitoidov stenic bodo prispevali k boljšemu poznavanju domorodne koristne favne ter njenega odziva ob vnosu in naselitvi nove tuje- rodne vrste (marmorirane smrdljivke). Eno izmed alter- nativnih rešitev bi lahko predstavljalo tudi sajenje priva- bilnih rastlin, s katerimi odvrnemo pojav škodljivca na glavni rastlini in njegovo zatiranje na privabilni rastlini z uporabo biotičnih agensov (entomopatogene glive, ento- Acta agriculturae Slovenica, 117/1 – 2021 9 Načini zatiranja marmorirane smrdljivke (Halyomorpha halys [Stål, 1855], Hemiptera, Pentatomidae) mopatogene ogorčice), sesalcev za mehansko odstranje- vanje žuželk z rastlin in ožiganja rastlin. Izkušnje iz tujine kažejo, da je obvladovanje mar- morirane smrdljivke izjemno zahteven proces, ki mora združevati številne ukrepe varstva rastlin. Temeljiti morajo na zanesljivih metodah spremljanja populacije škodljivca, predvidevanju nastanka škode ter pravoča- sni napovedi in izvedbi ukrepov. Poznavanje bionomije marmorirane smrdljivke v lokalnih razmerah, je pred- pogoj za načrtovanje ukrepov za prebivalce v naseljih ter preprečevanje škode v kmetijski pridelavi. 9 ZAHVALA Prispevek je nastal v sklopu projekta CRP V4-2002, ki je financiran s strani Javne agencije za raziskovalno dejavnost Republike Slovenije (ARRS) in Ministrstva za kmetijstvo, gozdarstvo in prehrano Republike Slovenije (MKGP). V projektu poleg vodilnega partnerja (UL – Bi- otehniška fakulteta) sodelujejo tudi KGZS – KGZ Nova Gorica in Maribor, UM – Fakulteta za kmetijstvo in bi- osistemske vede, Inštitut za hmeljarstvo in pivovarstvo Slovenije, Kmetijski inštitut Slovenije in UL – Fakulteta za strojništvo. 10 REFERENCE Abram, P.K., Hoelmer, K.A., Acebes-Doria, A. et al. (2017). 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