AActa agriculturae Slovenica • eISSN 1854-1941 • 119 – 3 • Ljubljana, oktober 2023 119•3 2023ACTA AGRICULTURAE SLOVENICA Acta agriculturae Slovenica Letnik / Volume 119 · Številka / Number 3 · 2023 eISSN 1854-1941 Glavna in odgovorna urednika / Editors-in-Chief Franc BATIČ, rastlinska pridelava / plant production Jernej OGOREVC, živalska prireja / animal production Področni uredniki / Section Editors Franc BATIČ (botanika in ekologija rastlin / botany and plant ecology), Majda ČERNIČ-ISTENIČ (agrarna ekonomi- ka 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 kmetij- skih zemljišč / agricultural land management), Andrej SIMONČIČ (varstvo rastlin / plant protection), Stanislav TRDAN (varstvo rastlin / plant protection), Andrej UDOVČ (agrarna ekonomika in razvoj podeželja / agricultural economics and rural deve- lopment), Andreja URBANEK-KRANJC (fiziologija rastlin / plant physiology), Rajko VIDRIH (živilstvo / food technology), Dominik VODNIK (fiziologija rastlin / plant physiology), Filip VUČANJK (kmetijsko strojništvo / agricultural machinery) Peter DOVČ (živalska biotehnologija / animal biotechnology, populacijske študije / population studies, genomika / genomics), Milena KOVAČ (selekcija in biometrija / selection and biometry), Janez SALOBIR (prehrana / nutrition) Mednarodni uredniški odbor / International Editorial Board Dunja BANDELJ (Koper, Slovenia), Iryna BANDURA (Melitopol, Ukraine), Michael BLANKE (Bonn, Germa- ny), Marko FLAJŠMAN (Ljubljana, Slovenia), Jürg FUHRER (Liebefeld-Bern, Switzerland), Helena GRČMAN (Ljubljana, Slovenia), Metka HUDINA (Ljubljana, Slovenia), Anton IVANČIČ (Maribor, Slovenia), Lučka KAJFEŽ BOGATAJ (Ljubljana, Slovenia), Damijana KASTELEC (Ljubljana, Slovenia), Iztok KOŠIR (Žalec, Slovenija), Chetan KESWANI (Varanasi, India), Ivan KREFT (Ljubljana, Slovenia), Jaromír LACHMAN (Prague, Czech Republic), Salim LEBBAL (Khenchela, Algeria), Mario LEŠNIK (Maribor, Slovenia), Zlata LUTHAR (Ljubljana, Slovenia), Ahad MADANI (Gonabad, Iran), Senad MURTIĆ (Sarajevo, Bosnia and Herzegovina), Alessandro PERESSOTTI (Udine, Italy), Hardy PFANZ (Essen, Germany), Slaven PRODANOVIĆ (Belgrade, Serbia), Naser SABAGHNIA (Maragheg, Iran), Olalekan Suleiman SAKARIYAWO (Abeokuta, Nigeria), Andrej SIMONČIČ (Ljubljana, Slovenia), Giuseppe SORTINO (Palermo, Italy), Bojan STIPEŠEVIĆ (Osijek, Croatia), Massimo TAGLIAVINI (Bolzano, Italy), Željko TOMANOVIĆ (Beograd, Serbia), Stanislav TRDAN (Ljubljana, Slovenia), Andrej UDOVČ (Ljubljana, Slovenia), Rajko VIDRIH (Ljubljana, Slovenia), Dominik VODNIK (Ljubljana, Slovenia), Alena VOLLMANNOVA (Nitra, Slovak Republic) Drago BABNIK (Ljubljana, Slovenia), Tomaž BARTOL (Ljubljana, Slovenia), Michel BONNEAU (Saint Gilles, Belgium), Milena KOVAČ (Ljubljana, Slovenia), Amarendra Narayan MISRA (Balasore, Orissa, India), Zdenko PUHAN (Zürich, Switzerland), Dejan ŠKORJANC (Maribor, Slovenia), Jernej TURK (Maribor, Slovenia) Tehnični uredniki / Technical Editors Karmen STOPAR, Jure FERLIN, Jože STOPAR Oblikovanje / Graphic art and design Milojka ŽALIK HUZJAN Jezikovni pregled / Proofreading Avtorji v celoti odgovarjajo za vsebino in jezik prispevkov / The authors are responsible for the content and for the language of their contributions. 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Ovitek: Učinki kadmija na rast čičerke (Cicer arietinum L.) v normalnih razmerah (kontrola) in pri različnih koncentracijah kadmija: a) sejanke, b) nadzemni deli, c) korenine, d) listna površina (kontrola, 2, 4 in 8 μg Cd g−1 perlita) (Foto: Maryam Kolahi, 1–18) Cover: Effect of cadmium on chickpea (Cicer arietinum L.) growth under normal and various concentrations of cadmium. a) Seedlings, b) Aboveground parts, c) Roots, d) Leaf areas (control, 2, 4 and 8 μg Cd g−1 perlite) (Photo: Maryam Kolahi, 1–18) Acta agriculturae Slovenica Volume / Letnik 119 · Number / Številka 3 · 2023 Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) produce high-quality flowers under high nitrogen fertilizer Pincirane sončnice (Helianthus annuus ‘Teddy Bear’) dajejo visoko kakovostna socvetja pri gnojenju z velimi količinami dušikovih gnojil Yahya SELAHVARZI , Maryam KAMALI, Sajede KARIMPOUR, Mahdiyeh KHARRAZI, Mohammad KARIMI 1–10 Study on the evolution of the fruit morphological and physico-chemical parameters of ‘Majhoul’ date palm during fruit growth Raziskava razvoja morfoloških in biokemičnih parametrov plodov dateljeve palme ‘Majhoul’ v rastni sezoni Mohamed ARBA, Iliass BERJAOUI, Ahmed SABRI 1–8 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) under cadmium stress and in silico features of HMAs proteins Preučevanje rastnih značilnosti, oksidativnega stresa in prevzema kovin pri čičerki (Cicer arietinum L.) v razmerah kadmijevega stresa in in silico lastnosti HMAs proteinov Maryam KOLAHI, Elham Mohajel KAZEMI, Milad YAZDI, Mina KAZEMIAN, Andre GOLDSON-BARNABY 1–18 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation Obsevanje semen jajčevca z ℽ-žarki vpliva na rast rastlin, pridelek in prehransko vrednost plodov v M1 generaciji Ekemini OBOK, Francis NWAGWU, Samuel AKPAN 1–13 Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators Razmerje med laboratorijskimi in poljskimi indikatorji kakovosti semen navadnega fižola (Phaseolus vulgaris L.) Albert MODI 1–8 Do mutations modifying the leaf area (nr3) and the number of potential seeds (dfc) influence photosynthetic gas exchange characteristics in common buckwheat Fagopyrum esculentum Moench? Ali mutaciji, ki spreminajata listno površino (nr3) in število potencialnih semen (dfc) vplivata na značilnosti fotosintezne izmenjave plinov pri navadni ajdi (Fagopyrum esculentum Moench)? Ivan N. FESENKO, Alexandr V. AMELIN, Aleksey N. FESENKO, Oksana V. BIRYUKOVA, Valeriy V. ZAIKIN, Evgeniy I. CHEKALIN, Roman A. IKUSOV 1–7 Results of testing of the efficacy of sublethal concentrations of bacterial-chemical insecticides combinations against cabbage moth larvae Poskusi s subletalnimi koncentracijami bakterijsko-kemijskih insekticidov na gosenice kapusnega molja Hrant TERLEMEZYAN, Masis SARGSYAN, Harutyun HARUTYUNYAN, Noushig ZARIKIAN, Sona SARGSYAN, Gabriel KARAPETYAN, Habetnak MKRTCHYAN 1–6 Review Article / Pregledni znanstveni članek Mycoviruses: trends in plant-fungus-mycovirus interactions and ‘biocontrol’ prospects in agriculture and the environment Mikovirusi: trendi v interakcijah rastlina-gliva-mikovirus in izgledi ‘biokontrole’ v kmetijstvu in okolju Elias Mjaika NDIFON, Gilbert Nchongboh CHOFONG 1–11 Acta agriculturae Slovenica, 119/3, 1–10, Ljubljana 2023 doi:10.14720/aas.2023.119.3.2556 Original research article / izvirni znanstveni članek Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) produce high- quality flowers under high nitrogen fertilizer Yahya SELAHVARZI 1, 2, Maryam KAMALI 1, Sajede KARIMPOUR 3, Mahdiyeh KHARRAZI 4, Moham- mad KARIMI 1 Received February 13 2022; accepted July 27, 2023. Delo je prispelo 13. februarja 2022, sprejeto 27. julija 2023 1 Department of Horticultural Science and Landscape Engineering, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran 2 Corresponding author, e-mail: selahvarzi@um.ac.ir 3 Department of Horticultural Science and Landscape Engineering, Shirvan Faculty of Agriculture, University of Bojnord, Bojnord, Iran 4 Ornamental Plants Biotechnology Research Department, Research Institute for Industrial Biotechnology, Iranian Academic Centre for Education, Culture and Re- search (ACECR), Mashhad, Iran Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) pro- duce high-quality flowers under high nitrogen fertilizer Abstract: This study was investigated the effect of remov- ing the central bud (pinching) and different levels of nitrogen fertilizer urea on some morphological and physiological traits of ornamental sunflower. This study was conducted as a factori- al experiment in a randomized complete block design with four replications on ornamental sunflower (Helianthus annuus ‘Ted- dy Bear’) at Horticulture Farm, Department of Horticulture, Ferdowsi University of Mashhad, Iran, in 2020-2021. The first factor was pinching in two levels (pinching and non-pinching) and the second factor was using urea at four levels (0, 200, 300, and 400 kg ha-1) in the form of water-soluble fertilizer. Results showed that the highest flower dry mass (59.25 g) was observed in pinched plants fertilized by 400 kg ha-1 of urea. Besides, the application of a high level of urea fertilizer and pinching treat- ment increased the amount of total chlorophyll and chlorophyll b. By removing the central bud, the amount of N, P, K, Ca, Zn, and Fe elements in the leaf increased by 1.5, 1.6, 1.3, 1.9, 1.4, and 1.5 times, respectively. Therefore, pinching and the adding of urea fertilizer at 400 kg ha-1is recommended for the produc- tion of high-quality sunflower plant ‘Teddy Bear’. Key words: flowering period, head diameter, nutrient ele- ments, photosynthesis, plant height Pincirane sončnice (Helianthus annuus ‘Teddy Bear’) dajejo visoko kakovostna socvetja pri gnojenju z velimi količinami dušikovih gnojil Izvleček: V raziskavi je bil preučevan učinek odstranje- vanja (pinciranja) osrednjega socvetja in različnih odmerkov gnojenja z ureo na nekatere morfološke in fiziološke lastnosti okrasnih sončnic. Raziskava je bila izvedena kot popolni fak- torski bločni poskus s štirimi ponovitvami na okrasnih sonč- nicah (Helianthus annuus ‘Teddy Bear’) na Horticulture Farm, Department of Horticulture, Ferdowsi University of Mashhad, Iran, v rastni sezoni 2020-2021. Prvi dejavnik je obsegal dve ravni pinciranja (pincirano in ne pincirano), drugi dejavnik pa štiri različne odmerke uree (0, 200, 300, and 400 kg ha-1) v obliki vodotopnega gnojila. Rezultati so pokazali, da je bila dosežena največja suha masa socvetij (59,25 g) pri pinciranih rastlinah in uporabi 400 kg ha-1 of uree. Večji odmerek uree je pri pinciranih socvetjih povečal vsebnost celokupnega klorofila in klorofila b. Pri odstranitvi osrednega socvetja se je vsebnost N, P, K, Ca, Zn in Fe v listih povečala za 1,5; 1,6; 1,3; 1,9; 1,4 in 1,5 krat. Zaradi naštetega priporočamo pinciranje in gnojenje s 400 kg ha-1 uree za vzgojo kakovostnih sončnic ‘Teddy Bear’. Ključne besede: cvetenje, premer koška, hranila, fotosin- teza, višina rastlin Acta agriculturae Slovenica, 119/3 – 20232 Y. SELAHVARZI et al. 1 INTRODUCTION Sunflower (Helianthus annuus L.) is an annual plant belonging to the Asteraceae family. This plant is native to North America and has medicinal, nutritional, and or- namental uses (Sehrawat et al., 2003) beside of its usage as a biodegradable source in biodiesel fuels (Saba et al., 2016). According to the specialized institute of cut flow- ers, in some sunflower cultivars, such as ‘Pro-Cut Gold’ and ‘Sunrich Lemon’, the stems are so long. In contrast, other sunflower cultivars produce lateral shoots and have short stems and uniform flowers (Dole, 2002). Removing the central bud (pinching) is considered one way to stim- ulate the plant to produce lateral branches and increase the number of stems per plant (Wien, 2015; Cheema, 2018). Depending on the stage of plant growth, pinching can be beneficial or harmful for plants (Smakel, 2006), as pinching of the different sunflower cultivars at the right time enhanced flower production three to four times (Wien, 2012a). However, pinching delays flowering and reduces flower size (Cheema, 2018) and the formation of flowers for 7-10 days (Wajid et al., 2007). Wien (2016b) reported that pinching the ‘Sunrich Orange’ cultivar, led to the production of smaller flowers but appropriate stem length. The smaller size of the flower, but with the mar- ketable stem length, allows the florists to use them in ar- ranging the flower bouquets properly. The study results of Badge and Panchbhai (2018) revealed that pinching the African marigold (Tagetes erecta L.) plants (15 days after transplanting) lead to the production of maximum flower yield in comparison to other treatments. The maximum nitrogen, phosphorus, and potassium content and uptake, as well as yield parameters, were obtained by pinching the plants 15 days after transplanting and foliar application of gibberellic acid at 300 mg l-1 (Badge et al., 2015). Prakash et al. (2016) reported that pinching the African marigold (Tagetes erecta) affects the plant height, number of lateral branches, number of flowers, and num- ber of days to 50 % of flowering. Adequate nutrition with essential elements, espe- cially with nitrogen, is very important for the successful development of plants. Nitrogen is an essential nutrient that plays a role in the structure of various proteins, en- zymes, coenzymes, nucleic acids, and cytochromes (Has- segawa et al., 2008), as well as, involving in the cell divi- sion and expansion, thereby increasing leaf length and width (Kumari, 2011, Lehri et al., 2011). Besides, this ele- ment plays a crucial role in the formation of chlorophyll and has a vital function in supplying carbohydrates and photosynthesis (Wajid et al., 2007). The effect of nitrogen on plant growth and development has often been linked to increased photosynthesis because the appropriate amount of nitrogen determines plant yield (Mekonnen et al., 2002). Studies indicated that the increase in growth and yield of the sunflower plant is dependent upon the adequate supply of nitrogen (Ali et al., 2004; Ali, 2015). The results of Oad et al. (2018) study indicated that sun- flower plants treated with foliar application of urea (1 %) after 35 days of sowing in addition to recommended soil applied urea (130 kg ha-1) led to the highest plant height, head diameter, grains per head, seed index, and grain yield. Ali et al. (2014) reported that the application of 80 kg ha-1 nitrogen fertilizer resulted in an increased plant height and head diameter of the sunflower plants. In an- other study, a significant increment in crop growth, bio- mass, dry matter production, and biological yield result- ed in 100 kg ha-1 of N rate application (Saifullah, 1996), but Handayati and Sihombing (2019) recommended the application of 150 kg ha-1 nitrogen for the cultivation of this plant. Considering the effect of pinching and nitrogen on the reproductive and vegetative traits of the sunflower plant, the present experiment was aimed to investigate the effect of removing the central bud (pinching) and different levels of nitrogen fertilizer (urea) on flowering, flower size, plant height, and other morphological and physiological traits of ornamental sunflower (Helianthus annuus ‘Teddy Bear’). 2 MATERIALS AND METHODS The field experiment was conducted at Research Farm, Department of Horticulture, Ferdowsi University of Mashhad, Iran, in 2020-2021. Before planting, chem- ical analysis of the soil was done at an upper 0-30 cm zone, the results of which are shown in Table 1. This experiment was conducted as a factorial ex- periment in a randomized complete block design with four replications on ornamental sunflower (Helianthus annuus ‘Teddy Bear’). The first factor was removing or not removing the central bud (pinching); and the second factor was applied in four levels of adding urea fertilizer (CO (NH2)2): 0, 200, 300, and 400 kg ha -1 in the form of water-soluble fertilizer. The sunflower seeds were purchased from the Dutch Hemogenetic Company and sown in April 2020. Four weeks later, the seedlings with four true leaves were planted at spacing 50 × 20 cm. Ten days after transplanting, urea fertilizer was applied three times (weekly) with irrigation water according to the mentioned levels. Then, one month after transplanting, the pinching treatment was applied. During the experiment, the number of days to flowering (vegetative period), and the duration of the flowering period (flowering period) were recorded. The number of flowers per plant was counted, and the head Acta agriculturae Slovenica, 119/3 – 2023 3 Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) produce high-quality flowers under high nitrogen fertilizer diameter and the stem diameter of each treatment were measured with a digital caliper. In 50 % of the flowering stage, the number of leaves per plant and the plant height were calculated. At this stage, the rates of photosynthesis and transpiration were also measured using a portable photosynthesis system (Li-6400) from 9:00 to 11:00 AM under natural conditions. Fresh leaf tissue was used for the measurement of chlorophyll contents. 0.2 g fresh leaf was crushed in 10 ml of methanol 96 %. The resulted solution was filtered through Whatman filter paper and then centrifuged at 2500 rpm for 10 minutes. The supernatant optical ab- sorption was then read at 653, and 666 nm using a spec- trophotometer (model CE2502, BioQuest, UK) method (Sukran et al., 1998). Finally, the chlorophyll pigments were obtained using the following equations: Chla (µg.ml -1) = 15.65 A666 - 7.340 A653 Chlb (µg.ml -1) = 27.05 A653 - 11.21 A666 ChlTotal = Chla + Chlb After applying the treatments, at the beginning of the reproductive phase, N, P, K, Zn, Fe, and Ca elements in the sunflower leaf were measured. The amount of nitrogen in the plant was measured using the Kjeldahl method (Bremmer and Mulvaney, 1982). Concentrations of P, K, Ca, Zn, and Fe were analyzed by an inductively coupled plasma optical emission spectrometer (ICP- OES, Perkin-Elmer Optima 5300 DV) in plant samples (Van de Wiel, 2003). The flowers were collected and dried during the flowering period to record the flower dry mass. After flowering, the leaf area was measured using the leaf area meter (Model Li-Cor-1300, USA). The specific leaf area (SLA = leaf area leaf dry mass-1) and the leaf area ratio (LAR = leaf area total dry mass-1) were also calculated. To measure the dry mass of plant components (stems, roots, leaves, and flowers) and the total dry mass, the plant sam- ples were dried at 70 °C until the sample mass was held constant. Then the dry mass of different plant parts was recorded. 2.1 DATA ANALYSIS Data were analyzed with One Way ANOVA us- ing JMP® (v.8) software (SAS institute, 1989-2021), and means were compared based on the LSD test at the 5 % of probability level. 3 RESULTS The results of ANOVA revealed that urea applica- tion and pinching have significant effects on different traits of the sunflower plant including mineral uptake, vegetative and generative traits, photosynthesis and tran- spiration rate, chlorophyll contents, and dry mass of dif- ferent parts of sunflower (data not shown). 3.1 ELEMENT UPTAKE The element content of sunflower shoots was af- fected by urea application and pinching, and not by their interaction. The use of urea led to an increase in N con- tent in shoots as well as P, K, Ca, Zn, and Fe contents. As the amount of urea fertilizer increased, the accumulation of these elements in the shoots also increased. Using urea fertilizer at 400 kg ha-1 induced mineral accumulation 2.4, 2.6, 1.7, 2.3, 4.1, and 4.2 times more than the control for N, P, K, Ca, Zn, and Fe, respectively (Table 2). Contra- riwise, these element contents decreased when pinching was applied. Pinched plants had 1.5, 1.6, 1.3, 1.9, 1.4, and 1.5 times less amount of N, P, K, Ca, Zn, and Fe than non- pinched sunflower plants, respectively (Table 2). 3.2 VEGETATIVE TRAITS The interaction of pinching × urea fertilizer had a significant effect on vegetative traits including plant height, stem diameter, leaf number, leaf area, SLA, and LAR. Sunflower plants had the biggest height (153 cm) when grown using 400 kg ha-1 of urea fertilizer and not pinched, while pinched plants without urea fertilizing showed the lowest height (129 cm). The same results were obtained for stem diameter growth with 28.98 and 20.90 mm, respectively. The leaf number increased by urea application and pinching (27.5-30.0), whereas, the lowest number of leaf production (13.0) was recorded in non-pinched plants without urea. The biggest leaf area (16130.25 cm2) showed in 400 kg ha-1 of urea applica- Table 1: The physical and chemical properties of the soil CaZnFeKPNECpHLoamClaySandSoil TextureDepth (mg kg-1)(dS m-1) (%)(cm) 29371522471662516066101.37.527 3340Sandy loam0-30 Acta agriculturae Slovenica, 119/3 – 20234 Y. SELAHVARZI et al. tion and pinching treatment, while non-pinched plants grown without urea fertilizer expanded their leaf to the minimum amount (8953.65 cm2). SLA was the highest when 400 kg ha-1 of urea fertilizer with pinching (302.46 cm2 g-1) and 300 kg ha-1 of urea fertilizer without pinch- ing (301.37 cm2 g-1) was applied and the lowest amount of SLA was shown in the 300 kg ha-1 of urea application with pinching (266.60 cm2 g-1) treatment (Table 3). The high- est amount of the LAR was obtained in two treatments include 400 and 300 kg.ha-1 of urea fertilizer + pinching (73.64 and 72.97 cm2.g-1, respectively), and the lowest amount was recorded for plants with no urea fertilizing with (62.11 cm2.g-1) or without (62.36 cm2 g-1) pinching (Table 3). 3.3 GENERATIVE TRAITS We obtained the highest number of flowers (77.75) in pinched plants fertilized by 400 kg ha-1 of urea ferti- lizer, and non-pinched plants produced the less flower number (21.25-26.00) in all levels of urea fertilizer (Ta- ble 4, A). The head diameter had the highest amount (146.68-15.1.59 mm) when pinching was not applied in plants of urea fertilizer in 0, 200, and 300 kg ha-1, and the lowest amount (97.42 mm) was recorded in the pinched plants with no urea using. The number of days to first flower appearance and the duration of the flowering stage were affected by urea fertilizer, that is, the increase in urea levels led to prolongation of the vegetative and gen- erative period and low levels of urea stimulate the enter- ing to and shortening of the generative stage. Duration of the flowering stage also was increased by pinching up to 6 days compared to the non-pinched plants (Table 4, B). 3.4 CHLOROPHYLL CONTENTS, PHOTOSYN- THESIS, AND TRANSPIRATION RATE The content of chlorophyllb and total chlorophyll was affected by the interaction of urea fertilizer × pinch- ing, while the chlorophyll content was not influenced by interaction but was affected by simple effect of them. The plants which were grown under 400 kg ha-1 of urea ferti- lizer with (0.28 µg g-1 FM) or without pinching (0.24 µg Table 2: The simple effect of pinching and urea fertilizer treatments on element content in sunflower shoots FeZnCaKPNTreatmentsFactors (mg kg-1) 93.000d8.0000d9167.0d12346.0d820.00d940.00d*0Urea fertilizer (kg ha-1) 108.500c11.3750c11939.3c16934.0c968.00c1088.00c100 333.875b17.8750b20584.0b19746.0b1094.00b1212.50b200 393.125a32.8750a20798.0a20847.8a2172.00a2292.00a400 277.000a20.3750a20623.1a19499.4a1539.50a1659.50a-Pinching 187.250b14.6875b10621.1b15437.4b987.50b1106.75b+ *Means followed by similar letters in each trait and for each factor didn’t have any significant difference based on LSD test (p ≤ 0.01) Table 3: The interaction effect of pinching × urea fertilizer on vegetative traits of the sunflower plant Pinching Urea fertilizer (kg ha-1) Plant height (cm) Stem diameter (mm) Leaf number Leaf area (cm2) SLA** (cm2 g-1) LAR** (cm2 g-1) - 0 139.33abc* 25.49abcd 13.00b 8953.65d 294.81bc 62.36c - 200 143.66abc 26.25abc 22.00ab 9976.30cd 298.51ab 69.07ab - 300 147.00ab 26.37abc 21.00ab 12407.76b 301.37a 72.97a - 400 153.00a 28.98a 30.33a 9856.37cd 273.78cd 64.12bc + 0 129.00c 20.90d 22.33ab 11416.25bc 276.35c 62.11c + 200 133.00bc 21.45cd 27.50a 12969.25b 296.16ab 69.29ab + 300 138.00abc 23.02bcd 26.66a 12813.25b 266.60e 65.21b + 400 147.33ab 28.01ab 30.00a 16130.25a 302.46a 73.64a *Means followed by similar letters in each trait do not have any significant difference based on the LSD test (p ≤ 0.01) **SLA: The specific leaf area, LAR: The leaf area ratio Acta agriculturae Slovenica, 119/3 – 2023 5 Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) produce high-quality flowers under high nitrogen fertilizer g-1 FM) had the highest amount of chlorophyll b and the lowest was related to not using urea fertilizer for pinched and non-pinched plants (0.11-0.13 µg g-1 FM). In the same manner, total chlorophyll content was the highest in non-pinched plants treated by 400 kg ha-1 of urea ferti- lizer (0.45 µg g-1 FM), and the lowest amount was record- ed in the pinched and non-pinched plants without urea fertilizing (0.25-0.26 µg g-1 FM)(Table 5, A). Unlike the chlorophyllb and total chlorophyll, the amount of chlo- rophyll only was affected by urea fertilizer and pinching. Urea fertilizer at 300 kg ha-1 (0.19 µg g-1 FM) and pinch- ing (0.17 µg g-1 FM) provoked chlorophylla accumula- tion. There was a trend for photosynthesis and transpira- tion rate, increasing urea levels from zero to 400 kg ha-1 enhanced the amounts of photosynthesis from 6.19 to 11.39 µmol mol-1 CO2 and transpiration rate from 1.44 to 2.65 mmol.mol-1 H2O, respectively. Pinching significantly led to a decrease in photosynthesis and transpiration rate (Table 5, B). 3.5 DRY MASS OF PLANT ORGANS Leaf, head, root, and total dry mass of sunflower was affected by the interaction of urea fertilizer × pinch- ing, as the highest amount of them was recorded on pinched plants were fertilized by 400 kg ha-1 of urea ferti- lizer, 53.33, 59.25, 39.99, and 219.04 g, respectively. Non- Table 4: The interaction effect of pinching × urea fertilizer (A) and simple effect of them (B) on generative traits of the sunflower plant (A) (B) Pinching Urea fertilizer (kg ha-1) Flower number Head diameter (mm) Day to1st flowering (day) Duration of flowering (day) - 0 23.50d* 146.68a Urea fertilizer (kg ha-1) 0 44.0000D 32.3750D - 200 21.25d 151.59a 200 46.1250C 34.1250C - 300 26.00d 148.68a 300 48.7500B 36.7500B - 400 25.50d 119.27b 400 52.7500A 40.7500A + 0 45.50c 97.42c Pinching + 200 46.75c 106.84bc - 44.9375A 32.9375B + 300 63.50b 111.24bc + 50.8750A 39.0625A + 400 77.75a 110.04bc *Means followed by small (interaction effect) and capital (simple effect) letters in each trait does not have a significant difference based on the LSD test (p ≤ 0.01) Table 5: The interaction effect of pinching × urea fertilizer on chlorophyll b and total chlorophyll content (A) and simple effect of them on photosynthesis, transpiration rate and Chlorophyll a content (B) in sunflower plant (A) (B) Pinching Urea fertilizer (kg ha-1) Chlorophyllb content (µg g-1 FM) Total chloro- phyll content (µg g-1 FM) Chlorophylla content (µg g-1 FM) Photosynthesis (µmol.mol-1 CO2) Transpiration rate (mmol mol-1 H2O) - 0 0.11c* 0.25d Urea fertilizer (kg ha-1) 0 0.13C 6.19D 1.44D - 200 0.15bc 0.30cd 200 0.15BC 7.67C 1.78C - 300 0.11c 0.37b 300 0.19A 8.96B 2.08B - 400 0.24a 0.45a 400 0.17AB 11.39A 2.65A + 0 0.13c 0.26d Pinching + 200 0.16bc 0.31c - 0.15B 9.07A 2.09A + 300 0.18b 0.31c + 0.17A 8.09B 1.88B + 400 0.28a 0.42ab *Means followed by small (interaction effect) and capital (simple effect) letters in each trait does not have a significant difference based on the LSD test (p ≤ 0.01) Acta agriculturae Slovenica, 119/3 – 20236 Y. SELAHVARZI et al. pinched plants had the lowest amount of leaf (30.37 g), head (33.75 g), root (22.78 g), and total (143.57-153.70 g) dry matter. Indeed, the total dry matter was not much significantly affected by urea fertilizer levels (Table 6, A). Urea fertilizer and pinching had a significant ef- fect on stem dry mass as a simple effect. The application of urea at 300 kg ha-1 induced the highest dry matter in the stem, while the lowest amount was detected when no urea fertilizer was used. Pinching increased stem dry mass to 1.2 times (62.03 g) in comparison with non- pinching (Table 6, B). The percentage of dry matter allocation in differ- ent parts of the plant showed no distinctive difference between treatments. On average, the highest to the low- est percentage of dry matter allocation were for stem (32.7 %), head (26.0 %), leaf (23.4 %), and root (17.9 %), respectively (Table 7). 4 DISCUSSION Besides the high cost of chemical fertilizer, the envi- ronmental impacts of their application are the important reason for the need to determine the exact amount of fertilizers. In our experiment, the interaction of different amounts of urea fertilizer (CO (NH2)2) and the removal of apical bud (pinching) had distinctive results on sun- flower ‘Teddy Bear’ growth and development. The nitro- gen fertilizer that used in this experiment is quite soluble and converts to ammonia in several days. So, as expected, a rise in the urea levels caused an increase in the nitrogen uptake. Similarly, the uptake of nitrogen is enhanced in broccoli plants when nitrogen fertilizer amounts increase (Vagen, 2003). In addition, the amount of P, K, Zn, Fe, and Ca was enhanced in the sunflower shoots by increas- ing the urea levels (Table 2). Confirmed results were re- ported by Karitonas (2003) and Yildirim et al. (2007) on broccoli plant, that an increase in the uptake of P, K, Fe, and Ca were shown by adding nitrogen fertilizer. Similar- ly, lettuce and tomato plants which were foliar sprayed by urea had higher amounts of N and K (Padem and Alan, 1995), and N, K, and Fe (Alan and Padem, 1994), re- spectively. All studied nutrient elements (i.e., N, P, K, Ca, Zn, and Fe) play several important functions and criti- cal roles within plants; metabolism, and catabolism pro- cesses, so, increasing in their uptake by plants can explain the significant differences in the studied traits in this ex- periment. The availability of nitrogen in the soil increase Table 6: The interaction effect of pinching × urea fertilizer (A) and simple effect of them (B) on the dry mass of different parts of the sunflower plant (A) (B) Pinching Urea fertilizer (kg ha-1) Leaf dry mass (g) Head dry mass (g) Root dry mass (g) Total dry masst (g) Stem dry mass (g) - 0 30.37e* 33.75e 22.78e 143.57c Urea fertilizer (kg ha-1) 0 53.83B - 200 33.42de 37.14de 28.50cde 144.42c 200 55.60AB - 300 41.17ab 45.75bcd 30.88bcd 170.03bc 300 60.87A - 400 36.00cde 40.00cde 25.07de 153.70c 400 57.19AB + 0 41.31bcd 45.90bcd 32.84bc 183.78b + 200 43.79bc 48.66bc 36.04ab 187.16b Pinching - 51.71B + 300 48.06ab 53.40ab 35.70ab 196.48ab + 62.03A + 400 53.33a 59.25a 39.99a 219.04a *Means followed by small (interaction effect) and capital (simple effect) letters in each trait does not have any significant difference based on the LSD test (p ≤ 0.01) Table 7: The percentage of dry matter allocation in differ- ent parts of the sunflower plant under different levels of urea fertilizer and pinching Pinching Urea fertilizer (kg ha-1) Dry mass (%) Leaf Head Stem Root - 0 20.5 27.0 33.2 19.2 - 200 20.6 28.2 32.2 19.0 - 300 25.6 23.1 35.7 15.6 - 400 25.6 24.0 34.1 16.2 + 0 22.8 26.8 32.3 18.1 + 200 22.8 27.8 30.7 18.8 + 300 22.7 28.0 30.4 18.9 + 400 26.5 22.8 33.0 17.7 Mean 23.4 26.0 32.7 17.9 Acta agriculturae Slovenica, 119/3 – 2023 7 Pinched sunflowers (Helianthus annuus ‘Teddy Bear’) produce high-quality flowers under high nitrogen fertilizer RuBisCO contents in leaves, even though some climate and soil factors including light, air humidity, and soil pH showed considerable influences on the fraction of nitro- gen allocated to RuBisCO regionally (Luo et al., 2021). Many scientists believe that the higher uptake of essential nutrients by plants as a result of the urea application is re- lated to the positive influence of nitrogen on the chemi- cal properties of the soil (Malhi et al., 2006; Haydon et al., 2007; Choudhury et al., 2011; Ai et al., 2017; Adekiya et al.,2018; Pasley et al., 2019). Ewulo et al. (2009) stated the possible reason for this is related to more microbial soil activity induced by urea application that causes more production and mineralization of organic matter in the soil. The reduction in the soil pH is another probable rea- son for higher element uptake by urea application that is shown in the Adekiya et al. (2018) report. As the sun- flower plants like the slightly acidic soils, this reduction in pH can improve elements uptake as the soil pH of the experiment site was close to neutral, 7.5 (Table 1). ‘Teddy Bear’ cultivar of sunflower is a dwarf cultivar and mature plants grow up maximum 140 cm. Urea ap- plication up to 400 kg ha-1 had a positive influence on plant height, and pinching reduced its effect. The sup- pressive effect of pinching on the plant height has been previously reported for different cultivars of sunflow- ers (Wien, 2016b; Cheema, 2018). Increasing the plant height and the stem diameter by using urea fertilizer is related to more leaf area production (Milford et al. 2000), while increasing the amount of chlorophyll in the sun- flower leaves, followed by increasing photosynthesis and dry matter production, is closely related to higher uptake of various elements, including iron and zinc. It has been reported that iron is involved in the structure of chloro- phylls, cytochrome, and nitrogenase enzymes, and zinc is involved in the activity of enzymes associated with chlorophyll formation and consequently increase photo- synthesis, accelerating the formation of growth composi- tions such as tryptophan as the raw material of auxins (Haydon et al., 2007). Enhanced dry matter production in non-pinched plants under more urea fertilizer, is probably due to increased water and mineral absorption by extended roots and rapid growth (Solangi et al., 2015). Steer et al. (1986), also reported an increscent in N up- take and dry matter production by enhancing nitrogen fertilizer levels, as the application of low amounts of ni- trogen fertilizer reduced leaf expansion and also the ac- cumulation of dry matter in sunflower. We also obtained the higher dry matter of leaf, root, and head amounts in pinched plants in positive relation with urea levels, while there was an optimum level at 300 kg ha-1 in non-pinched plants (Table 6). Leaf area, SLA, and LAR traits were the highest in 400 kg ha-1 of urea fertilizer application with pinching (Table 3). Leaf area is a critical index for plant growth as it is associated with important criteria including light in- terception, photosynthesis, transpiration, and evapotran- spiration rates (Goudriaan and Van Laar, 1994; Zahoor et al., 2010). Leaf growth in earlier stages needs more nitro- gen amounts (Evans, 1989; Johnson et al., 2010) and leaf area is limited when nitrogen is deficient by affecting cell division and enlargement (Roggatz et al., 1999). Pinching also had a positive effect on leaf area expansion in chry- santhemum ‘Snowball’ (Ona et al., 2015). An increase in leaf number after pinching is reported by others on dif- ferent herbaceous plants (Sehrawat et al., 2003; Tomar et al., 2004; Sudarshan, 2004; Salyh, 2013; and Ona et al., 2015). It seems that it might be related to the fact that pinching alters the direction of growth from upward to lateral parts of the plant (Salyh, 2013). The findings of this study indicated that pinching and urea application extended vegetative and flowering stages up to eight more days (Table 4. B). The number of days to flowering increased up to 70 days in pinched or- namental sunflowers, while non-pinched plants started to flower after 63 days (Wien et al., 2016). The same re- sults were reported by Ona et al. (2015) for chrysanthe- mum ‘Snowball’ and other species (Ahmad et al., 2007; Ryagi et al., 2007; Salyh, 2013). The pinching effect on delayed flowering is due to delay in flower initiation and bud physiological maturity (Naresh and Singh, 2012) because the growth rate in axillary buds is slower than apical buds. In pinched plants, flower number was affected by urea levels in a positive trend, while head diameter in- dicated a negative trend. Flower diameter was indepen- dently by cultivar decreased by pinching intensity (Bur- nett, 2017; Cheema, 2018). Other studies also confirmed these results (Ryagi et al., 2007; Habiba, 2012; Salyh, 2013; Ona et al., 2015). The removal of the shoot apex leads to the activation of dormant axillary buds below it to form branches Naresh and Singh (2012). Flower disk diameter of sunflower (Wien, 2016b) and the flower size of chry- santhemum (Ona et al. 2015) were reduced by pinching due the competition between branches and flowers. They have revealed that the number of the branches in a unit area has a negative linear relationship with head size in sunflower (Majid and Schneiter, 1987; Robinson et al., 1980; Wien, 2016b). 5 CONCLUSION This study demonstrated that adding urea in the soil and pinching improved photosynthetic traits by increasing leaf area and number, SLA, LAR, and total chlorophyll content. The findings also revealed that the Acta agriculturae Slovenica, 119/3 – 20238 Y. SELAHVARZI et al. interaction of pinching × urea application at 400 kg ha-1 is the best combination of investigated variation sources for the cultivation of sunflower ‘Teddy Bear’. The suitable amount of dry matter production (219.04 g), number of flowers (77.7), head diameter (110.04 cm), and plant height (147.3 cm) are the important reasons for this rec- ommendation. 6 AUTHOR CONTRIBUTION STATEMENT All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. 7 ACKNOWLEDGMENTS We would like to thank Ferdowsi University of Mashhad, Mashhad, Iran for their support to perform this study. 8 CONFLICT OF INTEREST The authors certify the following: - This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue; - The authors have no affiliation with any organi- zation with a direct or indirect financial interest in the subject matter discussed in the manuscript. 9 DATA AVAILABILITY The data used to support the findings of this study are available from the corresponding author upon re- quest. 10 REFERENCES Adekiya, A.O., Aboyeji, C.M., Dunsin, O., Adebiyi, O.V., & Oyinlola, O.T. 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Delo je prispelo 23. decembra 2022, sprejeto 6. avgusta 2023 1 Alex Ekwueme Federal University Ndufu-Alike, Faculty of Agriculture, Department of Crop Science, Abakaliki, Nigeria 2 Corresponding author, e-mail: emndi4nn@yahoo.com 3 Julius Kühn Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany Mycoviruses: trends in plant-fungus-mycovirus interactions and ‘biocontrol’ prospects in agriculture and the environment Abstract: Mycoviruses are cosmopolitan in plants, ani- mals, fungi, bacteria, in soils, and water. There is a scarcity of information about them, which necessitated this review to pro- vide some leads on where research should focus. Mycoviruses are able to persist in disparate types of hosts by utilizing diverse measures. They may engage either parasitic, pathogenic, or mutualistic tendencies. Mycoviruses employ many existential strategies that can be utilized by man. Hypovirulence may be induced in fungal hosts by mycoviruses via RNA silencing, al- teration of genetic expression, and disruption of the transcrip- tome. Mycoviruses interact with killer phenotypes of yeasts and Ustilago spp. and proffer advantages to these fungi. Mycovirus interaction with some plants result in provision of thermal tol- erance to plants. Based on their mode of microbe destruction mycoviruses may be used for waste disposal and termination of some life processes. For instance, grazer viruses completely oxidize the organic content of their host into carbon dioxide and inorganic nutrients, while lytic viruses release the organic material from their hosts without modification. Viruses may be utilized to facilitate the exchange of genetic material from one host to another. However, pathogenic mycoviruses exist espe- cially in mushrooms. Key words: control, disease complex, fungi synergy, in- tegrated pest management, phage, relationship Mikovirusi: trendi v interakcijah rastlina-gliva-mikovirus in izgledi ‘biokontrole’ v kmetijstvu in okolju Izvleček: Mikovirusi so kozmopoliti v rastlinah, živalih, glivah, bakterijah, v tleh in vodi. O njih je le malo informacij, kar je bilo vodilo za ta pregled kot smernico za bodoče raziska- ve. Mikovirusi so sposobni bivati v različnih gostiteljih z različ- nimi načini preživetja. Uporabljajo lahko zajedalske, patološke ali mutualistične strategije, ki jih lahko koristimo tudi ljudje. Hipovirulenca je v glivnem gostitelju lahko vzpodbujena z mi- kovirusi preko RNA utišanja, spremembe izražanja genov in razgradnje transkriptoma. Mikovirusi sodelujejo z ubijalskimi fenotipi kvasovk in sneti (Ustilago spp.), kar daje prednosti tem glivam. Sodelovanje mikovirusov in nekaterih rastlin rezultira v njihovi toleranci na termperaturne spremembe. Na osnovi njihovega uničevanja mikrobov bi lahko mikoviruse uporabi- li za razgradnjo odpadkov in za zaključek nekaterih bioloških procesov. Na primer, virusi, ki se “pasejo” na mikrobih (grazer viruses) popolnoma oksidirajo organsko vsebino gostitelja do ogljikovega dioksida in anorganskih hranil med tem, ko litični virusi sproščajo organske snovi iz njihovih gostiteljev. Virusi se lahko uporabljajo za olajševanje izmenjave dednine iz enega gostitelja v drugega. Še posebej veliko patogenih mikovirusov živi v gobah. Ključne besede: nadzor, bolezenski kompleks, glivno so- delovanje, integrirano uravnavanje škodljivcev, fag, odnosi Acta agriculturae Slovenica, 119/3 – 20232 E. M. NDIFON and G. N. CHOFONG 1 INTRODUCTION The discovery of bacteriophages and ultimately of mycoviruses/mycophages has been a great leap forward for researchers. Mycoviruses (mycophages) are a group of viruses that are naturally associated with fungi (in- cluding fungi associated with plants, mushrooms, mi- crobes, soil, and water) (SDSU, 2021; Hu et al., 2022). Mycoviruses interact with four phyla of true fungi (eu- fungi): the Chytridiomycota (chytrids), Zygomycota (bread molds), Ascomycota (yeasts and sac fungi), and the Basidiomycota (club fungi). The relation of myovi- ruses with Pseudofungi like those in the Phyla Oomycota and Hyphochytridiomycota (in Kingdom Chromista i.e. some water moulds or Straminipila) and as well as slime moulds - other fungi-like organisms (Ghabrial and Suzu- ki, 2009; Pearson et al., 2009; Beakes et al., 2014; Xie and Jiang, 2014; Zhong et al., 2016; Calvalier-Smith, 2018; Myers et al., 2020; Zhou et al., 2021; Hough et al., 2023) was not covered in this review. Fungi are frequently in- fected with two or more unrelated viruses (Ghabriel and Suzuki, 2008; Howitt et al., 2006). Fungi may also act as vectors of viruses of higher life forms (Adams, 1991). The mycovirus-host fungus relationship take the form of mu- tualistism, commensalism, or parasitism. Viruses associated with fungi or mycoviruses as- sociated with higher life forms usually do not induce symptoms in their host fungi, except in the case of hyper- virulence (increase in virulence of the symptoms of the infection of the fungus on its host: extremely or unusu- ally virulent) and hypovirulence (decrease of the symp- toms of the infection of the fungus on its host: extremely or unusually reduced virulence) (Ghabrial and Suzuki (2009). On the other hand, the diseases on some fungi and mushrooms/macrofungi are caused by the mycovi- ruses themselves. Ghabrial and Suzuki (2009) reported that mycoviruses are associated with latent infections of all major groups of plant pathogenic fungi. Some myco- viruses cause debilitating diseases and/or reduce the vir- ulence of their phytopathogenic fungal hosts and these may lead to attenuation (hypovirulence) or enhancement of fungal virulence (hypervirulence). Kong et al. (1997), Nuss (2005), Ong et al. (2016), García-Pedrajas et al. (2019), and Siddique (2020) reiter- ated that some mycoviruses reduce the virulence of the host fungus (hypovirulence), which can make the fungus less harmful to plants, whereas other mycoviruses have been shown to enhance the virulence of the host fungus (hypervirulence). However mycoviruses may be patho- genic on their hosts. For instance, la France virus disease of cultivated mushrooms (Agaricus bisporus (J.E. Lange) Pilat was first reported in the late 1940s (Hollings, 1962; Ghabrial and Suzuki, 2009). Alvarez-Jubete et al. (2011) reported that Mushroom Virus X affects important traits associated with mushroom quality (including colour and appear- ance). Another instance is the effective virus-control of chestnut blight (caused by the fungus - Cryphonectria parasitica (Murrill) M.E. Barr) as a consequence of the infection of the fungus by the mycovirus - Cryphonec- tria parasitica hypovirus 1 (CHV1) in Europe (Hollings, 1962). The natural distribution of mycoviruses seems to follow a normal distribution spectrum with avirulent, mutualistic, and virulent members being commonplace. Many mycoviruses have been shown to be mutualists. Mycoviruses can alter host’s tolerance to environ- mental stresses, e.t.c. Most of these mycoviruses have not been described to date or are unrelated to any known vi- ruses. According to the PVEN (Plant Virus Ecology Net- work) (2011) viruses are widely distributed entities that can cause substantial mortality of plants and animals. Secondly, viruses can move genetic elements between hosts e.g. potentially between genetically engineered plants and non-target species. Studies of host–mycovirus–vector interactions in nature offer both opportunities and challenges that will ultimately produce multi-faceted understanding of the role of mycoviruses in shaping ecological and evolution- ary dynamics (Fargette et al., 2006; PVEN, 2011). Studies of pathogenic viruses have probably left out a vast ma- jority of viruses. Mycovirus diversity is another area of mycovirology that has barely been explored. Virtually all plant (and perhaps all animal) species harbor pathogenic or mutualistic fungi in their tissues. Kotta-Loizou (2019) pointed out that our current understanding of mycoviruses is not as detailed as in other fields of virology and currently not based on cut- ting-edge methodology. The general assumption is that much information is yet to be generated on mycoviruses especially considering that the majority of these myco- viruse are viruses of microorganisms (VOMs). With the advent of high-throughput sequencing and bioinformat- ics analysis pipelines in mycovirology, different types of mycoviruses are being discovered in all the four phyla of true fungi. Recent research has revealed an unexpected diversity of these mycoviruses, their interactions with plants, and modulation of some plant biotic and abiotic stresses. Mycoviruses can be useful in molecular biology and biotechnology. We are just beginning to tap this poten- tial. This appraisal was set up to document the literature on mycoviruses, diversity of currently known host-par- asite interactions and biocontrol prospects possible in agriculture and the environment. Acta agriculturae Slovenica, 119/3 – 2023 3 Mycoviruses: trends in plant-fungus-mycovirus interactions and ‘biocontrol’ prospects in agriculture and the environment 2 PLANT-FUNGI-MYCOVIRUS INTERAC- TIONS Recently, researchers reported that viruses are the most abundant and dynamic entities in the hydro- sphere (Weinbauer, 2004; Suttle, 2007) although Payet et al. (2014) contested that little is known about viruses in these water habitats. Viruses are major agents of mi- crobial mortality and account for about 50% of bacterial mortality in the hydrosphere (Kirchman, 2018). Daily, between 20–50% of heterotrophic bacteria, cyanobacte- ria and phytoplankton are infected by viruses (Brussaard, 2004; Suttle, 2007). Viral lysis releases organic cellular content and nu- trients necessary for autotrophic and heterotrophic mi- crobial life forms (Shelford et al., 2012). This essentially result in major changes in the biogeochemical nutrient (carbon, nitrogen and phosphorus) cycles and flow of en- ergy in the oceans (Suttle, 2007; O‘Malley, 2016). Kirch- man (2018) stated that apparently viruses infecting fungi do not lyse their host and are rather transmitted from one fungus to another intracellularly, without being re- leased into the external environment. True mycoviruses demonstrate an ability to be transmitted and infect other still healthy fungi cells. The interaction between the mycovirus (Cryphonectria para- sitica hypovirus 1 (CHV1)) with Cryphonectria parasitica (the causative agent of chestnut blight)), in Europe re- sulted in hypovirulence in the fungus. Thus the blight was controlled whenever a virulent strain of the virus at- tacked the plant. However, this ‘biocontrol’ is restricted to a small number of plant vegetation compatibility groups (pVCGs). For instance, in North America plant vegeta- tion incompatibility reactions prevent plant roots from fusing and exchanging their cytoplasmic content, thus hypovirulent strains of mycoviruses are hindered from spreading (See Anagnostakis et al., 1998). Hence in the USA, China and Japan this ‘biocontrol’ measure tends to fail due to a large number of different plant VCGs (Liu and Milgroom, 2007). The natural host range of a mycovirus is supposed to be confined to taxa performing cytoplasmic fusion (Buck, 1986) but some mycoviruses can replicate in un- related taxa not allowing anastomosis of the fungal hy- phae. This is the case with two fungal species (Sclerotinia homoeocarpa Benn. and Ophiostoma novo-ulmi Braiser) associated with chestnut tree (Deng and Boland, 2003; Nuss et al., 2005). Chen et al. (1994) extended the natural host range of CHV1 to several phylogenetically unrelated fungal species associating with chestnut and supported their hypothesis using in vitro virus transfection tech- niques. In line with this, CHV1 can also propagate in the genera Endothia Murrill species (Cryphonectriaceae) and Valsa Fr. species (Diaporthales, Valsaceae) (Ghabriel and Suzuki, 2008). Various studies revealed that the same mycovirus can be transmitted between different species of the same genus found in the same habitat. For instance the same mycovirus was transmitted between Cryphonectria spp. (i.e.; Cryphonectria parasitica and Cryphonectria sp.), Sclerotinia spp. (i.e.; Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotinia minor Jagger), and Ophiostoma spp. (Ophiostoma ulmi (Buism.) Nannf. syn. Ceratocystis ulmi (Buism.) C. Moreau and Ophiostoma novo-ulmi) (Liu et al., 2003; Melzer et al., 2005). Moreover, interspecies transmission has been re- ported between Fusarium poae (Peck) Wollenw and Aspergillus species (van Diepeningen et al., 2006). The mode of transmission in these instances is unknown and is still subject to guess work. Mycovirus infections are common even in humans as is the case with the mycovi- ruses in Aspergillus fumigatus Fresenius (i.e. AfuPmV-1) and Talaromyces marneffei Segretain, Capponi & Sureau) Samson, Yilmaz, Frisvad & Seffert (i.e. TmPV1) (Kotta- Loizou and Coutts, 2017; Lau et al., 2018). Research on mycoviruses is hindered by many fac- tors amongst which is the lack of appropriate infectiv- ity assays (McCabe et al., 1999) and mixed infection or unknown numbers of infecting viruses. These situa- tions make it difficult to ascribe a particular phenotypic change in the host to a particular virus under investiga- tion. Moreover, neutral co-existence (likely due to co- evolutionary processes) may be in operation in a virus- fungus interaction (Araújo et al., 2003). These difficulties have hindered the studies on hypovirulent strains of my- coviruses. This is often due to lack of correlation between phenotypes and specific genomes or particular metabolic pathways (Xie et al., 2006). Equilibrium offsetting conditions could also be responsible for changes in host-parasite relationships. Possibly, this is due to changes from mutual to neutral then to deleterious, and so on. Other relationships exist in the same habitat. Vidhyasekaran (2004) reported that satellite viruses are dependent on other viruses to sup- ply the enzyme replicase and other enzymes necessary for replication. A satellite virus associated with Tobacco necrosis is not serologically related to Tobacco necrosis virus (TNV). TNV multiply indefinitely without causing the production of a satellite virus. However, the satellite virus is entirely dependent on TNV for its multiplication. The satellite virus has a viral coat and a small genome of its own. Both viruses are transmitted among roots by the fungus Olpidium brassicae (Woronin) P.A. Dang. Sometimes satellite viruses also have satellite RNAs e.g., the satellite of Tobacco necrosis virus (TNV) has a Acta agriculturae Slovenica, 119/3 – 20234 E. M. NDIFON and G. N. CHOFONG small satellite RNA that is dependent on Tobacco ne- crosis virus for replication and on the satellite virus for encapsulation (Vidhyasekaran, 2004). Moreover, various plant viruses (of the Tombusviridae) generate defective interfering RNA viruses during replication (Rubio et al., 1999). This new relationship may result in viral symptom amelioration (Roux et al., 1991; Kong et al., 1997) or in- tensification as observed in the case of the Turnip crin- kle virus (Li et al., 1989; Kong et al. 1997). Hough et al. (2023) stated that mycoviruses have the ability to reduce the virulence of their hosts. Rowley (2016), and Moonil et al. (2015) reported that asymptomatic associations with fungi and by myco- viruses are very common. Furthermore, fungi are often associated with unrelated viruses or ‘defective dsRNA’ and/or satellite dsRNA (Howitt et al., 2006; Ghabrial and Suzuki, 2009). Moreover, some viruses simply use fungi as vectors (which differentiate them from mycoviruses) since they do not replicate inside the fungus (Adams, 1991). Tran et al. (2019) reported that very little is known about mycoviruses infecting Monilinia species although virus-like particles (VLPs) resembling those of partiti- viruses, totiviruses, tobraviruses, and furoviruses have been reported from these hosts. McCabe et al. (1999) and Rowley (2016) argued that the virulence of a virus is ultimately limited by the need for the host to survive and thus permit the virus to replicate and continue to exist. This has not been proven. Based on the obligate parasitic nature of viruses, the majority of mycoviruses should have some negative effect(s) on fungal growth or survival. This depends on the mode of infection and the population of the viruses. More than 250 mycoviruses infect true fungi in the afore- mentioned phyla (Bozarth, 1972; Rochon et al., 2004; Hacker et al., 2005; Ghabrial and Suzuki, 2009; Rowley, 2016; Tran et al., 2019; Xia et al., 2020). Many viruses can simultaneously infect a single fungus (Hollings, 1962). Based on O‘Malley (2016) viruses may operate in hosts with or without being pathogenic. De Filippis and Villarreal (2000) stated that a competition between diffe- rent viral strains or individuals inside a host may result in selection of the fittest. Viruses have both general and specific requirements for replication and existence. The direction and extent of this change is determined by a combination of stochastic and environmental factors that are specific for a given time, space, and taxon. Though viruses of plants have long been recognized as important components of plant ecosystems, only a few notable mycovirus have been studied in detail. Marzano et al. (2015) reported that a comprehensive picture of mycoviral diversity is lacking. Tran et al. (2019) lamented that the influence of mycoviruses on the ecosystem has not been well studied. For instance, the lack of studies on how some mycoviruses reduce the ability of their fungal host to cause plant diseases. Besides, it has been assumed that the natural host range of mycoviruses is confined to closely related vegetation-compatibility groups (VCGs) which allow fusion of cytoplasm (Buck, 1986). These assumptions may or may not be true, and are based on assumptions. Zhang et al. (2020) attested that it is unclear how mycovirus that cause hypovirulence prevail in the field. Myers and James (2022) suggested the presence of mutu- alism between mycoviruses and their hosts. Pearson et al. (2009) agreed that our understanding of the interaction between mycoviruses and their hosts is largely limited to a few well‐studied, possibly atypical systems. Coupled with the problem of mixed infections by multiple viruses (for example the mixed infection of Botrytis cinerea virus F (BCVF) and Botrytis virus X (BVX) in Botrytis cinerea Pers.) it may not be easy to ascribe a definite role to a mycovirus (Howitt et al., 2006). De Filippis and Villarreal (2000) emphasized that viral infection of a host may not necessarily involve tissue destruction, mortality or even full/partial mobilization of host antiviral mechanisms. Indeed, virus association with hosts may result in mutu- alistic relationships. Most mycoviruses do not cause symptomatic in- fections in their hosts (Ghabrial et al., 2015; Khan et al., 2022). Symptom expression usually occur when there is hypersensitive reaction or incompatibility of the host and parasite. Rowley (2016) reported that fungal hosts defend themselves from mycoviruses using RNA interfe- rence (RNAi), which inhibit mycovirus replication. This may result in cell death thus blocking mycovirus tran- smission. De Filippis and Villarreal (2000) reported that disabling antiviral systems in fungi improves the chances of virus continuity. Bacteria hosts can employ abortive infection as a last resort to escape from the effects of bac- teriophages (Weinbauer 2004). However, many mycovi- ruses interfer with fungal RNAi to prevent the inhibition of their replication. Interactions between vegetatively incompatible plants and fungal isolates culminate in programmed cell death (PCD) thus hindering any ex- change of infected cellular contents (Nuss, 2011). Biella et al. (2002) affirmed that mycovirus infec- tion is influenced by the rate of PCD which could mean that mycoviruses may have developed mechanisms for delaying or hindering occurrence of PCD. RNA silenc- ing (as a defence mechanism in fungi) invoked by fungi against viruses may be made inefficient by some viruses including mycoviruses (Segers et al., 2007). Furthermore, Moonil et al. (2015), and Rowley (2016) pointed out that some mycoviruses are associated with killer satellite vi- rus particles which induce their fungus host to secrete Acta agriculturae Slovenica, 119/3 – 2023 5 Mycoviruses: trends in plant-fungus-mycovirus interactions and ‘biocontrol’ prospects in agriculture and the environment toxins that kill competing fungi. This host fungus ben- eficial mechanism is exhibited by the budding yeasts (Sacharomyces cerevisiae (Desm.) Meyen) in fermented foodstuffs. These dsRNA satellite viruses are dependent on the Totiviridae mycoviruses for their stability. Alone, totivi- ruses have a minimal impact upon S. cerevisiae, but the additional presence of satellite RNAs provide additional capabilities to the virus which is an important example of a beneficial virus system. In fact, these killer systems are so beneficial to their hosts that in some cases, they have resulted in the loss of host RNAi systems (Drinnen- berg et al., 2011; Moonil et al., 2015). Thus symptomless or latent mycoviruses may have unknown functions in their hosts. Somehow, some mycoviruses may act as ex- tra‐chromosomal genes that confer an advantage to the host as can be observed with the killer systems in yeast (Schmitt and Breinig, 2006). Another example of beneficial relationship with a mycovirus, is a three-way symbiosis (among a mycovi- rus, an endophytic fungus, and tropical panic grass). The endophytic fungus (Curvularia protuberata Boedijn), pa- nic grass (Dichanthelium lanuginosum (Elliott) Gould), and other plants can only survive high soil temperatures in the presence of the mycovirus (Márquez et al., 2007; Moonil et al., 2015). The mycovirus in turn obtains its basic necessities from its hosts. The mechanisms invol- ves two distinct viral dsRNAs. A mutualistic relationship is also found in an interaction among Trichoderma Pers. species and their mycoviruses, and the host plant (Beilei et al., 2020). The fungus is required for thermal tolerance of the plants. A parasite often tend to reduce its impact on its host, thus many parasites have co-evolved to an equilib- rium state resulting in minimal impact. Therefore there is great variability in reactions between a single host and different viruses or dsRNAs. Furthermore, Khan et al. (2022) reported that se- veral types of virus-virus interactions (i.e.; synergistic, antagonistic, and mutualistic interactions) have been reported in fungal hosts. Co-infections of single fungal strains by over ten mycoviruses has been reported for several phytopathogenic fungi, which implies that much work has to be carried out to determine the type of re- lathionships that are created in such co-infections. The effects of a mycovirus seems to be dependent on other factors like environment and presence of other invaders. For instance, Chu et al. (2002) reported a wide spectrum of reactions: reduced growth, increased pigmentation, reduced virulence, and a 60‐fold decrea- sed production of trichothecene mycotoxins associated with a dsRNA during a study of Fusarium graminearum Schwabe (syn Gibberella zeae (Schwein.) Petch) on whe- at. Fine (1975) assumed that mycoviruses may be una- ble to persist if they lower the fitness of their hosts, be- cause they are limited to vertical transmission only. In a detailed study of the effects of dsRNA on the fitness of asexual Aspergillus species, no beneficial effects were ob- served (Van Diepeningen et al., 2006) in vitro. In contrast Tran et al. (2007) observed higher growth rates of BVX‐ infected fungus compared to the same uninfected isolate. It has been postulated that the virus environment is both multidimensional and continually changing thus constantly driving the increase in population fitness. It could also be argued that based on quantity of variables in the environment, viruses exhibit greater mobility through the space of their selective or adaptive environ- ments than do more complex organisms (Moya, 1997). De Filippis and Villarreal (2000) reported that the many levels of viral characters (point mutations, coding region products, multigene assemblages, behavioral traits, and even populational characters) can be conside- red as adaptations and may all endow their possessors with replication advantages. The adaptive viral characters favored within the relatively closed system of one indi- vidual host arise and persist due to intra-host selection pressure, the nature and strength of which is determined by the environmental conditions and other virus strains contained therein. De Filippis and Villarreal (2000) reported that the host’s cellular, tissue, and organismal environments are vitally important selective realms that contribute pro- foundly to the adaptation and diversity of viruses inclu- ding mycoviruses. Also by disabling antiviral systems the virus reduces its own population decline. In the eco- system the fittest mycovirus optimizes its utilization of host resources and does not maximize the utilization of host resources. This permits them to continue to persist despite the intrahost selection pressure. Thus the fittest individuals are not the ones that maximizes the use of host resources, rather the fittest individuals are those that optimizes the utilization of host resources. To ensure continuity in most viral infections, less than 1 % of the susceptible host tissue is actually infec- ted/harvested (Griffin, 1997). Such a host-parasite inte- raction could persist and be observed as any of the for- ms of guilds depending on the colorations and flavours added to it. In micro-ecosytems, the essential portion of the environment that is of most concern is the inorga- nic nutrients and energy derivable from the hosts. The mycovirus should therefore be properly adapted to avo- id depleting these resources unnecessarily. In the case of bacteriophages, they impact the movement of nutrients and energy within the micro-ecosystems primarily by ly- sing bacteria and secondarily by encoding of exotoxins (a subset of which are capable of solubilizing the biolo- Acta agriculturae Slovenica, 119/3 – 20236 E. M. NDIFON and G. N. CHOFONG gical tissues of living hosts/animals) (Weinbauer, 2004). Much has been reported already about viruses of plants, humans and animals so this will only be discussed brie- fly as antagonistic components of the micro-ecostystem. Kazinczi et al. (2004) pointed out that weeds, as alterna- tive hosts of plant viruses can act as alternative nutrient sources for viruses and virus vectors. Weeds play im- portant role in virus ecology and epidemiology. Alemu et al. (2002) reported that chronic infection with viruses is a major constraint that often force farmers to ban hot pepper production. This can result in decrease in the po- pulation of virus and mycovirus entities in an area. The presence of infected weeds throughout the year means, that they are reservoirs and sources of viruses for secon- dary spread. Yudin et al. (1986) reported that western flower thrips (Frankliniella occidentalis Pergande, 1895 - a known vector of tomato spotted wilt virus, was found to be associated with 48 plant species growing within the Kula vegetable-growing region on the island of Maui, Hawaii. This type of vector can be very vital for continual existence of mycoviruses even when the host plant and fungus are facing difficult times in the dry season. Weeds are widely infected by viruses. For instance, McGovern et al. (2008) reported that Solanum viarum Dunal (the invasive tropical soda apple) in Florida was infected by nine viruses which can in turn infect solanaceous crops. 3 IMPLICATION OF MYCOVIRUS IN- TERACTIONS WITH PLANTS IN CROP PROTECTION: TRENDS IN RESEARCH, APPLICATIONS, AND ‘BIOLOGICAL’ CONTROL POTENTIALS USING THESE AGENTS We have just seen how the killer phenotypes can provide some advantages to yeasts and Ustilago (Pers.) Roussel species due to their interactions with viruses (Schmitt and Breing, 2002; Marquina et al., 2007). Killer isolates secrete proteinous toxins (mostly cell wall de- grading enzymes) against sensitive cells of the same or closely related species, while the producing cells them- selves are immune. These types of killer isolates could be beneficial in medicine, agriculture and industry (Schmitt and Breing, 2002). We have also seen that three-part interaction pro- vide thermal tolerance by the plant (Marquez et al., 2007). Another example is the A78 virus of Aspergillus fumigatus Fresen causing mild hypervirulence on Gal- leria mellonella (L., 1758) (Greater wax moth) (Ozkan and Coutts, 2015). Likewise, TmPV1 associated with T. marneffei caused hypervirulence on T. marneffei in the mouse host (Lau et al., 2018). Liu et al. (2022) reported that mycovirus Stemphyli- um lycopersici alternavirus 1 (SlAV1) from a necrotroph- ic plant pathogen (Stemphylium lycopersici) that causes altered colony pigmentation and hypovirulence by spe- cifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. Li et al. (2019) reported that most Fusarium myco- viruses establish latent infections, but some mycoviruses such as Fusarium graminearum virus 1 (FgV1), Fusarium graminearum virus-ch9 (FgV-ch9), Fusarium gramine- arum hypovirus 2 (FgHV2), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1) cause hypovirulence. Khan et al. (2023) emphasized that among members of the genus Sclerotinia, a huge number of mycoviruses have been identified; some of them have a hypovirulent effect on the fitness of their fungal hosts. Zhou et al. (2021) revealed that mycoviruses have been associated with plant adaptation to extreme envi- ronments, conferring heat tolerance to plants that con- tain fungal endophytes. They reported that endophytic fungi, can confer fitness to the host plants. It is unclear whether biological factors can modulate the parasitic and mutualistic traits of a fungus. Kotta-Loizou (2021) affirmed that in fungus-mycovirus-environmental in- teractions, the environment and both abiotic and biotic factors play crucial roles in whether and how mycovirus mediated phenotypes are manifest. Connor (2021) reported that soybean leaf-associ- ated gemycircularvirus-1 (SlaGemV-1) is capable of in- ducing hypovirulence in the highly pathogenic fungus Sclerotinia sclerotiorum as does the hypovirus 1 (CHV1) controlling C. parasitica in chestnut in Europe. It is an excellent model organism for studying hypovirulence in fungi (Anagnostakis et al., 1998; Liu and Milgroom, 2007). Kirchman (2018) pointed out that viruses infecting fungi do not appear to lyse their host. The use of myco- virus can open many avenues for handling waste and de- composition, or terminating some life processes. For in- stance grazers completely oxidize the organic content of their host into carbon dioxide and inorganic nutrients. A third mode of employing viruses may theoretically be to facilitate the exchange of genetic material from one host to another. Most of these processes have been relatively poorly studied (Pearson et al., 2009). Hypovirulence may be induced in hosts by myco- viruses via RNA silencing, alteration of genetic expres- sion, and disruption of the transcriptome that can re- sult in phenotypic changes like reduction in growth or changes in pigmentation (Nuss, 2005). Alterations of miRNAs expressions using viral suppressors of RNA si- lencing (VSRs) occurs by applying papain-like protease p29 (Segers et al., 2006) and potyvirus HC-Pro (Maia et Acta agriculturae Slovenica, 119/3 – 2023 7 Mycoviruses: trends in plant-fungus-mycovirus interactions and ‘biocontrol’ prospects in agriculture and the environment al., 1996). Also, C. parasitica when infected by the hy- povirulence-inducing mycovirus undergoes RNA silenc- ing thereby affecting the MAPK cascade and G-protein signaling. Moreover, direct disruption of the fungal tran- scriptome may occur (Nuss, 2005). Proof of the ability of a mycovirus being able to con- trol a pathogen in the field is either scarse or unavailable (Griffin 1986, MacDonald et al. 1991) but mycoviruses have been shown to be able to control fungi in modified environments (MacDonald et al., 1991; Milgroom et al., 2004). Two major forms of defense signaling include: sys- temic acquire resistance (SAR) and induced systemic re- sistance (ISR). (Vidhyasekaran, 2015). Another theoriti- cal approach usable to increase a plant resistance against pathogenic infection is resistance priming like that in- volved in SsHADV-1 allowing S. sclerotiorum to induce priming in plants. ‘Priming is the process of inoculating plants, often the seeds, with beneficial microorganisms to improve nutrient use efficiency and to potentially improve resistance to pathogens’ (Rakshit et al., 2015). Actually, Qu et al. (2020) demonstrated that SsHADV-1-infected, hypovirulent S. sclerotiorum is reprogrammed to act as a beneficial, bio-priming mycorrhiza in rapeseed due to Sclerotinia Fuckel stem rot reduction and improved yield. Mycoviruses have been shown to be involved in all forms of interactions (e.g. mutualism) with fungi hosts. In the future, mycoviruses may be required for manipulating micro-ecosystems within plants, humans, animals and so on. They are simple enough for direct insertion and removal of genes here and there if the right equipment is available. However, pathogenic mycoviruses have been reported and they can severely ravage host populations especially domesticated mushrooms e.g. la France dis- ease on Agaricus bisporus. Thus, mycoviruses have to be controlled in fungus-fungus, fungus-plant, fungus-ani- mal systems, etc. Ruiz-Padilla et al. (2021) propounded that products based on microorganisms (including mycoviruses senso lato) can be used in biocontrol strategies alternative to chemical control. Keçeli (2017) reported that the use of mycoviruses in the treatment of invasive fungal infec- tions in humans has not been suggested yet. Xie and Jiang (2014) suggested that fungal vegetative incompatibility is likely to be the limiting factor in the widescale utilization of mycoviruses to control crop diseases. 4 CONCLUSION Past, present and future trends in mycovirus re- search are of interest to humans. They can reveal the prospects of mycoviruses in agriculture and the environ- ment in terms of pathogen control and amelioration of the environment. Use of mycoviruse to induce hypoviru- lence in fungi host isolates has shown great potentials e.g. using the A78 virus of Aspergillus fumigatus, TmPV1 on T. marneffei, soybean leaf-associated gemycircular- virus-1 (SlaGemV-1) in Sclerotinia sclerotiorum, the hypovirus 1 (CHV1) in Cryphonectria parasitica. Hypo- virulence may be induced in fungi hosts by mycoviruses via RNA silencing, alteration of genetic expression, and disruption of the transcriptome which can result in phe- notypic changes like reduction in growth or changes in pigmentation. Moreover, direct disruption of the fungal transcriptome may occur. Another approach to increase a plant’s resistance against pathogenic infection is resist- ance priming that may be required for manipulating mi- cro-ecosystems within the plants. However, pathogenic mycoviruses have been reported and they can severely ravage host populations especially domesticated mush- rooms 5 REFERENCES Adams, M. J. (1991). 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ISME Journal, 15(7), 1893–1906. https:// doi.org/10.1038/s41396-021-00892-3 Acta agriculturae Slovenica, 119/3, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.3.12436 Original research article / izvirni znanstveni članek Study on the evolution of the fruit morphological and physico-chemical parameters of ‘Majhoul’ date palm during fruit growth Mohamed ARBA 1, 2, Iliass BERJAOUI 3, Ahmed SABRI 4 Received February 16, 2023; accepted June 18, 2023. Delo je prispelo 16. februarja 2023, sprejeto 18. junija 2023 1 Plant ecophysiology and cultures of arid zones laboratory, Hassan II Institute of Agronomy and Veterinary Medicine, Agadir, Morocco 2 Corresponding author, e-mail: arbamohamed@yahoo.fr 3 SYGENTA company (Seed distribution and plant protection), Marrakech, Morocco 4 National Institute of Agricultural Research (INRA), Draa-Tafilalet Agricultural Research Center (CRA), Errachidia, Morocco Study on the evolution of the fruit morphological and physi- co-chemical parameters of ‘Majhoul’ date palm during fruit growth Abstract: Date palm is an economically important species in the Middle East and North Africa. In Morocco, date palm is the main crop in the southeastern region, mainly in Draa- Tafilalet area. The ‘Majhoul’ is ranked among the worldwide best quality dates due to its large size and good texture. This work aimed to study the effect of three phases of flowering (early flowering, seasonal and late) on fruit quality of ‘Majhoul’ during its development. Experiments were carried out on an adult plantation in a modern palm grove in Tafilalet. Obtained results showed that, except for the chemical parameters of the fruit, there is a significant difference (p ≤ 0.01) between the three flowering phases for the morphological parameters stud- ied (fruit mass, size, and dimensions) during all the fruit de- velopment stages. The early flowering phase yielded fruits with higher parameters than the other flowering phases. The mean fruit size (volume) for all the fruit development stages was 22 cm3 for the early flowering phase, whereas it was only 12.86 and 10 cm3, respectively, for the seasonal and late flowering phases. The final fruit size was 19.70, 13.55, and 9.97 cm3, respectively, for the early, seasonal, and late flowering phases. Key words: Tafilalet area, date palm ‘Majhoul’, flowering phase, fruit development, fruit morphological and chemical parameters Raziskava razvoja morfoloških in biokemičnih parametrov plodov dateljeve palme ‘Majhoul’ v rastni sezoni Izvleček: Dateljeva palma je ekonomsko pomembna vr- sta v Bližnjem vzhodu in severni Afriki. V Maroku je dateljeva palma glavna kulturna rastlina na jugovzhodnih območjih, v glavnem na območju Draa-Tafilalet. Sorta Majhoul je uvrščena med najboljše na svetu zaradi svoje kakovosti, velikih plodov in njihove dobre teksture. V raziskavi je bil preučevan učinek treh obdobij cvetenja (zgodnje cvetenje, cvetenje v glavni sezoni in pozno cvetenje) na razvoj in kakovost plodov. Poskus je pote- kal v odraslem nasadu z moderno vzgojno obliko v Tafilaletu. Rezultati so pokazali, da so bile z izjemo kemijskih parametrov plodov, značilne razlike (p ≤ 0,01) med tremi obdobji cvetenja v vseh preučevanih morfoloških parametrih plodov (masa, ve- likost in dimenzije plodov) v vseh fazah razvoja. Zgodnja faza cvetenja je dala plodove, ki so imeli vrednosti vseh merjenih pa- rametrov večje kot plodovi, nastali iz poznejših cvetenj. Popreč- na vrednost velikosti plodov (volumen) nastalih po zgodnejm cvetenju je bila 22 cm3 med tem, ko sta bili velikosti sezonskih in poznih plodov samo12,86 in 10 cm3. Končne velikosti plodov so bile 19,70; 13,55 in 9,97 cm3, za plodove nastale iz zgodnjega, sezonskega in poznega cvetenja. Ključne besede: območje Tafilalet , dateljeva palma ‘Majhoul’, faze cvetenja, razvoj plodov, morfološki in kemični parametri plodov Acta agriculturae Slovenica, 119/3 – 20232 M. ARBA et al. 1 INTRODUCTION Date palm (Phoenix dactylifera L.) is a perennial monocotyledon plant, which is part of the family of Pal- maceae and the genus Phoenix, which includes 14 spe- cies that are native to tropical and subtropical regions of South Asia or East and North Africa (Dransfield et al., 2008; Shengji et al., 2010). It has been currently grown in the Middle East, North Africa, parts of Central and South America, India, and Pakistan (Al-Shahib & Mar- shall, 2003) and recently introduced in some African countries such as Namibia. Date palm has been an im- portant fruit species in the Middle Eastern and North Af- rican countries for a long time (Marondedze et al., 2014). In Morocco, date palm occupies an area of around 52.000 ha and represents the backbone of agriculture of the Oasian regions, mainly Draa-Tafilalet area, which is the main production area in the country. The genetic diver- sity of date palm in Morocco consists of more than 223 varieties which are well known and represent 52 % of the total population. The rest (48 %) consists of ‘khalts’, hy- brid seedlings. Traditional commercial varieties of good quality represent only 36 % of the national heritage. They consists of the varieties ‘Majhoul’, which represents 9 % of the national heritage, ‘Bouffegous’, which represents 15 %, ‘Jihel’ 12 % and ‘Bouskri’ which represents only 0.1 % (ORMVAT, 2015). Dates fruit are oblong drupes or stone fruits with more or less fleshy and fibrous flesh, which represents 85-90 % of the total fruit mass and contains a single seed (Mansour, 2005; Lobo et al., 2014). They are a funda- mental nutrient for the oasis populations. They are an important food source rich in sugars, proteins, dietary fiber, antioxidants, and minerals (magnesium, iron, po- tassium, etc.) (Amira et al., 2011; Rastegar et al., 2012). With an average annual production of 92976 tons in Mo- rocco, the dates provide an average yearly value of 743.8 million dirhams and contribute 40 to 60 % of the income of the Oasian farms. Dates are the engine of the economy of the producing regions and an important cash source for the farmers of these regions and for the financing of their agricultural activities (ORMVAT, 2015). Dates have reached the international market with famous commer- cial varieties like ‘Bouffegous’ and ‘Majhoul’ (Chafi et al., 2015). Several studies have been conducted on the phys- ico-chemical, biochemical, and biological constituents of date varieties (Hasnaoui et al., 2010; Elguerrouj et al., 2011; Chafi et al., 2015), and their results have classified the dates of the ‘Majhoul’ among the good quality dates with a large size and high sugar content (more than 70 %) (Acourene et al., 2001). After the fruit set, there are five development stages in date palm, which are based on changes in fruit size, color, texture, and chemical composition. These develop- ment stages are known internationally as ‘’Hababouk’’ (immature fruits in the form of peas), ‘’Kimri’’ (large and green fruits), ‘’Khalal’’ (color stage of the fruit which be- comes crisp when eaten), ‘’Rutab’’ (fruit ripening stage, soft fruit, and succulent texture) and ‘’Tamar’’ (full rip- ening stage and less humid flesh) (Al-Shahib & Marshall, 2003; Fadel et al., 2006). Marondedze et al. (2014) also reported that fruit development of date palm consists of morphological and physiological changes in the fruit, which occur as biological processes associated with cel- lular metabolic activities. Fruit growth and development in date palm also leads to morphological, physiological, and biochemical changes after fruit set (Lobo et al., 2013). Date palm is a species where flowering does not oc- cur simultaneously because the spathe emission is done gradually. Consequently, the flowering and pollination of date palm will also occur progressively over time. The growers in the producing regions distribute the flower- ing in three phases: an early flowering phase, a seasonal, and a late one. Therefore, fruit quality of these flowering phases have not been studied, and very little research has been carried out. However, in modern date palm groves in date palm growing regions of Morocco, producers of the ‘Majhoul’ have always used the practice of limiting clusters on clusters that are produced from early and late flowering phases and have always opted to maintain the seasonal flowering regimes in their production system. This research work aimed to study the effect of the three flowering phases on fruit development and quality of ‘Majhoul’ date palm during fruit growth, by harvesting fruit samples over time. 2 MATERIALS AND METHODS 2.1 THE SITE OF TRIALS The experiment was set up in a modern date palm grove located in the Goulmima region, Tafilalet area (31°41’ N, 4°57’ W, and 1028 m elevation), and the trials were carried out on a 13-year-old plantation of ‘Majhoul’ date palm with an IGP (geographical protection index). The planting density is 7 x 6 m (238 palms per hectare). The irrigation system used on the farm is drip irrigation with two drip ramps per planting row and two drips per palm (one drip per ramp). Plants are irrigated once a week during January and February, twice a week during September, October, November, December, March, and April, three times a week during May, and once a day during June, July, and August.The irrigation dose is 500 l per date palm tree.The fertilization program used on the farm is presented in Table 1. Acta agriculturae Slovenica, 119/3 – 2023 3 Study on the evolution of the fruit morphological and physico-chemical parameters of ‘Majhoul’ date palm during fruit growth The pollinating variety is a ‘khalt’ which is also 13 years old, and the pollination is carried out manually by placing 5 to 7 spikelets of mature male inflorescence in the middle of the female inflorescence, which is slightly attached with a lace of leaflets to maintain the pollen in- side the female inflorescence. The pollination period of each flowering phase of date palm in the farm of trials is presented in Table 2. 2.2 PARAMETERS STUDIED AND MEASURES AND OBSERVATIONS REALIZED Morphological parameters studied included fruit size (volume), dimensions (length and diameter), and fruit mass. Fruit size is determined with a graduated cylinder of 100, 250 and 1000 ml, fruit dimensions are measured with a caliper and fruit, pulp mass and seed mass are measured with an electronic balance having an accuracy of 0.01 g. Fruit shape and color are determined by visual observation. The percentage of pulp relative to fruit and seed mass is determined according to Acourene et al. (2001): % pulp = pulp mass/fruit mass x 100 Seed mass = fruit mass - pulp mass The determination of the fruit dry mass is carried out on fruits; which are devoid of their seeds and dried in the oven at a temperature of 70 °C for 48 hours (Achour et al., 2003). 2.3 CHEMICAL ANALYSIS OF THE FRUITS Chemical analysis of the fruits was carried out on the pH of the fruit juice and the content of total sugars in the fruits. The juice was extracted from the fruits accord- ing to the method of Chafi et al. (2015). The fruits were washed with ordinary water, and their seeds were re- moved. They were then ground very finely with a mortar, and the resulting crusher was added twice its mass in dis- tilled water. The mixture was centrifuged for 20 minutes in a centrifuge; the supernatant was recovered and then filtered using a vacuum quenching. The filtrate was then adjusted with distilled water to 200 ml, and the resulting solution constituted the raw juice to be analyzed. The pH of the juice was determined using a pH meter, and the content of total sugars in the fruits was determined with a digital refractometer. 2.4 THE EXPERIMENTAL DESIGN AND STATISTI- CAL ANALYSIS OF DATA Adopted experimental design was a completely random design with a single factor; the flowering phase with three repetitions on five date palm trees, which were randomly selected on the farm and pollinated homo- Table 1: Fertilization program used in the farm of trials on a 13-year old plantation of ‘Majhoul’ date palm in the Goul- mima region, Tafilalet area, Morocco Intake period Fertilizer used Dose provided (kg per ha per month) December January February Sulfuric acid 10 Compost 5000 Acide Humique 5 March April Hydrocomplex 50 Phosphoric Acid 5 May June Hydrocomplex 50 Phosphoric Acid 15 Humic Acid 5 July August Ammonium Sulphate 20 Sulfiric Acid 10 Potassium Sulfate 45 Table 2: Pollination period of each one of the three flowering phases (early, seasonal and late flowerings) of ‘Majhoul’ date palm in the Goulmima region, Tafilalet area, number of clusters used per palm and dates of harvesting fruits for morphological measures and chemical analyzes Flowering phase Pollinating period Number of clusters used per palm tree of the study Dates of harvesting fruitsP1 P2 P3 P4 P5 Early flowering From 23 to 28 February 2016 2 5 2 2 3 06/02/2016 ; 06/22/2016 ; 07/02/2016 ; 07/13/2016 ; 07/31/2016 ; 08/10/2016 ; 09/03/2016 Seasonal flowering From 9 to 13 March 2016 3 4 5 3 3 Late flowering From 25 to 29 March 2016 3 4 0 4 0 P = date palm tree Acta agriculturae Slovenica, 119/3 – 20234 M. ARBA et al. geneously for each flowering phase. Twenty fruits were randomly chosen per flowering phase and fruit harvest- ing stage, which coincides with a fruit development stage to make measures and analyses. The fruits were selected at a rate of 3 to 5 fruits per cluster at different heights and orientations of the cluster, and the harvested fruits were deprived of their scars. The aim was to carry out the measures of the morphological parameters and the chemical analysis of the fruits in the laboratory to fol- low the evolution of these morphological and chemical parameters from fruit set to fruit ripening. Table 2 shows the number of clusters selected per date palm of the study and per flowering phase, the number of fruit samples tak- en, and the dates of harvesting fruits. Fruit samples col- lected per fruit development stage and flowering phase were placed in white plastic bags, labeled and placed in an isothermal container, and brought back to the labora- tory for analysis. Statistical analysis of data was performed with the Minitab 16 software, the determination of the mean was made by ANOVA with a single factor, and the compari- son of the means was performed with the Tukey test with an error of 5 %. 3 RESULTS AND DISCUSSION 3.1 EVOLUTION OF THE MORPHOLOGICAL PA- RAMETERS OF THE FRUITS DURING THEIR DEVELOPMENT 3.1.1 Evolution of the fruit size and dimensions The evolution of fruit size and dimensions (length and diameter) in the three flowering phases (early flow- ering, seasonal and late) of ‘Majhoul’ date palm during fruit development in Tafilalet area is presented in Figure 1. It shows that fruit size and dimensions are higher in the early flowering phase than in the other phases. This is because the fruits of the early flowering phase have an 11 to 15 days growth advance compared to fruits of the seasonal flowering phase and 28 to 31 days compared to fruits of the late flowering phase. The mean and final val- ues of the fruit size and dimensions in the three flowering phases and for all the fruit harvesting dates are presented in Table 3, and statistical analysis of data has shown that for these parameters, there is a significant difference (p ≤ 0.001) between the three flowering phases. Several authors have also reported that the stages of fruit devel- opment in date palm lead to physical and physiologi- cal changes in the fruit, and modifications in color and texture of the fruit from fruit set to fruit ripening (Al- Shahib & Marshall, 2003; Fadel et al., 2006; Lobo et al., 2014). These morphological and physiological changes in the fruits of date palm provide a promising approach for characterizing their development and quality parameters (Marondedze et al., 2014). For the sixth (July 31 2016) and seventh (August 10 2016) fruit harvesting dates fruit size is not different between the seasonal and late flowering phases, while it is different between these phases for all the other fruit harvesting dates. This convergence in fruit size between these two flowering phases results in low fruit growth in the seasonal flowering phase and high fruit growth in the late flowering phase (Figure 1a). Whereas the differ- ence in fruit size between the seasonal and late flowering phases during the first six fruit harvesting dates (from Figure 1: Evolution of the fruit size (volume) (a) and dimen- sions (b and c) in the early flowering phase, seasonal and late one during fruit growth in ‘Majhoul’ date palm in the Goul- mima region, Tafilalet area, Morocco Acta agriculturae Slovenica, 119/3 – 2023 5 Study on the evolution of the fruit morphological and physico-chemical parameters of ‘Majhoul’ date palm during fruit growth Ta bl e 3: M ea n an d fin al v al ue s o f f ru it siz e an d di m en sio ns in th e th re e flo w er in g ph as es (e ar ly fl ow er in g, se as on al a nd la te p ha se s) o f ‘ M aj ho ul ’ d at e pa lm in th e G ou lm im a re gi on , T afi la le t a re a. Ea rly fl ow er in g ph as e Se as on al fl ow er in g ph as e La te fl ow er in g ph as e Fr ui t l en gt h (c m ) Fr ui t d ia - m et er (c m ) Fr ui t s iz e (c m 3 ) Fr ui t le ng th (c m ) Fr ui t d ia - m et er (c m ) Fr ui t s iz e (c m 3 ) Fr ui t le ng th (c m ) Fr ui t d ia - m et er (c m ) Fr ui t s iz e (c m 3 ) M ea n va lu e of th e fr ui t p ar am et er fo r al l t he fr ui t h ar ve st in g st ag es 4. 43 ± 3 2. 87 ± 2 22 .0 2 ± 4 3. 47 ± 3 2. 43 ± 2 12 .8 6 ± 3 2. 95 ± 2 2. 15 ± 2 9. 55 ± 2 .5 ** Fi na l v al ue o f t he fr ui t p ar am et er on S ep te m be r 3 2 01 6 48 .7 6 ± 6 26 .0 7 ± 5 19 .7 0 ± 5 39 .7 8 ± 5 23 .2 9 ± 4 13 .5 5 ± 3 36 .1 0 ± 5 21 .4 4 ± 5 9. 97 ± 2 .5 ** ** S ig ni fic at iv e di ffe re nc e at p ≤ 0 .0 01 June 2 to July 31 2016) is due to difference in fruit growth between the two flowering phases. Moreover, the dif- ference in the final fruit size between the two flowering phases on September 3 2016 (Figure 1a) is due to the loss of water in the fruits as they are in the ripening phase. Regarding fruit dimensions, fruit length is also the same for the seasonal and late flowering phases at the time of the sixth fruit harvesting stage (Figure 1b), and fruit diameter during the sixth and seventh fruit harvesting stages is also the same for these flowering phases (Figure 1c). This overlap at the time of the sixth fruit harvest- ing date can be only explained by the difference in fruit growth between these flowering phases, which is due to a delay of about 16 days between the two flowering phases. 3.1.2 Evolution of the fruit, pulp mass and seed mass Figure 2 presents the evolution of the fruit mass, and pulp mass and seed mass in the three flowering phases during fruit development. It shows that for all the fruit harvesting stages, the mass of fruit, pulp and seed in the early flowering phase is higher than the mass of these ele- ments in seasonal and late flowering phases. This is due to fact that the fruits of the early flowering phase have an 11 to 15 days growth advance compared to the sea- sonal flowering phase and 28 to 31 days growth advance compared to the late flowering phase. The mean and final values of fresh mass of the fruit, pulp and seed and the mean and final dry mass of the fruit of the three flower- ing phases for all the fruit harvesting stages are presented in Table 4. Moreover, statistical analysis of data showed that for these parameters of the fruit, there is a significant difference (p ≤ 0.01) between the fruits of the three flow- ering phases. During the sixth (July 31 2016) and seventh (Au- gust 10 2016) fruit harvesting dates, the seasonal and late flowering phases yielded fruits with similar fruit and pulp fresh mass, whereas they were different during the other fruit development stages (Figure 2a and b). In the case of seeds, it is only during the last fruit harvesting stage (September 3 2016) that their mass is similar in the three flowering phases. However, it is different between the flowering phases in the other fruit harvesting dates, except for the seventh fruit harvesting date where seed mass of the seasonal flowering phase is close to that of the late flowering phase (Figure 2c). This is due to favora- ble climatic conditions for fruit development during the early flowering phase, which are favorable to fruit de- velopment during the early stages of fruit growth. Some authors have also reported that favorable climatic con- ditions, which coincide with the early flowering phase, promote the development of growth hormones, mainly Acta agriculturae Slovenica, 119/3 – 20236 M. ARBA et al. Ta bl e 4: M ea n an d fin al v al ue s o f t he fr ui t a nd p ul p fr es h m as s, se ed m as s a nd fr ui t d ry m as s o f t he fr ui ts o f t he e ar ly, se as on al a nd la te fl ow er in g ph as es o f ‘ M aj ho ul ’ d at e pa lm in th e G ou lm im a re gi on , T afi la le t a re a Ea rly fl ow er in g ph as e Se as on al fl ow er in g ph as e La te fl ow er in g ph as e Fr ui t f re sh m as s ( g) Pu lp fr es h m as s ( g) Se ed m as s (g ) Fr ui t d ry m as s (g ) Fr ui t f re sh m as s ( g) Pu lp fr es h m as s ( g) Se ed m as s (g ) Fr ui t d ry m as s ( g) Fr ui t f re sh m as s ( g) Pu lp fr es h m as s ( g) Se ed m as s (g ) Fr ui t d ry m as s ( g) M ea n va lu e of th e fr ui t p ar am et er fo r al l t he fr ui t h ar ve st in g st ag es 22 ,4 3 ± 3 20 ,9 5 ± 2. 51 ,4 2 ± 1. 2 5, 73 ± 2 .5 13 ,3 2 ± 3 12 ,2 1 ± 2 1, 11 ± 1 .2 3, 65 ± 2 9, 44 ± 2 8, 56 ± 1 .5 0, 91 ± 0 .5 2, 70 ± 1 .4 * Fi na l v al ue o f t he fr ui t p ar am et er o n Se pt em be r 3 2 01 6 23 .3 3 ± 3 21 .8 8 ± 2. 51 .4 2 ± 1. 2 16 .4 4 ± 3 16 .0 1 ± 3. 51 4. 85 ± 2 .5 1. 40 ± 1 .2 10 .7 2 ± 3 12 .1 5 ± 2. 51 1. 08 ± 2 1. 38 ± 1 7. 72 ± 2 * * S ig ni fic at iv e di ffe re nc e at p ≤ 0 .0 1 gibberellic acid, which induces the accumulation of re- serves in the fruit pulp (El-Otmani et al., 2015). Fruit dry Figure 2: Evolution of the fruit fresh mass (a), pulp fresh mass (b), seed mass (c) and fruit dry mass (d) of the fruits of the early, seasonal and late flowering phases during fruit develop- ment of ‘Majhoul’ date palm in the Goulmima region, Tafilalet area Acta agriculturae Slovenica, 119/3 – 2023 7 Study on the evolution of the fruit morphological and physico-chemical parameters of ‘Majhoul’ date palm during fruit growth mass is almost similar during the first four harvesting stages in the seasonal and late flowering phases (Figure 2d). This is due to fruit development of these flowering phases, which took the same pace during the early stages of fruit development because the two flowering phases are separated only for a short period. 3.2 EVOLUTION OF THE CHEMICAL COMPO- SITION OF THE FRUITS DURING THEIR DEVELOPMENT The evolution of the chemical composition of the fruits during their development is presented in Figure 3. It shows that the pH of the fruit juice has a similar evolu- tion for the three flowering phases from the second fruit harvesting stage to the last, while it’s different between the flowering phases for the first fruit harvesting stage (Fig- ure 3a). The content of total sugars in the fruits also has a similar evolution for the three flowering phases during all the fruit harvesting stages (Figure 3b). The mean and final values of the pH of the fruit juice and the content of total sugars in the fruits of the three flowering phases and for all the fruit harvesting stages are presented in Table 5. Moreover, statistical analysis of data showed that there is no significant difference (p ˃ 0.05) between the three flowering phases for the two parameters. This is probably because the flowering phase does not affect the pH of the fruit juice and the content of total sugars in the fruits; however, the fruit harvesting stage affects these param- eters in the three flowering phases. Several authors have also reported that the chemical composition of the fruits varies according to the stages of fruit development (Sal- man Haidar et al., 2013), and fruit development in date palm consists of biological processes which are associat- ed with chemical changes in the cell from fruit set to rip- ening stage (Lobo et al., 2013; Marondedze et al., 2014). Figure 3: Evolution of the pH of the fruit juice (a) and the content of total sugars in the fruits (b) of the three flowering phases (early flowering, seasonal and late) of ‘Majhoul’ date palm in the Goulmima region, Tafilalet area Table 5: Mean and final values of the pH of the fruit juice and the content of total sugars in the fruits of the three flowering phases (early flowering, seasonal and late) of ‘Majhoul’ date palm in the Goulmima region, Tafilalet area Early flowering phase Seasonal flowering phase Late flowering phase pH of the fruit juice Content of total sugars in the fruits (%) pH of the fruit juice Content of total sugars in the fruits (%) pH of the fruit juice Content of total sugars in the fruits (%) Mean value of the fruit chemical parameter for all the fruit harvesting stages 5.86 ± 3 25.89 ± 3 5.68 ± 2.5 25.49 ± 4 5.74 ± 2.5 25.36 ± 4 ns Final value of the fruit chemical parameter on September 3 2016 6.90 ± 4 77.80 ± 11 6.80 ± 3.5 76.90 ± 11 6.90 ± 4 76.00 ± 11 ns ns: No significant difference at p ˃ 0.05 4 CONCLUSIONS For all studied morphological parameters of the fruit (fruit size and dimensions and fruit mass), there is a difference in their evolution between the three flower- ing phases during fruit development, and along this evo- lution, the parameters of the early flowering phase are higher than those of the other flowering phases. This is partly because the fruits of the early flowering phase have Acta agriculturae Slovenica, 119/3 – 20238 M. ARBA et al. a remarkable 11 to 31 days of growth advance compared to other flowering phases. On the other hand, favorable climatic conditions for fruit growth (mild temperatures and long days) during the spring season which coincides with the early stages of fruit development of date palm in the region of study. However, for the content of total sugars in the fruits and the pH of the fruit juice, their evolution during the fruit harvesting stages is similar for the three flowering phases, while their values vary from one fruit harvesting stage to another and for the three flowering phases. This is because the flowering phase does not affect these parameters, while the fruit develop- ment stage affects these parameters. Based on these results, we can suggest that grow- ers keep only the early flowering phase clusters for their cluster-limiting operation when the number of clusters of this flowering phase is sufficient. Moreover, when the number of clusters of the early flowering phase is not suf- ficient, the choice of clusters to be retained in the limita- tion operation can be made on the clusters of the early and seasonal flowering phases to obtain a good fruit yield and quality and an early entry into production. 5 ACKNOWELGEMENTS Many thanks to Charouit family in Goulmima. They have made at our disposal their date palm farm for the trials. Many thanks also to the Agricultural Research Center (CRA) of Errachidia and Hassan II Institute of Agronomy and Veterinary Medicine for their support. 6 REFERENCES Achour, M., Ben Amara, S., Ben Salem, N., Jebali, A., Hamdi, M. (2003). Effet des conditionnements sous vide et sous atmosphère modifiée sur la conservation des dattes De- gletNor en Tunisie. 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Agricultural Engineering International CIGR Journal, 3, 1-9. Hasnaoui, A., Elhoumaizi, M. A., Hakkou, A., Wathelet, B., Sindic, M. (2010). Physico-chemical characterization, clas- sification and quality evaluation of date palm fruits of some Moroccan cultivars. Biology Journal of Scientific Research, 3(1), 139-149. https://doi.org/10.3329/jsr.v3i1.6062 Lobo, M. G., Elhadi, M. Y., Kader, A. A. (2013). Biology and postharvest physiology of date Fruit. In M. Siddiq, S. M. Aleid & A. A. Kader (Eds.), Dates: Postharvest sci- ence, processing technology and health benefits (pp. 57- 80). UK, John Wiley & Sons Ltd Publisher. https://doi. org/10.1002/9781118292419.ch3 Mansour, H. M. (2005). Morphological and genetic characteriza- tion of some common Phoenix dactlifera L. cultivars in Is- mailia region. M.Sc. Thesis. Suez Canal University, Faculty of Science, Department of Botany, Egypt. Marondedze, C., Gehring, C., Thomas, L. (2014). Dynamic changes in the date palm fruit proteome during develop- ment and ripening. Horticulture Research, 1, 1-14. https:// doi.org/10.1038/hortres.2014.39 ORMVAT (2015). Bilan Phoenicicole au titre de la compagne agricole 2014-2015. Office Régional de Mise en Valeur Agri- cole de Tafilalet (ORMVAT). Rastegar, S., Rahemi, M., Baghizadeh, A., Gholami, M. (2012). Enzyme activity and biochemical changes of three date palm cultivars with different softening pattern during ripening. Food Chemistry, 134, 1279-1286. https://doi. org/10.1016/j.foodchem.2012.02.208 Salman Haidar, M., Iqrar, A. K., Summar, A. N., Jaskani, M. J., Rashad, W. K., Nafees, M., … Pasha, I. (2013). Fruit devel- opmental stages effects on biochemical attributes in date palm. Pakistan Journal of Agricultural Scences, 50(4), 577- 583. Shengji, P., Sanyang, C., Lixiu, G., Henderson, A. (2010). Phoe- nix Linnaeus. Flora China, 23,143-144. Acta agriculturae Slovenica, 119/3, 1–18, Ljubljana 2023 doi:10.14720/aas.2023.119.3.12555 Original research article / izvirni znanstveni članek Investigating the growth characteristics, oxidative stress, and metal ab- sorption of chickpea (Cicer arietinum L.) under cadmium stress and in silico features of HMAs proteins Maryam KOLAHI 1, Elham Mohajel KAZEMI 2, Milad YAZDI 3, Mina KAZEMIAN 2, 4, Andre GOLDSON- BARNABY 5 Received March 01 2023; accepted September 23, 2023. Delo je prispelo 1. marca 2023, sprejeto 23. septembera 2023 1 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2 Department of Plant, Cell and Molecular Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran 3 Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4 Corresponding author, e-mail: Mina.kazemian69@gmail.com 5 Department of Chemistry, University of the West Indies, Mona, Jamaica Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) under cad- mium stress and in silico features of HMAs proteins Abstract: Heavy metal contamination can have a strong effect on the morphological and physiological characteristics of plants. In the present study, Cicer arietinum L. (chickpea) was exposed to different concentrations of cadmium (control, 2, 4, 8 μg Cd g-1 perlite) and the effect on plant growth and antioxidant enzymes were evaluated. The observed morphological changes in chickpea plant included stunted growth, reduced root system development and plant color change. A significant increase in enzyme activity of peroxidase, superoxide dismutase, catalase, and ascorbate peroxidase was observed at 4 μg Cd g-1 perlite, with a subsequent decrease when concentration was increased to 8 μg Cd g-1 perlite in the leaves of the plants. The highest cadmium levels were determined at a concentration of 8 μg Cd g-1 perlite. With the addition of 2 μg Cd g-1 perlite, manganese uptake in the aboveground part of the plant increased signifi- cantly, but then decrease at higher cadmium concentrations. In addition, zinc and copper levels decrease in the presence of cadmium. These results indicate that chickpea has a relatively high adsorption capacity for cadmium in aboveground tissues and special precautions should be taken when growing chick- pea. In silico analysis led to the identification of 13 heavy metal ATPases (HMAs) in chickpea. These proteins contain 130 to 1032 amino acids with 3 to 18 exons. They are involved in the transfer of cadmium and zinc and help in heavy metal detoxifi- cation of plants. Bioinformatics studies have been conducted to better understand the mechanism by which the plant is able to combat heavy metal stress. Key words: cadmium, chickpea, HMAs, oxidative stress Preučevanje rastnih značilnosti, oksidativnega stresa in pre- vzema kovin pri čičerki (Cicer arietinum L.) v razmerah kad- mijevega stresa in in silico lastnosti HMAs proteinov Izvleček: Onesnaženje s težkimi kovinami ima lahko mo- čan učinek na morfološke in fiziološke lastnosti rastlin. V razi- skavi je bila čičerka (Cicer arietinum L.) izpostavljena različnim koncentracijam kadmija (kontrola, 2, 4, 8 μg Cd g-1 perlita). Ovrednoteni so bili učinki na rast rastlin in na antioksidacijske encime. Opažene morfološke spremembe čičerke so bile zavr- ta rast, zmanjšan razvoj koreninskega sistema in spremembe v barvi rastlin. Značilna porast aktivnosti encimov peroksidaze, superoksid dismutaze, katalaze in askorbat peroksidaze je bila opažena pri 4 μg Cd g-1 perlita s posledičnim upadom, ko se je koncentracija povečala na 8 μg Cd g-1perlita v listih tretiranih rastlin. Največja vsebnost kadmija je bila določena pri obravna- vanju z 8 μg Cd g-1 perlita. Pri dodatku 2 μg Cd g-1 perlita se je privzem mangana v nadzemnih delih rastlin značilno povečal a se je pri večjih koncentracijah kadmija zmanjšal. Dodatno so se v prisotnosti kadmija vsebnosti cinka in bakra zmanjševale. Ti izsledki kažejo, da ima čičerka relativno veliko sposobnost pri- vzema kadmija v nadzemna tkiva in moramo na to biti pozorni, če jo gojimo v s kadmijem onesnaženem okolju. In silico ana- lize so vodile k prepoznavanju 13 ATPaz (HMAs), povezanih s težkimi kovinami. Ti proteini vsebujejo 130 do 1032 amino kislin s 3 do 18 eksoni. Vključeni so v prenos kadmija in cinka in pomogajo v rastlinah pri detoksikaciji težkih kovin. Za bolje razumevanje mehanizmov s katerimi rastline premagujejo stres težkih kovin so bile izvedene tudi bioinformacijske raziskave. Ključne besede: kadmij, čičerka, HMAs, oksidativni stres Acta agriculturae Slovenica, 119/3 – 20232 M. KOLAHI et al. 1 INTRODUCTION Chickpea (Cicer arietinum L.) is a major legume crop that is consumed globally especially on the Africa and Asia continents (Kaur et al., 2022). Chickpea has a very high nutritional content and is one of the cheapest sources of protein and an important source of minerals (manganese, molybdenum, phosphorus and potassium) and vitamins (Mohanty et al., 2022), so measures need to be taken to avoid its contamination with heavy metals such as cadmium. Cadmium (Cd) is one of the most important con- taminants due to its high toxicity and high water solubil- ity and is readily absorbed by the root system of many plants (Zulfiqar et al., 2022). High levels of Cd can have detrimental effects on plant physiological and biochemi- cal processes, leading to reduced growth, impaired nutri- ent uptake, and disruption of cellular functions. More- over, Cd toxicity inhibits plant growth by affecting cell division, cell elongation, and differentiation processes (Tuver et al., 2022). It disrupts hormone balance, lead- ing to stunted root and shoot growth, reduced biomass production, and impaired reproductive development. Cd toxicity can interfere with the uptake and transport of essential nutrients such as iron, calcium, magnesium, and zinc (Zhou et al., 2022). It can bind to transporters, enzymes, and carrier proteins, thereby disrupting nutri- ent homeostasis and causing nutrient deficiencies. Fur- thermore, Cd toxicity negatively impacts photosynthe- sis, reducing the efficiency of light absorption, electron transport, and carbon assimilation (Zulfiqar et al., 2022). Plants have evolved several mechanisms to mitigate the toxic effects of cadmium (Cd) and minimize its ac- cumulation in their tissues. One crucial strategy is the sequestration of cadmium into vacuoles, which serves as a storage site for toxic metals (Jogawat et al., 2021). On the other hand, Cd toxicity leads to the generation of reactive oxygen species (ROS) in plant cells, causing oxi- dative stress (Zhang et al., 2019). Antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxi- dase (APX), and catalase (CAT), scavenge and neutral- ize ROS, protecting cellular components from oxidative damage (Faria et al., 2022). Moreover, Plants possess transporters that can efflux Cd ions from the cytoplasm to the extracellular space or restrict their entry into spe- cific tissues. ATP-binding cassette (ABC) transporters and heavy metal ATPases (HMAs) are involved in Cd transport across cell membranes. These transporters play a crucial role in minimizing the accumulation of Cd in sensitive tissues and facilitating its sequestration (Tian et al., 2023). HMAs belong to the P-type ATPase superfamily and are localized in the plasma membrane or tonoplast (vac- uolar membrane) of plant cells. HMAs play a crucial role in the detoxification of cadmium by actively transporting it out of sensitive cellular compartments or sequester- ing it into vacuoles. This process contributes to reducing the concentration of free cadmium in the cytoplasm and minimizing its toxic effects on plant growth and devel- opment. HMAs function as efflux pumps, actively trans- porting Cd ions out of the cytoplasm and extruding them from the cell or into specific compartments, such as the vacuole (Fang et al., 2016). By pumping Cd out of sensi- tive cellular regions, HMAs reduce the concentration of free cadmium in the cytoplasm and minimize its toxic ef- fects on cellular processes (Satoh-Nagasawa et al., 2012). HMAs participate in the regulation of metal ion homeo- stasis in plants. They are involved in maintaining the bal- ance between essential metals (such as zinc and copper) and non-essential heavy metals (such as Cd) (Fang et al., 2016). This regulation ensures that essential metals are properly acquired and utilized while minimizing the up- take and accumulation of toxic metals like Cd. HMAs in- teract with metal chelators, such as phytochelatins (PCs), which are small peptides that bind to heavy metal ions, including Cd. This process contributes to the detoxifica- tion and sequestration of cadmium in less sensitive cel- lular compartments (Tian et al., 2023). The purpose of the study is to get insights how chickpea plants respond to cadmium, a harmful heavy metal that can contaminate soil and negatively affect plant health .Understanding the mechanisms of Cd tox- icity in plants is crucial for developing strategies to miti- gate its adverse effects. The first objective is therefore to examined the impact of Cd on the growth characteristics, activity of oxidative enzymes, Cd, zinc (Zn), copper (Cu) and manganese (Mn) content in chickpea. The next objective of this study was to gain a better understanding of the role that HMAs play in chickpea, particularly under conditions of cadmium stress and to provide insights into how chickpea plants respond to cadmium by Bioinformatics analyses such as number of genes, proteins, gene loci, cellular location, phylogenetic relationship, three-dimensional protein structure, con- served domains, similar template and catalytic site. 2 MATERIALS AND METHOD 2.1 PROPAGATION AND CADMIUM EXPOSURE Chickpea (Cicer arietinum L.) seeds were germinat- ed in sterilized Cucupite and Perlite in a greenhouse on the photoperiod of 8 h light and 16 h darkness. Seedlings with leaves were planted in pots (diameter, 12 cm and height 15 cm) under controlled conditions and watered Acta agriculturae Slovenica, 119/3 – 2023 3 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... with distilled water every 3 days. Cadmium chloride were added in four concentrations (control, 2, 4, and 8 μg Cd g-1 perlite) calculated per g of perlite. Plants were watered with Hoagland nutrient solution (Hoagland and Snyder 1933) without cadmium chloride (field capacity was considered). After 10 days of Cd treatment plants were harvested for further investigations. 2.2 GROWTH PARAMETERS The fresh and dry mass of the roots and above ground parts were determined (mg). Plantlet height, leaf area, root length, shoot length and internode length were measured. Stomatal densities on the lower and upper epi- dermis were evaluated. 2.3 ENZYME ASSAYS Enzyme extracts were prepared from fresh chickpea leaves (1 g) with phosphate potassium buffer (5 ml). Ho- mogenous samples were prepared by pulverizing followed by centrifugation (4 °C, 25 min, 15000 rpm) and storage at -80 °C. Catalase enzyme activity was determined by mixing phosphate buffer (2.5 ml, pH 7.5) and hydrogen peroxide (1%, 0.1 ml) in an ice bath and the addition of enzyme extract (0.1 ml) and the rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240nm via spectrophotometer. Peroxidase enzyme activity was determined based on the method by Koroi (1989). The reaction mixture consisted of acetate buffer (0.2 M, 2 ml, pH 5), benzidine (0.02 M, 100 ml), hydrogen peroxide (3 %, 200 µl) and enzyme extract (25 µl). The absorption was determined at 530 nm. Ascorbate peroxidase (EC11.1.11.1) activity was determined spectrophotometrically (Nakano and Asada, 1987). To the enzyme extract (100 µl) was added K2HPO4 (0.5 M, 2.5 ml), ascorbate (0.5 mM, 0.1 ml), EDTA (0.1 mM, 0.1 ml) and H2O2 (1 %, 0.2 ml) and the absorbance read at 290 nm. Specific enzyme activity was reported as units/g fresh mass (Nakano and Asada 1987). Total soluble protein was determined utilizing the Brad- ford assay with bovine serum albumin (BSA) as standard. The absorbance was read at 595 nm (Bradford, 1976). 2.4 CADMIUM AND OTHER ELEMENTS MEAS- UREMENT Plant samples were oven dried (72 h, 60 °C) and the dry mass determined. Dried samples were ashed (550 °C, 8 h). The digested extract (1N HCl, 1 mL; nitric acid, 97 %, 1 ml, 1 h) was made to a final volume of 20 ml and the cadmium, zinc, copper and manganese content of the samples measured (Chellaiah, 2018) utilizing a Flame Atomic Absorption Spectrometer (GBC, SAVANTAA scientific equipment, Australia) which has a detection limit of 0.007 µg ml-1. Cd (II), Zn (II), Cu (II) and Mn (II) standard solution were prepared using their nitrate salts in nitric acid. Bioconcentration factor (BCF) computed as heavy metal accumulated in each plant tissue to that dissolved in the soil medium (Bose and Bhattacharyya 2008). Root bioconcentration factor: BCF = root/soil Shoot bioconcentration factor: BCF = shoot/soil TF = BCFshoot/BCFroot 2.5 BIOINFORMATICS ANALYSIS The gene database of NCBI was searched utilizing the keyword „HMA“. Gene characteristics included lo- cation, exon count and conserved domain. Protein se- quences were used for localization prediction from the Localizer and protein tertiary structure predicted by Phyre2. Potential tunnels within each protein and cata- lytic pocket were predicted utilizing CAVER Web. The Jones-Taylor Thornton model was selected to obtain the phylogenies tree of HMAs from chickpea and Arabidop- sis using the neighbor-joining (NJ) method, with a boot- strap test performed using 1000 iterations in MEGA5 (Tamura et al., 2007). Multiple sequence alignments were performed utilizing the muscle algorithm of mega 7 soft- ware to detect conserved residues (Kumar et al., 2016). HMAs from Arabidopsis were highlighted in green. Some information has been mentioned below: XP_004509102.1: Probable cadmium/zinc-trans- porting ATPase HMA1, chloroplastic [Cicer arietinum], P_004487939: Cadmium/zinc-transporting ATPase HMA3-like isoform X1 [Cicer arietinum], XP_027189340: Cadmium/zinc-transporting ATPase HMA2-like isoform X2 [Cicer arietinum], XP_012573401: Putative inactive cadmium/zinc-transporting ATPase HMA3 [Cicer arieti- num], XP_004488108: Cadmium/zinc-transporting AT- Pase HMA3-like [Cicer arietinum], XP_012573132: Cop- per-transporting ATPase HMA4-like [Cicer arietinum], XP_012574029: Copper-transporting ATPase HMA4- like isoform X1 [Cicer arietinum], XP_027192934: Copper-transporting ATPase HMA4-like isoform X2 [Cicer arietinum], XP_004500941: Cation-transporting ATPase HMA5-like [Cicer arietinum], XP_004511583: Probable copper-transporting ATPase HMA5 [Cicer ari- etinum], XP_004504792: Copper-transporting ATPase PAA1, chloroplastic [Cicer arietinum], XP_004504659: Acta agriculturae Slovenica, 119/3 – 20234 M. KOLAHI et al. Copper-transporting ATPase RAN1 [Cicer arietinum], XP_004501429: Copper-transporting ATPase PAA2, chloroplastic [Cicer arietinum]. Q9SH30 (Protein: Probable copper-transporting ATPase HMA5, Gene: HMA5, Organism: Arabidopsis thaliana (L.)Heynh., P0CW78 (Protein: Cadmium/zinc- transporting ATPase HMA3, Gene: HMA3, Organism: Arabidopsis thaliana, Q9SZW4 (Protein: Cadmium/ zinc-transporting ATPase HMA2, Gene: HMA2, Organ- ism: Arabidopsis thaliana, Q4L970 (Protein: Copper-ex- porting P-type ATPase, Gene: copA, Organism: Staphy- lococcus haemolyticus Schleifer & Kloos, 1975 (strain JCSC1435), O32220 (Protein: Copper-exporting P-type ATPase, Gene: copA, Organism: Bacillus subtilis (Ehren- berg 1835) Cohn 1872 (strain 168), Q9S7J8 (Protein: Copper-transporting ATPase RAN1, Gene: RAN1, Or- ganism: Arabidopsis thaliana), Q9M3H5 (Protein: Prob- able cadmium/zinc-transporting ATPase HMA1, chloro- plastic, Gene: HMA1, Organism: Arabidopsis thaliana). 2.6 STATISTICAL ANALYSES Data analyses were performed using the SPSS 20 software package (SPSS Inc., Chicago, USA). All experi- mental data were presented as the mean ± SD. One-way ANOVA was used to test differences between various means followed by the post hoc Tukey test (homogeneity of variances and data normally distributed). The level of significance was set at p < 0.05 for all tests. 3 RESULTS 3.1 GROWTH CHARACTERISTICS IN THE ABOVEGROUND PARTS OF CHICKPEA SEED- LINGS AFFECTED BY CADMIUM Observed morphological changes in chickpea seed- lings exposed to cadmium included changes in plant length, coloration and leaf size. Results indicated that stem color changed to a bright green-yellow. Moreo- ver, changes were observed in leaf color (yellow) due to cadmium exposure. There was a significant reduction in shoot and root length. Shorter and less dense roots were observed in the treated samples (Table 1). The fresh and dry mass of the shoots and roots in chickpea plants were also significantly affected by cadmium with the lowest seedling mass being observed at high cadmium concen- trations. Plants treated with 2 μg Cd g-1 perlite had a de- cline in leaf area which was less than half that of the con- trol. At cadmium levels of 2 μg Cd g-1 perlite, the length of the first internodes increased, whereas at higher con- centrations, there was a decrease, while the length of the Fig. 1: Effect of cadmium on chickpea (Cicer arietinum L.) growth under normal and various concentrations of cadmium. a Seed- lings, b Aboveground parts, c Roots, d Leaf areas (control, 2, 4 and 8 μg Cd g-1 perlite) Acta agriculturae Slovenica, 119/3 – 2023 5 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... peroxidase enzyme activity showed that this enzyme was also affected by cadmium exposure. The highest ascor- bate activity was observed in cadmium treatments with 4 and 8 μg Cd g-1 perlite (Fig. 2d). Oxidative enzyme activity (SOD, APX or CAT) was shown to increase in the leaves of plants exposed to cadmium. Increased SOD activity is associated with an increase in the formation of superoxide, which activates gene expression by signal induction. 3.3 MEASUREMENT OF CADMIUM CONTENT AND ELEMENTAL CHANGES IN THE AERIAL PARTS OF CHICKPEA SEEDLINGS AFFECTED BY CADMIUM The cadmium content in aerial parts of chickpea grown in different concentrations of cadmium chloride increased significantly. The highest concentrations were observed at cadmium chloride concentration of 8 μg Cd g-1 perlite. A doubling of cadmium accumulation was observed in the aerial parts of the plant when the cad- mium content of the medium was increased from 2 to 4 μg Cd g-1 perlite (Fig. 3a). Moreover, elemental compo- sition was significantly affected by cadmium levels (Fig. 3). Chickpea cultivated in cadmium-containing media showed a significant difference in the amount of manga- nese present in the aerial part of the plant. With the addition of cadmium, manganese uptake increased significantly by approximately three times, second internodes showed only a significant reduction at high concentrations of cadmium (Fig. 1, Table 1). Fur- thermore, with the addition of cadmium (4 μg Cd g-1 per- lite), stomatal densities on the lower epidermis increased significantly but subsequently declined while higher con- centrations of cadmium (Table1). 3.2 EFFECT OF CADMIUM ON SOD, POD AND CAT ACTIVITIES IN THE AERIAL PARTS OF CHICKPEA SEEDLINGS Cadmium stress resulted in a significant increase in POD enzyme activity. The highest ascorbate activity was observed in cadmium treatments at 4 and 8 μg Cd g-1 perlite. Further increase in cadmium exposure result- ed in a decline in POD activity which was however still significantly higher than that of the control and plantlets treated with 4 μg Cd g-1 perlite. The lowest enzyme activ- ity was observed in the controls (Fig. 2a). SOD enzyme activity significantly increased in chickpea with the high- est enzyme activity being observed in plantlets treated with 4 μg Cd g-1 perlite with the lowest enzyme activity being observed in the control (Fig. 2b). There was a sig- nificant increase in catalase enzyme activity. The high- est catalase activity was also observed in plants treated with 4 μg Cd g-1 perlite with a subsequent decline when cadmium chloride concentration was increased to 8 μg Cd g-1 perlite. The lowest level of enzyme activity was ob- served in the control (Fig. 2c). Investigation of ascorbate Table 1: Effect of Cd (Control, 2, 4 and 8 μg Cd g-1 perlite) on morphometric features in chickpea (Cicer arietinum L.) Values with different letters are significantly different at p < 0.05 Parameters Control 2 μg Cd g-1 perlite 4 μg Cd g-1 perlite 8 μg Cd g-1 perlite Plant length (cm) 62.76 ± 1.36a 58 ± 0.0709a 42.56 ± 1.78b 37.93 ± 1.78b Shoot length (cm) 29.33 ± 0.66a 25.65 ± 0.779b 22.55 ± 1.35bc 21.16 ± 1.092c Root length (cm) 35 ± 0.57a 30.86 ± 0.69b 18.56 ± 0.92c 16.6 ± 0.83c Plant fresh mass (g) 4.0367 ± 0.043a 3.442 ± 0.238b 3.084 ± 0.169b 1.715 ± 0.042c Shoot fresh mass (g) 2.291 ± 0.11a 1.6317 ± 0.14b 1.297 ± 0.061c 0.682 ± 0.014d Root fresh mass (g) 2.24 ± 0.078a 1.9 ± 0.1b 1.3167 ± 0.109c 0.99 ± 0.003d Shoot dry mass (g) 1.987 ± 0.01a 1.4783 ± 0.11b 1.0447 ± 0.029c 0.606 ± 0.002d Root dry mass (g) 2.01 ± 0.04a 1.696 ± 0.063b 1.123 ± 0.069c 0.823 ± 0.062d Leaf area (mm2) 103.33 ± 1.76a 48.33 ± 2.18b 20.66 ± 0.666c 18.33 ± 1.201c First internode length (cm) 1.1 ± 0.264 b 1.766 ± 0.0577 a 1.3 ± 0.3 b 0.833 ± 0.838 c Second internode length (cm) 2.433 ± 0.513 a 2.266 ± 0.503 a 1.766 ± 0.808 b 1.633 ± 0.850 b Stomatal densities on the upper epidermis 35 ± 0.545 ab 31 ± 0.564 b 24.33 ± 0.413 c 39.33 ± 0.633 a Stomatal densities on the lower epidermis 29.6667 ± 0.448 c 39 ± 0.653 b 46 ± 0.765 a 37.33 ± 0.985 b Acta agriculturae Slovenica, 119/3 – 20236 M. KOLAHI et al. while higher concentrations of cadmium reduced the amount of manganese in chickpea plants (Fig. 3b). In- crease in the levels of cadmium in the culture also caused changes in the amount of zinc present in the aerial parts of pea plants. Increasing the levels of cadmium in the medium resulted in a decline in zinc (Fig. 3c). Increasing cadmium concentration, also decreased the levels of cop- per present in the aerial parts of chickpea seedlings. The lowest amount of copper was observed in high-cadmium seedlings (Fig. 3d). The BCF and TF values is greater than one at 8 μg Cd g-1 perlite (Fig. 3 e,f). 3.4 BIOINFORMATICS In the current bioinformatics study of chickpea un- der cadmium stress, HMA proteins were chosen. In silico analysis of chickpea HMAs showed that of the 13 HMA identified, there were three proteins for each HMA3 and HMA4, two proteins for HMA5 and one protein for HMA 2, 6, 7, 8 (Table 2). The ATPase PAA2, chloro- plastic, copper-transporting ATPase RAN1, and copper- transporting ATPase PAA1, chloroplastic identified in chickpea were identified as HMA6, HMA7, HMA8, in Arabidopsis, respectively. HMA7 and HMA8 all contrib- ute to copper transport. The HMA 1, HMA 3, g HMA 2, HMA 4, HMA 5, PAA1, RAN1 and PAA2 genes are located on chromosomes 7, 1 and 7, 1, 6 and 7, 5 and 8, 6, 6, 5 respectively (Table 2). These proteins contain 130 to 1032 amino acids with 3 to 18 exons. The confidence level of predicting the three-dimensional structure of chickpea HMAs proteins is shown in Table 3. Their cel- lular locations are often in the nucleus and chloroplast. Using phyre2, their three-dimensional structure was de- termined. The protein templates and organisms used to predict the three-dimensional structure of these proteins are listed in Appendix 1. Among these templates, c3rfuC was used to predict all 13 proteins in a study related to copper-transporting PIB-type ATPase from the gram- negative bacterium Legionella pneumophila subsp. pneu- mophila Brenner DJ, Steigerwalt AG, McDade JE 1979. The patterns of c3j08A and c3j09A are also related to the p-type ATPase copper transporter CopA. Five (5) tem- plates including copper-transporting proteins ATPase ATP7A, apoWLN5-6, domains 3 and 4 of human ATP7B, apo HMA domain of copper chaperone for superoxide dismutase and C2H2 type zinc finger (region 641-673) of human zinc finger protein 473 belong to humans. In to- tal, the HMA studied in chickpeas were found to contain nine domains which are common in the 13 HMAs. The COG4087 domain is listed as Soluble P-type ATPase and pfam00122 as E1-E2_ATPase are present Fig. 2: The activities of a Peroxidase (POD), b Superoxide dismutase (SOD), c Catalase (CAT) and d Ascorbate peroxidase en- zymes in aboveground parts of chickpea (Cicer arietinum L.). Values with different letters are statistically significantly different at p < 0.05 (One-way ANOVA, post hoc Tukey test) Acta agriculturae Slovenica, 119/3 – 2023 7 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... in ten HMAs. Following the prediction of the three- dimensional structure for chickpea HMAs, the longest tunnels for each protein and catalytic pocket predicted by CAVER Web for ion passing was determined. The longest and shortest tunnels predicted belonged to cad- mium/zinc-transporting ATPase HMA3-like and cation- transporting ATPase HMA5-like, respectively. The puta- tive inactive cadmium/zinc-transporting ATPase HMA3 was the largest HMA with 1032 amino acids and a short tunnel having a length of 41.7. No tunnel was predicted for copper-transporting ATPase PAA2, chloroplastic and copper-transporting ATPase PAA1, chloroplastic with 934 and 884 amino acids. The three-dimensional structure with the longest predicted tunnel allowing for the passage of ions rep- resented in color is illustrated in Fig 4. Based on the software used to analyze 8 of the 13 HMA chickpeas, the catalytic site was determined. From the proposed envelope for the HMAs the catalytic position for inter- action with ions was determined. For XP_027192934, three catalytic sites with Asp residues at positions 522, 729, 733 with 40 % similarity over a specific reference of active site type and metal ion-binding site were identi- fied. These catalytic sites can be evaluated and compared based on their pocket score. The neighboring residues of the catalytic position are also presented in the Table 3. In Fig. 3: Effects of different cadmium treatments on a Accumulation of cadmium, b Manganese, c Zinc, d Copper content in the aerial parts of chickpea seedlings after 10 days of cadmium treatment. Values with different letters are statistically significantly different at p < 0.05. (One-way ANOVA, post hoc Tukey test). Bioconcentration factor (e) and translocation factor (f). BCF values > 1 indicate that the concentration in the organism is greater than that of the medium. Translocation factor (TF) values more than one can be considered potential as Cd accumulators for phytoremediation. Mean plant tissues BCF are averages of five BCF values (n = 5) ± SEM Acta agriculturae Slovenica, 119/3 – 20238 M. KOLAHI et al. Table 2: An overview of the features of chickpea HMAs proteins structure, genes loci, conserved Protein Domain Family, cellular location, Phyre2 confidence (residues modelled at > 90 % confidence), templates used for 3D prediction and longest tunnel pre- dicted by the Caver Web for transport ions Protein Length Gene Exon count Conserved domain Location Template pattern Longest tunnel XP_004509102.1 839 101490857 Chromosome: Ca7 13 COG4087 TIGR01512 pfam00122 Chloroplast c3rfuC,c1mhsA,c3j08A,c5 mrwF,c4umwA,c3j09A 70.1 XP_004487939 834 101492022 Chromosome: Ca1 9 COG2608 COG4087 pfam00122 Nucleus c3rfuC, c3j08A, c4umwA, c3j09A 85.5 XP_027189340 569 101492022 chromosome: Ca1 9 cl21460 COG2608 - c4umwA, c3rfuC, c3j08A, c3j09A 29 XP_012573401 1032 101505376 Chromosome: Ca7 11 COG2608 TIGR01512 pfam00122 Nucleus c3rfuC,c2emcA, c3j08A,c4umwA,c3j09A 41.7 XP_004488108 832 101497233 Chromosome: Ca1 9 COG2608 COG4087 pfam00122 Nucleus c3rfuC, c3j08A, c4umwA, c3j09A 107.3 XP_012573132 853 101504726 Chromosome: Ca6 7 COG2217 cd00371 pfam00122 - c3rfuC, c4u9rA, c3j08A, c3j09A 43.8 XP_012574029 958 101515614 Chromosome: Ca7 10 COG2217 COG2608 COG4087 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c3j09A 72 XP_027192934 849 101515614 Chromosome: Ca7 10 cd02094 cd00371 cl00207 Nucleus c3rfuC, c4u9rA, c3j08A, c3j09A 95.6 XP_004500941 130 101507723 Chromosome: Ca5 3 pfam00122 - c3rfuC, c3j08A, c3j09A, c2kijA, c2hc8A 11.3 XP_004511583 998 101498342 Chromosome: Ca8 7 COG2217 COG4087 cd00371 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c3j09A 94.3 XP_004504792 934 101496348 Chromosome: Ca6 17 COG2217 COG4087 cd00371 pfam00122 Chloroplast c3rfuC, c4u9rA, c3j08A, c3j09A - XP_004504659 995 101509532 Chromosome: Ca6 10 COG2217 COG4087 pfam00122 Nucleus c2ew9A, c3rfuC, c2rmlA, c2ropA, c3j08A, c2crlA, c3j09A 95.9 XP_004501429 884 101500347 Chromosome: Ca5 18 COG2217 COG4087 cd00371 Nucleus, Chloroplast c3rfuC, c3j08A, c3j09A - Acta agriculturae Slovenica, 119/3 – 2023 9 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... most cases, the amino acid Asp residue is introduced. For XP_012574029 and XP_004504659 the predicted pocket score was 100  % with XP_004504659 having an active site and three metal ion-binding sites (Table 3). In the phylogenic tree of the HMAs (Fig. 5), com- parison of the protein sequences of chickpea HMA with Arabidopsis revealed great similarity between these pro- teins in chickpea and Arabidopsis. HMA 2 and 4 are very similar to Arabidopsis and are next to HMA 3 chickpeas. HMA 3 chickpea is adjacent to HMA 3 Arabidopsis. HMA 1 2 3 chickpea are all involved in cadmium and zinc transfer and are in close proximity to each other in the tree. The P-type ATPases of Arabidopsis are very simi- lar to the copper-transporting ATPase PAA2 chickpeas. Copper-transporting ATPase PAA1 pea is very similar to Arabidopsis P-type ATPases. In chickpea, copper-trans- porting ATPase RAN1 resembles copper-transporting ATPase HMA5, which is adjacent to copper-transporting ATPase RAN1 Arabidopsis. Cation-transporting ATPase HMA5-like and copper-transporting ATPase RAN1 are also in the vicinity of copper-transporting ATPase RAN1 Arabidopsis. 4 DISCUSSION Heavy metal pollution is a significant environmen- tal problem. Increasing our knowledge of the mecha- nisms by which plants are able to mitigate heavy metal stress could assist in creating new tools applicable to Fig. 4: An overview of the 3D model of chickpea HMAs generated by Phyre2 software. The structures were predicted using coordinate templates represented in Table 2. Colored regions in 3D structure represent the longest tunnel. a XP_004509102.1, b XP_004487939, c XP_027189340, d XP_012573401, e XP_004488108, f XP_012573132, g XP_012574029, h XP_027192934, i XP_004500941, j XP_004511583, k XP_004504792, l XP_004504659, and m XP_004501429 Acta agriculturae Slovenica, 119/3 – 202310 M. KOLAHI et al. Table 3: Index, residue, accession code of the reference entry, sequence identity to the reference entry, type, description, neighbor- hood and pocket score features of chickpea HMAs proteins structure Protein accession number Index Residue Accession code of the reference entry Sequence identity Type Description Neighbor- hood Pocket score XP_004511583 656 Asp Q9SH30 73.8 % active site 4-aspartylphosphate inter- mediate VFDKT VFDKT 100 % 860 Asp Q9SH30 73.8 % metal ion- binding Magnesium VGDGI VGDGI 864 Asp Q9SH30 73.8 % metal ion- binding Magnesium INDSP INDSP XP_004488108 591 Asp P0CW78 50.2 % metal ion- binding Magnesium VGDGI VGDG 33 % XP_012573401 590 Asp Q9SZW4 54.6 % metal ion- binding Magnesium LGDGL VGDGL 28 % XP_012574029 838 Asp Q9SH30 56.4 % metal ion- binding Magnesium VGDGI VGDGI 100 % XP_012573132 522 Asp Q4L970 41.3 % active site 4-aspartylphosphate inter- mediate VFDKT VFDKT 6 % 730 Asp Q4L970 41.3 % metal ion- binding Magnesium VGDGI VGDGI XP_027192934 522 Asp O32220 41.6 % active site 4-aspartylphosphate inter- mediate VFDKT VLDKT 68 % 729 Asp O32220 41.6 % metal ion- binding Magnesium VGDGI VGDGI 733 Asp O32220 41.6 % metal ion- binding Magnesium INDSP INDAP XP_004487939 392 Asp P0CW78 49.8 % active site 4-aspartylphosphate inter- mediate AFDKT AFDKT 13 % 591 Asp P0CW78 49.8 % metal ion- binding Magnesium IGDGI VGDGL XP_004504659 649 Asp Q9S7J8 73.4 % active site 4-aspartylphosphate inter- mediate IFDKT IFDKT 100 % 138 Cys Q9S7J8 73.4 % metal ion- binding Copper AACVN AACVN 869 Asp Q9S7J8 73.4 % metal ion- binding Magnesium VGDGI VGDGI 873 Asp Q9S7J8 73.4 % metal ion- binding Magnesium INDSP INDSP XP_004509102 467 Asp Q9M3H5 68.2 % active site 4-aspartylphosphate inter- mediate AFDKT AFDKT 25 % 701 Asp Q9M3H5 68.2 % metal ion- binding Magnesium INDAP INDAP Acta agriculturae Slovenica, 119/3 – 2023 11 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... phytoremediation. It is important to further research processes involved in heavy metal detoxification and signaling pathways in plants so as to identify useful tar- gets for biotechnological applications thereby increasing plant fitness in heavy metal polluted sites (Dala-Paula et al., 2018). Cadmium exposure reduced leaf area, shoot and root length. The effect of cadmium ion suppression on root expansion extends through its effect on cell growth (Hassan et al., 2008). Cadmium attaches to the cell wall and the middle lamella, increasing the bonding between the wall components, ultimately leading to growth in- hibition and a decline in cell and organ development. Cadmium also alters water proportions in plants caus- ing physiological dryness, which leads to metabolic dys- function and production of ROS. These factors reduce growth and impact on plant length and mass (Zulfiqar et al. 2022). Many studies on the mechanism of cadmium blockage on cell growth have shown degradation of cell membranes by cadmium and changes in the degree of cell exchange and cellular depletion (Bücker-Neto et al., 2017). The observed changes in plants exposed to cadmi- um may be as a result of multiple nutritional deficiencies being experienced by the plant. Nutrients serve an essential role in the formation, expansion, and operation of chloroplasts. Cd-phytotox- icity affects the synthesis and extensibility of cell walls (Gomes et al., 2011). Cell wall thickening in root endo- dermal tissue affords a greater surface area over which cadmium accumulation can occur thereby limiting its transportation to the shoot (Zulfiqar et al., 2022). Chlo- rosis observed in the leaves of bean plants exposed to Fig. 5: Phylogenic tree of HMAs from chickpea and Arabidopsis. A phylogenetic tree was constructed using the neighbor-joining (NJ) method, with a bootstrap test performed using 1000 iterations in MEGA5 with the amino acid sequences of HMAs. HMAs from Arabidopsis are highlighted in green cadmium may be due to loss of magnesium which is an integral structural feature of the porphyrin ring present in chlorophyll. Physiological changes observed in leaves are due to the associated toxic effects of cadmium includ- ing mesophyll curvature, decreased leaf thickness and a reduction in the composition of intercellular spaces of spongy parenchyma (Tuver et al., 2022). At higher doses of cadmium, the thickness of palisade and spongy tissues is reduced. A decline in the dimensions and composition of the main mid-vein bundle suggests that cadmium al- ters leaf expansion (Cregeen et al., 2015). A study of the effect of heavy metals on the cell death of Halophila stipulacea (Forssk.) Asch leaves showed that high concentrations of metal causes necrosis of the epi- dermal cells and mesophyll, inhibiting surface growth of the leaves. High levels of heavy metal accumulation in plant cells inhibits the process of respiration and energy reactions, which are associated with cell growth (Ayang- benro, 2017). A decline in cell division and growth could also be a contributing factor to the observed morpholog- ical changes. Additionally, a decrease in photosynthetic rates has been observed in plants exposed to elevated lev- els of heavy metals. Higher concentrations of cadmium commonly result in root injury, damage to photosynthet- ic machinery, inhibition of plant growth, reduced nutri- ent and water uptake (Tuver et al., 2022). Cadmium may exert its inhibitory effect in different ways, namely bind- ing specific groups of proteins and lipids thereby inhibit- ing normal function and possibly inducing free radical formation due to oxidative stress. The former may occur at transport and channel proteins of cell membranes dis- turbing the uptake of many other macro- and microele- Acta agriculturae Slovenica, 119/3 – 202312 M. KOLAHI et al. ments whereas the latter is due to the inactivation of anti- oxidant enzymes by cadmium (Long et al., 2017). The results showed that oxidative enzymes activ- ity (SOD, APX, POD and CAT) increased in the leaves of chickpea exposed to cadmium. Similar observations have been observed in CAT and POD enzymes present in cereals and squash (Ashraf, 2003). Increased activ- ity of these enzymes is a consequence of lipid peroxida- tion. The effect of cadmium on growth and antioxidant enzymes in two varieties of Brassica napus showed that cadmium decreased the growth indices, nitrate reduc- tase activity and leaf water potential while antioxidant enzyme activities increased. The highest level of enzyme activity was in relation to SOD enzymes, which showed more than 80  % increase in activity. The least increase in enzyme activity was observed in the catalase enzyme (Irfan et al., 2014). Increasing the absorption and accu- mulation of heavy metals in plants causes changes in cell metabolism, oxidative stress and cell destruction which is induced by ROS. Cadmium can induce mineral stress that reduces plant dry mass (Zhou et al., 2022). Tabarzad et al. (2017) showed that wheat seedlings grown in the presence of cadmium had changes in the level of SOD and POD activity. The observed decline in enzyme activ- ity suggests a weakening of the oxygen and superoxide water scavenging system. Reduced activity of the other antioxidant enzymes in some tissues, is due to poor performance in oxygenate decomposition in cadmium treated tissues. ROS activity increased significantly un- der cadmium stress due to an increase in wall oxidation. Reduced SOD activity is justifiable as cadmium is known to be an enzyme inhibitor (Tabarzad et al., 2017). Schutzendubel (2001) showed the inhibition of SOD, POD and total inactivation of APX in pine roots after 48 days of cadmium treatment. An increase in the activity of these enzymes under cadmium stress has been observed in other studies (Schutzendubel et al., 2001). Li et al. (2013) examined the effect of cadmium stress on growth and antioxidant enzymes and lipid oxidation in two Kenaf (Hibiscus cannabinus L.) species. In the study, glutathione reductase activity (GR) was greater than that of the control. The general trend was that of an increase in SOD, CAT and POD activities in the roots of cadmi- um-stressed plants followed by a decline. POD activity however remained relatively unchanged at all stress lev- els (Zhou et al., 2022). Ulusu et al. (2017) investigated the antioxidant capacity and cadmium accumulation of stressed parsley. In the study, enzyme activity increased for catalase and ascorbate peroxidase, (75 to 150 μM cad- mium), while decreasing at 300 μM. The results showed that antioxidant enzymes activity was suppressed due to the accumulation of cadmium in parsley leaves and in- creased non-enzymatic antioxidant activity (Ulusu et al., 2017). Pereira et al. (2002) studied the activity of anti- oxidant enzymes in Crotalaria juncea L. which showed that under the influence of cadmium, catalase activ- ity did not show any significant changes in the root. At concentrations of 2 mM cadmium, catalase activity in the leaves increased 6 fold compared to the control. In- creased activity of some antioxidant enzymes exposed to metals reveal the crucial role that these enzymes play in detoxification (Pereira et al. 2002). Various antioxidant cycles under normal physiological metabolism, results in the production and scavenging of reactive oxygen spe- cies which is in a state of dynamic equilibrium (Zhou et al., 2022). Kisa (2018) studied the response of anti- oxidant systems to stress induced by heavy metals in the leaves and roots of tomato which showed that cadmium treatment significantly increased the activity of the APX and SOD enzymes. Antioxidant scavenging systems are connected with ROS detoxification which is a defense mechanism employed by plant tissue to combat oxidative stress (Kisa, 2018). Tomato plants exposed to cadmium showed significantly higher SOD. Catalase activity was however reduced. The cadmium content in aerial parts of chickpea grown in different concentrations of cadmium increased significantly. Research conducted by Tang et al. (2022) revealed that cadmium concentrations in the seeds of beans from different regions and varieties is based on complex genetic factors and the environment. For dif- ferent legume varieties, environmental factors such as climate, soil, agricultural and geological techniques, in comparison to genetic factors, are more important in the accumulation of heavy metals such as Cd. Compared to the genus and plant species, the accumulation of heavy metals seems to be more influenced by the genetic poten- tial of the plant (Tang et al. 2022). The ability to absorb and distribute cadmium to the aerial regions of the plant is related to its attachment to the extracellular matrix, root flow, intracellular detoxification and transfer effi- ciency (Akhtar and Macfie, 2012). Cadmium is absorbed in the root of the plants subsequently accumulating in the aerial parts, which often limits the absorption and distri- bution of other elements (Gomes et al., 2013). Cadmium binds to the functional epidermis through direct bind- ing to ion carriers via production of oxygen species that are associated with membrane affects (Altaf et al., 2022). Ling Liu et al. (2012) showed that legumes can increase the accumulation of cadmium in adjacent plants. Cad- mium increase in plants was a direct result of planting crops in proximity to legumes. The study suggests that the system of cultivation of beans should be redesigned to prevent food contamination with cadmium (Liu et al., 2012). Vijendra et al. (2016) showed that in Moth bean (Vigna aconitifolia L.) cadmium concentrations in- Acta agriculturae Slovenica, 119/3 – 2023 13 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... creased significantly in the leaves and roots. Cadmium reaches the aerial sections via the xylem of the plant (Vi- jendra et al., 2016). At concentrations of 0.04 to 0.32 mM, cadmium is non-polluting in soil. Knowledge about the distribution of cadmium in plant tissues is important to better understand the tolerance mechanism and accu- mulation of heavy metals in plants. Cadmium in plants is transferable through apoplast pathways of the stems and leaves (Benavides et al., 2005). Cadmium affects mem- brane potential, protein pump activity and can limit corn growth (Karcz & Kurtyka, 2007). The result indicated that increasing cadmium con- centration, also decreased the levels of copper and zinc present in the aerial parts of chickpea seedlings. Further studies also showed that zinc and copper along with cad- mium have an antagonistic effect and that these minerals act in a competitive manner in relation to the transfer processes. Heavy metals, such as copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe), serve as essential mi- cronutrients for an array of metabolic processes. These micronutrients serve as cofactors, participate in cellular redox reaction and affects protein structure (Schutzen- dubel & Polle, 2002). At toxic levels Cu will however interfere with physiological processes. Zn also serves as a micronutrient but can be toxic if present at high con- centrations (Schutzendubel & Polle, 2002). To minimize the potential effects of excess metal contaminants, the plant utilizes various homeostasis mechanisms which include the use of specialized transport proteins which serve as carriers mediating the transfer of heavy met- als across cell membranes (Lee et al., 2007). Cadmium has a negative effect on the absorption of essential nu- trients. It reduces ATPase activity and decreases the ex- change of ion H+/K+ in the plasmalema surface (Brzoska & Moniuszko-Jakoniuk, 2001). Page and Feller (2005) showed that the transfer of zinc, manganese, cobalt and cadmium in the leaves and roots of wheat were selective. When other minerals are in close proximity to cadmium, the amount of zinc in the root decreases (Page and Feller, 2005). Santos et al. (2014) showed that in the family of legumes, lead and cadmium adsorption was competitive. In this study, the concentration of zinc was eight times higher than that of cadmium, which indicates that zinc adsorption is preferable to cadmium. In plants treated with zinc and lead, lower concentrations of cadmium were observed in plant tissues in comparison to plants treated with cadmium alone. Zinc and lead along with cadmium compete for the sites of absorption and transfer (dos Santos et al., 2014). Chen et al. (2007) showed that manganese reduces the toxic effects of cadmium in corn. This suggests that manganese can be utilized to manage cadmium contamination (Chen et al., 2007). Zinc acts as a micro-element that is essential for plant growth and is part of the structure of regulatory enzymes and proteins. Zinc is very important in reducing cadmium toxicity and decreases the oxidative stress induced by cadmium. Some studies describe zinc and phosphorus interactions in plants (Marques et al., 2013). The phosphorus content in the aerial parts of plants treated with cadmium is relat- ed to the low zinc content in these sections. The negative correlation between zinc and phosphorus content in the shoots of cadmium treated plants explains the high con- tent of phosphorus in these plants (Sarwar et al., 2010). Analysis of cadmium and manganese content in this study supports the competitive theory of absorption of these two elements. The precise mechanism for promot- ing growth and reducing the toxic effects of cadmium is not well known. The uptake of various cations (K+, Ca2+, Mg2+, Mn2+, Zn 2+, and Fe2+) is severely affected by the presence of cadmium (Linger et al., 2005). Different types of proteins and adsorption carriers for cadmium are known such as NRAMP family (Thom- ine et al. 2000), P-type ATPase (Morel et al., 2009), ABC transporter (Kim, Gustin et al., 2004), CAX family, ZIP family (Pence et al., 2000), LCT transporter and CE fam- ily (Guerinot, 2000). Researchers report that cadmium has an antagonistic and synergistic effect on the micro- elements and macro elements in wheat. Many studies on the effect of cadmium inhibition on cell growth suggests the destruction of cell membranes and changes in min- eral levels (Rietra et al., 2017). Jibril et al. (2017), showed that the content of micronutrients and macro elements in different varieties of lettuce is significantly affected by cadmium levels. The study showed that cadmium (12 mg l-1) reduced essential elements by 72, 69, 56, 61 and 52 % (nitrogen, phosphorus, potassium and calcium, respec- tively). Copper content was higher in the root than the shoot of cadmium treated plants. This therefore reduces the effect of cadmium toxicity. Indeed, cadmium increas- es the absorption of copper, but prevents it from transfer- ring to the shoots (Jibril et al., 2017). Gomez et al. (2013) examined the effect of cadmium on nutrient distribution in Pfaffia glomerata (Spreng.) Pedersen. Plants were cul- tured with different minerals and cadmium concentra- tion was simultaneously increased over a 20 day period. The study showed that cadmium strongly affects the dis- tribution of microelements and macroelements in the roots and shoots. Despite the high toxicity of cadmium, the micro and macro nutrients present in plants are able to survive in contaminated environments (Gomes et al., 2013). Present study detected that at low concentrations of cadmium, the amount of manganese increased. With an increase in cadmium concentration, the level of manganese decreased in chickpea. Manganese plays a role in many biochemical functions, such as activating Acta agriculturae Slovenica, 119/3 – 202314 M. KOLAHI et al. enzymes involved in respiration, redox reactions, intra- cellular electron transfer systems, and the Hill reaction in chloroplasts, amino acid synthesis, and regulation of hormones (He et al., 2022). Manganese concentration was higher in the shoots than the root of plants treated with cadmium. The transfer of manganese to the shoot may in fact be a tolerance mechanism that reduces the effects of cadmium toxicity on photosynthesis. Research suggests that cadmium and manganese compete for the same membrane carriers (Socha & Guerinot, 2014). Dias et al. (2013) showed that at cadmium concentrations of 5 and 10 μm there was a significant decline in the min- eral content of lettuce leaves. At high concentrations of cadmium, a significant decline in manganese in the roots was observed. Cadmium appears to interfere with the transmission of macro and micro elements in the leaf (Dias et al., 2013). According to Guerinot, members of the ZIP and NRAMP or Ca channels and transporters which are responsible for the uptake of essential ele- ments are involved in the transport of cadmium via the same route (Guerinot, 2000). Imbalance in nutrient level and growth inhibition is ultimately due to competition between nutrients and toxic metals for binding sites in the cell. Sun and Shen (2007) explained that the decrease in concentrations of Mn, Fe, Mg, S, and P in the leaves of Cd-sensitive cultivars under cadmium stress is a con- tributing factor to the decline in photosynthesis and the decrease of cabbage growth (Sun & Shen, 2007). Heavy metal ATPases (HMAs), belong to the large P-type ATPase family located in the plasma membrane or tonoplast. They play an important role in the transport of metals in plants and provide resistance to the uptake and transportation of metals. The identified HMAs may contribute to the mechanisms by which chickpea plants manage, detoxify, or tolerate cadmium exposure. Under- standing the structure, function, and localization of these HMAs could offer new strategies for enhancing cadmium tolerance in chickpea, a crucial crop in many parts of the world. HMAs are classified based on substrate binding with one group bound to copper and silver and the oth- er to cadmium, lead and cobalt (Chkadua et al., 2022). HMAs 9 and 8 have been studied in rice and Arabidopsis, respectively. AtHMA1–4 in A. thaliana and OsHMA1–3 in Oryza sativa L. are in the first group and AtHMA5–8 and OsHMA4–9 in the second group. The expression of each of these genes is sensitive to heavy metals as indi- cated by mutagenesis. Typical P1B-ATPase proteins have been studied in various barley plants, Arabidopsis and poplar as well as in Thlaspi caerulescens J.Presl & C.Presl (Takahashi et al. 2012). In poplar (Populus trichocarpa Torr. & A.Gray ex. Hook), seventeen HMAs are known. PtHMA1 – PtH- MA4 belong to the subgroup of metals on cadmium, lead and cobalt. PtHMA5 – PtHMA8 belonging to the silver and copper groups have been identified. Most of these genes are located on chromosome 1 and 2 of poplar. On both sides of the P1B-ATPase C and N terminals there is also a metal binding site HMA4 in poplar which pro- duces mature RNA transcripts during alternative splic- ing of mRNA, containing approximately six hundred and twenty-six amino acids with an amino acid aver- age of ninety-eight. PtHMA in poplar are all in plasma membrane except PtHMA1 and PtHMA5.1 which are located in the cytoplasm. Poplar HMAs have 5 to 16 in- trons, PtHMA6, 5 introns, 8 PtHMA has 16 introns and 1 PtHMA has 5 introns with the remaining possessing 10 introns (Li et al., 2015). PtHMA1 – PtHMA4 belong to the subgroup of metals consisting of cobalt and cad- mium with the rest belonging to lead, silver and copper. There are 10 HMA genes related to silver and copper in poplar that are significantly higher than those in rice and Arabidopsis.2 OsHMA plays an important role in trans- mitting cadmium entry from the root to the stem and especially to rice grains (Li et al., 2015). OsHMA3 trans- ports cadmium to root cell vacuoles. Manipulating and altering the expression of these genes is a useful tool for reducing cadmium concentration in the seeds. AtHMA1 is within the chloroplast and zinc anti-toxic while AtH- MA 3 is present in the vacuolar membrane with zinc and cadmium playing a role. The motifs of poplar HMA are very similar to Arabidopsis and rice proteins and it seems that family members of these genes may be functionally divergent due to differences in gene organization and ex- isting motifs (Tian et al. 2023). AtHMA 1 and 2 are in the plasma membrane and in zinc and cadmium fluxes. OsHMA 1 is involved in zinc transfer. No HMA 4 type has been reported in rice. The number of HMA genes in the soybean genome is higher than that in Arabidop- sis and rice, probably due to duplication of the soybean genome. Phylogenetic study of these genes divides them into six groups, based on their divergent gene structure, conserved segments or protein motif patterns. Examina- tion of the cellular location of these proteins indicates that only GmHMA1 is involved in the secretion pathway while 1, 16, 17, 20, 20 peptides are mitochondrial targets, whereas 1, 2, 2, and 2 GmHMA2 are chloroplast peptides (Fang et al., 2016). Researchers have identified nine typi- cal P1B-ATPase in barley. HvHMA2, a P (1B)-ATPase is highly conserved among cereal crops with functionality in the transportation of zinc and cadmium. Addition- ally, HMA4 (Heavy Metal ATPase 4) has a key role in the translocation of cadmium in non-hyperaccumulating dicots, such as Arabidopsis thaliana (Mills et al., 2012). Acta agriculturae Slovenica, 119/3 – 2023 15 Investigating the growth characteristics, oxidative stress, and metal absorption of chickpea (Cicer arietinum L.) ... 5 CONCLUSION Chickpea seedlings exposed to cadmium exhibited changes in their morphological features which included changes in plant length, coloration and leaf size. The re- sults indicated that shoot and root length were signifi- cantly reduced. With the addition of cadmium (4 μg Cd g-1 perlite), stomatal densities on the upper epidermis decreased significantly but subsequently increased while higher concentrations of cadmium. Oxidative enzyme activities were also affected by cadmium stress. Oxida- tive enzyme activity (peroxidase, superoxide dismutase, catalase, ascorbate peroxidase) increased in the leaves of plants exposed to cadmium suggesting that these en- zymes play an integral role in combatting heavy metal contamination. Cadmium content in aerial parts of chickpea increased significantly. The study also revealed that by increasing cadmium concentration there was a significant reduction in the amount of copper and zinc transported to the aerial regions of the plant. Moreo- ver, at low concentrations of cadmium, the amount of manganese increased It has been suggested that there is a competitive mechanism for mineral uptake in plants. One may therefore be able to manage cadmium accumu- lation by varying the type of fertilizers utilized in culti- vating plants. In silico analysis led to the identification of 13 Heavy Metal ATPases (HMAs) in chickpea. These proteins contain 130 to 1032 amino acids with 3 to 18 exons. Comparison of the protein sequences of chickpea HMA with Arabidopsis indicated that there was great similarity between these proteins. The presence of a va- riety of genes indicates the various mechanisms utilized by chickpeas to combat heavy metal stress. 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Delo je prispelo 16. aprila 2023, sprejeto 31. avgusta 2023 1 Crop Improvement Unit, Crop Science Department, Faculty of Agriculture, University of Calabar, Calabar, Nigeria 2 Corresponding author, e-mail: e.e.obok@unical.edu.ng Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation Abstract: The study examines agromorphological traits and nutrient compositions in three genotypes of eggplants (Solanum melongena ‘African Beauty F1’ and ‘Melina F1’ and S. aethiopicum ‘Kotobi’) grown from seeds irradiated by gamma rays (γ-ray) with 100 Gy. Experiments were carried out in the screenhouse and experimental field of Crop Science Depart- ment, University of Calabar, Nigeria. Completely randomised design with four replications and randomised complete block design with three replications was used in the screenhouse and field experiments respectively. Eggplant × γ-ray effect re- duced (p ≤ 0.05) seedling emergence, plant height and number of leaves in the nursery at 2 and 4 weeks after sowing. In the field, these traits were consistently lower for irradiated Melina F1 and Kotobi (p > 0.05) at ten weeks after transplanting. Irradi- ated African Beauty F1 had the highest (p ≤ 0.05) upper can- opy leaf area (429.54 cm2), higher (p > 0.05) plant height and stem width; lower (p > 0.05) number of branches and leaves. Un-irradiated and irradiated Kotobi had the highest (p ≤ 0.05) fruit load, lower (p ≤ 0.05) fruit volume, weight and yields over four harvest intervals. Carbohydrate and energy contents of Kotobi fruits grown from 100 Gy gamma-ray irradiated seeds were concurrently improved (p ≤ 0.05). Gamma ray irradia- tion had both positive and negative influences on the agromor- phological traits, mineral composition and nutrient profile of eggplants. However, 100 Gy dose of irradiation had a negative effect on fruit characteristics in general. From the results of this study, inconsistent variations in the agromorphological traits of the irradiated eggplants of the three varieties were reported. Therefore, the goal of mutation breeding in eggplant should not undermine the importance of the eggplant genotype as well as the actual radiation dose. Key words: ℽ-ray, eggplant, fruits, induced mutation, ir- radiation, Solanaceae Obsevanje semen jajčevca z ℽ-žarki vpliva na rast rastlin, pri- delek in prehransko vrednost plodov v M1 generaciji Izvleček: Raziskava preučuje agromorfološke lastnosti in prehransko sestavo treh sort jajčevca (Solanum melongena ‘African Beauty F1’ and ‘Melina F1’ in S. aethiopicum ‘Kotobi’) vzgojenih iz semen obsevanih z gama žarki, jakosti 100 Gy. Po- skusi so bili izvedeni v rastlinjaku in na poskusnem polju usta- nove Crop Science Department, University of Calabar, Nigeria. V obeh primerih je bil poskus zasnovan kot popolni naključni bločni poskus s štirimi ponovitvami. Obsevanje semen jajčevca z gama žarki je zmanjšalo vznik sejank (p ≤ 0,05), višino rastlin in število listov v rastlinjaku dva in štiri tedne po setvi. V polj- skem poskusu so bile vrednosti teh parametrov vedno manj- še pri obsevanih sortah Melina F1 in Kotobi (p > 0,05) deset tednov po presaditvi. Rastline obsevane sorte African Beauty F1 so imele največjo listno površino (p ≤ 0,05; 429,54 cm 2), ve- čjo višino (p > 0,05) in večjo debelino stebla, a manjšo število stranskih poganjkov in listov (p > 0,05). Neobsevane in obse- vane rastline sorte Kotobi so imele največ plodov (p ≤ 0,05), manjši volume plodov (p ≤ 0,05), manjšo maso in pridelek v vseh štirjih obdobjih pobiranja plodov. Vsebnosti ogljikovih hi- dratov in energetska vrednost plodov sorte Kotobi, zrasle iz se- men obsevanih z 100 Gy gama žarki sta se izboljšali (p ≤ 0,05). Obsevanje semen jajčevca z gama žarki je imelo pozitivne in negativne učinke na agromorfološke lastnosti, mineralno sesta- vo in na prehranski profil plodov jajčevca. Doza obsevanja 100 Gy je imela nasplošno negativni učinek na lastnosti plodov. Iz rezultatov raziskave je razvidno, da so spremembe agromorfo- loških lastnosti jajčevca vseh treh obravnavanih sort, vzgojenih iz obsevanih semen nekonsistetne. Iz tega sledi, da cilji žlahtne- nja z mutacijami ne smejo prezreti pomena genotipa jajčevca kot tudi ne dejanskih doz obsevanja. Ključne besede: ℽ-žarki, jajčevec, plodovi, inducirane mutacije, obsevanje, Solanaceae Acta agriculturae Slovenica, 119/3 – 20232 E. OBOK et al. 1 INTRODUCTION Eggplant is a vegetable crop mostly cultivated in tropical and subtropical regions of the world. It belongs to the Solanaceae family and the genus Solanum with more than 90 genera comprising nearly 3,000 species (Melissa, 2017; Singh et al., 2006). Eggplant has been rec- ognized as the fifth most economically important Solan- aceous crop after potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and tobacco (Nicotiana tabacum L.) (FAO, 2014). Eggplant has a very low caloric value and is considered among the healthiest vegetables with high vitamin, min- eral and bioactive compounds (Raigon et al., 2008; Plazas et al., 2013; Docimo et al., 2016). It is very common in rich dishes such as stews and soups (Edem et al., 2009; Chinedu et al., 2008). The need for improved eggplant varieties for sustainable production and adaptation to cli- mate change challenges cannot be overemphasized. The low yielding ability of the crop has been attributed to lack of varietal replacement through development of hybrid and persistent use of traditional practices coupled with the influence of environmental degradation (Chinedu et al., 2008). Increasing crop yields is a major demand for assuring food security and as such mutagenesis is an important tool to improve crops (Beyaz et al., 2017). As an alternative to natural mutation, which can take years, inducing mutations with different mutagens has greatly aided breeding projects in a variety of ways. Many stud- ies have reported that genetic variability for numerous desired traits may be successfully created through mu- tations, and its application in plant development pro- grammes is well known (Chopra, 2005). Because of its penetrating capabilities, gamma irradiation is one of the most successful techniques of creating genetic diversity in plants when compared to other ionising radiations (Moussa, 2006), as well as in the production of new vari- eties (Animasaun, 2014; Mohamad et al., 2006). Gamma- ray photons have the shortest wavelength in the electro- magnetic spectrum, and therefore possess more energy which gives them the ability to penetrate deeper into the plant tissues (Amano, 2006). Accordingly, gamma irra- diation has been used to induce mutation and still shows great potential for improving vegetative plants (Predieri, 2001). Mutation breeding is utilised in addition to tradi- tional plant breeding because it has a stronger potential for enhancing plant architecture and resulting in im- proved crop development (Khin, 2006). Gamma rays are used in inducing mutations in seeds, and other planting materials such as cuttings, pollens or callus cultures (Ali et al., 2015). Gamma rays are also being widely used as mutation techniques in an attempt to improve morpho- logical and plant growth characteristics. For example, gamma ray irradiation was used to extend the shelf life of tomatoes (Antaryami et al., 2016) and to improve potato storage capacity (Nouani et al., 1987) as well as the mor- phological traits in pepper (Abu et al., 2020). This can also be of great value and benefit for the improvement of eggplant. The improvement of eggplants through crea- tion of variability using gamma rays would enable the selection of high yielding genotypes with improved agro- morphological characters and increase the crop’s agricul- tural productivity. Thus, the objective of this study was to assess the effect of gamma irradiation on the growth and yield traits of three varieties of eggplants. 2 MATERIALS AND METHODS 2.1 SOURCE OF SEEDS Seeds of three varieties of eggplants, African Beauty F1 (Solanum melongena L.), Kotobi (Solanum aethiopi- cum L.) and Melina F1 (Solanum melongena L.), were purchased from Technisem® (Longue-Jumelles, France). 2.2 IRRADIATION OF SEEDS Protocol for the irradiation of eggplant seeds was followed according to the National Institute of Radiation Protection and Research at the University of Ibadan, Ni- geria. The irradiator used was a Gamma-Photon Irradia- tor with model GammaBeamTM X200 (Best Theratronics Ltd., Canada). The samples were irradiated with 100 Gy of gamma rays. 2.3 EXPERIMENTAL SITE AND DESIGN The study was conducted at the University of Cala- bar Teaching and Research Farm, Calabar in two phases – the screenhouse and the field. Completely randomised design with four replications was used for the potted experiment in the screenhouse comprising while ran- domised complete block design with three replications was used in the field. 2.3.1 Screenhouse experiment The top-soil (15–20 cm depth) used for the screen- house experiment was obtained from the earmarked experimental field site. The friable, humus-rich topsoil was properly sieved, uniformly mixed, weighed and then transferred into conically shaped base-perforated plastic Acta agriculturae Slovenica, 119/3 – 2023 3 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation pots with the following dimensions: 20 cm – height, 8 cm – base radius and 10 cm – rim radius. The total volume of each pot was 5108 cm3. Three-quarters of the total vol- ume of each pot was filled with the prepared topsoil (i.e., 3831 cm3 of topsoil). The potted soil was sufficiently and uniformly wetted with 500 ml of irrigation water the after preparation of the seed bed. The seeds were primed in distilled water for 24 h prior to sowing on 2 March 2020. A total range of 30 seeds were sown in each pot. The total number of pots was 24. Seedlings of irradiated and un- irradiated seeds were raised indoors in potted nursery in the screenhouse. Subsequent irrigation was done for 22 days: at germination (thrice, 50 ml at three days interval) and emergence (thrice, 100 ml at two days interval). At full emergence and growth (≥ 22 days after sowing), 500 ml of the irrigation water was applied at two days interval for three weeks. Transplanting of vigorous seedlings was done at six weeks after sowing (WAS). The seedlings were transplanted at a height of 10–15 cm on 13 April 2020 us- ing the ball-of-earth method. The plant spacing was 0.6 m × 0.6 m and the gross treatment plot size was 2.4 m × 3.0 m (for 20 seedlings) giving a total plant population of 27,777 stands per hectare. 2.3.2 Field experiment The net plot size of 1.2 m × 1.8 m, comprising of six tagged stands of eggplants, was earmarked for growth and yield data collection. Organic fertilizer, poultry ma- nure (15 t ha-1), was applied by broadcasting to the soil at two weeks before transplanting. Inorganic fertilizer, NPK 15:15:15 (120 kg ha-1), was applied by ring method at 10 cm away from the base of the plants at two weeks after transplanting (WAT). Pest was controlled using a sys- tematic pyrethroid insecticide, Fighter 35 EC (Lambda- Cyhalothrin 15 g l-1 + Acetamiprid 20 g l-1). Foliar appli- cation at the rate of 640 ml ha-1 was done at two and four WAT; manual weeding (hand-hoeing and hand-rouging) was done concurrently. Manual harvesting of mature fruits was done between 65 and 95 days after transplant- ing (DAT) (Mahanta and Kalita, 2020). The fruits were hand-picked four times at 10 days intervals. 2.4 DATA COLLECTION Growth and yield data were collected on the num- ber of germinated seedlings that emerged in the screen- house, plant height, number of fully-opened leaves per plant, number of branches (primary and secondary), leaf area according to Rivera et al. (2007), stem width, fruit load (i.e., number of mature fruits per plant), fruit vol- ume based on water displacement method, fruit mass, and fruit yield (per plant and per hectare). 2.5 PROXIMATE ANALYSIS Moisture, crude protein (Kjeldahl method), fat (Soxhlet method), crude fibre and ash contents of the harvested fruits (mean of the four harvests) were deter- mined according to the standard procedures of Asso- ciation of Official Analytical Chemists (AOAC) (2010). Content of carbohydrates was calculated by percentage difference between 100 % (accepted total value of nutri- tional status) and the sum of the moisture, fat, ash, crude protein and crude fibre (Ovenuga, 1986). Calorific value (Kcal 100 g-1) was determined from crude protein, crude fat and carbohydrate values accordingly: [(Crude protein × 4.0) + (Crude fibre × 9.0) + (Car- bohydrate × 3.75)] (FAO, 2003). 2.6 DETERMINATION OF ESSENTIAL MINERALS Analysis of essential minerals in fruit samples were performed in three replicates, and data are presented as mean ± SD. Iron was determined following the method of Pearson (1976). Phosphorus was determined by mo- lybdate method as described by Onwuka (2005). Flame photometer was used to determine potassium by the pro- cedure described by Osborne & Voogt (1978). Calcium (extracted by the titrimetric method with EDTA) and Zinc were determined by atomic-absorption spectro- photometry (David, 1958; David, 1959). Magnesium was determined with disodium ethylenediaminetetra-acetate (Smith & McCallcum, 1956) and sodium was determined using ion chromatography (Basta & Tabatabai, 1985). 2.7 DATA ANALYSIS Treatments and replicates mean values of all the nursery and field data obtained were subjected to a two- way analysis of variance (ANOVA) using software Gen- Stat 16th Edition (VSN International, 2013). Turkey’s Acta agriculturae Slovenica, 119/3 – 20234 E. OBOK et al. Honest Significant Difference test (HSD) was used for significant treatment means separation at 95 % confi- dence limit. 3 RESULTS AND DISCUSSION 3.1 RESULTS 3.1.1 Soil physical and chemical properties Soil properties for the two experiments are pre- sented in Table 1. The screenhouse soil texture was sandy loam while the field had a loamy sand soil texture. Soil pH ranged from strongly acidic (4.9) to moderately acidic (5.9). Overall, screenhouse soil had higher cation- exchange capacity (CEC) and base saturation (BS) com- pared to the soil in the field. Both soils were suitable for the cultivation of eggplant. 3.1.2 Effects of irradiation (γ-ray) on growth of egg- plants in the nursery The effects of γ-ray radiation, eggplant variety and their interactions on seedling emergence, height and number of leaves were examined at two and four weeks after sowing (Table 2). At 2 weeks after sowing (WAS), the eggplant variety, Melina F1 had the highest seedling emergence (p ≤ 0.05) followed by Kotobi and African Beauty F1 varieties. ‘Kotobi’ was shorter in height (p ≤ 0.05) than ‘Melina F1’ and ‘African Beauty F1’. There was no significant difference (p > 0.05) in the average number of leaves for the three eggplant varieties. In general, control (no irradiation) had significantly (p ≤ 0.05) higher effect on seedling emergence and plant height, but no significant (p > 0.05) influenced on the number of leaves borne by each of the eggplant varieties. Following the interaction effect, un-irradiated Melina F1 eggplant variety had a 100 % seedling emergence while irradiated ‘African Beauty F1’ had the lowest seedling emergence (31.1 %). In terms of plant height, irradiated Melina F1 eggplant variety was the tallest (4.85 cm) and significantly (p > 0.05) different from irradiated ‘Kotobi’, the shortest (2.98 cm) eggplant variety. There was no sig- nificant difference in the number of leaves for eggplant × γ-ray interaction effect at 2 WAS. At 4 WAS, single ef- fects of eggplant and γ-ray radiation were significant (p ≤ 0.05) for plant height. γ-ray radiation did not lead to a significant (p > 0.05) variation in the number of leaves. However, the eggplant × γ-ray interaction effect showed that un-irradiated and irradiated ‘Melina F1’ plants were the tallest, similar to irradiated and un-irradiated ‘Afri- can Beauty F1’, but significantly different (p > 0.05) from irradiated ‘Kotobi’. Meanwhile, the number of leaves ranged from 3.72 (irradiated ‘Kotobi’) to 4.78 (un-irra- diated ‘Melina F1’). All other eggplant × γ-ray effects, ex- cept ‘Melina F1’, were similar (p > 0.05) to irradiated ‘Ko- tobi’ in terms of the average number of leaves per plant at 4WAS. 3.1.3 Effects of irradiation (γ-ray) on growth of egg- plants in the field The single effects of γ-ray radiation, eggplant vari- ety and their interactions on plant height, stem width, number of branches, number of leaves and leaf area (up- per, middle and lower canopies) were assessed at ten weeks after transplanting to field (Table 3). Varietal effect was only significant (p ≤ 0.05) for leaf area of the upper canopy while radiation effect was significant (p ≤ 0.05) for stem width and number of branches. ‘African Beauty F1’ had the largest leaves (382.85 cm 2) and was not sig- nificantly (p > 0.05) different from those of ‘Melina F1’ (339.75 cm2). Plants from un-irradiated eggplant seeds had thicker stems and more leaves than its counterparts from irradiated seeds. The eggplant × γ-ray interaction only had significant influence on leaf area of the upper canopy of the eggplants. The largest upper canopy leaf area was obtained from irradiated ‘African Beauty F1’ (429.54 cm2) while irradiated ‘Kotobi’ had the lowest up- per canopy leaf area (267.77 cm2). The general observa- tion was that all growth traits of un-irradiated ‘Melina F1’ were consistently higher than its irradiated group. A similar trend was observed for un-irradiated ‘Kotobi’, ex- cept for leaf area of the middle canopy where the irradi- ated group led with larger leaves. 3.1.4 Effects of irradiation (γ-ray) on yield of egg- plants at harvests Four harvests were made and at each of these har- vests, records were taken on several yield and yield-re- lated characters of the three eggplants varieties obtained from their irradiated and un-irradiated seeds. There were highly significant (p ≤ 0.05) variations observed for all the characters (Table 4). Fruit load (number of mature whole fruits per plant) ranged from 5.5 (‘African Beauty F1’ and ‘Melina F1’) to 51.5 (‘Kotobi’). Up to the third har- vest, with the exception of Melina F1, all eggplant varieties from un-irradiated seeds had either similar or higher fruit load in comparison with the irradiated group. At fourth harvest, all irradiated varieties had higher fruit load, ‘Af- rican Beauty F1’ recorded its highest. Volume and mass Acta agriculturae Slovenica, 119/3 – 2023 5 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation Ta bl e 1: S oi l p hy sic al a nd ch em ic al p ro pe rt ie s pH in H 2O (1 :2 5) Sa nd Si lt C la y O C TN Av ai la bl e P K + C a2 + M g2 + N a+ A l3+ H + C EC BS (g k g- 1 ) (% ) (m g kg -1 ) (c m ol (+ ) k g- 1 ) (% ) Sc re en ho us e 5. 9 69 0 15 0 16 0 2. 89 0. 24 82 .5 0 0. 13 6. 2 2. 2 0. 10 1. 16 0. 56 10 .1 5 83 .0 0 Fi el d 4. 9 84 3 54 10 3 1. 10 0. 14 23 .1 7 0. 10 1. 4 1. 2 0. 09 0. 56 1. 76 5. 11 54 .5 9 Ta bl e 2: S in gl e an d in te ra ct io n eff ec ts o f r ad ia tio n (γ -r ay ) a nd v ar ie ty o n gr ow th o f e gg pl an ts at tw o an d fo ur w ee ks a fte r s ow in g in th e nu rs er y Tr ea tm en t Em er ge nc e (% ) Pl an t H ei gh t ( cm ) N um be r o f L ea ve s Pl an t H ei gh t ( cm ) N um be r o f L ea ve s Tw o W ee ks A fte r S ow in g Fo ur W ee ks A fte r S ow in g Eg gp la nt A fr ic an B ea ut y F 1 46 .4 b 4. 64 a 2. 28 a 7. 66 a 4. 03 b Ko to bi 54 .2 b 3. 41 b 2. 09 a 5. 34 b 3. 97 b M el in a F 1 77 .9 a 4. 67 a 2. 17 a 8. 33 a 4. 72 a H SD 0. 05 <0 .0 1 <0 .0 1 0. 14 <0 .0 1 <0 .0 1 γ- ra y Ir ra di at io n 36 .6 7 b 4. 00 b 2. 13 a 6. 44 b 4. 15 a N o irr ad ia tio n 82 .3 2 a 4. 49 a 2. 22 a 7. 78 a 4. 33 a H SD 0. 05 <0 .0 1 0. 03 0. 25 <0 .0 1 0. 18 Eg gp la nt  × γ -r ay Ir ra di at ed A fr ic an B ea ut y F 1 31 .1 d 4. 52 a 2. 22 a 7. 16 a b 4. 06 b c U n- irr ad ia te d A fr ic an B ea ut y F 1 61 .7 c 4. 77 a 2. 33 a 8. 17 a b 4. 00 b c Ir ra di at ed K ot ob i 25 .6 d 2. 98 b 2. 07 a 4. 44 c 3. 72 c U n- irr ad ia te d Ko to bi 82 .8 b 3. 84 a b 2. 11 a 6. 24 b c 4. 22 a bc Ir ra di at ed M el in a F 1 53 .3 c 4. 49 a 2. 11 a 7. 72 a b 4. 67 a b U n- irr ad ia te d M el in a F 1 10 0. 0 a 4. 85 a 2. 22 a 8. 93 a 4. 78 a H SD 0. 05 0. 01 <0 .0 1 0. 92 <0 .0 1 <0 .0 1 H SD 0. 05 = T uk ey ’s ho ne st ly si gn ifi ca nt d iff er en ce te st at 9 5  % co nfi de nc e le ve l Acta agriculturae Slovenica, 119/3 – 20236 E. OBOK et al. Ta bl e 3: S in gl e an d in te ra ct io n eff ec ts o f r ad ia tio n (γ -r ay ) a nd v ar ie ty o n gr ow th o f e gg pl an ts at te n w ee ks a fte r t ra ns pl an tin g in th e fie ld Tr ea tm en t Pl an t H ei gh t (c m ) St em W id th (m m ) N um be r o f Br an ch es N um be r o f Le av es Le af A re a (U pp er C an op y) Le af A re a (M id dl e C an op y) Le af A re a (B ot to m C an op y) (c m 2 ) Eg gp la nt A fr ic an B ea ut y F 1 63 .7 2 a 17 .8 5 a 16 .2 6 a 42 .2 3 a 38 2. 85 a 30 0. 34 a 32 7. 08 a Ko to bi 63 .0 0 a 16 .9 5 a 16 .5 1 a 44 .5 6 a 27 8. 65 b 24 1. 82 a 26 6. 44 a M el in a F 1 61 .9 3 a 15 .0 0 a 12 .5 3 a 30 .5 6 a 33 9. 75 a b 26 2. 79 a 25 6. 35 a H SD 0. 05 0. 94 0. 11 0. 12 0. 14 0. 02 0. 24 0. 19 γ- ra y Ir ra di at io n 60 .2 3 a 15 .3 1 b 13 .1 7 b 33 .9 7 a 33 9. 08 a 27 5. 06 a 27 7. 98 a N o irr ad ia tio n 65 .5 3 a 17 .8 9 a 17 .0 4 a 44 .2 6 a 32 8. 42 a 26 1. 57 a 28 8. 60 a H SD 0. 05 0. 22 0. 03 0. 04 0. 10 0. 67 0. 62 0. 74 Eg gp la nt  × γ -r ay Ir ra di at ed A fr ic an B ea ut y F 1 65 .3 5 a 18 .0 2 a 12 .5 0 a 34 .4 6 a 42 9. 54 a 33 6. 85 a 32 4. 50 a U n- irr ad ia te d A fr ic an B ea ut y F 1 62 .0 8 a 17 .6 9 a 20 .0 2 a 50 .0 0 a 33 6. 15 a b 26 3. 84 a 32 9. 67 a Ir ra di at ed K ot ob i 56 .1 6 a 14 .3 8 a 15 .0 6 a 38 .4 3 a 26 7. 77 b 25 1. 25 a 25 6. 61 a U n- irr ad ia te d Ko to bi 69 .8 5 a 19 .5 1 a 17 .9 5 a 50 .6 8 a 28 9. 53 a b 23 2. 38 a 27 6. 27 a Ir ra di at ed M el in a F 1 59 .1 9 a 13 .5 3 a 11 .9 4 a 29 .0 3 a 31 9. 93 a b 23 7. 08 a 25 2. 84 a U n- irr ad ia te d M el in a F 1 64 .6 7 a 16 .4 6 a 13 .1 3 a 32 .0 8 a 35 9. 57 a b 28 8. 50 a 25 9. 85 a H SD 0. 05 0. 28 0. 14 0. 29 0. 65 0. 01 0. 21 0. 98 H SD 0. 05 = T uk ey ’s ho ne st ly si gn ifi ca nt d iff er en ce te st at 9 5  % co nfi de nc e le ve l Acta agriculturae Slovenica, 119/3 – 2023 7 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation Ta bl e 4: E ffe ct s o f r ad ia tio n (γ -r ay ) × v ar ie ty × h ar ve st in te rv al o n yi el d of e gg pl an ts Tr ea tm en t C om bi na tio n  Fr ui t L oa d Fr ui t V ol um e (m l3 ) Fr ui t M as s ( g) Fr ui t Y ie ld (k g pl an t-1 ) Fr ui t Y ie ld (t h a-1 ) Fi rs t H ar ve st Ir ra di at ed A fr ic an B ea ut y F 1 5. 50 i 9. 35 a b 50 5. 05 a 2. 30 cd 62 .5 5 e U n- irr ad ia te d A fr ic an B ea ut y F 1 5. 50 i 8. 15 d 32 0. 05 f 1. 55 e 41 .7 2 i Ir ra di at ed K ot ob i 23 .5 0 d 3. 85 j 17 .4 1 r 0. 42 ij kl 10 .2 3 q U n- irr ad ia te d Ko to bi 46 .6 4 b 3. 68 jk l 18 .2 3 q 0. 69 h i 17 .9 1 n Ir ra di at ed M el in a F 1 5. 50 i 4. 92 g h 24 1. 72 n 1. 05 f 27 .8 3 k U n- irr ad ia te d M el in a F 1 7. 90 h i 5. 31 e f 29 6. 48 k 1. 79 e 48 .3 8 g Se co nd H ar ve st Ir ra di at ed A fr ic an B ea ut y F 1 5. 50 i 9. 25 a b 49 0. 05 b 2. 45 c 66 .7 1 c U n- irr ad ia te d A fr ic an B ea ut y F 1 5. 50 i 8. 85 c 45 0. 05 d 2. 15 d 58 .3 8 f Ir ra di at ed K ot ob i 13 .3 0 f 3. 85 j 17 .2 2 r 0. 23 l 5. 05 t U n- irr ad ia te d Ko to bi 51 .5 0 a 3. 78 jk 15 .7 6 u 0. 75 g h 19 .4 9 m Ir ra di at ed M el in a F 1 11 .5 0 fg 5. 05 fg 27 7. 55 l 2. 15 d 58 .3 8 f U n- irr ad ia te d M el in a F 1 5. 50 i 4. 65 h i 27 1. 30 m 1. 03 fg 27 .1 3 l Th ird H ar ve st Ir ra di at ed A fr ic an B ea ut y F 1 5. 50 i 9. 15 b 45 0. 05 d 2. 15 d 58 .3 8 f U n- irr ad ia te d A fr ic an B ea ut y F 1 9. 50 g h 8. 81 c 38 4. 05 e 2. 95 b 80 .6 0 b Ir ra di at ed K ot ob i 18 .0 0 e 3. 45 l 15 .4 6 v 0. 28 k l 6. 30 s U n- irr ad ia te d Ko to bi 36 .8 3 c 3. 45 l 16 .1 2 t 0. 55 h ijk 13 .9 4 p Ir ra di at ed M el in a F 1 5. 50 i 4. 95 g 30 5. 05 h 1. 15 f 30 .6 0 j U n- irr ad ia te d M el in a F 1 11 .5 0 fg 4. 53 i 18 6. 72 o 1. 75 e 47 .2 7 h Fo ur th H ar ve st Ir ra di at ed A fr ic an B ea ut y F 1 17 .5 0 e 8. 32 d 31 8. 38 g 4. 55 a 12 5. 05 a U n- irr ad ia te d A fr ic an B ea ut y F 1 8. 50 h 9. 47 a 48 0. 05 c 4. 55 a 12 5. 05 a Ir ra di at ed K ot ob i 36 .1 7 c 3. 51 k l 16 .4 1 s 0. 58 h ij 14 .8 6 o U n- irr ad ia te d Ko to bi 21 .3 2 d 3. 71 jk l 21 .3 0 p 0. 40 jk l 9. 90 r Ir ra di at ed M el in a F 1 11 .5 0 fg 5. 37 e 29 8. 18 j 2. 38 cd 64 .6 3 d U n- irr ad ia te d M el in a F 1 10 .0 0 gh 5. 52 e 30 3. 26 i 2. 15 d 58 .3 8 f H SD 0. 05 <0 .0 00 1 <0 .0 00 1 <0 .0 00 1 <0 .0 00 1 <0 .0 00 1 H SD 0. 05 = T uk ey ’s ho ne st ly si gn ifi ca nt d iff er en ce te st at 9 5% co nfi de nc e le ve l Acta agriculturae Slovenica, 119/3 – 20238 E. OBOK et al. Fi gu re 1 : C om pa ra tiv e pr ox im at e co m po sit io ns o f γ -r ay ir ra di at ed a nd u n- irr ad ia te d eg gp la nt s Acta agriculturae Slovenica, 119/3 – 2023 9 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation Fi gu re 2 : C om pa ra tiv e m ic ro nu tr ie nt p ro fil es o f γ -r ay ir ra di at ed a nd u n- irr ad ia te d eg gp la nt s Acta agriculturae Slovenica, 119/3 – 202310 E. OBOK et al. of eggplant fruits from irradiated seeds were the highest for ‘African Beauty F1’ up to the third harvest. Fruits of ‘Melina F1’, from both irradiated and un-irradiated seeds, were significantly (p ≤ 0.05) lighter in mass and smaller in volume than ‘African Beauty F1’. Characteristically, ‘Kotobi’ had higher fruit bearing attribute indicated by its high fruit load but with smaller fruit volume and lighter mass. The average fruit yield per plant ranged from 0.23 kg to 4.55 kg. The highest fruit yield on plant basis was recorded from ‘African Beauty F1’ (irradiated and un-irradiated seeds) at fourth harvest. Fruit yield (plant-1 and hectare-1) of ‘Kotobi’ was generally lower than other eggplant varie- ties across the four harvest intervals. ‘African Beauty F1’ maintained the same trend for fruit yield on per hectare basis. 3.1.5 Effects of irradiation (γ-ray) on comparative proximate composition of eggplants Proximate analysis of freshly harvested fruits of the three eggplants varieties in our study showed that fruits obtained from plants grown from γ-ray irradiated seeds had significantly (p ≤ 0.05) lower moisture content, crude protein, crude fibre, crude fat and ash content (Figure 1). In the other hand, all eggplants fruits from un-irradiated seeds had lower carbohydrate across the three varieties. Fruits of Kotobi variety had the lowest moisture content (70–77 %) followed by African Beauty F1 (85–89 %) and Melina F1 (89 – 92 %). Crude protein ranged from 17 % (irradiated ‘Kotobi’) to 28 % (un-irradiated ‘Melina F1’). Irradiated ‘Kotobi’ also had the lowest crude fibre (2.95 %) and crude fat (3.06 %), but had a significantly higher carbohydrate content (71.88 %). Although un-ir- radiated ‘Melina F1’ had the lowest carbohydrate content (55.09 %), its energy value was the highest (385.9 Kcal 100 g-1) which was not significantly (p > 0.05) differ- ent from the un-irradiated ‘Melina F1’ (385.72 Kcal 100 g-1). Irradiated (378.68 Kcal 100 g-1) and un-irradiated (378.88 Kcal 100 g-1) fruits of ‘African Beauty F1’ had the lowest energy values (p > 0.05). Overall, ‘Kotobi’ had the highest ash content (4.72–4.92 %) followed by ‘Melina F1’ (3.61–3.81 %) and ‘African Beauty F1’ (3.04–3.15 %). 3.1.6 Effects of irradiation (γ-ray) on macro- and micro-nutrients profile of eggplants There were significant (p ≤ 0.05) differences among the three eggplant varieties in micronutrient profiles of fruits obtained from γ-ray irradiated and un-irradiated seeds (Figure 2). In general, un-irradiated Kotobi egg- plant variety had the richest nutrient contents: sodium (47.18 mg 100 g-1), calcium (8.11 mg 100 g-1), magnesium (14.36 mg 100 g-1), phosphorus (21.02 mg 100 g-1), potas- sium (196.15 mg 100 g-1) and zinc (0.47 mg 100 g-1). With exception of zinc (varieties: Kotobi > African Beauty F1 ≥ Melina F1), the micronutrient profile richness followed the varietis order: Kotobi > Melina F1 > African Beauty F1. It was observed that Na, Mg and K contents followed the same trend in the eggplant fruits for grown from both irradiated and un-irradiated seeds. 4 DISCUSSION The use of gamma ray irradiation on eggplant va- rieties has helped in recent years to induce favourable mutation and improve agronomic attributes of the crop. There are reports that gamma ray could affect the growth and yield of eggplant. Contrary to Zanzibar and Sudrajat (2016) report that gamma ray irradiation could improve seed metabolism and stimulate seed germination, our re- sults consistently showed that seedling emergence in the nursery was however higher from un-irradiated (100 Gy) eggplant seeds of African Beauty F1, Kotobi and Melina F1 varieties. In comparison, Rozman (2014) found that the percentage of germination of barley (Hordeum vulgare L.) seeds irradiated with 100 Gy did not differ from the un-irradiated in the first year, it was significantly higher in the fifth year. Also, Suparno (2018) conducted a study on the phenotypic diversity of eggplant (S. melongena L.) resulting from various doses of gamma-ray irradiation (0, 100, 150 and 200 Gy). The results showed that gamma ray irradiation resulted in high significant differences in seedling growth, 100 Gy giving the highest percentage of seedlings emergence (77.5 %) and contrary to our report where we had a range of 31.1 to 53.3 %. However, our results on the effect of gamma ray on plant height of egg- plant (14 and 28 days after planting) grown from irradi- ated seeds was in consonant with Suparno (2018) who reported taller plants from un-irradiated seeds (4.19– 10.45 cm) over 100 Gy irradiated seeds (3.99–10.38 cm). Another study conducted by David et al. (2018) on the effects of gamma irradiation on the agromorphologi- cal traits of two eggplant (S. aethiopicum L.) accessions conforms with our findings which showed reductions in germination percentage (in the nursery) and plant height (nursery and field) at irradiation dose of 100 Gy when compared with plants grown from un-irradiated seeds (control). Although David et al. (2018) had reported that irradiation doses of 40 Gy and 60 Gy were appropriate in creating beneficial agronomic traits in S. aethiopicum L. accessions, these were not consistent between the egg- plant accessions. In support of our findings, Muhammad et al. (2021) reported that the growth, development, and survival rate of Bambara groundnut (Vigna subterranea Acta agriculturae Slovenica, 119/3 – 2023 11 Gamma irradiation of eggplant seeds influences plant growth, yield and nutritional profile in M1 generation (L.) Verdc.) increased with a decrease in gamma-irra- diation. In our study, we also observed similar varietal differences for ‘Kotobi’ (S. aethiopicum L.) in terms of stem width, number of branches, number of leaves and leaf area at different canopy heights in the field. Eggplant fruits have high contents of carbohydrates, proteins and some minerals such as Ca, Mg, and P (Kowalski et al., 2003; El-Nemr et al., 2012) and have low calories (25 kcal 100 g-1) (Aly et al., 2019). We reported higher amount of mineral composition in the un-irradiated eggplants group over the irradiated ones. Aly et al. (2019) reported that eggplant growth increased when using a dose of 50 Gy gamma rays while increasing irradiation dose level to 100 Gy reduced phenyl alanine ammonia-lyase (PAL) enzyme and polyphenol oxidase enzyme activities which influences plant growth and invariably its accumulation of some active compounds. Additionally, these enzymes could also have a role in mineral accumulation. Gamma rays are one type of ionising radiation that interact with atoms or molecules to produce free radicals in cells, ac- cording to Aly et al. (2019). These radicals can change essential constituents of plant cells. In contrast to Hus- sein et al. (2012) that treating seeds before sowing by gamma radiation (40–80 Gy) generally increased Na and K in growing damsisa plant (Ambrosia maritima L.) com- pared by its corresponding un-irradiated control, our Na and K contents were higher in un-irradiated eggplants. Also, our results on Ca content in fruits of plants pro- duced from irradiated seeds (except for ‘Melina F1’) was similar to Hussein et al. (2012) for Ca in A. maritima at fruiting even under salinity stress. It was reported that exposure of red radish (Raphanus sativus L.) seeds to gamma irradiation before cultivation improved the root contents of the elements (N, K, S, P, Ca, and Mg) (El- Beltagi et al., 2022). In the study, it was clear that the dose of 100 Gy had different effects on the fruit characteris- tics of eggplants according to the genotypes. For exam- ple, while 100 Gy increased the amount of carbohydrate and energy in ‘Kotobi’ genotype, Ca content increased in Melina F1 genotype, Zn content increased in African Beauty F1 genotype. Our research clearly shows that gam- ma-ray irradiation of eggplant is genotype dependent as a technique of producing variation for the generation of new genotypes. Our findings correspond with those of Ulukapi et al. (2015), who discovered inconsistencies in determining optimal gamma radiation dose in eggplant mutation breeding. The preservation, decrease, or in- crease in agromorphological traits, mineral and nutrient compositions of plants grown from irradiated eggplant seeds compared to the control plants made it a problem- atic task to clearly highlight the behaviour of eggplants in response to gamma ray irradiation. Consistent with our study, studies in different plants show that variations in growth and yield traits in response to gamma ray irradia- tion is dependent on the crop variety as well as the radia- tion dose (Rozman, 2014; Majeed et al., 2018; Aparecida Costa Nobre et al., 2022; Puripunyavanich et al., 2022; Saibari et al., 2023) and as such it is difficult to establish a standard dose for mutation breeding in eggplants. 5 CONCLUSIONS Though variations were created, gamma ray irra- diation dose of 100 Gy had inconsistent influences on the agromorphological traits and nutritional profile M1 generation of ‘African Beauty F1’ (Solanum melongena L.), ‘Kotobi’ (Solanum aethiopicum L.) and ‘Melina F1’ (Solanum melongena L.) eggplant varieties. Plants grown from irradiated eggplant seeds were negatively affected in terms of the following fruit characteristics: fruit load, fruit volume, fruit mass and fruit yield compared to egg- plants grown form un-irradiated seeds. 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Proceedings of the 4th In- ternational Conference on Energy Systems, Environment, Entrepreneurship and Innovation. Athens: WSEAS Press. VSN International (2013). GenStat for Windows 16th Edition. VSN International, Hemel Hempstead, UK. Zanzibar, M. & Sudrajat, D. (2016). Effect of gamma irradiation on seed germination, storage, and seedling growth of Mag- nolia champaca L. Indonesian Journal of Forestry Research, 3, 95–106. https://doi.org/10.20886/ijfr.2016.3.2.95-106 Acta agriculturae Slovenica, 119/3, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.3.13508 Original research article / izvirni znanstveni članek Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators Albert T. MODI 1, 2 Received March 05, 2023; accepted August 06, 2023. Delo je prispelo 5. marca 2023, sprejeto 6. avgusta 2023 1 School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, Pietermaritzburg, South Africa 2 Corresponding author, e-mail: albertmodi6@gmail.com Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators Abstract: The objective of this study was to extend the measure of seed quality beyond seed germination using three common bean (Phaseolus vulgaris. L) cultivars. Under labora- tory conditions, total seed germination was included in calcu- lation of other seed performance measures, mean germination rate and germination vigour index. These parameters were used to produce a new parameter, total potential value for germina- tion. The laboratory measures were duplicated under field con- ditions over two seasons to produce comparable data for seed- ling emergence, mean emergence rate and emergence vigour index. Consequently, total potential value for emergence was derived. The crop was grown under field conditions at three seeding rates (177 000 plants ha-1, 150 000 plants ha-1 and 115 000 plants ha-1). Prediction of seed performance under field conditions was extended by measuring plant size from the first trifoliate to initiation of reproductive stage. During this period, new measures comparable to those of laboratory seed vigour and emergence vigour were derived on the basis of vegetative growth vigour, resulting in total potential value of plant growth. The study revealed that germination and plant growth can be correlated using vigour indices. Key words: emergence, germination, growth index, seed vigour Razmerje med laboratorijskimi in poljskimi indikatorji kako- vosti semen navadnega fižola (Phaseolus vulgaris L.) Izvleček: Namen raziskave je bil določiti poleg kalitve še dodatne kakovostne indikatorje semen treh sort navadnega fi- žola (Phaseolus vulgaris. L). V laboratorijskih razmerah je bila celokupna kalivost semen vključena v izračun dodatnih meril določanja kakovosti semen kot sta poprečna kalivost in indeks kalitvenega vigorja. Ti parametri so bili uporabljeni za izdelavo novega parametra, imenovanega celukopni potencial kalitve. Laboratorijski postopki so bili podvojeni v razmerah poljske- ga poskusa v dveh rastnih sezonah za pridobitev primerljivih podatkov za vznik kalic, poprečno vrednost vznika in indeks vigorja vznika. Iz teh podatkov je bila izračunana poprečna ce- lokupne vrednost vznika. Posevek je rastel v treh gostotah (177 000 rastlin ha-1, 150 000 rastlin ha-1 in 115 000 rastlin ha-1). Na- poved uspešnosti rasti v poljskih razmerah je bila narejena na meritvah velikosti rastlin of prvega trojnatega lista do začetka reproduktivne faze razvoja. V tem obdobju so bila pridobljena merila za vigor rasti in celokupno potencialno vrednost rasti podobna tistim v laboratoriju, ki so določala vigor semen in vznika. Raziskava je pokazala, da bi kalitev in rast rastlin lahko korelirali z uporabo indeksov vigorja. Ključne besede: vznik, kalitev, indeks rasti, vigor semen Acta agriculturae Slovenica, 119/3 – 20232 A. T. MODI 1 INTRODUCTION Seed quality is an important determinant of plant genetic material performance under a wide range of en- vironmental conditions. Its assessment can be done to fit the purpose of the researcher in the laboratory where quick physiological responses can be adequate. However, for purposes of commercial crop production and long- term genetic preservation, it is necessary to use a reliable method whose results can be interpreted meaningfully and linked to crop or germplasm performance to pro- tect the plant breeder, the seed market, and the farmer (Bishaw & Turner, 2008; Fajardo et al., 2010; Francki et al., 2021). Seed germination has been tested and accepted as a reliable method to test seed quality. To accommodate its limitations, which can be linked to the effects of envi- ronment e.g., temperature, moisture as well as pre-and post-harvest growth and management conditions (Lou- waars & Manicad, 2022), international standards accept seminal root protrusion as a baseline indicator of quality in the context of laboratory seed germination. However, good seed quality is expected to provide a significant genotypic contribution of crop resilience to environmental conditions associated with soil, weather and management conditions of the farm. Seed germina- tion is the basic measure of seed quality recognised by scientists and producers. Previous studies have shown that seed germination response can be expanded to de- termine other laboratory related parameters, mainly seed vigour, which is based on germination rate and seedling size (Farshid et al., 2019; Hassani et al., 2019). Hence, germination and vigour are commonly used to- gether for quality determination because they are linked. However, seed germination can be used independently to recommend crop potential performance under field conditions (Beveridge, 2020). For example, it is estimat- ed that rapid seminal root protrusion under laboratory conditions must be a minimum of 90 % for it to be con- sidered for optimum production, but 100 % seed germi- nation is required (Allen & Meyer 1990, 1998; Rajendn- dra, 2023). However, there is no conclusive evidence that seed germination parameters are always linked to crop establishment, growth and final yield (Ellis, 1992). It is generally accepted that relating seed quality directly with plant performance parameters is difficult to achieve. This relationship may be implicitly indicated by special defini- tion of vigour. Previous studies have indicated that this relationship can be shown in theory. It was suggested that seed germination, vigour and size are three aspects of quality that may indirectly influence percentage emer- gence and time from sowing to emergence (Ellis, 1992). These factors may implicitly influence yield by altering plant population density, spatial arrangement, and crop performance. Seed vigour has become a reliable seed quality measure to confirm results of seed quality, includ- ing genetics, for both cultivated and other plant species (Priyanka et al., 2019). Previous studies have shown that plant population affects growth and yield (Ihsanullah et al., 2002). In view of rapidly changing environmental rigours for crop production, due to climate change, it is important to trace and relate seed quality aspects from the laboratory to a wide range during crop growth and development (Akinci et al. 2008; Singh, 2014). Hence, the objective of this study was to provide a practical method of explaining the concept of seed quality based on labo- ratory and field-based methods in order to produce new indices that have not been shown in seed science studies before. 2 MATERIALS AND METHODS 2.1 LABORATORY SEED QUALITY DETERMINA- TION Fresh seeds of three common bean cultivars, Ukul- inga, Gadra and Mthatha were donated by Pro-seed cc (https://www.africanadvice.com) from a plant breeding stock. Seed germination percent (G), shown as seminal root protrusion, was determined according to Interna- tional Seed Testing Association guidelines (ISTA, 2013) for a total period of seven (7) days. The paper towel method was used at 25 oC and replicated four times, with 25 seeds per replication. In addition, mean germination rate (MGR) and germination vigour index (GVI) were determined according to modification of Thanuja et al. (2019). For mean germination rate: MGR = (Σ D n)/ (Σ n) (Equation 1) Where, MGR is mean germination rate, D is the number of days from the beginning of germination, and n is the number of seeds that have germinated on day D. This value indicates the average rate of seed germination as indicated by seminal root protrusion. For germination vigour index (GVI): GVI = G1/N1 + G2/N2 +… + Gn/Nn (Equation 2) Where, G1, G2…Gn = number of germinated seeds in the 1st, 2nd… last count (n), and N1, N2…Nn = num- ber of germination days at the 1st, 2nd… last count (n). This value indicates the rate of germination daily. Labo- ratory seed quality was taken to have different aspects from minimum (G), moderate (MGR) and high (GVI) Acta agriculturae Slovenica, 119/3 – 2023 3 Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators indicator, respectively. The reason for this was that the value of germination is incrementally improved by con- sidering the rate (MGR) and then the rate combined with potential impression of physiological factors (Taiz et al., 2018, Takahashi et al. 2018) that affect robustness (GVI). To complete that seed quality view of aspects, the study calculated a new parameter, total potential value for ger- mination (TPVg). TPVg = GVI/N (Equation 3) Where, GVI is germination vigour index (see equa- tion 2 above) and N is the total germination period (7 days). This value indicates the number of seeds in rela- tion to vigour. 2.2 FIELD TRIAL FOR SEED QUALITY DETERMI- NATION AND CROP GROWTH A field trial was undertaken (29o37’45 S 30o 24’ 17” E) and repeated during the normal planting season. The experimental design was a split-plot in a randomised complete block, replicated three times. There were two factors used, namely three intermediate growth cultivars (Ukulinga, Gadra and Mthatha) and three plant densi- ties [high (177 000 plants/ha), medium (150 000 plants/ ha) and low (115 000 plants/ha)]. The variables were emergence and above ground plant size (mm). Prior to planting, soil analysis was performed to determine suit- able fertiliser application for common bean (Liebenberg, 2010). 2.3 SEED QUALITY DETERMINATION UNDER FIELD CONDITIONS Seedling emergence was monitored daily for a pe- riod of seven (7) days from planting. Total emergence percent (E), mean emergence rate (MER), daily rate of emergence EVI and total potential value for seedling emergence (TPVe) were determined using the same for- mulae as for laboratory seed quality. 2.4 PLANT GROWTH PARAMETERS From emergence, non-destructive evaluation of plant growth (cm) was determined. An average of five randomly selected plants per plot (two middle rows of a 2 m2 area used as one replicate) was used to measure plant growth weekly, between VE (emergence) and R1 (initia- tion of flowering) (Rahman et al., 2011). Accordingly, plant growth (P) was monitored for seven (7) growth stages from the first trifoliate (V1) to initiation of repro- ductive stage (R1). Plant size (MPI) and plant growth vigour index (PVI) were used to calculate potential value for plant growth (TPVp) using the same formulae as for laboratory seed and emergence quality. 2.5 STATISTICAL ANALYSIS Data were subjected to analysis of variance using GenStat® Version 18 (VSN International, United King- dom) at the 5  % probability level (p ≤ 0.05). Duncan’s Multiple Range test was used to compare means. There were no significant differences between seasons. There- fore, no data to compare the two growth seasons are shown. 3 RESULTS AND DISCUSSION Cultivar differences with respect to germination were significant (p = 0.03), with ‘Ukulinga’ showing com- plete germination by the fourth day. ‘Gadra’ was better than ‘Mthatha’, but both of these cultivars did not rich 100 % germination. The trend of differences between cul- tivars was consistent throughout the germination period (Table 1). With respect to germination rate, differences between cultivars diminished over time, so that by the fourth day there were no significant differences (Table 1). High germination was associated with a steady germina- tion rate, whereas delayed germination continued to have a high germination rate until the end of incubation pe- riod (Table 1). Cultivar differences with respect to emer- gence followed a similar trend to that of germination (p = 0.01). ‘Ukulinga’ showed complete emergence five days after planting, but all cultivars emerged completely sev- en days after planting (Table 1). Rate of emergence also showed a similar trend to that of germination (Table 1). Seeding rate had no effect on emergence. For all cultivars germination was highly signifi- cantly correlated with germination rate index (Figure 1A). Emergence was highly significantly correlated with emergence rate index (Figure 1B). Plant growth was highly significantly correlated with plant growth index (Figure 1C). Plant growth from the first plant trifoliate to initial reproduction stage showed no significant differences be- tween seeding rates and cultivars, overall (Figure 2). When the total potential seed value for germination was compared with that for emergence and plant growth to flowering, it was clear that both emergence and plant growth are highly correlated with seed quality (Figure 3). Acta agriculturae Slovenica, 119/3 – 20234 A. T. MODI Seed germination has been a reliable measure of seed quality in science and for agricultural production regardless of system (Sako et al., 2001). Both controlled environment nursery production and widely variable field conditions rely mainly on seed germination percent as the primary indicator of seed quality (Rahman et al., 2011). Over time, science has developed other measures of seed quality to test seed response to factors associat- ed with harsh conditions for growth, including imbibi- tion, high mineral content and suboptimal temperature and water conditions (Priyanka et al., 2019). This led to seed vigour being an additional seed quality measure closely associated with seed germination (ISTA, 2013). The usefulness of other seed quality measures associated with germination is generally limited to laboratory ex- periments and decisions for micro-level interpretation of seed quality (Ellis, 1992; Farshid et al., 2019). This study attempted to expand the meaning of seed quality beyond relying on seed germination as the most important meas- ure (Beveridge, 2020; Kildisheva et al., 2019). Advantage was taken of seed vigour determined by seed germina- tion rate, which can also be linked to seedling size in the laboratory (Grafton et al., 1988). Seed germination was found to be directly linked to emergence, but it does not guarantee a perfect match in that emergence can be over- estimated if one uses germination alone (Ambika et al., 2014). However, it was found useful to continue to use both germination (a clear indicator of seed viability in terms of seminal root protrusion under favourable con- ditions) and emergence (a clear indicator of the ability of seed to produce a seedling under optimum soil and climate conditions). A comparison of data in Table 1 with Figures 1A and 1 B clearly confirms this argument. Fur- ther, when plant size was related to growth rate (Figure 1C), there was a consistent comparison with what hap- pened when germination and emergence were similarity related to what would be a direct physiological response to them, mean germination rate and mean emergence rate, respectively. This comparison allowed the basic measure of seed quality, germination to be indirectly linked to measures of plant performance under field con- ditions, regardless of cultivar or seeding rate (Figure 2). Further consideration of all known laboratory seed quality indicators, germination, rate of germination and germination vigour index led to a new index of seed qual- ity, total potential value for germination (TPVg). This in- dex was directly comparable to those for field emergence (TPVe) (Figure 3). From these results, it can be assumed that the direct relationship between laboratory seed qual- ity index with plant performance in the field is possible. The study showed that it is possible to replicate seed vigour measures during the early stages of crop estab- lishment. This was shown by the significant similarity of mean germination test results to mean emergence test, as well as germination vigour index and emergence vig- our index. The reliability of these new comparisons be- tween simple measures of seed quality (germination and emergence) led to the interest in producing more indirect measures of seed value that may be implicitly related to seed quality beyond germination and emergence. This study expanded the concept of emergence to crop estab- lishment as well as growth and development under field Table 1: Comparison common bean cultivars (Gadra, Mthatha and Ukulinga) with respect to germination (G) and emergence (E) as well as their respective daily rates (MGR and MER) over a period of seven days (Day 1 to Day 7) of laboratory incubation and field planting, respectively. Values sharing the same letters are not significantly different (p ≤ 0.05) Variable Cultivar Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 G Gadra 0a 50b 80b 90b 95b 95b 95b Mthatha 0a 20a 60a 80a 90a 90a 90a Ukulinga 20b 60c 95c 100c 100c 100c 100c MGR Gadra 0a 0.16a 0.15a 0.18ab 0.21a 0.25a 0.29a Mthatha 0a 0.4b 0.2b 0.2b 0.22a 0.27a 0.31a Ukulinga 0.2b 0.13a 0.13a 0.16a 0.2a 0.24a 0.28a Variable Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 E Gadra 0a 0a 20a 60a 80a 100a 100a Mthatha 0a 0a 40b 85c 95b 100a 100a Ukulinga 0a 30b 60c 80b 100c 100a 100a MER Gadra 0a 0a 1c 0.44b 0.42b 0.4a 0.47a Mthatha 0a 0a 0.5b 0.31a 0.35a 0.4a 0.47a Ukulinga 0a 0.44b 0.33a 0.33a 0.33a 0.4a 0.47a Acta agriculturae Slovenica, 119/3 – 2023 5 Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators Figure 1: Comparison of germination (A) (Note: Germ = germination; Rate = germination rate), emergence (B) and plant size (C) with rates of seed germination, seedling emergence and plant growth, respectively. Germination and emergence occurred over a period of seven days (D1 to D7). Growth occurred over seven stages from the first trifoliate (V1) to initial reproduction (R1). Cor- relation across all parameters was indicated using cultivar Ukulinga mean values to represent the general trend Acta agriculturae Slovenica, 119/3 – 20236 A. T. MODI conditions using different cultivars and seeding rates (Grafton et al., 1988). Hence, the potential value of seed performance beyond germination was shown when a new measure of total potential value under germination (TPVg) was comparable to that derived for emergence (TPVe) and plant growth (TPVp). Both indicators were comparable to germination and emergence performanc- es of the crop. Further, the study was able to show that following the monitoring of vegetative growth stages, a new index of perceived seed potential can be determined based on crop growth vigour sub-indices. The vegetative growth rate index and growth range index were derived simply from measurement of plant size in the field. In combination, they were useable to predict growth poten- tial index. 4 CONCLUSION The results of this study confirm the parameters for seed quality determination which are already accepted by the International Institute for Seed Testing Association Figure 2: Plant size of three common bean cultivars (Gadra, Mthatha and Ukulinga) from first trifoliate (V1) to initiation of reproductive phase (R1) under different seeding rates (Low = 115 000 plants ha-1, Medium = 150 000 plants ha-1 and High = 177 000 plants ha-1) Figure 3: Relationship of total potential value of seed germination (TPVg; y-axis, 0 to 0.35) with those of emergence (TPVe; y- axis, 0 to 0.9) for three common bean cultivars Acta agriculturae Slovenica, 119/3 – 2023 7 Relationship between laboratory and field assessments of common bean (Phaseolus vulgaris L.) seed quality indicators (ISSTA) based on the general parameters of germination and vigour. The combination of existing parameters us- ing a simple model suggests that there may be three op- tions to relate seed quality to potential seed performance. These indices have different values, but TPVe and TPVg are not very different in terms of linkage to basic seed quality, germination and emergence. The index associ- ated with field growth and development indicated great- er differences between cultivars. It may be less effective than the indices done closer to early stages of biological activity for seed. This study showed that there is consist- ency of the relationship between laboratory seed quality determination, seedling establishment, shown as emer- gence, and plant growth following emergence. It is a sim- ple study that was designed to minimise variation in that related common bean genotypes and one site were used under a limited range of management conditions. The relative control of variation is necessary in experiments where new findings are a focus, instead of testing existing knowledge. The study concludes that there is a potential to expand seed quality determination beyond the simple germination and vigour indices under laboratory condi- tions. Total value potential (TPV) under laboratory and field conditions could be a new way of increasing knowl- edge about seed vigour. Expansion of this seed quality determinant to link it to field performance of the crop was encouraging. More research is needed to confirm the results under a wide range of genotypes and environ- mental or management conditions. Future studies should also determine the relationship between relevant crop performances that can be directly linked to interaction of plant physiology and yield. 5 REFERENCES Akinci C., Yildirim M. & Bahar B. (2008). The effect of seed size on emergence and yield of durum wheat. Journal of Food Agriculture and Environment, 6(6), 234-237. 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Effect of pre-sowing seed treatments on seed germination and seed- ling growth in Rakta Chandana (Pterocarpus santalinus L.): An endangered medicinal plant. International Journal of Chemical Studies, 7(3), 1577-1580. Acta agriculturae Slovenica, 119/3, 1–7, Ljubljana 2023 doi:10.14720/aas.2023.119.3.14538 Original research article / izvirni znanstveni članek Do mutations modifying the leaf area (nr3) and the number of potential seeds (dfc) influence photosynthetic gas exchange characteristics in com- mon buckwheat Fagopyrum esculentum Moench? Ivan N. FESENKO 1, 2, Alexandr V. AMELIN 3, Aleksey N. FESENKO 1, Oksana V. BIRYUKOVA 1, Valeriy V. ZAIKIN 3, Evgeniy I. CHEKALIN 3, Roman A. IKUSOV 3 Received June 12, 2023; accepted July 03, 2023. Delo je prispelo 12. junija 2023, sprejeto 3. julija 2023 1 Laboratory of Buckwheat Breeding, Federal Scientific Center of Legumes and Groats Crops, Orel, Russia 2 Corresponding author, e-mail: ivanfesenko@rambler.ru 3 Orel State Agrarian University, Orel, Russia Do mutations modifying the leaf area (nr3) and the number of potential seeds (dfc) influence photosynthetic gas exchange characteristics in common buckwheat Fagopyrum esculentum Moench? Abstract: Contemporary buckwheat breeding in Russia is based mainly on a Mendelian mutation det. Some additional mutations are being considered for inclusion in buckwheat breeding programs. Among them are the nr3 (narrow leaf 3) and dfc (determinate floret cluster). We evaluated the effects of the mutations on both the characteristics of photosynthetic gas exchange and the number of seeds per plant. The nr3 reduces the leaf surface area by 1.4 times. The mutant plants show some compensatory increase in photosynthesis rate, which, however, is not enough to reach the level of the source ability as in the wild type since the number of seeds per plant is significantly decreased. The possibility of using this mutation in buckwheat breeding depends on the accumulation of modifiers that in- crease either leaf size or photosynthesis rate. The reduced num- ber of flowers of the dfs mutation is compensated by an increase in flower fertility, and the number of seeds per plant does not change compared to the wild type. It explains the absence of differences between the dfs and wild type in terms of the photo- synthesis rate. This experiment did not reveal any problems for using the dfc mutation in breeding. In general, the results of the work support the photosynthesis rate in buckwheat is regulated based on the source-sink ratio. Key words: common buckwheat, photosynthesis, leaf area, source-sink ratio, breeding Ali mutaciji, ki spreminajata listno površino (nr3) in število potencialnih semen (dfc) vplivata na značilnosti fotosintezne izmenjave plinov pri navadni ajdi (Fagopyrum esculentum Moench)? Izvleček: Sodobno žlahtnjenje ajde v Rusiji temelji v glavnem na Mendlovi mutaciji det a so bile za vključitev v žlahtniteljske programme predlagane še dodatne mutacije. Med njimi sta mutaciji nr3 (ozki listi 3) in dfc (determinantno socvetje). V raziskavi smo ovrednotili vplive obeh mutacij na značilnosti fotosintezne izmenjave plinov in na število semen na rastlino. Mutacija nr3 zmanjša listno površino za 1,4 krat. Mutantne rastline kažejo nekatere kompenzacijske mehanizme v velikosti fotosinteze, ki pa ne zadoščajo za doseganje ravni pri divjem tipu, kar kaže značilno zmajšanje števila semen na rastlino. Možnost uporabe te mutacije v žlahtniteljskih pro- gramih ajde je odvisna na kopičenju sprememb, ki povečujejo listno površino ali velikost fotosinteze. Zmanjšano število cve- tov pri mutaciji dfs je kompenzirano s povečanjem plodnosti cvetov, pri čemer število semen na rastlino ni spremenjeno v primerjavi z divjim tipom. To razloži tudi odsotnost razlike v velikosti fotosinteze med dfs in divjim tipom. V poskusu tudi ni bilo ugotovljenih nobenih problemov v uporabi fc mutacije pri žlahtnenju. Na splošno rezultati raziskave kažejo, da je ve- likost fotosinteze pri navadni ajdi uravnavana z razmerjem vir : ponor. Ključne besede: navadna ajda, fotosinteza, listna površi- na, razmerje vir-ponor, žlahtnenje Acta agriculturae Slovenica, 119/3 – 20232 I. N. FESENKO et al. 1 INTRODUCTION Common buckwheat, Fagopyrum esculentum Moe- nch, is a grain and groats crop widespread throughout Eurasia (Kreft et al., 2003; Fesenko et al., 2006). Since the 1960s, buckwheat breeding in Russia has been based on mutations that are sometimes accumulated in popu- lations not affected by scientifically based selection, i.e. approved by natural selection (Fesenko, 1983; Fesenko et al., 2006). For example, det-mutation causing deter- minate growth habit (Fesenko, 1968; Ohnishi, 1990) is a core of most contemporary Russian buckwheat varieties (Fesenko & Fesenko, 2019). The use of the det mutation made a local green revolution since the mass distribution of the determinate varieties doubled the average yield of buckwheat in Russia (Fesenko & Fesenko, 2019). An assessment of the groups of both determinate and inde- terminate buckwheat varieties according to the intensity of photosynthesis revealed the advantage of the deter- minate ones at a stage of mass seed filling (Amelin et al., 2020). However, analysis of the effect of det-mutation per se using a segregated hybrid population did not reveal a significant difference with the wild type, i.e. indeter- minate one. On the one hand, it clarifies the role of the det-mutation in control of the photosynthesis intensity is not entirely clear. But it is evident that on its background, some other complexes of genes are formed, including ones determining the photosynthesis characteristics (Amelin et al., 2020). At present, some additional mutations are being considered for inclusion in buckwheat breeding pro- grams. Two of them are dfc and nr3 mutations (Figure 1). One of the significant aspects of the effects of the mutations on plants in the context of their breeding ap- plication is their influence on the characteristics of pho- tosynthesis. The nr3 reduces the leaves area surface, and is considered as the basis for creating varieties with re- duced self-shading. At the same time, reducing leaf area changes the source potential (using the terminology of photosynthesis researchers). The dfc mutation drastically, by 4-5 times, reduces the number of flowers in an inflo- rescence (Fesenko et al., 2010). It can change the sink potential (i.e. demand for assimilates), firstly by reduc- ing the assimilates demand for flower production, and secondly by reducing the fruiting potential. However, the latter can be leveled by increasing the fertility of flowers. So, the mutations can affect the source-sink ratio, which is the key to regulating the intensity of photosynthesis (Paul et al., 2001; McCormick et al., 2008; Katoh et al., 2015). An objective of this work was to evaluate both the characteristics of photosynthetic gas exchange (which re- veal the source ability) and the number of seeds per plant (which shows the sinking ability) of dfc and nr3 mutants vs wild type. Figure 1: A) Mutant nr3; B) Mutant dfc at a stage of almost mature seeds; C) Wild type (both normal leaves and normal flower number) Acta agriculturae Slovenica, 119/3 – 2023 3 Do mutations ... influence photosynthetic gas exchange characteristics in common buckwheat Fagopyrum esculentum Moench? 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL Mutants analized were next: - Mutant dfs (determinate floret cluster) leads to a sharp, 4-5 times reduction in the number of flowers in the inflorescence. The dfc plants participating in crosses were determinate (genotype det det). - Mutant nr3 (narrow leaf 3) causes a change in leaf geometry due to a decrease in its width and significantly reduces the leaves’ surface area of a plant. It reduces the plant’s photosynthetic potential in morphological terms. The nr3 plants participating in the work were determi- nate (genotype det det). Both dfc and nr3 mutants were isolated in the lab of buckwheat breeding, Federal Scientific Center of Leg- umes and Groats Crops. To level the possible influence of some unidentified genes on the parameters of photosynthetic gas exchange F2 hybrids ‘mutant × wild type’ were used for the analy- ses. As a wild type the next varieties were used: - Dikul, a determinate common buckwheat variety bred in Federal Scientific Center of Legumes and Groats Crops. The variety was registered in 1999. - Bogatyr, an indeterminate common buckwheat va- riety bred on Shatilovskaya Experimental Station (Orel region, Russia). It is the first commercial buckwheat vari- ety in Russia which was registered in 1938. F2 hybrids analized were next: (1) ‘dfc dfc/det det × Dikul’. (2) ‘nr3 nr3/det det × Dikul’. (3) ‘nr3 nr3 / det det × Bogatyr’. Since Dikul is a determinate variety, the F2 hybrids (1) and (2) manifest segregation only according to dfc and nr3 alterations, respectively. F2 hybrids with inde- terminate variety Bogatyr shown expected segregation comprising four phenotypical classes (Table 1). 2.2 EXPERIMENTAL APPROACHES The photosynthesis and transpiration intensities were evaluated on intact plants in real-time regime with a portable gas analyzer Li–COR – 6400 using the original methodology of the company Li–COR. The WUE (water use efficiency) was calculated for each plant analyzed us- ing the formula WUE = photosynthesis rate/transpiration intensity. The evaluations of photosynthesis and transpira- tion intensities were conducted in 2017, 2018 and 2021. All experimental plants were labeled and numbered. The measurements within single mutant segregations (dfc or nr3) were made in order “mutant - wild type - mutant - etc” with alternation on each plant. The measurements within a segregation for the two recessives (nr3 and det) were conducted in order “nr3 (non-det) - det (non-nr3) - wild type (both non-nr3 and non-det) - nr3+det - etc” with regular alternation in such order. To measure the leaf size of F2 hybrids ‘nr3 × Dikul’ with both narrow leaves and normal leaves the largest leaf from each plant was photographed with a scale in 2021. Leaves sizes were measured on the photos using Axio Vision Software. 25 plants of both types were taken randomly. Sowing dates were June 1in 2017, May 23 in 2018 and May 27 in 2021. Blossom beginning dates were July 6-7 in 2017, June 26-27 in 2018 and July 1-2 in 2021. The dates of photosynthesis assessment (which are men- tioned in the Table 1) fell on the period of mass filling of seeds. The number of filled grains per plant was evaluated on August 15 in 2017, on August 14 in 2018, and on Au- gust 10 in 2021. The significance of differences was assessed using ANOVA (Software Statistica 7). 3 RESULTS 3.1 ANALYSES OF PHOTOSYNTHETIC GAS EXCHANGE CHARACTERISTICS AND SEED PRODUCTIVITY OF INDIVIDUAL PLANTS WITHIN POPULATIONS SEGREGATED AC- CORDING TO DFC, NR3, AND NR3+DET ALTERATIONS 3.1.1 dfc-mutant The experiment revealed no differences between the mutant and non-mutant plants in photosynthesis and transpiration intensities (Table 1). Also, there were no significant differences in the number of seeds per plant between the groups of plants with normal (wt) and re- duced (dfc) number of flowers. Apparently, it points out that at this ontogenesis stage, the demand for assimilates is mainly formed by developing seeds. This mutation is considered the basis for creating varieties both with more simultaneous maturation and more early ripening. According to the results of the work dfc- mutation does not disturb the system of regulation of physiological processes associated with photosynthesis. It simplifies any application of the mutant for buckwheat breeding. Acta agriculturae Slovenica, 119/3 – 20234 I. N. FESENKO et al. H yb rid co m bi na tio n (d at e of a na ly sis ) Ph en ot yp e cl as s N t ( le af ),° C Ph ot os yn th es is (μ m ol m -2 s- 1 ) Tr an sp ira tio n (μ m ol m -2 s- 1 ) W at er u se e ffi ci en cy Se ed s p er p la nt F 2 (D ik ul × d fc ) (J ul y 28 , 2 01 7) w t 42 24 .7  ±  0 .6 2 8. 32 ± 3 .8 5 5. 84 ± 1 .7 3 1. 40 ± 0 .5 6 44 .8 7 ± 10 .3 1 df c 42 8. 69 ± 3 .6 3 6. 12 ± 1 .9 2 1. 40 ± 0 .4 9 45 .8 0 ± 6. 84 O ne -w ay A N O VA N S N S N S N S F 2 (D ik ul × n r3 ) (J ul y 26 , 2 02 1) w t 40 30 .6 ± 3 .9 7 8. 88 ± 4 .4 4 2. 51 ± 1 .1 8 4. 06 ± 2 .4 0 51 .2 0 ± 5. 14 nr 3 40 11 .1 1 ± 4. 93 2. 81 ± 1 .3 4 4. 76 ± 3 .0 0 46 .5 3 ± 6. 82 O ne -w ay A N O VA p < 0. 05 N S N S p < 0. 00 1 F 2 (D ik ul × n r3 ) (J ul y 12 , 2 01 8) w t 50 24 .8 ± 0 .6 3 12 .5 8 ± 4. 17 2. 14 ± 0 .5 7 6. 38 ± 4 .4 2 42 .9 2 ± 4. 36 nr 3 50 13 .7 5 ± 4. 88 2. 16 ± 0 .3 5 6. 20 ± 2 .7 1 38 .9 6 ± 5. 24 O ne -w ay A N O VA N S N S N S p < 0. 00 1 F 2 (B og at yr × nr 3) (J ul y 13 , 2 01 8) w t 30 25 .0 ± 0 .3 5 11 .4 8 ± 3. 84 2. 70 ± 1 .1 0 5. 13 ± 3 .2 2 35 .1 7 ± 2. 96 nr 3 30 10 .3 7 ± 3. 43 2. 08 ± 0 .7 7 6. 03 ± 4 .3 5 35 .7 3 ± 2. 77 de t 30 9. 73 ± 4 .4 5 2. 47 ± 0 .7 3 4. 13 ± 2 .0 6 38 .0 3 ± 3. 13 nr 3 + de t 30 10 .3 8 ± 3. 81 2. 21 ± 0 .8 8 5. 17 ± 2 .3 7 37 .2 3 ± 3. 02 Tw o- w ay A N O VA nr 3 N S p < 0. 02 N S N S de t N S N S N S p < 0. 00 1 nr 3 × de t N S N S N S N S Ta bl e 1: C ha ra ct er ist ic s o f m ut an t a nd n on -m ut an t ( w t) cl as se s i n se gr eg at ed p op ul at io ns in te rm s o f p ho to sy nt he tic g as e xc ha ng e an d se ed p ro du ct iv ity (M ea n ± SD ) Acta agriculturae Slovenica, 119/3 – 2023 5 Do mutations ... influence photosynthetic gas exchange characteristics in common buckwheat Fagopyrum esculentum Moench? 3.1.2 nr3-mutant This single-gene mutation makes leaves narrow and reduces their area by 1.4 times, 38.4 ± 12.2 vs 27.1 ± 7.4 (mean ± SD). Apparently, it should cause a shift within the “source-sink” balance toward a source deficiency. A priori, there can be two ways to compensate for such a shift. The first is a decrease in the number of seeds, i.e. a decrease in sink strength. The second is an increase in the intensity of photosynthesis per leaf area unit. In the experiments, we observed both types of effects (Table 1). In two experiments carried out in different years on the same hybrid material, the same patterns were obtained in terms of both the changes in the number of seeds per plant and the gas exchange parameters. In both cases, the number of seeds on narrow-leaved plants was significantly less compared to plants with normal leaves. Photosynthesis rate in both cases was higher for the narrow-leaved plants, on average, and in one of the two experiments, the difference was significant. Water use ef- ficiency (WUE) have tends to slight growth when photo- synthesis rate is significantly higher. Thus, this mutation can be considered as a model in which the sink potential is not fully realized due to the source insufficiency. Since this work shows the nr3-mutant has insuffi- cient leaf area resulting in the source deficiency, an es- sential aspect of its use for breeding commercial varieties should most likely be an increase in leaf size or/and an additional increase in photosynthesis rate due to the se- lection of some hypothetical modifiers that can be able to compensate the effect of the mutation. 3.1.3 nr3 + det We have previously reported that determinate varie- ties manifest higher photosynthesis rates at the seed-fill- ing stage than indeterminate varieties. However, the det mutation itself does not affect the gas exchange intensity. An experiment was conducted to evaluate the joint effect of det and nr3 mutations on photosynthesis parameters. The less number of grains per plant in the experi- ment compared to other ones in this work is due to the old low-yielding variety with normal leaves and indeter- minate growth habit Bogatyr has been used in crosses with determinate plants with narrow leaves (genotype det det/nr3 nr3). The det mutation did not affect the rates of photosynthetic gas exchange and had a certain effect on the number of seeds. However, the difference revealed was at a low level of significance (Table 1). The photo- synthesis rate did not differ significantly also between nr3 mutants and plants with normal leaves. It should be noted that nr3 homozygotes, both determinate and in- determinate, showed a significantly lower transpiration intensity. However, it could not be considered as a trend since in the experiments with F2 hybrids ‘nr3 × ‘Dikul’ the differences between mutants and non-mutants in transpiration intensity were not significant (Table 1). Wa- ter use efficiency also was not significantly affected by the mutations across all the experiments. 4 DISCUSSION There are many evidences suggesting the photosyn- thetic gas exchange intensity is a function of the source- sink interaction. The source potential is not realized at full capacity, and the value of photosynthesis rate per unit of leaf area can be increased if the leaves area of the plant is reduces by any way. Thus, soybean having leaves with smaller surface areas manifests a higher photosynthesis rate per unit of leaf area than those with larger leaves (Sung, Chen, 1989). Similar effect can be observed on a rice mutant NAL1 with more narrow leaves compared wild type (Takai et al., 2013). In addition, the excision of several leaves from trees of Eucalyptus globulus Labill. resulted in the growth of photosynthesis rate in the re- mained leaves (Eyles et al., 2013). Since photosynthesis rate usually does not reach the maximum possible values, there are some factors restrict- ing it. Matsuda et al. (2011) discussed the hypothesis for sink-limitation suggesting the photosynthesis rate is lim- ited by the demand for assimilates. This hypothesis was tested on two varieties of tomato and was not fully con- firmed (Matsuda et al., 2011).Thus, when several fruits at the early developmental stage were removed from plants, the remaining ones became larger. On the other hand, on intact plants all the fruits were smaller. It suggests rather a lack of source ability in this case. The examples when assimilate demand was increased due to experimental manipulation also are known. So, nitrogen application can provide higher sink strength (Pissolato et al., 2019; Chen et al., 2022). Inoculation with nodule bacteria also resulted in a higher photosynthesis rate which could be explained in two ways: 1) it is able to provide additional nitrogen and 2) growing nodules requires additional as- similates (Kaschuk et al., 2012). On potatoes it was shown the possibility to increase both source and sink ability using transgenic manipula- tions: it sufficiently increased the yield of starch in tubers (Jonik et al., 2012). Also, it was revealed the genetically controlled mechanisms influencing the translocation of assimilates toward developing seeds (Phung et al., 2019). In addition, it was hypothesized the buckwheat varie- ties with determinate growth habit manifest higher pho- Acta agriculturae Slovenica, 119/3 – 20236 I. N. FESENKO et al. tosynthesis due to optimizing the assimilates logistics (Amelin et al., 2020). We have analyzed the effect of well-distinguishable morphological mutations of two types on the photosyn- thetic gas exchange parameters. One of them, the dfc mu- tation, reduces the number of flowers within cyme (i.e., partial inflorescence) by 4-5 times. It can be assumed it forms some tendency to reduce sink potential. However, our experiment with this mutation shows the demand for assimilates is not different between non-mutant and dfc- mutant plants. The number of formed seeds on mutant and non-mutant plants also did not differ significantly. Thus, the increasing proportion of flowers setting seeds compensates for reducing flowers number on dfc-plants and allows them to form sufficient number of seeds and maintain the demand for assimilates. Mutation nr3 reduces leaf area by 1.4 times and in- creases photosynthesis rate, sometimes significantly. It can be interpreted as compensation for the decrease in leaf area. Also, it allows us to understand that in buck- wheat, the physiological processes associated with pho- tosynthesis are not at full capacity, and there are some possibilities to increase its intensity. Since the mutants typically produced fewer seeds compared to the wild type, it can be concluded that the compensatory increase in photosynthesis rate is unsufficient to reach the wild type source levels. Our experiments to assess the effects of mutations on the intensity of photosynthesis, both presented in this article and previously published, make it clear that buckwheat plants have a particular source limit per unit of leaf area which, however, is usually not reached, i.e. the source ability is not fully realized. It is due to certain limitations in the sink ability, an example of overcoming which, however, exists. This is a significant increase in the photosynthesis intensity within varieties with deter- minate growth habit at the stage of seed filling. The det mutation per se does not affect photosynthesis charac- teristics. Therefore, the higher photosynthesis rate of the determinate varieties is due to the accumulation of some additional genes, the role of which in physiology we do not yet understand (Amelin et al., 2020). There are no commercial varieties based on the nr3 and dfc mutations yet. If (or when) such varieties appear, it will be possible to evaluate their difference from varie- ties that do not carry these mutations, and, accordingly, it is possible to obtain additional cases of modifying the regulation of photosynthesis. This is especially true for the nr3 mutation, which application for buckwheat breeding can be successful only with the accumulation of certain modifiers. 5 ACKNOWLEDGMENTS The work was supported by grant of Russian Sci- ence Foundation № 22-26-00041, https://rscf.ru/pro- ject/22-26-00041 6 REFERENCES Amelin, A.V., Fesenko, A.N., Chekalin, E.I., Fesenko, I.N., & Zaikin, V.V. (2020). 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Scien- tific Reports, 3, 2149. https://doi.org/10.1038/srep02149 Acta agriculturae Slovenica, 119/3, 1–6, Ljubljana 2023 doi:10.14720/aas.2023.119.3.14714 Original research article / izvirni znanstveni članek Results of testing of the efficacy of sublethal concentrations of bacterial- chemical insecticides combinations against cabbage moth larvae Hrant TERLEMEZYAN 1, Masis SARGSYAN 1, Harutyun HARUTYUNYAN 1, Noushig ZARIKIAN 2, 3, Sona SARGSYAN 1, Gabriel KARAPETYAN 1, Habetnak MKRTCHYAN 1 Received June 26, 2023; accepted September 14, 2023. Delo je prispelo 26. junija 2023, sprejeto 14. septembra 2023 1 Research Centre of Risk Assessment and Analysis in Food Safety Area, Yerevan, Armenia 2 Department of Experimental Zoology, Scientific Center of Zoology and Hydroecology of NAS RA, Yerevan, Armenia 3 Corresponding author, e-mail nzarikian@gmail.com The experiments of sublethal concentrations of bacterial- chemical insecticides against cabbage moth larvae Abstract: Using chemical pesticides has adverse effects on the environment and humans. Bacterial preparations may pro- vide an alternative to chemical pesticides. The study aims to test different combinations of sublethal concentrations of bacterial and chemical preparations against cabbage moth larvae. During 2020-2022 different combinations of sublethal concentrations of bacterial (Lepidocide) and chemical (Arrivo, Voliam Flexi, Proclaim Fit) preparations were tested in labora- tory and field conditions, against cabbage moth young larvae (stage I-II). The combinations of insecticides with bacterial and chemical sublethal concentrations show high biological effi- ciency against the cabbage moth larvae. No statistical difference was found between the efficiency indicators of the combined and standard chemical (Arrivo, Voliam Flexi, Proclaim Fit) op- tions and the significance level was generally between 2.0 and 5.9 %, showing that the results of the scientific experiments are reliable. Key words: biological effectiveness, cabbage moth larvae, insecticides, laboratory and field tests, statistical analysis Poskusi s subletalnimi koncentracijami bakterijsko-kemij- skih insekticidov na gosenice kapusnega molja Izvleček: Uporaba kemijskih insekticidov ima škodljive učinke na okolje in ljudi. Pripravki iz bakterij so lahko alterna- tiva kemijskim pesticidom. Namen raziskave je bil preiskusiti različne kombinacije subletalnih koncentracij bakterijskih in kemijskih pripravkov proti gosenicam kapusnega molja. V letih 2020-2022 so bile v laboratoriju in poljskih razme- rah preiskušene različne kombinacije subletalnih koncentracij bakterijskih (Lepidocide) in kemijskih (Arrivo, Voliam Flexi, Proclaim Fit) pripravkov za zatiranje mladih gosenic kapusne- ga molja (razvojni štadij I-II). Kombinacije bakterijskih in kemijskih insekticidov v su- bletalnih koncentracijah so pokazale veliko biološko učinko- vitost na tretiranih gosenicah. V kazalnikih učinkovitosti ni bilo statistično značilne razlike med kombiniranimi pripravki in standardnimi kemijskimi insekticidi (Arrivo, Voliam Flexi, Proclaim Fit). Raven značilnosti je bila nasplošno med 2,0 in 5,9 %, kar kaže, da so izsledki poskusov zanesljivi. Ključne besede: biološka učinkovitost, gosenica kapu- snega molja, insekticidi, laboratorijski in poljski poskusi, sta- tistična analiza Acta agriculturae Slovenica, 119/3 – 20232 H. TERLEMEZYAN et al. 1 INTRODUCTION The soil and climatic conditions of Armavir region of the Republic of Armenia are favorable for the culti- vation of white-head cabbage (Brassica oleracea L. ssp. oleracea convar. capitata (L.) Alef f. alba). The increase of the yield of this plant is often hindered by the cabbage moth Plutella maculipennis (Curtis, 1832) which belongs to the Plutellidae family of the insect order Lepidoptera. The damage caused by its larvae reduces the yield and lowers the quality of the crop. Hatched larvae eat the parenchyma of the leaves, leaving the epidermis intact, resulting in the formation of areas covered with a thin membrane, called “windows” (Avetyan & Marjanyan, 1976), and the more mature lar- vae open through holes on the leaves. The damage be- comes more dangerous when they feed on the young leaves forming the head of the plant (Safaryan, 1968; Terlemezyan, 1996; Philips et al., 2014; Andreeva et al., 2021). Besides the white-head cabbage, phytophagous lar- vae also damage other economic importance crucifer- ous plants, for example cauliflower, broccoli, rapeseed, etc. (Tsedeler, 1931; Harcourt, 1957; Terlemezyan, 1996; Churikova & Silaev, 2010; Shpanev, 2015; Kholod & Korenyuk, 2016; Tuleeva & Sarmanova, 2019). Therefore, it is extremely important to implement effective, environ- mentally safe control measures against harmful larvae. In the integrated pest control system, the preference is given to the use of bacterial preparations based on Bacillus thuringiensis Berliner, 1915 (Bt) species, which have high biological efficiency against leaf-eating harm- ful insects and, unlike chemical preparations, are safe for humans, warm-blooded animals, entomophages and fish. (Talekar & Shelton, 1993; Belyaev & Nozdrenko, 2004; Ivantsova, 2004; Sarantseva & Bobreshova, 2006; Sargsyan, 2013; Fathipour & Mirhosseini, 2017; Semer- enko, 2019; Zakharova et al, 2022). Currently, the implementation of economically justified control of phytophagous larvae through com- binations of sublethal concentrations (used in small quantities) of bacterial and chemical insecticides is also emphasized (Mesropyan, 2011; Avagyan, 2012; Chapan- yan, 2022). Based on the above, we aimed to test different com- binations of sublethal concentrations of bacterial and chemical preparations against cabbage moth larvae in laboratory and field conditions. 2 MATERIALS AND METHODS The scientific experiments were carried out during 2020-2022, in the laboratory conditions at the Scientific Center for Risk Assessment and Analysis of Food Safety and cabbage plantations of Nalbandian community of Armavir region. The research materials were: the young cab- bage moth larvae (stage I-II), the cabbage plant (variety: Slava), the commercial bacterial lepidocide preparations KA 3000 IU mg-1 in the powder for liquid suspension: the usage rate is 1.0 kg ha-1 (Russian Federation), chemi- cal preparations: 25 % concentrated emulsion Arrivo: the usage rate is 0.3 l ha-1 (FMC, USA), 30 % concentrated suspension Voliam Flexi: the usage rate is 0.3 l ha-1, and 45  % water-soluble granules of Proclaim fit: the usage rate is 0.1 l ha-1 (Syngenta, Switzerland). All the above-mentioned preparations are al- lowed to be used against harmful insects in the Republic of Armenia. Cabbage plantations, where the number of moth larvae was at the threshold of economic damage of a specified pest (that is: 2-5 larvae per plant), when 10 % or more of the plants in the experimental site are occupied by them (Polyakov, 1984), were selected as ex- perimental sites. The biological effectiveness of insecticides com- bined with sublethal concentrations (Lepidocide + Ar- rivo, Lepidocide + Voliam Flexi, Lepidocide + Proclaim Fit) was determined according to the methodological manual (Methodological guidelines for testing biologi- cal products for plant protection from pests’ diseases and weeds, 1973). The lethal concentrations of 3 (in case of lepiocide: 0.33 kg ha-1) and 10 dilutions (in case of Arrivo and Voliam Flexi: 0.03 l ha-1, in case of Proclaim Fit: 0.01 l ha-1) of bacterial (lepidocide) and chemical (Arrivo, Voliam Flexi and Proclaim Fit) insecticides, respectively, were combined. The samples sprayed with different solvents (Lepidocide: the usage rate is 1.0 kg ha-1, Arrivo: the us- age rate is 0.3 l ha-1, Voliam Flexi: the usage rate is 0.3 l ha-1, Proclaim Fit: the usage rate is 0.1 l ha-1) were taken from the plantations naturally inhabited by moth larvae. During small-scale and production experi- ments, cabbage plants grown under laboratory condi- tions (in camps) and artificially inhabited by moths were sprayed with a hand-held sprayer full of working fluid, using backpack AO - 2 and motorized K-14 sprayers. The working fluid consumption was 400 l ha-1. In small-scale experiments, the size of the experimental area for each (sampled separately and experimentally combined) op- tion was 100 m², as for large-scale spraying, it was 0.2 ha. Each option included in the experiments had 3 replicates. In laboratory conditions, 30 larvae were in- cluded in each option (10 larvae in each replicate), and Acta agriculturae Slovenica, 119/3 – 2023 3 Results of testing of the efficacy of sublethal concentrations of bacterial-chemical insecticides combinations against cabbage moth larvae in two-year small-scale and production experiments, the number of phytophagous larvae was generally between 51 and 70, and 55 and 77, in certain cases. The numbers of alive and dead larvae in the experimental plots were counted before spraying (base- line), and 3, 5 and 7 days after spraying, also before the mating phase. In laboratory conditions when experimenting the options with the sub-threshold concentrations, the mi- crobiological isolation of Bacillus thuringinsis var. kursta- ki Bulla et al. 1979 pathogens which are the basis for the production of the sprayed commercial lepidocide bacte- rial preparations, were isolated according to the meth- odological manual (Netrusov et al., 2005). The statistical analysis of the results of the scientific experiments was carried out according to the protocol presented by (Ashmarin and Vorobyev, 1962; Bernstein, 1968). 3 RESULTS AND DISCUSSION According to the results of scientific experiments carried out in laboratory conditions in 2020, it was proved that the combinations of standard lepidocide bacterial (3 dilutions of the lethal concentration) and di- luted lethal chemical (Arrivo, Voliam Flexi and Proclaim Fit) concentrations (10 dilutions of the lethal concentra- tion) have shown a high biological efficiency against phy- tophagous larvae (stage I-II) just in 7 days after spraying, generally ranging from 93.3 % to 96.7 %. The indicators of biological efficiency of the sample options for the same recording period were also high, ranging from 93.3 to 100 %. No mortality of phytophagous larvae was observed on the sprayed sample, during the observation period. The high rates of biological efficiency recorded in laboratory conditions made it possible, as well, to test the insecticides individually (standard/sample options) and in combination with sublethal concentrations (experi- mental options) against the cabbage moth larvae under field conditions (field and production experiments). According to the results of the partial (small-scale) research, it was demonstrated that even 7 days after spraying, the indicators of biological efficiency of com- bined options, such as Lepidocide + Arrivo, Lepidocide + Voliam Flexi, Lepidocide + Proclaim Fit, were still high, generally ranging from 91.5 % to 94.3 % (Table 1). As it is presented in Table 1, Lepidocide (sample), Arrivo (sample), Voliam Flexi (sample) and Proclaim Fit (sample) options have also demonstrated high biological efficacy (overall 85.7 % - 96.1 %). The pattern of high biological efficiency in small- scale experiments demonstrated by individual and sub- lethal concentrations of insecticides against the cabbage moth larvae was maintained during production experi- ments conducted in 2021-2022 (Table 2). According to the two-year data from Table 2, the in- dicators of biological efficiency (7 days after spraying) of the standard lepidocide were 82.4 % and 85.4 %. As for the combined options of sublethal concentrations, such as Lepidocide + Arrivo, Lepidocide + Voliam Flexi, and Lepidocide + Proclaim Fit, those were 89.1 % and 90.1 %, 92.2% and 93.0 %, and 91.3 % and 91.5 %, respectively. The indicators of biological efficiency recorded for all standard (sample) chemical insecticides were between 86.3 % and 95.2 % for the same period of observation. Moreover, the above-mentioned indicators recorded on the 7th day were constant for all tested options before the mating period. From the data in Tables 1 and 2, it is clear that the indicators of biological efficiency recorded in the experi- ments conducted 3 and 5 days after spraying were rela- tively low compared to those recorded on the 7th day, Table 1: The indicators of biological effectiveness of standard (sample) and combined insecticides against cabbage moth larvae (stage I-II) (small-scale experiments, Nalbandyan, 2020) Options The number of larvae on the plant 20 option, quantity Biological efficiency according to accounting days, % 3 5 7 Lepidocide + Arrivo 59 57.6 78.0 91.5 Lepidocide +Voliam Flexi 53 62.3 84.9 94.3 Lepidocide + Proclaim Fit 67 56.7 80.6 92.5 Lepidocide (Sample) 70 51.4 77.1 85.7 Arrivo (Sample) 58 67.2 84.5 89.7 Voliam Flexi (Sample) 51 78.4 88.2 96.1 Proclaim Fit (Sample) 62 62.9 87.1 93.5 Acta agriculturae Slovenica, 119/3 – 20234 H. TERLEMEZYAN et al. which is apparently due to the specificity of the mecha- nism of action of insecticides on larvae. Compared to the water-spraying practices, when the experimental options, i.e., the combined bacterial and chemical sublethal concentrations, were applied, the cabbage larvae gradually refused to feed on plants. Moreover, no response to contact or any other mechani- cal stimuli was observed, which, eventually, contributed and led to larval death. The bodies of dead larvae, com- pared to healthy larvae, have become grey and have re- duced in size. Microbiological studies have confirmed that the gut cavity and decayed tissues of dead moth larvae were full of vegetative cells of the Bacillus thuringiensis pathogen, as well as insecticidal spore-crystal components. Using the Student’s t-test criteria, it was proved (Ta- ble 3) that the two-year indicators of biological efficiency of the experimental options, when the lepidocide was combined with sublethal concentrations of insecticides, significantly exceeded those of the standard lepidocide samples, because in the first case with p = 0.95 and n = 3, the student’s t-test scores, generally ranging from 3.601 to 7.095, were higher than the tabulated Student’s t-test score of 3.182. It was also statistically confirmed that there was no significant difference between the indica- tors of biological effectiveness recorded in the combined versions, on the one hand, and in the standard individual options of Arrivo, Voliam Flexi and Proclaim Fit, on the other hand (with p = 0.95 and n = 3, the calculated two- year average scores of the student’s t-tests were between 0.056 and 1.756, which were less than its table (3.182) index). In the two-year production studies, the statistical error was generally ranging from 2.0  % to 5.9  %, con- firming that the results of the scientific experiments are reliable (Table 4). 4 CONCLUSIONS Based on the results of the experiments, we came to a conclusion that the combinations of insecticides with bacterial and chemical sublethal concentrations show high biological efficiency against the cabbage moth lar- vae. The efficiency indicators for the latter are statistically different from those of the standard bacterial lepidocide. However, no statistical difference was found between the efficiency indicators of the combined and standard chemical (Arrivo, Voliam Flexi, Proclaim Fit) options. The statistical error indicators prove that the results of the scientific experiments are accurate. Table 2: The indicators of biological effectiveness of standard (sample) and combined insecticides against cabbage moth larvae (stage I-II) by years (production experiments, Nalbandian, 2020) Options The number of larvae on the plant 20 option, quantity Biological efficiency according to accounting days, % 3 5 7 During 2021 Lepidocide + Arrivo 55 56.4 76.4 89.1 Lepidocide + Voliam Flexi 64 60.9 84.4 92.2 Lepidocide + Proclaim Fit 59 50.8 78.0 91.5 Lepidocide (Sample) 55 49.1 76.4 85.4 Arrivo (Sample) 73 64.4 83.6 86.3 Voliam Flexi (Sample) 61 77.0 85.2 95.1 Proclaim Fit (Sample) 62 61.3 85.5 93.5 During 2022 Lepidocide + Arrivo 71 54.9 77.5 90.1 Lepidocide + Voliam Flexi 57 61.4 82.4 93.0 Lepidocide + Proclaim Fit 69 53.6 79.7 91.3 Lepidocide (Sample) 74 50.0 75.7 82.4 Arrivo (Sample) 77 66.2 81.8 88.3 Voliam Flexi (Sample) 63 76.2 87.3 95.2 Proclaim Fit (Sample) 70 60.0 84.3 91.4 Acta agriculturae Slovenica, 119/3 – 2023 5 Results of testing of the efficacy of sublethal concentrations of bacterial-chemical insecticides combinations against cabbage moth larvae Table 3: The comparative assessment of indicators of biological efficiency recorded in experimental and standard (sample) options during production experiments verified by Student‘s t test criteria (by years) Options  Indicators of biological efficiency 7 days after spraying, % Student’s t test scores Indicators of biological efficiency 7 days after spraying, % Student’s t test scores During 2021 During 2022 Lepidocide + Arrivo 89.1 3.601*1.756 90.1 4.424*1.208 Lepidocide + Voliam Flexi 92.2 4.687**1.756 93.0 7.095**1.535 Lepidocide + Proclaim Fit 91.5 4.643***1.628 91.3 4.537***0.056 Lepidocide (Sample) 85.4 - 82.4 - Arrivo (Sample) 86.3 - 88.3 - Voliam Flexi (Sample) 95.1 - 95.2 - Proclaim Fit (Sample) 93.5 - 91.4 - Note. * in the numerator: the combined experimental and lepidocide (sample) options, in the denominator: the comparative indicators of biologi- cal efficiency recorded in the experimental and Arrivo (sample) options, ** in the numerator: experimental and lepidocide (sample) options, in the denominator: the comparative indicators of the biological efficiency recorded in the experimental and Voliam Flexi (sample) options and *** in the numerator: the comparative indicators of biological efficiency recorded in the experimental and lepidocide (sample) options, in the denominator: the experimental and Proclaim Fit (sample) options Table 4: The statistical indicators of the average number of dead cabbage moth larvae (stages I-II) per replicate, 7 days after spray- ing, by years (production experiments) Options The average number of dead larvae per replicate, quantity Statistical indicators The squared deviation The coefficient of variation, % The average error The statistical error, % During 2021 Lepidocide + Arrivo 16.33 0.575 3.52 0.332 2.0 Lepidocide + Voliam Flexi 19.67 1.661 8.44 0.959 4.9 Lepidocide + Proclaim Fit 18.00 1.414 7.85 0.816 4.5 Lepidocide (Sample) 15.67 1.205 7.69 0.696 4.4 Arrivo (Sample) 21.00 1.633 7.78 0.943 4.5 Voliam Flexi (Sample) 19.33 1.737 8.99 1.003 5.2 Proclaim Fit (Sample) 19.33 1.009 5.22 0.583 3.0 During 2022 Lepidocide + Arrivo 21.33 1.741 8.16 1.005 4.7 Lepidocide + Voliam Flexi 17.67 1.199 6.79 0.692 3.9 Lepidocide + Proclaim Fit 21.00 2.160 10.29 1.247 5.9 Lepidocide (Sample) 20.33 1.739 8.55 1.004 4.9 Arrivo (Sample) 22.67 1.185 5.23 0.684 3.0 Voliam Flexi (Sample) 20.00 1.633 8.17 0.943 4.7 Proclaim Fit (Sample) 21.33 1.303 6.11 0.752 3.5 Acta agriculturae Slovenica, 119/3 – 20236 H. 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