AActa agriculturae Slovenica • eISSN 1854-1941 • 119 – 4 • Ljubljana, december 2023 119•4 2023 ActA AgrIcuLturAe SLoveNIcA Acta agriculturae Slovenica Letnik / Volume 119 · Številka / Number 4 · 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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Trenutno revija ne zaračunava stroškov za predložitev in obdelavo vključenih prispevkov. / The journal does not charge APCs or submission charges. Acta agriculturae Slovenica izhaja s finančno pomočjo / is published with the financial support: Javne agencije za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije / Slovenian Research and Innovation Agency. Acta agriculturae Slovenica je vključena v / is included into: Scopus (SJR, SNIP), DOAJ, WOS Zoological Records, CrossRef, CAB Abstracts, FSTA, Google Scholar, dLib, COBISS. Ovitek: Novoletno voščilo izdajatelja Cover: New Year’s greeting from the issuer Acta agriculturae Slovenica Volume / Letnik 119 · Number / Številka 4 · 2023 Table of Contents / Kazalo Original Scientific Article / Izvirni znanstveni članek Using pyrimidinecarboxylic acids as growth stimulants for Rhododendron ledebourii Pojark and Rhododendron smirnowii Trautv. Uporaba pirimidin karboksilne kisline kot rastnega vzpodbujevalca za vrsti rododendrona, Rhododendron ledebourii Pojark in Rhododendron smirnowii Trautv. Tatiana V. VOSTRIKOVA, Andrey Yu. POTAPOV, Nadezhda V. STOLPOVSKAYA, Khidmet S. SHIKHALIEV 1–10 Application of fluorescence spectroscopy as a field method in the determination of varietal differences after tomato harvesting Uporaba fluorescentne spektroskopije kot metode za ugotavljanje razlik med sortami paradižnika na polju po obiranju Veselina MASHEVA, Vanya SLAVOVA 1–5 Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture Preučevanje gojišč za gojenje nezrelih embrijev ječmena (Hordeum vulgare L.) Abdullah Hassn MOHAMMED 1–8 Physical and aerodynamic properties of date palm pollen grains Fizikalne in aerodinamične lastnosti pelodnih zrn dateljeve palme Mohamed M. IBRAHIM, Mohamed GHONIMY, Eid ABD EL RAHMAN 1–11 Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara Fitokemični profil in alelopatski potencial vrste Haloxylon scoparium Pomel (Chenopodiaceae) iz alžirske Sahare Reguia OTMANI, Bachir KHENE, Abdellah KEMASSI, Fatna ARABA, Mohamed HARRAT, Mohamed YOUSFI 1–11 Effects of particle size on determination of the contents of grain and legume dietary fibre and resistant starch Vpliv velikosti delcev na določitev vsebnosti prehranske vlaknine in rezistentnega škroba v žitih in stročnicah Blaž FERJANČIČ, Mojca KOROŠEC, Saša PISKERNIK, Jasna BERTONCELJ 1–9 The development of agricultural use in the area of the photovoltaic power plant D3 next to the flow accumulation of the HE Brežice Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice Jože PODGORŠEK 1–10 Influence of mulches on soil moisture and water infiltration in the tomato crop Vpliv mulčenja na vlažnost tal in infiltracijo vode v nasadu paradižnika Imerina TANKEUOO KOPA, Mohamed MEDDI, Abdelhadi AMMARI 1–16 Effect of the alcohol content on sensory perception of the fruit spirits Učinek vsebnosti alkohola na senzorično zaznavanje žganih pijač Arman ŠEKERIĆ , Milenko BLESIĆ, Pakeza DRKENDA, Nermina SPAHO 1–7 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay Hitra in učinkovita metoda DNK ekstrakcije iz semen kloščevca (Ricinus communis L.), bogatih na oljih, primerna za PCR analizo Elham R. S. SOLIMAN 1–8 Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) Učinek časa setve in gnojenja na vsebnost beljakovin v semenu in pridelku beljakovin v suhem fižolu (Phaseolus vulgaris L.) Andrea GYÖRGYINÉ KOVÁCS, Gabriella TÓTH, Tamás SIPOS, Béla SZABÓ, István HENZSEL 1–11 Effectiveness of local biopesticides in the control of Diamondback Moth (Plutella xylostella L.) in cabbage production in Zanzibar, Tanzania Učinkovitost lokalnih biopesticidov za uravnavanje kapusne sovke (Plutella xylostella L.) pri gojenju zelja v Zanzibarju, Tanzanija Ahmed Gharib KHAMIS, Ali Abdulla ALI, Mwanamanga Haji ALI, Ali Seif NASSOR, Mohammed Omar HAMAD, Salma Omar MOHAMMED, Mohammed Dhamir KOMBO 1–6 Review Article / Pregledni znanstveni članek Herbicides weed management in changing environmental conditions Uravnavanje plevelov s herbicidi v razmerah spreminajočega se okolja Zvonko PACANOSKI, Arben MEHMETI 1–11 Non-chemical control options against the virus vector nematodes Xiphinema index Thorne & Allen, 1950 Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphinema index Thorne & Allen, 1950 Julija POLANŠEK, Franci Aco CELAR, Saša ŠIRCA 1–12 Acta agriculturae Slovenica, 119/4, 1–10, Ljubljana 2023 doi:10.14720/aas.2023.119.4.2762 Original research article / izvirni znanstveni članek Using pyrimidinecarboxylic acids as growth stimulants for Rhododen- dron ledebourii Pojark and Rhododendron smirnowii Trautv. Tatiana V. VOSTRIKOVA 1, 2, Andrey Yu. POTAPOV 1, Nadezhda V. STOLPOVSKAYA 1, Khidmet S. SHI- KHALIEV 1 Received July 05, 2022; accepted October 19, 2023. Delo je prispelo 5. julija 2022, sprejeto 19. oktobra 2023 1 Voronezh State University, Voronezh, Russian Federation 2 Corresponding author, e-mail: tanyavostric@rambler.ru Using pyrimidinecarboxylic acids as growth stimulants for Rhododendron ledebourii Pojark and Rhododendron smirnowii Trautv. Abstract: Synthesised organic compounds of pyrimidi- necarboxylic acids are characterised by high biological activ- ity, even when their concentrations are low. These compounds, when applied to the seeds of Rhododendron, a genus of woody plants, with concentrations of 0.01, 0.05, and 0.1 %, stimulate the growth of the plants. The effect is more obvious 3 months af- ter the start of the experiment, rather than 7 months after. Thus, Rhododendron ledebourii Pojark. seedlings grew by 13.3-33.5 %, and Rhododendron smirnowii Trautv. seedlings grew by 29.6- 48.1 %. Rhododendron ledebourii and Rhododendron smirnowii seedlings demonstrated similar direct correlations: when the concentration of 2-benzylamino-4-methyl-pyrimidine-5-car- boxylic acid and 4-methyl-2-morpholin-4-pyrimidine-5-car- boxylic acid (for Rhododendron ledebourii seeds only) rose from 0.01 to 0.1 %, the height of the seedlings increased. It is suggest- ed using 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid, 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid, and 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid at concentrations of 0.01, 0.05, and 0.1 % as growth stimulants for the species of Rhododendron genus. Key words: growth stimulants, synthesized organic com- pounds, pyrimidine carboxylic acids, Rhododendron Uporaba pirimidin karboksilne kisline kot rastnega vzpod- bujevalca za vrsti rododendrona, Rhododendron ledebourii Pojark in Rhododendron smirnowii Trautv. Izvleček: Za sintetične organske spojine pirimidin kar- boksilnih kislin je značilna velika biološka aktivnost, tudi pri majhnih koncentracijah. Tretiranje semen rododendrona s temi spojinami v koncentracijah 0,01; 0,05 in 0,1 %, stimulira rast rastlin. Ta učinek je jasnejši 3 mesece po začetku poskusa kot po 7 mesecih. Sejanke vrste Rhododendron ledebourii Po- jark. so rastle pri koncentracijah 13,3-33,5 %, sejanke vrste Rhododendron smirnowii Trautv. pa pri koncentracijah 29,6- 48,1 % stimulanta. Sejanke obeh vrst so se na tretiranje odzvale podobno in sicer: kadar se je koncentracija 2-benzilamino- 4-metil-pirimidin-5-karboksilne kisline in 4-metil-2-morfolin- 4-pirimidin-5-karboksilne kisline (le za semena vrste Rhodo- dendron ledebourii) dvignila iz 0,01 na 0,1 %, se je višina sejank povečala. Priporočamo uporabo 4-metil-2-piperidin-1-il-pi- rimidin-5-karboksilne kisline, 2-benzilamino-4-metil-pirimi- din-5-karboksilne kisline in 4-metil-2-morfolin-4-pirimidin- 5-karboksilne kisline v koncentracijah 0,01; 0,05 in 0,1 % kot rastne vzpodbujevalce za vrste iz rodu Rhododendron. Ključne besede: rastni vzpodbujevalci, sintetične organ- ske spojine, pirimidin karboksilne kisline, Rhododendron Acta agriculturae Slovenica, 119/4 – 20232 T. V. VOSTRIKOVA et al. 1 INTRODUCTION Over the past few years, the cultivation of plants has been facing a number of challenges, including a dramatic increase in temperatures, lack of soil water, and the need for more resistant and hardy plants, which grow even un- der the harshest conditions. It is therefore vital to mini- mise the time required for the growth of planting mate- rial, which can be done using growth and germination stimulants, including synthesised chemical compounds Pentelkina, 2003; Vasin et al., 2008, 2009; Ostroshenko and Ostroshenko, 2011; Schuchka, 2006; Baranova, 2013a; Khodaei-Joghan et al., 2018; Nesterkina et al., 2019) and other original modes (Shibaeva et al., 2018). It has been noted high biological activity of pyridines and pyrimidines. For example, 1, 4-dihydropyridines have received large attention because of their fundamen- tal role in different biological processes (Goldmann and Stoltefuss, 1991; Litvinov, 1998; Balalaie et al., 2008). It has been reported about the wide biological activity of dihydropyridine derivatives (Ghoneim and Assy, 2015). But a promising area of the study, related to the growing need for the development of effective and safe drugs, is the synthesis of new heterocyclic systems, containing a pyrimidine fragment. Pyrimidine fragments and pyrimi- dine base are included in DNA. So pyrimidine deriva- tives show different biological activity. There is another mode for applying chemical compounds, containing py- rimidine fragments, for instance, as mutagens. The composition of diethyleneimide-2-amidopy- rimidyl phosphoric acid (phosphazine, phosphomid, syn. phosphemide - phospemidum) includes two ethylene imine groups connected to phosphorus and a pyrimidine base. Ethyleneimine causes mutations, the pyrimidine base is included in the chromosome during DNA syn- thesis, determining the specificity of the effect (Weisfeld, 2015). Laboratory seed germination and morphomet- ric parameters of seedlings (length of roots and shoots, number of leaves) were studied after seed treatment with heterocyclic sulfur-containing compounds on the exam- ple of woody plants (Vostrikova et al., 2020) and agricul- tural crop (Vostrikova et al., 2021). As the composition of the phosphemide mutagen, pyrimidine-carboxylic acids contain a pyrimidine base. Sulfur (in the composition of alkylating compounds: diethyl- and dimethylsulfonate, ethylmethanesulfonate) enhances biological activity. It is known that mutagens in low concentration have stimu- lating effect for plant objects. A necessary working step is the determination of concentration ranges (Bome et al., 2017). Over the last years, attempts have been made to syn- thesise new organic compounds, derivatives of pyrimi- dines (Dlugosz and Dus, 1996; Elkholy and Morsy, 2006; El-Gazzar et al., 2009; Marjani et al., 2011; Tkachenko et al., 2013, 2018; Azizian et al., 2014), which can be used as growth regulators. Such compounds should be more effective than the existing commercial formulations be- cause of their different concentrations that may have ei- ther stimulating or inhibiting activity (Dlugosz and Dus, 1996; Gavrilov et al., 1988; Litvinov, 1998; Brown et al., 2004; Balalaie et al., 2008; Moiseeva et al., 2012a). The effect of pyrimidine carboxylic acids on seed germination and plant height was studied using another annual flower – spreading marigold (Tagetes patula L.) (Vostrikova et al., 2012; Kalaev et al., 2013a). However, the effect of pyrimidinecarboxylic acids on the growth of other plants has not been studied yet. Up to the present time, there have also been no stud- ies considering the results of application of synthesised chemical compounds of pyrimidinecarboxylic acids to the seeds of woody plants, which grow much slower than annual plants. It is therefore of great importance to conduct a longitudinal study and measure the height of ornamental woody plants over long time intervals (eg. 3 and 7 months after the application of the growth regula- tor) in order to determine, whether the growth stimulat- ing effect lasts or deteriorates over time. Two ornamental woody plants, namely Rhododen- dron ledebourii Pojark. and Rhododendron smirnowii Trautv. were used. The long history of studying these species (Moiseeva et al., 2012b; Baranova, 2013b) at the B.M. Kozo-Polyansky Botanical Garden of Voronezh State University has demonstrated, that Rhododendron ledebourii is a winter-hardy, drought-resistant, and fruit- bearing shrub. Rhododendron smirnowii is also quite winter-hardy, though less drought-resistant. It also grows slower than Rhododendron ledebourii (Alexandrova, 2003; Vostrikova, 2011). It has been reported about the antioxidant activity in Rhododendron leaves, connec- tions between this characteristic in maternal plant and the cytogenetic structures in the seedlings of the Rho- dodendron species (Vostrikova et al., 2022). Cytogenetic polymorphism of seed progeny of introduced plants has been studied on the example of Rhododendron ledebourii (Burmenko et al., 2018). It has been revealed the high seeds quality. So the representatives of the Rhododendron genus are valuable resource plant. The aim of our research was to study the effect from pre-sowing seed treatment of the following woody plants: Rhododendron ledebourii and Rhododendron smirnowii by pyrimidine carboxylic acids. 2 MATERIALS AND METHODS The research was conducted at the B.M. Kozo-Pol- Acta agriculturae Slovenica, 119/4 – 2023 3 Using pyrimidinecarboxylic acids as growth stimulants ... yansky Botanical Garden of Voronezh State University (geographic coordinates: 39°22’ N, 51°40’ E; 168.2 metres above sea level). It has been focused on the effect of synthesised or- ganic compounds of pyrimidinecarboxylic acids on the height of seedlings of Rh. ledebourii, and Rh. smirnowii. The following compounds were used: 4-methyl-2-pi- peridin-1-yl-pyrimidine-5-carboxylic acid, 2-ben- zylamino-4-methyl-pyrimidine-5-carboxylic acid, and 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid synthesised at the Department of Organic Chemistry of Voronezh State University. Prior to the sprouting process, the seeds of Rh. lede- bourii and Rh. smirnowii were soaked in a water suspen- sion of the above listed compounds with concentrations of 0.01 %, 0.05 %, and 0.1 % for 18 hours. The control group consisted of the same type of seeds soaked in tap water solution of a commonly used growth stimulator, Epibrassinolide (commercial fraction Epin Extra pro- duced by NNPP NEST M, Russia), with the concentra- tion of 0.05 % (in accordance with the instruction). In case of each of the studied concentrations of the acids, as well as the control group, the experiment was conducted three times using a set of 100 seeds. After soaking, the rhododendron seeds were placed in Petri dishes contain- ing blotting paper, and germinated in the laboratory con- ditions at a constant temperature 22 оС. On the 21st day, the sprouts were planted in containers filled with high- moor peat and then kept in a greenhouse. The height of the seedlings of Rhododendron ledebourii was measured with a ruler, 3 and 7 months after the start of the experi- ment. Since Rhododendron smirnowii grows slower, the height of its seedlings was measured 7 months after the start of the experiment. Sprouts are formed during the early stage of plant ontogenesis, which starts after the germination stage, i.e. when the seed coat develops, and finishes, when the first leaf of the hypocotyledonous stem (the shoot rising from the plumule) develops (Korovkin, 2007). After the first true leaves appear, young plants are considered seedlings (Korovkin, 2007). The results were statistically processed using the STADIA software package. The procedures of data grouping and processing were described by A. P. Kulai- chev (2006). The mean values were compared using Stu- dent’s t-test. Coefficient of variation (Cv) was counted according to G. F. Lakin (1990). If Cv was below 10 %, it meant that the degree of variation was low, with Cv be- tween 10 and 25 % it was medium, and when Cv was over 25 % - the degree of variation was high (Lakin, 1990). Av- erage values of seedlings (plants) height were compared using Student’s t-test. The seed germination in control and experimental variants were compared using Z-test for equality of frequencies. To estimate the influence of various concentrations of the chemical compounds on the height of the plants, one-way analysis of variance was used. The power of influence was calculated according to Snedecor (in %). 3 RESULTS AND DISCUSSION The standard growth stimulant hasn’t shown influ- ence of the seed treatment on the height of Rhododen- dron ledebourii seedlings. It hasn’t been revealed the significant difference between Сontrol group and Epin group. That means special stimulant impact for sowing qualities in Rhododendron seeds or specific seeds reac- tion for the treatment. The seeds germination wasn’t strongly increased (Table 1-3). But there were significant differences between control and experimental groups in this parameter (Table 1-3). Suspensions of 2-benzylamino-4-methyl-pyrimi- dine-5-carboxylic acid at concentrations between 0.01 and 0.1 % proved to be more effective than other growth stimulators (Table 1). When suspensions of 4-methyl- 2-morpholin-4-pyrimidine-5-carboxylic acid at con- centrations of 0.05 and 0.1 % were used, the seedlings of Rhododendron ledebourii grew up to 55.6–116.7 % higher (Table 1). Within 7 month after the start of the experiment all the studied compounds at any concentration proved to be effective for Rhododendron ledebourii (Table 2). Within 7 month after the start of the experiment, the height of the Rhododendron smirnowii seedlings also increased (Table 5). The parameter “height of the seed- lings” varied greatly for the control group of Rhododen- dron ledebourii 3 months after the start of the experiment. This is indicated by Cv  =  44 %. For the experimental groups, Cv was medium (Table 1), which indicated that the reactions of individual seedlings to the compounds used were similar. 7 month after the start of the experi- ment, the degree of variation was medium in the con- trol group, and even low in the experimental group. This might mean that older seedlings adapt better to experi- mental conditions. Changes of the seedlings growth are illustrated in Fig. 1-3. For the seedlings of Rhododendron smirnowii, a me- dium coefficient of variation was observed (Table 3). Be- cause of the slow growth and the small size, differences between experimental and control groups of Rhododen- dron smirnowii are not significant 3 month after the start of the experiment. Acta agriculturae Slovenica, 119/4 – 20234 T. V. VOSTRIKOVA et al. Table 1. The seedlings height of Rhododendron ledebourii seedlings 3 months after the seed were treated with the studied synthe- sized organic compounds Concentration, % Average height of the plants, cm Min - max, cm Cv, % The seeds germination, % Increase in the height of the plants, % Сontrol group Epin group 1.8 ± 0.2 1.9 ± 0.1 1.0–3.0 1.5–2.5 44 15.8 72 75 – – 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid 0,01 % 0,05 % 0,1 % 3.3 ± 0.1***3 2.8 ± 0.1**2 2.2 ± 0.11 3.0–3.5 2.5–3.0 2.0–2.5 9.1 10.7 13.6 78* 78* 79* 83.3 55.6 – 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 0,01 % 0,05 % 0,1 % 2.9 ± 0.2**2 3.4 ± 0.2***3 3.8 ± 0.2***3 2.5–3.0 3.0–3.5 3.5–4.0 12.5 13.5 11.9 78* 80* 82** 61.1 88.9 111.1 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid 0,01 % 0,05 % 0,1 % 2.3 ± 0.11 2.8 ± 0.1**2 3.9 ± 0.2**2 2.0–2.5 2.5–3.0 3.0–4.5 13.0 10.7 15.4 74 75 78* – 55.6 116.7 Designations: Cv – variation coefficient * – differences with the control group are significant (p < 0.05) ** – differences with the control group are significant (p < 0.01) *** – differences with the control group are significant (p < 0.001) 1 – differences with the Epin group are significant (p < 0.05) 2 – differences with the Epin group are significant (p < 0.01) 3 – differences with the Epin group are significant (p < 0.01) Fig. 1: The height of the seedling of Rhododendron ledebourii (in mm) seedlings after the seed were treated with 4-methyl-2-pi- peridin-1-yl-pyrimidine-5-carboxylic acid; 1 – seedlings (21 days), 2 – plants 3 months, 3 – plants 7 months Acta agriculturae Slovenica, 119/4 – 2023 5 Using pyrimidinecarboxylic acids as growth stimulants ... Designations: Cv – variation coefficient * – differences with the control group are significant (p < 0.05) ** – differences with the control group are significant (p < 0.01) *** – differences with the control group are significant (p < 0.001) 1 – differences with the Epin group are significant (p < 0.05) 2 – differences with the Epin group are significant (p < 0.01) 3 – differences with the Epin group are significant (p < 0.01) Table 2: The height of Rh. ledebourii seedlings 7 months after the seed were treated with the studied synthesized organic com- pounds Concentration, % Average height of the plants, cm Min - max, cm Cv, % The seeds germination, % Increase in the height of the plants, % Сontrol group Epin group 5.6 ± 0.2 5.7 ± 0.2 4.5–6.5 5.0–6.5 11.5 9.3 72 75 – – 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid 0,01% 0,05% 0,1% 7.7 ± 0.2***3 7.6 ± 0.2***3 6.8 ± 0.2***3 7.0–8.5 6.5–8.5 6.0–7.5 7.6 8.7 7.9 78* 78* 79* 22.9 27.5 33.5 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 0,01% 0,05% 0,1% 7.2 ± 0.2***3 7.7 ± 0.2***3 8.4 ± 0.2***3 6.5–8.0 7.0–8.5 7.5–9.0 7.5 7.5 6.9 78* 80* 82** 22.9 27.0 17.8 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid 0,01% 0,05% 0,1% 6.4 ± 0.2*1 6.5 ± 0.2**2 6.9 ± 0.2***3 5.5–7.5 5.5–7.5 6.0–7.5 11.5 11.5 6.9 74 75 78* 13.3 14.6 19.0 Fig. 2: The height of the seedling of Rhododendron ledebourii (in mm) seedlings after the seed were treated with 4-methyl-2-mor- pholin-4-pyrimidine-5-carboxylic acid, 1 – seedlings (21 days), 2 – plants 3 months, 3 – plants 7 months Acta agriculturae Slovenica, 119/4 – 20236 T. V. VOSTRIKOVA et al. The growth ratio of Rhododendron ledebourii seed- lings (in the experimental group as compared to the control group) after the seeds were treated with organic compounds of pyrimidinecarboxylic acids, demonstrates that the stimulating effect is most obvious 3 month after the start of the experiment. However, some treatment do not effect growth (Table 1). 7 month after the start of the experiment, the stimulating effect deteriorates, but all the compounds at any concentration studied in this paper still have a positive effect (Table 2). It was shown significant influence of the treatment of seeds with the synthesised organic compounds (as a factor) on the height of the Rhododendron seedlings. The power of influence of the treatment of seeds with the syn- thesised organic compounds on the height of the seed- lings was evaluated using the one-way analysis of vari- ance (Table 4-6). 4-methyl-2-piperidin-1-yl-pyrimidine-5-car- boxylic acid, 2-benzylamino-4-methyl-pyrimidine- 5-carboxylic acid, and 4-methyl-2-morpholin-4-py- rimidine-5-carboxylic acid with concentrations of 0.01, 0.05, and 0.1 % resulted in the increase in the height of Fig. 3: The height of the seedling of Rhododendron ledebourii (in mm) seedlings after the seed were treated with 2-benzylamino- 4-methyl-pyrimidine-5-carboxylic acid1, 1 – seedlings (21 days), 2 – plants 3 months, 3 – plants 7 months Designations: Cv – variation coefficient * – differences with the control group are significant (p < 0.05) ** – differences with the control group are significant (p < 0.01) *** – differences with the control group are significant (p < 0.001) 1 – differences with the Epin group are significant (p < 0.05) 2 – differences with the Epin group are significant (p < 0.01) 3 – differences with the Epin group are significant (p < 0.01) Table 3: The height of Rh. smirnowii seedlings 7 months after the seed were treated with the studied synthesized organic com- pounds Concentration, % Average height of the plants, cm Min - max, cm Cv, % The seeds germination, % Increase in the height of the plants, % Сontrol group Epin group 1.4 ± 0.1 1.4 ± 0.1 1.0–2.0 1.0–2.0 24.1 28.1 58 60 – – 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 0,01 % 0,05 % 0,1 % 1.8 ± 0.1*1 1.9 ± 0.1**1 2.0 ± 0.1**2 1.5–2.0 1.5–2.0 1.5–2.5 14.7 12.7 20.4 64* 68* 72** 29.6 37.0 48.1 Acta agriculturae Slovenica, 119/4 – 2023 7 Using pyrimidinecarboxylic acids as growth stimulants ... Rhododendron ledebourii seedlings. A direct correla- tion can be observed: the higher the concentration of 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid (within the range between 0.01 and 0.1  %), the higher the seedlings of Rhododendron ledebourii and Rhodo- dendron smirnowii are. The same correlation is observed for Rhododendron ledebourii seedlings, when 4-methyl- 2-morpholin-4-pyrimidine-5-carboxylic acid is applied. When Rhododendron ledebourii seeds were treated with 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid at the concentration between 0.01 and 0.1 %, a negative correlation was observed: the lower the concentration, the higher the seedlings. We can thus say, that the studied synthesised organic compounds of pyrimidinecarboxylic acids are character- ised by high biological activity and stimulate the growth of ornamental woody plants when their concentrations are low. The stimulating effect is most obvious 3 months after the seed treatment (the seedlings are 55.6–116.7 % higher than the seedlings in the control group). 7 month after the start of the experiment the stimulating effect starts deteriorating. Compounds of pyrimidinecarbox- ylic acids increase the height of Rhododendron ledebourii seedlings by 13.3-33.5 %, and the height of Rhododen- dron smirnowii seedlings by 29.6-48.1 %. Apparently, pyrimidinecarboxylic acids are more biologically active than other chemical substances, as their chemical struc- ture is similar to the structure of a natural growth stimu- lator - indole acetic acid (heteroauxin). It can be assumed that auxin activity was kept better within 3 month, than during 7 month after the start of the experiment. Earlier research studied the stimulating effect of 0.01–0.05 % 4-methyl-2-piperidin-1-yl-pyrimidine- 5-carboxylic acid on the germination and growth of the seedlings of spreading marigold: with the concentration of 0.03-0.05 %, the height of the plants increased (dif- ferences with the control group are reliable, p < 0.001). However, other compounds of pyrimidine-5-carboxylic acids at the studied concentrations demonstrated strong- er stimulating effects (Vostrikova et al., 2012; Kalaev et al., 2013 a). Obtained results are consistent with earlier studies by R. G. Gafurov and co-workers on carbon N- and O- benzyl-containing compounds that have bright auxin ac- tivity, which is ensured by the presence of a benzyl group at the nitrogen or oxygen atom (Gafurov and Makhmu- Table 4: The power of influence (in %) of the seed treatment on the height of Rhododendron ledebourii seedlings on the 3 month after the start of the experiment Stimulator as compared to the control group as compared to the Epin group as is 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid 5.0*** 8.8*** 8*** 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 7.4*** 7.9*** 8.3*** 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid 7.2*** 8.9*** 8.2*** Designations: *** - the influence of the factor is significant (p < 0.001) Table 5: The power of influence (in %) of the seed treatment on the height of Rhododendron ledebourii seedlings on the 7 month after the start of the experiment Stimulator as compared to the control group as compared to the Epin group as is 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid 6.7*** 7.5*** 6.7*** 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 8.4*** 8.9*** 8.5*** 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid 4.9*** 3.3** 5.6** Designations: *** - the influence of the factor is significant (p < 0.001) *** - the influence of the factor is significant (p < 0.001) Table 6: The power of influence (in %) of the seed treatment on the height of Rh. smirnowii seedlings on the 7 month after the start of the experiment Stimulator as compared to the con- trol group as compared to the Epin group as is 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid 4.3*** 4.2*** 6.2*** Designations: *** - the influence of the factor is significant (p < 0.001) Acta agriculturae Slovenica, 119/4 – 20238 T. V. VOSTRIKOVA et al. tova, 2003, 2005). These compounds contain effector fragments that together determine the stress protective activity, namely, the quaternary ammonium and benzyl groups and the hydroxyethyl group - an analog of the benzoxyethyl group (Budykina et al., 2005; Timeyko et al., 2005). Tested substance (2-benzylamino-4-methyl- pyrimidine-5-carboxylic acid) contains similar frag- ments, so it also shows bright auxin activity. Based on the literature data (Budykina et al., 2005; Timeyko et al., 2005) and the results of our research, it can be assumed that used compound has the stress-protective activity for valuable ornamental plants of Rhododendron species. 4 CONCLUSIONS In this paper, it has been demonstrated that the ef- fect of the same concentrations of synthesised organic compounds of pyrimidine carboxylic acids on the seed- lings of woody plants after the pre-sowing seed treatment doesn’t differ. Rhododendron ledebourii and Rhododen- dron smirnowii seedlings demonstrated similar direct correlations: when the concentration of 2-benzylamino- 4-methyl-pyrimidine-5-carboxylic acid and 4-methyl- 2-morpholin-4-pyrimidine-5-carboxylic acid (for Rho- dodendron ledebourii seeds only) rose from 0.01 to 0.1 %, the height of the seedlings increased. Therefore, 4-me- thyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid, 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid, and 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid at concentrations of 0.01, 0.05, and 0.1 % can be used as growth stimulators for species of Rhododendron genus. 2-benzylamino-4-methyl-pyrimidine-5-carboxyl- ic acid proved to be more effective at the initial develop- ment stages of Rhododendron seedlings. 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Periódico Tchê Química, 17(35), 781-790. https:// doi.org/10.52571/PTQ.v17.n35.2020.66_VOSTRIKOVA_ pgs_781_790.pdf Weisfeld L. I. (2015). About cytogenetic mechanism of chemi- cal mutagenesis. In A. I. Opalko, L. I. Weisfeld, S. A. Beku- zarova, N. A. Bome, G. E. Zaikov (Eds.), Ecological conse- quences of increasing crop productivity. Plant breeding and biotic diversity (259-269). Toronto-New Jersey, Apple Aca- demic Press Inc. Acta agriculturae Slovenica, 119/4, 1–5, Ljubljana 2023 doi:10.14720/aas.2023.119.4.12518 Original research article / izvirni znanstveni članek Application of fluorescence spectroscopy as a field method in the deter- mination of varietal differences after tomato harvesting Veselina MASHEVA 1, Vanya SLAVOVA 2, 3 Received February 14, 2023; accepted October 22, 2023. Delo je prispelo 14. februarja 2023, sprejeto 22. oktobra 2023 1 Department of Plant Genetic Resources, Institute of Plant Genetic Resources “Konstantin Malkov” – Sadovo, Agricultural Academy Bulgaria, Sadovo, Bulgaria 2 Department of Plant Breeding, Maritsa Vegetable Crops Research Institute, Agricultural Academy Bulgaria, Plovdiv, Bulgaria 3 Corresponding author, e–mail: vania_plachkova@abv.bg Application of fluorescence spectroscopy as a field method in the determination of varietal differences after tomato harvest- ing Abstract: The study’s purpose is to establish the applica- tion based on fluorescence spectroscopy as a field method in the determination of varietal differences after tomato harvest- ing. The tomato fruits will be compared to determine the spec- tral distribution due to the varietal differences of a particular genotype. This will allow the approach to be practiced non-in- vasively in the quality control of tomato production in unspeci- fied rooms and outdoors. The experimental studies have been conducted locally at the Institute of Plant Genetic Resources “K. Malkov” - Sadovo for three varieties. The spectral installation for the generation of emission fluorescence spectra is mobile. In its adjustment (optical ad- justment), a system engineering approach based on the classical principles of modern optoelectronics was applied. The results of the experiment can be used to optimize the time for the analysis of the varietal difference of tomato genotypes after harvesting, under uncontrolled conditions. This will support the process of determining the belonging of a specific аccession to a given variety (even for аccessions of unknown origin) when it is nec- essary to qualify a score of samples in a short time. Key words: tomato fruits, uncontrolled conditions, field method, fluorescence spectroscopy Uporaba fluorescentne spektroskopije kot metode za ugota- vljanje razlik med sortami paradižnika na polju po obiranju Izvleček: Namen raziskave je bil razviti poljsko metodo za ugotavljanje razlik med sortami paradižnika po obiranju z uporabo fluorescenčne spektroskopije. Razlike med sortami so določene na osnovi razlik med porazdelitavmi spektrov, ki so odvisni od genotipov. To bo omogočilo praktičen neinvazivni nadzor kakovosti pri pridelavi paradižnika na prostem in v ne- specializiranih skladiščih. Poskusi so bili izvedeni na ustanovi Institute of Plant Genetic Resources “K. Malkov – Sadovo, Bol- garija, na treh sortah. Naprava za generiranje emisijskega fluorescentnega spek- tra je mobilna. Za njeno nastavitev (optična nastavitev) je bil uporabljen klasični princip moderne optične elektronike. Re- zultati te raziskave bi se lahko uporabili za optimiziranje časa analize razlik med genotipi različnih sort paradižnika po obira- nju v nenadzorovanih razmerah. To bo podpora pri določanju akcesij, ki pripadajo določeni sorti paradižnika, tudi tistih ne- znanega izvora, kadar je potrebno pregledati kakovost vzorcev v kratkem času. Ključne besede: plodovi paradižnika, nenadzorovane razmere, poljska metoda, fluorescentna spektroskopija Acta agriculturae Slovenica, 119/4 – 20232 V. MASHEVA and V. SLAVOVA 1 INTRODUCTION The established function of fluorescence spectros- copy as a field method in the assurance of varietal dif- ferences after tomato harvesting is the aim of the present study. Various techniques have been investigated for non-invasive spectrometric analysis of tomatoes. Near- infrared spectroscopy is used to determine the content of soluble solids (Slaughter et al., 1996) and detect ca- rotenoids (Pedro and Ferreira, 2005) has been success- fully applied. Also, reflectance approaches (Polder et al., 2004) and fluorescence spectroscopy (Lai et al., 2007) have been well enforced to assess surface pigmentation. Raman spectroscopy is a proven technique in carotenoid research (Schulz et al., 2005; Pudney et al., 2011). In the assessment of the ripeness and firmness of to- matoes (Qin and Lu, 2008), the absorption and scattering properties are applied. Advances in fiber optic applied science attempt to provide outstanding conveniences for the development of an ample range of highly deftly fiber optic sensors in many modern application fields. Fiber optic insides have been successfully becoming assemblies with micro-optic pieces such as lenses, mirrors, prisms, gratings, and others (Dakin & Brown, 2006; Mitchke, 2010). In many analytical areas of science, fluorescence spectroscopy is an important research tool. It is currently the dominant methodology and is widely recycled in bio- technology, flow cytometry, medical diagnostics, DNA sequencing, agriculture, and genetic investigations, as well as in many other application areas. Methods using this light phenomenon are highly sensitive and rapid and do not require the expense and difficulty of using radio- active tracers (Becker et al., 2003; Albani, 2006). For quality control of vegetable crops, including to- matoes, the effects of light apply to spectral analysis such as fluorescence, transmission, and diffuse reflectance. Also, they can serve as a field method in the determina- tion of varietal differences after tomato harvesting, since fluorescence emission is visualized in the visible spectral range and from ultraviolet rays. The spectral distribution of the emission signal in tomato fruits consists mainly of two maxima in the visible range. The intensity and shape of the fluorescence emission spectrum at room tempera- ture depend mainly on the concentration of the fluoro- phores and to a lesser extent on the structure, photosyn- thetic activity, and arrangement of the cells in the tissue (National Research Council, 1968; Leo et al., 2007). In connection with the demands of consumers for high food quality, the conducted research can serve as a basis for the creation of mobile detecting devices with which to carry out instant analysis of warehouse produc- tion of tomatoes in uncontrolled conditions, both in pro- cessing plants and in food retail outlets. The present study aims to establish the function of fluorescence spectroscopy in the act of field method in the determination of varietal differences after tomato har- vesting. They will be compared to determine the spectral distribution due to the varietal differences of a particu- lar genotype. The specimens were grown under uncon- trolled field conditions. This will permit the technique to be applied non-invasively in the quality control of tomato production in unspecified rooms and outdoors. 2 MATERIALS AND METHODS The fruits that are the subject of the research are the varieties Local Dwarf (determinate), Pikador (deter- minate), and Ideal (indeterminate). The seeds that were sown to grow the accessions were taken from GenBank at the Institute of Plant Genetic Resources, Sadovo. The local Dwarf tomato was dense, planted up to 40–50 cm tall, had potato-type leaves, bright red fruits with a flat round shape, and a mass of 100–120 g. The determinate cultivar Pikador is a highly effi- cient variety, making fruit with fiercely red flesh that is considerable for preserving. Admirably, lightly stretched tomatoes that parallel pears in shape are firm and hard, with a mass of 50–60 g. The fruits of tomatoes of this va- riety are located in the form of a bunch on the plant. The variety is strongly resistant to mold. The indeterminate Ideal was a medium-early toma- to variety with considerable fruits, 130–180 g, flat-round to arced, kind of ridged, multi-chambered, orange-red in color, and an amiable taste. The indeterminate Ideal was a medium-early toma- to variety with considerable fruits, 130–180 g, flat-round to arced, kind of ridged, multi-chambered, orange-red in color, and an amiable taste. The experiment was conducted at the Institute of Plant Genetic Resources „Konstantin Malkov“ - Sadovo. The seeds are set in seedling plates filled with peat-pe- arlitic substrate and launched at a temperature of 25–28 degrees C for impregnation. Tomato seedlings are trans- planted once they are 15–25 cm in height and have 3–5 true leaves. Indeterminate ‘Ideal’ is planted at an area amid rows of 75-80 cm and 35-40 cm - betwixt plants in the row. The branches are attached to a supporting structure. Determinate ‘Pikador’ and ‘Local Dwarf were plan- ted 25–30 cm between tomato plants, and space rows 60–90 cm aside. The field experiment was block design with three repetitions, with ten (10) plants in repetition. Acta agriculturae Slovenica, 119/4 – 2023 3 Application of fluorescence spectroscopy as a field method in the determination of varietal differences after tomato harvesting The agrotechnical measures were carried out in the excellent terms for the crop. Fluorescence spectroscopy was applied to determi- ne the varietal differences after tomato harvesting under uncontrolled conditions. The spectral analysis was per- formed locally at the Institute of Plant Genetic Resources „Konstantin Malkov“ - Sadovo. The analysis was carried out with a fiber-optic spec- trometer, which enables the formation of emission fluo- rescence signals from 200 nm to 1200 nm. The unproved setup adds a laser diode (emis- sion wavelength 285 nm, optical power 16 mW, DC) and a compact spectrometer (exemplary AvaSpec-UL- S2048CL-EVO). The accessions are placed one by one on a duralumin plate. The experimental setup allows emission fluorescence signals to be detected through a Y-shaped optical fiber at 180 degrees to the sample and at a distance of 1.7 cm from it. The fruits are placed and arranged on a duralumin plate, which allows the encoun- ter of an emission signal in perpendicular geometry at 180 degrees and at a distance of 1.7 cm by Y-shaped op- tical fiber. This curtails aberrations and allows the for- mation of an exceptional emission fluorescence signal (Fig. 1). The resolution of the spectrometer can range from 0.06 - 20 nm, with that of the setup recycled for our experiment being 0.09 nm. The useful fluorescence signal is generated in a direction that is 180° from the excitation radiation, so as not to saturate the receiver. A laser diode (LED) was used as a source because its spec- tral width was too small. The source is high-power and more sensitive than other commercial LEDs. It generates a continuous or pulsed light signal. It has an SMA-905 connector for connecting optical fibers and works with a 5V or 1.6A power supply. LED has a relatively wide spec- tral width of emission (30–40 nm) and an angular distri- bution of emission in the range of +/–30о. The sensitivity of the spectrometer is in the range of 200 nm to 1200 nm. The mode of operation of the AvaSpec-ULS2048CL-EVO enables both the emission spectrum and the excitation source spectrum to be recorded. The emission signal re- presents the spectral distribution of the signal emitted by the accession after fluorescence has occurred. The excita- tion spectrum represents the dependence of the emission intensity measured for one scanning wavelength against the excitation wavelength of the LED. This spectrum is represented as a function of the wavelength of the light signal incident on the photodetector in the spectrometer. Complementary metal-oxide semiconductor (CMOS) instead of conventional charge-coupled device (CCD) technology, this spectrometer owes its key advantage over others with a similar configuration to the dominant position of the CMOS detector in its design. For this par- ticular circuit, the photodetector is of the CMOS model S9132 type. Its sensitivity is in the range of 200 nm to 1200 nm. Its resolution is δλ = 5 nm. S9132 was chosen because it could detect emission radiation from fruit with analogous cell morphology, biology, and chemical composition. The laser radiation is deflected against the source and hits the sample. Afterward, the sample fluoresces, and the emission signal is conducted on a U-shaped op- tical fiber with a core diameter of 200 µm, a step-index of refraction, and a numerical aperture of 0.22. It sends a signal to the detector. In the spectrometer, the light signal is converted to an electrical-digital signal using a USB 2.0 wire, downloaded to a computer with AvaSoft8 software, and exported to Excel. This allows analysis, processing, and visualization of the results of the conducted research. 3 RESULTS AND DISCUSSION The optical properties of tomato fruits are deter- mined by their energy structure, which includes both the occupied and free energy levels as well as the ener- gy levels of the atomic vibrations of the molecules in the crystal lattice. The achievable transitions between these energy levels, as a function of photon energy, are specific to the tomato, resulting in spectra and optical properties unique to it. Tomato fruits contain particles with sizes smaller than the wavelength of visible light. Particles in the turbid medium (such as tomato fruits) act as inde- pendent light sources, emitting incoherently, causing the samples to visibly fluoresce. Therefore, fluorescence spectroscopy finds applica- tion for analysis in this vegetable crop. The optical param- eters and spectral properties also change as a function of temperature, pressure, external electric and magnetic Figure 1: Accustomed aspect of the experimental installation used by fluorescence spectroscopy Acta agriculturae Slovenica, 119/4 – 20234 V. MASHEVA and V. SLAVOVA fields, etc. This allows obtaining essential information about changes in the chemical and cellular morphologi- cal composition of the tomato. The spectral distributions of tomato fruits of the Lo- cal Dwarf and Pikador varieties are presented in Fig. 2. A certain correlation is observed between them (the emis- sion wavelength of Local Dwarf t is 425 nm; the emission wavelength of Pikador is 421 nm). Their emission fluo- rescence signals are close in terms of peak wavelength and signal intensity level. The emission wavelengths are due to the content of certain fluorophoric compounds in tomatoes, for example, nucleocapsid (N) proteins, lectin, some carboxyl compounds, and others. This is because both varieties are determinant tomatoes. They are close in biological and cellular morphological composition. The method of fluorescence spectroscopy is applied in this study to distinguish the fruits of these two varieties since the correlation in the spectral distribution is suf- ficiently distinct and distinguishable. This fact is used in this study to determine the tomato fruit belonging to a given variety. Fig. 3 shows the spectral distributions of tomato fruits of the Local Dwarf and Ideal varieties; a significant correlation is observed between them (emission wave- length for Local Dwarf is 425 nm; emission wavelength for Ideal is 410 nm). Their fluorescence emission signals are not close and have a significant offset in wavelength localization and signal intensity level. The method of fluorescence spectroscopy can be applied to distinguish the fruits of these two cultivars because the correlation in the spectral distribution is of considerable distinctness and distinction. The method of fluorescence spectros- copy may practically be used to qualitatively resolve the belonging of fruits to a given variety. Fig. 4 shows the spectral distributions of tomato fruits of the Pikador and Ideal varieties. A significant dif- ference is observed between them (the emission wave- length for Pikador is 421 nm; the emission wavelength for Ideal is 410 nm). Their fluorescence emission signals are not close and have a significant offset in wavelength localization and signal intensity level. Since the differenc- es in the spectral distribution are available for these two cultivars, the method of fluorescence spectroscopy can be applied to distinguish their fruits. The method of fluo- rescence spectroscopy can practically be used to qualita- tively determine the belonging of fruits to a given variety. A literature survey was conducted using similar methods. It turned out that, until now, the described ex- perimental approach for the field method in the deter- mination of varietal differences after tomato harvesting has not been applied internationally. This gives us reason to claim that for the first time, fluorescence spectroscopy was used in the application of fluorescence spectroscopy as a field method in the determination of varietal differ- ences after tomato harvesting under uncontrolled condi- tions. The method is successfully applied to distinguish Figure 2: Emission wavelengths of tomato fruits Local Dwarf and Pikador varieties Figure 3: Emission wavelengths of tomato fruits Local Dwarf and Ideal varieties Figure 4: Emission wavelengths of tomato fruits Pikador and Ideal varieties Acta agriculturae Slovenica, 119/4 – 2023 5 Application of fluorescence spectroscopy as a field method in the determination of varietal differences after tomato harvesting fruit tomatoes from different varieties. Fluorescence spectroscopy can be applied to analyze the tomato fruit of unknown cultivars and establish its origin with a suf- ficiently well-structured data library. Because it can be applied topically to test specimens. It eliminates sample damage during transport and provides a highly sensitive assay. 4 CONCLUSIONS The fluorescence spectroscopy method is fast-acting in application as a field method in the determination of varietal differences after tomatoes harvesting locally un- der uncontrolled conditions. It has been proven that fluorescence spectroscopy will successfully apply as a rapid tool to establish the ori- gin of unknown tomato fruits in the presence of a rich library of spectra. The developed method can be used successfully in tomato breeding programs. The stabili- ty of the breeding line and its common blacks with an established cultivar of the same species can be observed by following the difference in the spectral distribution. The differentiation of related varieties is a laborious and time-consuming task. For these reasons, the develo- pment of techniques that could assist in an early, quick, and accurate differentiation of related varieties is of ut- most importance. A systematic engineering approach to the setup (op- tical setup) of a mobile fiber optic plant for fluorescence spectroscopy research was found to be applicable in de- termining varietal differences in tomato cultivation. 5 REFERENCES Albani, J. (2007). Principles and applications of fluorescence spectroscopy. Blackwell Science, 103-186. https://doi. org/10.1002/9780470692059 Becker, M., Christensen, J., Frederiksen, C. & Haugaard, V. (2003). Front-face fluorescence spectroscopy and che- mometrics in analysis of yogurt rapid analysis of ribofla- vin. Journal of Dairy Science, 86, 2508-2515. https://doi. org/10.3168/jds.S0022-0302(03)73845-4 Dakin, J. & Brown, R. (2006). Handbook of Optoelectronics, Tay- lor & Francis, 74-253. Lai, A., Santangelo, E., Soressi, G.P., Fantoni, R. (2007). 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Journal of Food Science, 61, 695–697. https://doi.org/10.1111/j.1365-2621.1996.tb12183.x Acta agriculturae Slovenica, 119/4, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.4.13264 Original research article / izvirni znanstveni članek Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture Abdullah Hassn MOHAMMED 1, 2 Received March 17, 2023; accepted October 13, 2023. Delo je prispelo 17. marca 2023, sprejeto 13. oktobra 2023 1 Field Crops Department, College of Agriculture, Tikrit University, Tikrit, Iraq 2 Corresponding author, e–mail: abdullah@tu.edu.iq Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture Abstract: Germination of immature embryos of interspe- cific crosses in vitro is the most important problem in barley breeding programs. The effect of four media types (MS, ½ MS, B-5, ½ B-5 on immature embryo culture of nine barley variet- ies (Sameer, Buraq, Aksad, Shuaa, Arevat, Alwarkaa, Alhadher, Amel, and Rihan) was studied by using completely random- ized design (C.R.D.) with six replicates. Analysis of variance showed highly significant effects due to varieties, media, and interaction for all studied characteristics, indicated high varia- tion among varieties and media. Aksad variety was significantly superior to all varieties for all studied traits except root number (RN). Medium of ½ B-5 was significantly superior to all media for all studied traits. Aksad interactions with media except B-5 media gave full germination with highly significant superiority over all interactions. Aksad x ½ B-5 interaction showed a highly significant superiority for shoot length (SL) and RL. As a result, ½ B-5 medium was the efficient medium for germination of im- mature embryos of most varieties. These varieties could be used in breeding program of interspecific crosses with wild species by using ½ B-5 medium. Key words: barley varieties, MS medium, Gamborg’s B-5 medium, final germination percentage, shoot length, root length, root number Preučevanje gojišč za gojenje nezrelih embrijev ječmena (Hordeum vulgare L.) Izvleček: Kalitev nezrelih embrijev in vitro je pri medvr- stnih križanjih največji problem pri žlahtnjenju ječmena. Preu- čevan je bil učinek štirih vrst gojišč (MS, ½ MS, B-5, ½ B-5) na kulture nezrelih embrijev devetih sort ječmena (Sameer, Buraq, Aksad, Shuaa, Arevat, Alwarkaa, Alhadher, Amel, in Rihan) v popolnem naključnem poskusu s šestimi ponovitvami. Anali- za variance je pokazala visoko značilne učinke sort, gojišč in njihovih interakcij za vse preučevane lastnosti, kar kaže na ve- like razlike med sortami in gojišči. Sorta Aksad je bila značilno superiorna napram vsem ostalim sortam za vse preučevane la- stnosti, razen za število korenin. Gojišče ½ B-5 je bilo značil- no najboljše za vse preučevane lastnosti. Sorta Aksad je imela popolno kalitev na vseh gojiščih, razen na gojišču B-5. Sorta Aksad je imela na gojišču ½ B-5 značilno najdaljše poganjke in korenine. Izkazalo se je, da je gojišče ½ B-5 najbolj učinkovito za kalitev nezrelih embrijev večine obravnavanih sort. Te sorte bi lahko bile uporabljene v žlahtniteljskih programih za medvr- stna križanja z divjimi vrstami ječmena z uporabo gijišča ½ B-5. Ključne besede: sorte ječmena, MS gojišče, Gamborg’s B-5 gojišče, odstotek končne kalitve, dolžina poganjka, dolžina korenine, število korenin Acta agriculturae Slovenica, 119/4 – 20232 A. H. MOHAMMED 1 INTRODUCTION Barley (Hordeum vulgare L.) is one of the most important cereal crops in the world. It is ranking as the fourth major crop in production and area next to wheat rice and maize (Goedeke et al., 2007, Blake et al., 2011). Barley is one of the earliest, the most domesticated food crop that humans have known since prehistoric times. It was the main source for making bread, soup and por- ridge dishes in the countries of the ancient world. Cur- rently, barley has many uses, whether in human nutrition or animal feed. Barley flour is used alone or mixed with wheat flour in the industry of various types of pastries, especially bread. Barley is also included in many food industries, such as baby food, breakfast cereals, biscuits, beer, other alcoholic and non-alcoholic beverages, malt syrup, malted milk, and chemical industries. Barley is also a rich source of vitamins, minerals and dietary fiber. Its high content of soluble dietary fiber has reinforced the importance of barley and its place as an important food ingredient. Many studies have shown that regular consumption of barley reduces the risk of certain dis- eases such as colon cancer, gallstones, high blood pres- sure and chronic heart disease (Idehen et al., 2017, Biel et al., 2020). In India, most of the barley produced (90 %) is used for human consumption. Barley is also used as animal feed. Its grain (rich in carbohydrates) and the re- sulting straw are used to feed animals. It is also grown in some countries for use as green fodder or as pasture for animals. Some countries, including Iraq, use almost 80 % of grown barley as fodder (FAO, 2009). The use of bar- ley as feed has a special value by giving desirable parts of visceral fat (Kumar et al., 2012). Barley is also grown as a cover crop to prevent soil erosion (water and air erosion) and curb weeds (Mohammed, 2018). Abiotic and biotic stresses lead to a decrease in crop production and nutri- tional value of crop grains over the world. Drought, sa- linity, and disease are among the most important stresses from an economic point of view and the most important in biology (Hussain, 2006; Mohammed et al., 2021). Due to the limited genetic variance in the cultivated varieties of barley, the use of large genetic variance that available in the wild species is necessary in the breeding programs (Ellis et al., 2000, Sreenivasulu et al., 2008). Therefore, interspecific crosses between domestic barley and wild species such as H. bulbosum L. and H. murinum L. are a major source of genetic differences and gives plant breed- ers a great opportunity to recombine the chromosomes set in order to take advantage of the positive aspects found in several individuals and collect them in one or- ganism (Araus et al., 2003, Nevo and Chen, 2010). Many researchers have pointed that the widespread of wild spe- cies of barley indicates the high potential for great genet- ic diversity and adaptation to biotic and abiotic stresses that serve as an important genetic resource for breeding programs. Where the different genotypes of wild species constitute an important source for plant breeders in im- proving the adaptive traits that contribute to increasing and stabilizing production under conditions of biotic and abiotic stresses, and can be an entry point for improving varieties (Suprunova et al., 2007; Alassaf, 2018).However, interspecific crosses between domestic and wild species of barley has many sexual barriers such as very low seed set and abort endosperm due to sexual incompatibilities. Therefore, plant tissue culture plays an important role in solving these problems that faced by plant breeders in breeding programs to improve barley varieties (Houben et al., 2011). Immature embryo rescue is one of the most important applications of tissue culture to solve the prob- lem of sexual incompatibilities. Embryo culture technol- ogy also has several uses such as producing rare species, haploid plants that use to produce homozygous diploid plants, testing seed viability, and breaking seed dorman- cy. In addition, embryo culture is used to produce large number of plants from one embryo by the callus method. However, mature embryos can grow in a simple nutrient medium, but immature embryos need a more complex nutrient medium. As a result, immature embryos need to be grown in efficient culture media to mature and give a complete and healthy natural plant (Burun and Poyra- zoglu, 2002; Pickering and Johnston, 2005; Chahal and Gosal, 2006). Crosses also differ significantly in the type of medium that suitable for each cross (Mohammed et al., 2020). Therefore, there is a need to determine the most suitable culture media for the largest number of genotypes that provide the regular growth of embryo in order to obtain successful breeding programs to improve barley varieties. 2 MATERIALS AND METHODS Experiments were carried out in the field and labo- ratory of College of Agriculture/ Tikrit University in or- der to study the effect of different types of media on im- mature seed germination of nine varieties of barley. Seed of varieties used in this study were obtained from Seed Technology Center/ Office of Agricultural Research/ Ira- qi Ministry of Science and Technology. Barley varieties were: (Sameer, Buraq, Aksad, Shuaa, Arevat, Alwarkaa, Alhadher, Amel, Rihan). Seed of each of the nine varie- ties were planted in the field during the winter session of 2020/2021 to obtain immature embryos for use in the me- dia assessment experiment. All crop service operations such as irrigation, fertilization and weed control were carried out according to researchers’ recommendations Acta agriculturae Slovenica, 119/4 – 2023 3 Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture for the barley crop. Spikes from each variety were har- vested 12-14 days after pollination which embryo length was approximately 1.5 mm. The harvested spikes (with peduncle) were placed vertically in a beaker containing a little amount of water which cover spike peduncle and then placed in the refrigerator until dissection (Devaux 2003). Seed were removed from spikes of each of the nine varieties then were sterilized in 70 % ethanol for a min- ute. After that seeds were disinfested with 1  % sodium hypochlorite solution (NaClO) for 6 minutes then rinsed three times with sterile distilled water then dried on ster- ile paper napkin. After that, immature embryos of each variety were dissected from their caryopses and placed in 100 ml jars (five embryos per jar) containing either full concentration of Murashige and Skoog medium (Sigma- Aldrich Co., M-5519, St. Louis, MO), half concentra- tion of MS medium, full concentration of Gamborg B-5 medium (Sigma-Aldrich Co., G-5893, St. Louis, M), or half concentration of Gamborg B-5 medium. All types of media were without plant growth regulators. Planted jars were labeled and wrapped with ParafilmM® PM-996 (Be- mis Company Inc., Neenah, WI). Process was conducted under a laminar flow hood (ENVIRCO, Environmental Air Control, Inc., Hatfield, PA) to control contamination. All types of media were supplemented with 0.8 % agar (Sigma-Aldrich Co., A-1296, St. Louis, MO); 3  % and 2  % sucrose (Fisher Scientific, S5-500, Fair Lawn, NJ) for MS and Gamborg B-5 media, respectively; pH of MS and Gamborg B-5 media was adjusted to 5.7 and 5.5, re- spectively by using 0.1 N NaOH (Burun and Poyrazoglu, 2002; Houben et al., 2011). Germination process was car- ried out in the growth chamber (Percival, Mod. 135LLVL, Controlled Environments. Boone, IA) in the darkness at a temperature of 25 °C ± 2 for ten days. The number of normal germinated embryos was only counted daily to determine final germination percentage by using the fol- lowing equation described by Scott et al. (1984): Final number of germinated embryos Total number of embryos planted Comparisons were made among the types of media for final germination percentage, shoot length (cm), root length (cm), and root numberfor the nine varieties of barley. Experiment was arranged as a completely rand- omized design (C. R. D.) with six replications (jars). Data were statistically analyzed as a CRD by using PROC MEANS and PROC GLM in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Significant mean separa- tion among types of media, varieties, and the interactions was determined using Fisher’sleast significant difference (LSD) at α = 0.05 and 0.01. Data were graphed usingSig- maPlot version 13 (Systat Software Inc., San Jose, CA). 3 RESULTS AND DISCUSSION Analysis of variance showed highly significant dif- ferences among barley varieties, media types, and barley varieties x media types of interaction for all studied char- acteristics (Table 1). The significant effects among barley varieties, and among media types indicated that there was a high ge- netic variation among varieties, and high variation among media. Furthermore, the significant interaction between varieties x media indicated that each variety re- sponded to each medium type independently. Previous studies have also found significant differences among varieties, media types, and barley varieties x media inter- action (Burun and Poyrazoglu, 2002; Hayes et al. 2003; Han et al. 2011; Houben et al. 2011; Kahani et al. 2012; Mohammed, 2018; Mohammed et al. 2020). Germination of immature embryos is the most important problem that plant breeder faces in breeding program of interspecific crosses between domestic bar- ley and wild barley to improve barley varieties. The final germination percentage (FGP) of immature embryos of nine barley varieties were evaluated based on four types of media (Table 2). The results showed a high significant differences among barley varieties for FGP trait. The Table 1: Analysis of variance of nine varieties of barley (Sameer, Buraq, Aksad, Shuaa, Arevat, Alwarkaa, Alhadher, Amel, Rihan), four types of media (MS, ½ MS, B-5, ½ B-5), and their interaction for final germination percentage (FGP), shoot length (SL), root length (RL), and root number (RN) Source of variation Degrees of freedom FGP SL RL RN Varieties (V) 8 *** *** *** *** Media (M) 3 *** *** *** *** V x M 24 *** *** *** *** Error 180 *** Significant at the 0.001 probability level NS, significance level p > 0.05 Acta agriculturae Slovenica, 119/4 – 20234 A. H. MOHAMMED mean of FGP of immature embryos of barley varieties ranged from 36.11 to 99.17 % at four types of media. Ak- sad variety gave the highest FGP value (99.17 %) and was highly significant superior to all varieties; it fallowed by Arevat variety with 86.67 % of FGP. While Rihan vari- ety had the lowest value of FGP (36.11 %). Table 2 also showed highly significant differences among media types for this trait and ranged from 64.33 to 77.69  % of im- mature embryos. The half concentration of Gamborg’s B-5 medium (1/2 B-5) showed the highest value of FGP (77.69 %) and was significant superior on all media types; while the half concentration of Murashige and Skoog (1/2 MS) medium gave the lowest value (64.33 %). Interaction between barley varieties x media types revealed highly significant difference for FGP trait. The value of FGP of combinations ranged from 23.33 to 100.00 % (Table 2). The interactions between Aksad va- riety with MS, ½ MS, and ½ B-5 media were superior by giving the full and highest value of FGP (100.00 %). While the interaction between (Rihan x ½ MS), (Rihan x B-5), and (Shuaa x ½ MS) showed the lowest average of FGP which amounted to 23.33 %, 26.67 %, and 28.33 %, respectively. These results confirmed the high significant differences among varieties as well as media types. Also, these data confirm that individual varieties of barley re- sponded differently.to each type of media. For example, Shuaa, Alwarkaa, Alhadher, and Rihan varieties gave the highest value of FGP of immature embryos when they cultured on ½ B-5 medium. While Buraq, Amel, and Arevat varieties had the highest value in ½ MS, B-5, and ½ MS & B-5 media, respectively. On the other hand, Are- vat variety was superior with complete FGP in MS, ½ MS, and ½ B-5 media. In conclusion, Aksad variety and half concentration of B-5 medium were the best for final germination trait. Many researchers have found that MS, ½ MS, B-5 and other media can be used for culturing immature and mature embryo. These results were con- firmed by previous studies. Mihailescu and Giura (1996) reported that germination of barley embryo was better in B-5 medium than MS. While Burun and Poyrazoglu (2002) found that MS and ½ MS media were better than B-5 medium when they evaluated the FGP of Kaya 7794 variety on MS, ½ MS, B-5, and RC (Randolph and Cox) media. Han et al. (2011) reported that FGP of barley va- rieties in B-5 medium was higher by 10 % than MS me- dium. Also, Chen et al. (2011) reported that is difficult to draw a conclusion about the most suitable medium for different barley genotypes. Chen’s result confirmed by Mohammed (2018) and Mohammed et al. (2020) found that each genotype of barley responded to each culture medium independently of germinated immature embryo of many crosses that obtained from interspecific crosses between domestic and wild barley. This result may be due to component of media, genotypes, embryo size, sucrose rate, and pH. As a result, these varieties could be used in breeding program of interspecific crosses with wild bar- ley species by using the ½ B-5 media to improve some quality and quantity traits of barley varieties. Table 3 showed the shoot length (SL) of ger- minated immature embryos of nine barley varieties that cultured at four types of media. The results showed high- ly significant differences among barley varieties for SL Table 2: Mean of final germination percentage (FGP) of nine barley varieties in four types of media Varieties of Barley Types of Media Mean (%)MS ½ MS B-5 ½ B-5 Sameer 67.67 62.22 74.17 82.22 71.57 Buraq 62.98 74.60 56.11 70.63 66.08 Aksad 100.00 100.00 96.67 100.00 99.17 Shuaa 58.89 28.33 52.67 73.33 53.31 Arevat 87.78 93.33 93.33 72.22 86.67 Alwarkaa 68.89 63.81 67.78 84.78 71.31 Alhadher 59.33 56.67 60.00 76.00 63.00 Amel 58.89 73.33 93.33 86.67 78.06 Rihan 41.11 26.67 23.33 53.33 36.11 L.S.D. (0.05) L.S.D. (0.01) 8.212 10.900 4.106 5.450 Mean (%) 67.28 64.33 68.60 77.69 L.S.D. (0.05) L.S.D. (0.01) 2.737 3.633 Acta agriculturae Slovenica, 119/4 – 2023 5 Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture trait. The mean of SL of germinated immature embryos of barley varieties ranged from 2.59 to 6.67 cm at four types of media. Aksad variety gave the highest SL value (6.67 cm) and was highly significant superior to all varieties; it fal- lowed by Amel and Arevat varieties with values of 5.83 and 5.65 cm of SL, respectively. Whilst, Shuaa and Al- warkaa varieties gave the lowest value 2.59 and 2.79 cm of SL. Table 3 also revealed highly significant differences among media types for SL trait and ranged from 3.12 to 5.92 cm. The type ½ B-5 medium gave the highest value of SL (5.92 cm) and was significant superior on all me- dia types; while the type 1/2 MS medium had the lowest value of SL (3.12 cm). The barley varieties x media types interaction showed highly significant difference for the trait of SL.The shoot length value of combinations ranged from 1.29 to 11.98 cm (Table 3).The interaction of Ak- sad variety x ½ B-5 medium was a high significant su- perior by giving the highest value of SL which amounted to 11.98 cm; it followed by Aksad x B-5 and Amel x ½ G-5 with 8.97 and 8.00 cm of SL, respectively.Whereas the interactions of Sameer, Shuaa, Alhadher, and Rihan varieties with ½ MS media gave the lowest values of SL. These results confirmed the high significant differences among varieties and among media types for the SL trait. These data confirm that individual varieties of barley re- sponded differently to each type of media for this trait. For instance, MS medium was suitable for Sameer and Arevat varieties as well as ½ MS medium for Arevat by giving the highest value of SL of germinated immature embryos. While ½ B-5 medium was suitable medium for Aksad, Shuaa, Rihan, Alhadher, and Amel varieties as well as B-5 medium for ‘Alhadher’ and ‘Amel’. On the other hand, Buraq and Alearkaa varieties did not show significant differences for SL trait in the four types of me- dia. For this trait, Aksad variety was also the best variety when its immature embryos cultured on 1/2 B-5 medium. The same result was found by Mohammed (2018) that some crosses were superior when cultured in MS, while other crosses were superior in B-5 medium for SL trait These data are consistent with previous findings which reported that genotypes responded to culture medium independently (Burun and Poyrazoglu, 2002; Russowski et al., 2006; Chen et al., 2011; Mohammed et al., 2020). The root length (RL) of immature embryos of nine barley varieties were evaluated based on four types of media (Table 4). The results showed a highly significant difference among barley varieties for RL trait. The mean of RL of immature embryos of barley varieties ranged from 1.45 to 4.68 cm at four types of media. Aksad variety had the highest value (4.68 cm) of RL and was highly significant superior to all studied varieties of barley for RL trait; Are- vat and Amel varieties followed Aksad variety in terms of superiority of RL trait (4.02 and 4.01, respectively). While Shuaa and Sameer varieties had the lowest RL values (1.45 and 1.79, respectively). Table 4 also showed highly significant differences among media types for RL trait and ranged from 2.34 to 3.66 cm of immature embryos of barley varieties. The medium of 1/2 B-5 had the highest value (3.66) of RL trait and was significantly superior on all types of media. Whilst the 1/2 MS medium showed Table 3: Mean of shoot length (SL) of nine barley varieties in four types of media Varieties of Barley Types of Media Mean (cm)MS ½ MS B-5 ½ B-5 Sameer 5.73 1.29 1.97 3.94 3.23 Buraq 5.07 4.58 4.92 5.30 4.96 Aksad 3.50 2.23 8.97 11.98 6.67 Shuaa 2.35 1.38 2.43 4.18 2.59 Arevat 6.97 6.97 3.06 5.58 5.65 Alwarkaa 2.06 2.95 2.99 3.17 2.79 Alhadher 1.87 1.53 5.18 5.18 3.44 Amel 2.43 5.38 7.50 8.00 5.83 Rihan 4.07 1.80 1.98 5.98 3.46 L.S.D. (0.05) L.S.D. (0.01) 1.670 2.217 0.835 1.108 Mean (cm) 3.78 3.12 4.33 5.92 L.S.D. (0.05) L.S.D. (0.01) 0.556 0.739 Acta agriculturae Slovenica, 119/4 – 20236 A. H. MOHAMMED the lowest RL value (2.34 cm). The combination between barley varieties x media types showed highly significant difference for RL trait. The value of RL of interactions ranged from 0.38 to 7.97 cm (Table 4). The combination between Aksad variety x 1/2 B-5 media were superior by giving the highest RL value (7.97 cm); it followed by the interaction of Amel and Aksad with B-5 medium which amounted to 5.61 and 5.48 cm, respectively. While Sa- meer variety x ½ MS interaction gave the lowest average of RL trait (0.38 cm). These results confirmed the high significant differences among varieties and among me- dia types. These data also confirm that individual barley varieties responded differently.to each type of media. For example, Sameer, Rihan, and Buraq varieties were supe- rior in RL when cultured on MS medium as well as B-5 medium for Buraq variety. While Alwarkaa and Arevat varieties had the highest value in ½ MS medium as well as MS medium for Arevat variety. On the other hand, ½ B-5 was suitable medium for Aksad variety as well as B-5 medium for Alhadher and Amel varieties. Whilst four types of media had no significant effect on RL of Shuaa variety. For this trait, Aksad variety was also the best va- riety when its immature embryos cultured on 1/2 B-5 medium. These results were confirmed by previous stud- ies (Burun and Poyrazoglu, 2002; Russowski et al., 2006; Han et al., 2011; Mohammed et al., 2020) that is difficult to draw a conclusion about the most suitable medium for different barley genotypes. Table 5 revealed the root number (RN) of germinat- ed immature embryos of nine barley varieties that cul- tured at four types of media. The results showed a highly significant differences among varieties of barley for RN trait. The mean of RN of germinated immature embryos of varieties of barley ranged from 2.88 to 4.32 at four media types. Aksad, Amel, Arevat, and Buraq varieties had the highest values of RN and were highly significant superior to all other studied of barley varieties with val- ues of 4.32, 4.29, 3.98, and 3.94 of RN trait, respectively. While the other five varieties gave the lowest values of RN. Table 5 showed highly significant differences among types of media for RN trait and ranged from 2.99 to 4.12 roots. The medium type of ½ B-5 gave the highest value of RN which amounted to 4.12 roots and was significant superior on all media types; whereas the medium type of 1/2 MS had the lowest RN value (2.99). The interaction between barley varieties x media types showed highly significant difference for the trait of RN. The root num- ber value of combinations ranged from 1.67 to 5.58 roots (Table 5). The interaction of Aksad and Amel varieties with ½ B-5 medium, Arevat x ½ MS medium, and Ak- sad x B-5 medium had the highest values of RN which amounted to 5.58, 5.27, 5.20, and 5.11 roots, respectively. While the interactions of Rihan and Sameer varieties with ½ MS medium, ‘Alhadher’ x MS medium, and ‘Sh- uaa’ x B-5 medium gave the lowest values of RN. These results confirmed the high significant differences among varieties and among media types for the RN trait. Also, these data confirm that individual varieties of barley re- sponded differently.to each type of media for this trait. For instance, MS medium was suitable for Sameer variety by giving the highest value of SL of germinated immature embryos. While ½ MS medium was suitable Table 4: Mean of root length (RL) of nine barley varieties in four types of media Varieties of Barley Types of Media Mean (cm)MS ½ MS B-5 ½ B-5 Sameer 3.07 0.38 1.40 2.30 1.79 Buraq 4.37 2.96 4.00 3.63 3.74 Aksad 3.62 1.66 5.48 7.97 4.68 Shuaa 1.75 1.30 1.00 1.76 1.45 Arevat 4.79 4.83 2.76 3.72 4.02 Alwarkaa 1.62 3.05 2.76 2.29 2.43 Alhadher 2.06 1.77 3.95 4.12 2.97 Amel 1.71 3.90 5.61 4.83 4.01 Rihan 4.25 1.22 2.27 2.37 2.53 L.S.D. (0.05) L.S.D. (0.01) 1.197 1.590 0.598 0.795 Mean (cm) 3.03 2.34 3.25 3.66 L.S.D. (0.05) L.S.D. (0.01) 0.399 0.530 Acta agriculturae Slovenica, 119/4 – 2023 7 Investigation of the medium for barley (Hordeum vulgare L.) immature embryo culture medium for Arevat variety. Aksad, Buraq, Shuaa, Amel, and Rihan varieties were superior in RN when cultured on ½ B-5 medium, while Alwarkaa and Alhadher varie- ties gave the highest value in B-5 medium. For this trait, Aksad variety was also the best variety when its imma- ture embryos cultured on 1/2 B-5 medium. These results are consistent with previous studies which reported that genotypes responded to culture medium independently (Burun and Poyrazoglu, 2002; Russowski et al., 2006; Chen et al., 2011; Han et al., 2011; Mohammed et al., 2020). 4 CONCLUSION There were highly significant differences among bar- ley varieties, media types, and interactions for all studied characteristics except the leaf number, which indicated that there was a high genetic variation among varieties, and high variation among media. Aksad variety, ½ B-5 medium, and Aksad x ½ B-5 gave the highest values for all studied traits. Varieties of barley responded to each type of media independently. 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Plant Molecular Biology, 64(1-2),17-34. https://doi.org/10.1007/ s11103-006-9131-x Acta agriculturae Slovenica, 119/4, 1–11, Ljubljana 2023 doi:10.14720/aas.2023.119.4.13421 Original research article / izvirni znanstveni članek Physical and aerodynamic properties of date palm pollen grains Mohamed M. IBRAHIM 1, 2, Mohamed GHONIMY 3, 1, Eid ABD EL RAHMAN 1 Received April 15, 2023; accepted October 02, 2023. Delo je prispelo 15. aprila 2023, sprejeto 2. oktobra 2023 1 Department of Agricultural Engineering, Faculty of Agriculture, Cairo University, Giza, Egypt 2 Corresponding author, e–mail: mohamed.ibrahim@agr.cu.edu.eg 3 Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University, Buraydah, Saudi Arabia Physical and aerodynamic properties of date palm pollen grains Abstract: The present study was designed to determine the effect of four moisture content levels (4, 5, 6, and 7 %) on the physical and aerodynamic properties of date palm pollen grain (DPP). The physical properties of DPP included pollen length (L), width (w), thickness (T), projected area (Ap), geo- metric mean diameter (dg), mass (m), sphericity (S), and bulk density (ρp). It was observed that the moisture content did not significantly influence the physical properties of the DPP. The aerodynamic properties of DPP included the terminal veloc- ity (Vt), drag coefficient (Dc), drag force (Df), and Reynolds number (Re). The pollen Reynolds number (Re) is significant at different pollen grain moisture content, and regression mod- els were developed in the form of polynomial and exponential relationships. Also, the 3rd order polynomial relationship was found between Re and Dc. The results showed that the average values of Vt, Dc, Df, and Re were about 0.6 m s -1, (0.38 to 0.45), 1.09E-11 N, and (0.29 to 0.42), respectively. The results of this study will be helpful in the performance of date palm pollina- tion machines. Key words: aerodynamic, date palm, pollen grain, prop- erties, terminal velocity Fizikalne in aerodinamične lastnosti pelodnih zrn dateljeve palme Izvleček: Namen raziskave je bil določiti učinek štirih vsebnosti vode (4, 5, 6, in 7  %) na fizikalne in aerodinamič- ne lastnosti pelodnih zrn dateljeve palme (DPP). Fizikalne lastnosti so obsegale dolžino peloda (L), širino (w), debelino (T), projekcijsko površino (Ap), poprečni geometrični premer (dg), maso (m), sferičnost (S), in gostoto (ρp). Ugotovljeno je bilo, da vsebnost vode ni značilno vplivala na fizikalne lastno- sti peloda. Aerodinamične lastnosti peloda so obsegale končno hitrost (Vt), koeficient upora (Dc), moč upora (Df) in Reynoldo- vo število (Re). Reynoldovo število peloda (Re) je bilo značilno različno pri različnih vsebnostih vode, razvit je bil regresijski model na osnovi polinomnih in ekspotencialnih razmerij. Med Re in Dc je bilo ugotovljeno polinomno razmerje tretjega reda. Rezultati so pokazali, da so bile poprečne vrednosti parametrov Vt, Dc, Df, in Re okrog 0,6 m s -1, (0,38 do 0,45), 1,09E-11 N in (0,29 do 0,42). Rezultati raziskave bodo pripomogli k boljšemu delovanju opraševalnih naprav. Ključne besede: aerodinamika, dateljeva palma, pelodna zrna, lastnosti, končna hitrost Acta agriculturae Slovenica, 119/4 – 20232 M. M. IBRAHIM et al. 1 INTRODUCTION Date palm is the source of a wide range of products and services, including many necessities of life. The pri- mary product of the date palm is fruit, which is rich in protein, vitamins, and mineral salts. The total date palm production in Egypt is about 1.64 million tons annually (FAOSTAT, 2019). Date palm pollination has been iden- tified as a major factor for successful date production since quality and fruit yield depend on the correct ap- plication of pollen grains. It has conclusively been shown that the benefits of the pollination process lie in obtain- ing large-sized, high-quality fruits, avoiding the presence of small fruits (Salomón-Torres et al., 2017). In Egypt, farmers select male palm trees and ex- change palm pollen between farms even over long dis- tances from the country (Bekheet and El-Sharabasy, 2015) Notably, the production of pollen from male palms differs significantly according to changing geographic regions. Generally, any male palm variety’s pollen can be used to pollinate any female variety. Either, to estab- lish a perfect pollination and achieve the highest yield with good fruit quality, it was advantageous to mix pol- len grains with different carriers, essential minerals and ascorbic acid. It also improves the effectiveness of pollina- tion (Radwan et al., 2022). However, as most male palms are seedlings, there are significant variances in the pollen quality of these plants (Salomón-Torres et al., 2021). In addition, pollen’s physical and aerodynamic properties are one of the most important properties that affect pol- lination. The characteristics of pollen can affect its trans- mission from date palm pollinating machines to female flowers. Alharbi and Mousa (2021) studied some physi- cal properties of palm pollen in the Qassim region, Saudi Arabia; they reported that date palm pollen grains come in a wide variety of shapes, sizes, and surface marking characteristics. The palm pollen sizes range from 3.3 to 704 μm. The pollen shape found in this study was spheri- cal. The aerodynamic properties are the most important in describing the movement of grains in the air. For de- signers and operators of agricultural machinery, knowl- edge of the aerodynamic properties of the grain (floating velocity, final velocity, aerodynamic coefficient of resist- ance) is important and essential (Polyák and Csizmazia, 2010). These properties are terminal velocity, drag force, drag coefficient, and Reynolds number (Khoshtaghaza and Mehdizadeh, 2006). Terminal velocity is the steady air velocity value at which an object or a material is sus- pended in the vertical air stream. In other words, the maximum or terminal speed attained in free fall before air resistance will keep it from falling faster (Mohsenin, 2020). The morphology of pollen grains may also affect the aerodynamic properties. The relationship between terminal velocity and moisture content of pistachio nut was studied by Unal et al. (2008), 14.2 % increasing per- centage in the terminal velocity was found depending on the change in moisture content from 5.83 to 30.73 % (wb). Morever, Matouk et al. (2008) mentioned that cof- fee cherries (‘Catual’) drag coefficient decreased from 0.05 to 0.03 as moisture content increased from 10.7 to 53.9 % (d.b). According to Eşref, and Nazmi (2016), as the moisture content of yellow lentil seeds increased the terminal velocity increased linearly from 1.5 to 2.09 m s-1 due to the increasing seed mass per unit frontal that faces the air stream. Abubakar et al. (2019) fabricated a measuring device to determine the terminal velocity of paddy rice, sorghum, and bean grains. Terminal veloci- ties results in that study were 6.95 ± 0.37, 4.71 ± 0.24, and 10.98 ± 0.27 m s-1 for paddy rice, sorghum, and beans, respectively. In addition, the device efficiency was found to be 70.06 %. Thus, the main objective of this article is to determine the physical and aerodynamic properties of date palm pollen grain because these properties signifi- cantly impact the performance of date palm pollination machines. 2 MATERIALS AND METHODS 2.1 DATE PALM POLLEN GRAINS (DPP) PREPA- RATION DPP collected from three resources of a local mar- ket in Siwa Oasis, Matrouh Governorate, Egypt, free of foreign matters, immature, and broken grains were se- lected for this study. The date palm pollen variety used in this research is not specified because commercial com- panies selling pollen have collected from more than one source and mixed them to improve the properties of the resulting crop. Traditionally, pollen grains for some varieties are mixed and then used for pollination. Moisture content is one of the most important factors affecting the quality of the pollination process. The moisture content of the pollen grains was determined using the oven method at 103 ± 2 °C until reaching a constant mass (AOAC, 2000) and as described in many investigational studies. Results of minimum and maximum moisture content values of DPP were 3.6 and 8.2 % respectively. Considering the significance of moisture content in pollination process. Four levels of moisture content (4 %, 5%, 6 %, and 7 % db.) were used to determine the aerodynamic proper- ties of the DPP. So far, each moisture content level was prepared using a pollen sample taken and placed in the oven at a temperature of 50 oC, then the sample mass was measured, and moisture content was calculated every 15 Acta agriculturae Slovenica, 119/4 – 2023 3 Physical and aerodynamic properties of date palm pollen grains minutes’ interval, in order to find out the time required to remove moisture to reach the different required mois- ture contents (4 %, 5 %, 6 %, and 7 % dry basis). After that, each sample was placed inside a desiccator in order to acclimatize it to the ambient environment (Coşkun et al., 2005). Conversely, the levels of chosen moisture con- tent cover the range often seen in stored DPP and was checked before each experiment. 2.2 PHYSICAL PROPERTIES OF DATE PALM POL- LEN GRAIN 2.2.1 Date palm pollen grain mass (m) The mass (m) of the date palm pollen grain was cal- culated by dividing the 1 g of pollen mass by the num- ber of pollen per one gram. Using the hemocytometer method, it is possible to count the number of pollens in a specific volume of water (suspensions solution: water + pollen) (Mahmoud-Aly et al., 2018). A hemocytometer method consists of microscope (QUANTA FEG250, Ja- pan) and a thick glass microscope slide (dimension 3×3 mm). The slide is divided into nine squares (dimension 1 × 1 mm), as shown in (Fig. 1). The volume covered by one square is calculated by multiplying the area of one square (1 mm x 1 mm = 1 mm2) by the depth of the square (0.1 mm). Hence, the final volume of each square is 0.0001 ml. The 2.5 g of pollen grains were added to 500 ml of distilled water and mixed for 5 min using mag- netic stiller, so the dilution factor is 200 ml of water 1 g-1 of pollen (500 ml of water 2.5 g-1 of pollen). The sample from suspension solution was loaded into slide by using a micropipette. Then the hemocytometer slide was placed under the microscope, so it could determine the pollens number. The average pollen per small square was calcu- lated (448 pollen). The number of pollen grains per one gram was calculated from equation (1). (1) Where; Npg is the number of pollen grains per one gram, Pss is the average number of pollens per small square (448 pollen), Du is the dilution factor (equal to 200 ml g-1), and vss is the volume of suspension upper small square (equal to 0.0001 ml). 2.2.2 Date palm pollen grain dimensions and pro- jected area The DPP dimensions were measured at different moisture content levels (4, 5, 6, and 7 %) using scan- ning electron microscopy (SEM, JSM-5200, Jeol Japan). Twenty date palm pollen were randomly selected from each moisture content level to determine grain dimen- sions and projected area (Ap). Three major dimensions of the pollen grain, namely length (L, µm), width (w, µm), and thickness (T, µm) were measured according to the biggest and smallest surface of the pollen grain (Obi and Offorha, 2015). The pollen samples were placed on a cop- per holder and coated with a fine gold layer using a fine coat (JFC-1100 E, Ion sputtering device, JEOL, Japan) before the observation to avoid electrostatic charging during observation. Then, the sample was observed un- der a high vacuum with acceleration voltage (25 kV) and at 500, 2000, and 5000-fold magnifications. The projected area of the pollen was determined by using the Image J program. Figure 1: Pollen number determination using hemocytometer methods: (a) hemocytometer slide, and (b) pollen under micro- scope Acta agriculturae Slovenica, 119/4 – 20234 M. M. IBRAHIM et al. 2.2.3 The bulk density (ρp) The bulk density (ρp) defined as the mass per unit volume of a particle, was determined for date palm pol- len grain at different moisture content levels according to Abdelhady et al. (2023). 2.2.4 The geometric mean diameter (dg) The geometric mean diameter (dg, µm) was calcu- lated from equation (2) according to Mohsenin (2020) (2) Where; L is the pollen grain length (µm), w is the pollen grain width (µm), and T is the pollen grain thick- ness (µm). 2.2.5 Sphericity (S) The sphericity expresses the shape character of the pollen relative to that of a sphere of the same volume. As- suming that the diameter of the circumscribed sphere is equal to the longest intercept L of the ellipsoid and that the volume of the pollen grain is equal to the volume of a triaxle ellipsoid with intercepts L, w, and T. Also, the degree of sphericity (S) was calculated from equation (3), according to Mohsenin (2020). (3) 2.3 AERODYNAMIC PROPERTIES OF DATE PALM POLLEN GRAIN 2.3.1 Terminal velocity (Vt) To evaluate the performance of pollination opera- tions and options involving the presence of air flow, it is necessary to determine the terminal velocity of the date palm pollen grain. It will be used in studying the pollen grain in the airflow. When the pollen grain is immersed into an ascendant air flow, the pollen is subjected to the action of two kinds of forces: gravitational force and re- sisting drag force as shown in Fig. (2). When these vector magnitudes are balanced (Fig. 2), the pollen grain begins a movement at a constant speed so-called terminal velocity that is depending on pollen mass (m), acceleration due to gravity (g), pollen grain density (ρp), air flow density (ρa), drag coefficient (Dc), and pollen grain projected area (Ap). The experimental DPP terminal velocity (Vt) was measured at different moisture content levels using a device illustrated in Fig. (3). It consists of a centrifugal fan connected with an electric motor (Dayton- 1/70 HP). The air velocity was controlled by a digital control switch/regulator inverter connected to the blower motor; it is allowed to change the speed of the motor (Obaia and Ibrahim, 2015). The next unit was a cylindrical tube, it is divided to three parts. The first part is a PVC pipe with a length of 200 mm and a diameter of 50 mm, connected at its upper end to a grid to homogenize airflow. The second part is a PVC pipe with a length of 700 mm and a diam- eter of 50 mm connected at its upper end to a perforated screen to carry pollen on it. This tube has two holes in the middle with a diameter of 5 mm to connect the sensors to measure the speed of the air stream. The third part of the tube is a transparent glass tube (acrylic) with a length of 900 mm and a diameter of 50 mm to watch the pol- len grains being carried by the air stream. The terminal velocity of the studied DPP could be obtained by meas- uring the air velocity required to suspend the particles Figure 2: Pollen weight and drag force acting on the date palm pollen grain Acta agriculturae Slovenica, 119/4 – 2023 5 Physical and aerodynamic properties of date palm pollen grains in the vertical air stream. The air velocity was measured by using the hot wire air velocity meter connected with a velocity probe (TENMARS TM-4001-the air velocity: 0.01 - 25 m s-1). 2.3.2 The drag coefficient (Dc) The drag coefficient (Dc) of date palm pollen grains was calculated from equation (4) according to Mohsenin (2020). (4) Where Dc is the drag coefficient (dimensionless), m is the date palm pollen grain mass (kg), g is the gravi- tational acceleration (9.81 m s-2), ρp is the particle bulk density (kg m-3), ρa is the air bulk density which equals to 1.206 kg m-3 at room temperature, Ap is projected area of date palm pollen grain (m2), and Vt is the pollen grain terminal velocity (m s-1). 2.3.3 The drag force (Df) When air flow occurs around the date palm pollen grain, the action of the forces involved can be illustrated by Fig. (4). The pressure on the upper side of the pollen grain is less than the pressure P in the undisturbed air stream and that on the lower side is greater than the pressure P in the undisturbed air stream. The results in a decrease of pressure, -P, on the upper side indicated by arrows drawn Figure 3: Experimental set-up used for measuring the terminal velocity Acta agriculturae Slovenica, 119/4 – 20236 M. M. IBRAHIM et al. away from the surface, and an increase of pressure, +P, shown by arrows drawn toward the object. In addition, these forces normal to the surface of the pollen, and there are shear stresses, τ, acting tangential to the surface in the direction of airflow and resulting from frictional effects. The resultant force Fr, may be resolved into components of drag force (Df) and lift force (Fh). The equations for calculating Df and Fh have been derived by dimensional analysis assuming the smooth pollen grain having a pro- jected area (Ap), moving through air flow density (ρa), drag coefficient (Dc), and air velocity (Vt). Therefore, the Df will be as the following equation, (5) To derive the drag force using the Kirchhoff and Gogh-Mann dimensional analysis method, the following steps are followed: 1. Determine the number of properties involved in the relation, n = 5 2. Write the dimensions of all the properties in- volved in the relationship are shown in table (1). 3. Choose three base quantities, in this case it is (ρa, Vt, Ap), which must satisfy the following condition: 4. Determine the number of criteria (k) K = n-3 = 5-3 = 2 5. The relation (5) can be writen as follows: (6) 6. On the basis of π theory, it can be writen: (7) 7. Estimating the values of μ, λ and τ for each cri- terion using dimensional analysis and solving equations algebraically and by substituting in the original exponen- tial functional relationship, we get that: (8) Where Df is the drag force (N), Ap is the date palm pollen grain projected area (m2), ρa is the air density which is equal to 1.206 kg m-3 at room temperature(Gupta, 2013), and Vt is the pollen grain terminal velocity (m s -1). Figure 4: Air flow rate and force analysis acting the pollen grain Table 1: Symbols and dimensions of the properties affecting the drag force of date palm pollen grain Property Symbol Dimension Mμ Lλ Tτ μ λ τ Drag force Df 1 1 -2 Air density ρa 1 -3 0 Air velocity Vt 0 1 -1 Projected area Ap 0 2 0 Drag coefficient Dc 0 0 0 Acta agriculturae Slovenica, 119/4 – 2023 7 Physical and aerodynamic properties of date palm pollen grains 2.3.4 Reynolds number (Re) Reynolds number (Re) is an essential aerodynamic attribute that represents the ratio of inertial effects to vis- cous effects on the particle, in similar words, it is the ratio between the product of the particle’s velocity and length scale to viscosity of the medium/fluid in which the parti- cle is moving in this case, air (Zare et al., 2013). In most recent methodology, the Reynolds number (Re) was cal- culated from equation (9) (Grega et al., 2013): (9) where ρa is the air density, which is equal to 1.206 kg m-3 at room temperature, Vt is the pollen grain terminal velocity (m s-1), dg is the geometric mean diameter of the seed (m), and μ is air viscosity (1.816 × 10–5 N s m-2 at room temperature). The tested values of moisture content (MC) were 4, 5, 6, and 7 % dry basis. 2.4 STATISTICAL PROCEDURE Measured data with all variables were statistically analyzed by a software program (CoStat ver. 6.400, 2008), applying a complete randomized design via analysis of variance. The means of the treatments were obtained, and Student-Newman-Keuls differences were tested at a 5 % level of probability. 3 RESULTS AND DISCUSSION 3.1 PHYSICAL PROPERTIES OF DATE PALM POL- LEN GRAIN The average values of the date palm pollen grain length (L), width (w), thickness (T), geometric mean di- ameter (dg), projected area (Ap), mass (m), sphericity (S) and bulk density (ρp) at four levels of moisture content 4, 5, 6, and 7 % are listed in Table (2) and Fig. (5). It is clear that the pollen grain dimensions L, w, T, dg, S, m, and ρs were insignificant at different pollen grain mois- ture content. The pollen grain length (L) increased by 1.0 and 2.03 % when the moisture content increased from 4 to 5, and 6 %, respectively while the L decreased by 1.5 % when the moisture content increased from 6 to 7 %. Also, the L increased by 1.0 % when the MC increased from 5 % to 6 %. The same trend was found for w, and T. The geometric mean diameter (dg) remained nearly con- stant (10.4 µm), with the MC increased from 4 to 7 %. The pollen mass (m), and sphericity (S) remained nearly constant at 1.1E-12 kg, and 0.52, respectively, with the MC increased from 4 to 7 %. The pollen grain projected area (Ap) increased by 1.4, 1.9, and 2.5 % when the mois- ture content increased from 4 to 5, 6, and 7 %, respec- tively. Also, the Ap increased by 0.5 and 1.1 % when the pollen grain moisture content increased from 5 % to 6 and 7 %, respectively. The bulk density (ρp) increased by 0.7, 1.4, and 2.6 % when the moisture content increased from 4 to 5, 6, and 7 %, respectively. Also, the ρp increased by 0.7 and 1.9 % when the pollen grain moisture content increased from 5 % to 6 and 7 %, respectively. The lack of increase in the dimensions and projected area of date pollen grain with the increase of moisture content is due to the hardness of the pollen cover and its lack of water absorption (Pope, 2010; Bunderson and Levetin, 2015). 3.2 AERODYNAMIC PROPERTIES OF DATE PALM POLLEN GRAIN The average values of aerodynamic properties in- cluded terminal velocity (Vt), drag coefficient (DC), drag force (Df), and Reynolds number (Re) of the date palm pollen grain (DPP) at different moisture content (4, 5, 6, and 7 %) are listed in Table (3). It’s clear that the Vt, DC, and Df were insignificant at different pollen grain mois- ture content. However, the pollen Reynolds number Re are significant at different pollen grain moisture content. Figure 5: Scanning electron microscopy of date palm pollen grain at different levels of moisture content Acta agriculturae Slovenica, 119/4 – 20238 M. M. IBRAHIM et al. For terminal velocity (Vt), the average values of Vt were about 0.59 and 0.6 m s-1. The terminal velocity (Vt) of pollen grains was thoroughly examined as it plays an important role in the performance of pollination ma- chines. The frequency distribution of the measured val- ues of Vt are shown in Figure (6). It is clear that the 25, 35, 30, and 30 % of the Vt measured values ranged from 0.59 to 0.6 m s-1 for 4, 5, 6, and 7 % (dry basis) of moisture content, respectively. Also, from Table (3), the average values of drag coef- ficient (Dc) were 0.45 ± 0.16, 0.41 ± 0.13, 0.39 ± 0.12, and 0.38 ± 0.10 for 4, 5, 6, and 7 % (dry basis) of moisture content respectively. The results confirmed an inverse relationship between Dc and pollen moisture content, as the moisture content of pollen grains increased from 4 to 7  %. (dry basis) the drag coefficient decreased from 0.54 to 0.38. The pollen grain Dc decreased by 8.0, 13.3, and 15.5 % when the moisture content increased from 4 to 5, 6 and 7 % (dry basis) respectively. While, the Dc decreased by 4.9, and 7.3 % when the moisture content increased from 5 to 6 and 7 % (dry basis) respectively. In addition, the Dc decreased by 2.6 % when the mois- ture content increased from 6 to 7 % (dry basis). For drag force (Df), the Df remained nearly constant, 1.09E-11 N, with the MC increased from 4 to 7  % (dry basis). This means that the DPP mass is not affected by the increase in moisture content (From Table 3), and therefore, it does not need an increase in the drag force. The Reynolds number (Re) of date palm pollen grain increased by increasing moisture content. A direct relationship was observed for the Re as it increased from 0.29 to 0.42 as the moisture content increased from 4 to 7 % (dry basis) and Re increased by 6.9, 34.5, and 44.8 % when the moisture content increased from 4 to 5, 6, and 7 % (dry basis), respectively. While the Re increased by 25.8, and 35.5 % when the moisture content increased from 5 to 6 and 7 % (dry basis) respectively. In addition, the Re increased by 7.7 % when the moisture content in- creased from 6 to 7 % (dry basis). The maximum value of drag coefficient was found at moisture content level 4% (dry basis), while the highest value of Vt and Re were performed at 7 % (dry basis) moisture content level. The relation between Reynolds number (Re) and drag coefficient (Dc) is shown in Fig. (7). It’s clear that there is an inverse relationship between Re and Dc. The data of Re and Dc was analyzed to give the best fit relation of the 3rd order polynomial function: (10) The range of the Reynolds number (Re) into equa- tion (10) were 0.42 ≥ Re ≥ 0.29 while, the values of the Table 2: Physical properties of date palm pollen grain at different moisture contents MC,% dry basis Pollen grain dimensions S m, kg Ap, µm 2 ρp, kg m -3L, µm w, µm T, µm dg, µm 4 19.7 ± 8.6 a 8.6 ± 0.9 a 6.8 ± 2.6 a 10.4 ± 1.8 a 0.52 ± 0.1 a 1.1E-12 ± 6.2E-28 a 133.33 ± 58.0 a 692.9 ± 21.9 a 5 19.9 ± 4.1 a 8.8 ± 1.8 a 6.8 ± 1.7 a 10.4 ± 1.4 a 0.52 ± 0.1 a 1.1E-12 ± 6.2E-28 a 135.21 ± 41.9 a 697.6 ± 22.4 a 6 20.1 ± 4.2 a 8.8 ± 1.4 a 6.8 ± 1.4 a 10.5 ± 1.2 a 0.52 ± 0.1 a 1.1E-12 ± 6.2E-28 a 135.92 ±3 7.6 a 702.4 ±2 3.7 a 7 19.9 ± 4.3 a 8.9 ± 1.4 a 6.9 ± 1.4 a 10.4 ± 1.4 a 0.52 ± 0.1 a 1.1E-12 ± 6.2E-28 a 136.70 ± 38.1 a 711.2 ± 25.2 a Significant n.s n.s n.s n.s n.s n.s n.s n.s MC = Pollen moisture content, L = pollen length, w = pollen width, T = pollen thickness, dg = pollen geometric mean diameter, S = pollen sphericity, m = pollen mass, Ap = pollen projected area, ρp = pollen bulk density n.s. Not significant at the 0.05 and 0.01 probability levels Table 3: Aerodynamic properties of date palm pollen grain at different moisture content MC (% dry basis) Vt, m s -1 Dc Df, N Re 4 0.59 ± 0.03 a 0.45 ± 0.16 a 1.09E-11 ± 7.1E-16 a 0.29 ± 0.014 b 5 0.60 ± 0.02 a 0.41 ± 0.13 a 1.09E-11 ± 3.1E-16 a 0.31 ± 0.015 b 6 0.60 ± 0.03 a 0.39 ± 0.12 a 1.09E-11 ± 5.1E-16 a 0.39 ± 0.011 a 7 0.60 ± 0.03 a 0.38 ± 0.10 a 1.09E-11 ± 7.2E-16 a 0.42 ± 0.039 a Significant n. s n. s n. s * MC = Moisture content, Vt = Terminal velocity, Dc = drag coefficient, Df = drag force, Re = Reynolds number * Significant at the 0.05 probability level. n.s. Not significant at the 0.05 and 0.01 probability level Acta agriculturae Slovenica, 119/4 – 2023 9 Physical and aerodynamic properties of date palm pollen grains constants a, b, c, and d were (-) 50.821, 60.238, (-) 23.846, and 3.527 respectively. 3.3 REGRESSION MODELS Data from the research of the date palm pollen grain’s (DPP) aerodynamic characteristics at various MC levels were fitted and the best fit was chosen. The models created for the DPP’s terminal velocity (Vt), drag coef- ficient (Dc), and Reynolds number (Re) as functions of MC are shown in Table 4. The R2 varied between 0.93 and 1.00. The suitable models for predicting the aerody- namic characteristics of DPP as a function of moisture content were found to be polynomial regression models. As a function of moisture content, Pradhan et al. (2010) initiated a polynomial model for the terminal velocity of mung bean seeds, whereas linear models were created for the drag coefficient and Reynolds number. Additionally, Nalbandi et al. (2010) initiated a polynomial model for predicting Makhobeli seed terminal velocity as a func- tion of moisture content. A linear relationship was re- ported by Khodabakhshian et al. (2012) between the Vt of Tef grain and its MC, while for the terminal velocity of coffee cherries and beans as a function of their moisture content and true density, a non-linear equation was de- rived by Matouk et al. (2008). 4 DISCUSSION The present study evaluated and modeled the physi- cal and aerodynamic properties of date palm pollen grain (DPP) as a function of moisture content. In summary, the physical properties of DPP included pollen length (L), width (w), thickness (T), projected area (Ap), geometric mean diameter (dg), mass (m), sphericity (S), and bulk density (ρp). It was observed that the physical properties of the DPP were not significantly influenced by the mois- ture content. The aerodynamic properties of DPP includ- ed the terminal velocity (Vt), drag coefficient (Dc), drag force (Df), and Reynolds number (Re). The results indi- cated that the terminal velocity increased slightly from 0.59 to 0.6 m s-1 with an increase in moisture content from 4 to 7 % (dry basis). As it was noted by Nalbandi et al. (2010) the slight increase in the terminal veloc- ity of the DPP with an increase in the moisture content may be attributed to the formation of the pollen grain cover is a solid layer that does not absorb water, so there is no increase in the pollen grain mass thus the critical speed of pollen is not affected. Furthermore, Sharma et al. (2012) reported an increase in the terminal velocity of mung bean from 4.86 to 5.29 m s–1 as the moisture content increased from 7.28 to 17.77 % (db). Strong evi- dence was found according to Galedar et al. (2010) study, the terminal velocity (m s-1) of sunflower, soybean, and canola seeds increased by 10.67 %, 2.16 %, and 4.31 %, as the moisture content of the seeds increased from 7.35 to 23.7, 9.52 to 24.64, and 7.11 to 25.72 % (wb), respectively, these results agree with the present study findings. Also results showed that the Dc od DPP decreased from 0.45 to 0.38 with the increase in moisture content from 4 to 7 % (dry basis). The results of this investigation concur with the bulk of other studies conducted by various research- Figure 6: The frequency distribution of the pollen grain termi- nal velocity Figure 7: Drag coefficient (Dc) versus Reynolds number (Re) at four levels of moisture content Table 4: Regression models of aerodynamic properties of date palm pollen grain Regression models R2 Vt = - 0.0025 MC 2 + 0.0155 MC + 0.5775 0.93 Dc = 0.0075 MC 2 - 0.0605 MC + 0.5025 1.00 Df = Constant = 1.09E-11 Re = 0.249 e 0.134 MC 0.95 Acta agriculturae Slovenica, 119/4 – 202310 M. M. IBRAHIM et al. ers, which found an inverse relationship between the drag coefficient and the moisture content of seeds. The inverse connection could be explained by variations in the seeds’ surface characteristics, actual densities, mor- phologies, and sizes as the moisture content increased. Also, it was mentioned by Nalbandi et al. (2010) that Makhobeli, triticale, and wheat seeds’ drag coefficient decreased by increasing seeds’ moisture content.; while Mohsenin (2020) reported that as the MC increased from 6.2 to 14.4 % (dry basis) for the cultivars NSFH-36, PSFH-118, GKSFH-2002, and SH-3322, respectively, the drag coefficient of four different cultivars of unshelled sunflower seeds decreased from 0.23 to 0.18, 0.31 to 0.20, 0.27 to 0.16, and 0.36 to 0.12. The current study’s find- ings are consistent with those of Pradhan et al. (2010) for mung bean seed; the drag coefficient decreased as moisture content increased. The trend observed in the Reynolds number of DPP investigated in this study as the moisture content increased from 0.29 to 0.42 was ap- proximately similar to that reported by Schwendemann et al. (2007) for saccate pollen grains. It was reported that the Reynolds number of saccate pollen grains increased from 2.3E-2 to 6.5E-2 with increased moisture content. The Df of DPP remained constant (1.09E-11 N) at differ- ent moisture content levels. 5 CONCLUSION The physical and aerodynamic properties of date palm pollen grain (DPP) as a function of moisture con- tent were evaluated. The physical properties of DPP included pollen grain dimensions (length L, width w, thickness T), projected area (Ap), geometric mean diam- eter (dg), mass (m), sphericity (S), and bulk density (ρp). The results indicated that, the pollen grain dimensions’ L, w, T, and dg were insignificant at different pollen grain moisture content. Also, the pollen mass (m), and sphe- ricity (S) remained nearly constant 1.1E-12 kg and 0.52, respectively, with the moisture content increased from 4 to 7 %. While, the pollen grain projected area (Ap) in- creased by 1.4, 1.9, and 2.5 % when the moisture content increased from 4 to 5, 6, and 7 % respectively. The pollen grain bulk density increased from 692.9 to 711.2 when the moisture content increased from 4  % to 7  %. The aerodynamic properties of DPP included the terminal velocity (Vt), drag coefficient (Dc), drag force (Df), and Reynolds number (Re). The results of aerodynamic prop- erties indicated that the pollen grain Vt, Df, and Df were insignificant at different pollen grain moisture content. However, the pollen grain Reynolds number is signifi- cant at different pollen grain moisture content. There is an inverse relationship between the Re and Dc. The op- timal models for predicting the aerodynamic properties of DPP as a function of moisture content were found to be polynomial regression models. Finally, the results of this study will be helpful in the performance of date palm pollination machines. 6 ACKNOWLEDGMENTS The authors acknowledge the financial support from Cairo University, Egypt for funding the project titled “Design and manufacture of date palm pollination machine operated by tractor”. 7 REFERENCES Abdelhady, A., Ibrahim, M., Mansour, H., El-Shafie, A., & Abd El Rahman, E. (2023). Physico-mechanical properties of sugarcane stalks. Acta Technologica Agriculturae, 26(3), 142–151. https://doi.org/10.2478/ata-2023-0019 Abubakar, A.J., Iya, S.A., Kabri, H.U., & Abdulrafeeu, M.A. (2019). Development of a terminal velocity measuring de- vice for grains. Nigeria Journal of Engineering Science and Technology Research, 5(1), 76-83. Alharbi, A.B., & Mousa, H.M. (2021). 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Geometric and mechanical properties of mung bean (Vigna ra- diata L.) grain: Effect of moisture. International Jour- nal of Food Properties, 11(3), 585-599. https://doi. org/10.1080/10942910701573024 Zare, D., Bakhshipour, A., & Chen, G. (2013). Physical proper- ties of cumin and caraway seeds. International Agrophysics, 27, 491–494. https://doi.org/10.2478/intag-2013-0020 Acta agriculturae Slovenica, 119/4, 1–11, Ljubljana 2023 doi:10.14720/aas.2023.119.4.13609 Original research article / izvirni znanstveni članek Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara Reguia OTMANI 1, 2, Bachir KHENE 1, Abdellah KEMASSI 3, Fatna ARABA 1, Mohamed HARRAT 4, Mo- hamed YOUSFI 4 Received May 20, 2023; accepted October 27, 2023. Delo je prispelo 20. maja 2023, sprejeto 27. oktobra 2023 1 Laboratoire de Mathématiques et Sciences Appliquées, Université de Ghardaïa, Ghardaïa, Algeria 2 Corresponding author, e–mail: otmani.regaia@univ-ghardaia.dz 3 Laboratoire de Protection des Ecosystèmes en Zones Arides et Semi arides, Université Kasdi Merbah, Ouargla, Algérie 4 Laboratoire des sciences fondamentales (LSF), Université Amar Télidji, Laghouat, Algeria Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara Abstract: The aim of the present work is to study the chemical composition, to estimate the phenolic compounds content and to evaluate the potential allelopathic effects of the Haloxylon scoparium Pomel. Phytochemical tests revealed that Haloxylon scoparium contains tannins, saponins, coumarins, alkaloids, flavonoids and steroids. Furthermore, it contains high levels of total phenolic (588.33 mg GAE 100 g-1) and fla- vonoids (95.45 mg QE 100 g-1) contents. Moreover, LC-MS-MS analysis allowed us to determine their chemical composition. The results of this characterization confirm the presence of van- illin, naringenin, folic acid, maleic acid, benzoic acid, myric- etin, qwuercetin, beta-carotene, butylhydroxyanisole (BHA), butylated hydroxytoluene (BHT), rutin, cafeic acid, hydroxy- 4-coumarine, ascorbic acid, and gallic acid. The allelopathic ef- fect was studied on seed germination and seedling growth of four weed species. The bioassays were performed using differ- ent concentrations (1 %, 2.5 %, 5 % and 10 %) against a negative control. The seed germination, shoot and root length of weed species were completely inhibited at the highest concentrations (10 %, 5 %). However, the lower concentrations exhibited lesser inhibition percentages on the germination and the seedling growth. The phytochemical results and the significant allelo- pathic effects of the plant extract suggest that this species may offer new substances for the biocontrol of weeds. Key words: phytochemical profile, allelopathic potential, Haloxylon scoparium, LC-MS-MS analysis, allelochemicals, Al- gerian Sahara Fitokemični profil in alelopatski potencial vrste Haloxylon scoparium Pomel (Chenopodiaceae) iz alžirske Sahare Izvleček: Namen raziskave je preučiti kemijsko sestavo in vsebnost fenolnih spojin za ovrednotenje alelopatskega po- tenciala vrste Haloxylon scoparium Pomel. V raziskavi je bilo ugotovljeno, da vrsta vsebuje različne tanine, saponine, kuma- rine, flavonoide, alkaloide in steroide. Vsebuje velike količine celokupnih fenolov (588,33 mg GAE 100 g-1) in flavonoidov (95,45 mgQE 100 g-1). Podrobnejša kemijska LC-MS-MS anali- za je pokazala prisotnost vanilina, naringenina, folne, jabolčne in benzoične kisline, mircetina, kvercetina, beta-karotena, butil hidroksianizola (BHA), butiliranega hidroksitoluena (BHT), rutina, kavne kisline, hidroksi-4-kumarina, askorbinske in galne kisline. Alelopatski učinek je bil preučevan na kalitvi in rasti kalic štirih vrst plevelov. Preizkušene so bile različne kon- centracije alelopatskih snovi (1 %; 2,5 %, 5 % in 10 %) napram kontroli. Kalitev semen in dolžinska rast korenin in poganjkov plevelov je bila popolnoma zavrta pri največjih koncentracijah alelopatskih snovi (10 %, 5 %). Manjše koncentracije alelopat- skih snovi so pokazale manjši odstotek zaviranja kalitve in rasti sejank plevelov. Alelopatski učinek izvlečkov te rastline naka- zuje, da bi ta rastlina lahko bila vir novih učinkovin pri biokon- troli plevelov. Ključne besede: fitokemični profil, alelopatski potencial, Haloxylon scoparium, LC-MS-MS analiza, alelokemikalije, al- žirska Sahara Acta agriculturae Slovenica, 119/4 – 20232 R. OTMANI et al. 1 INTRODUCTION Weed interference in agricultural fields reduces the quantity and quality of crops, causing enormous eco- nomic losses for farmers (Sarić-Krsmanović et al., 2019). Control strategies of weed have relied mainly on the ap- plication of synthetic herbicides. However, the continu- ous and excessive application of these treatments cause environmental pollution, negative effects on human health and unsafe agricultural products. Moreover, this practice increases herbicide resistance in weeds (Batish et al., 2007). Therefore, in order to resolve this problem, and minimize the dependency on chemical herbicides for weed control, great efforts have been given to develop natural and eco-friendly alternatives (Bhadoria, 2011). Allelopathy refers to any process that involves al- lelochemicals produced by plants. Some plants may in- hibit seed germination, emergence and growth of other plants by exuding toxic substances. These substances are called allelopathic chemicals or allelochemicals (Monem, 2012). Biochemical compounds offer a great potential for the discovery of new environmentally safe herbicides, re- ferred to as “bioherbicides”. Thus, the usage of plant sec- ondary metabolites proved to be a promising solution in biological control (Weih et al., 2008). The Algerian Sahara, known for its richness in spontaneous plants, harbors about 500 species of higher plants, some of which are used as medicinal plants. The Haloxylon scoparium Pomel plant, belonging to the Che- nopodiaceae family, and locally named “remth”, is used in traditional medicine to treat eye disorders (Chehma, 2006; Salah et al., 2002). Several studies have been carried out on this spe- cies extract; mostly targeting its’ polyphenol contents or its’ biological activities. This plant has been reported to possess antidiabetic potential (Benkherara et al., 2021), antimicrobial and antiradical properties (Drioiche et al., 2019), antibacterial and antioxidant activities (Bouaziz et al., 2016), antidiabetic, antiseptic and anti-inflammatory effects (Ziyyat et al., 2014), anticancer activity (Bourogaa et al., 2014), anti-leukemic agent (Bourogaa et al., 2011), molluscicidal activity (Mezghani- Jarraya et al., 2009), anti-cancer and anti-plasmodial activities (Sathiyamoor- thy et al., 1999), larvicidal activity (Sathiyamoorthy et al., 1997). The allelopathic potential of Haloxylon scoparium on various weeds and crops have rarely been investigated (Salhi, 2011). Therefore, this study aims to evaluate the allelopathic effects of Haloxylon scoparium leaf extract on seed germination and seedlings growth. As well as to de- termine the chemical composition we carried out extrac- tion of phenolic compounds, then their qualitative and quantitative characterization. The analytical techniques used are phytochemical tests based on precipitation or coloration of extract by specific reagents; Determination of total polyphenol and total flavonoids contents was determined by spectrophotometry and identification of phenolic compounds by Liquid Chromatography- Mass Spectrometry (LC-MS-MS). 2 MATERIAL AND METHODS 2.1 EXTRACT PREPARATION The leaves of Haloxylon scoparium were harvest- ed from the plant in its’ natural habitat, located in the northeastern region of the Algerian Sahara, during its’ vegetative stage. The leaves were subsequently washed with tap water then shade dried. The dried leaves were ground using an electric blender and stored in glass jars until the extraction process. The plant extract was ob- tained using reflux extraction. In a flask, 50 g of plant material were added to a hydro-methanolic solution. Us- ing a flask heater, the mixture was heated at 60 °C for six hours. Filtration was then carried out using Whatman No. 1 filter paper. The collected filtrate underwent treat- ment under reduced pressure in a rotary evaporator in order to eliminate the methanol. The recovered aqueous extract was subsequently stored at 4 °C in a refrigerator until used for the biological testing (Kemassi et al., 2019). To evaluate the dose dependent effect on the germi- nation and seedling growth of weed, different concentra- tions were prepared from the stock solution, diluted with distilled water (10 %, 5 %, 2.5 % and 1 %). Distilled water served as control. 2.2 PHYTOCHEMICAL TESTS The leaf extract was examined for the presence of the following phytochemical classes, using the numerous standard methods of evaluation described by various au- thors in the scientific literature 2.2.1 Tannins A solution of FeCl3 (5 %) was added to the crude extract. The presence of tannins was indicated by the ap- pearance of a black or a blue-green color (Lerato et al., 2017). Acta agriculturae Slovenica, 119/4 – 2023 3 Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara 2.2.2 Anthocyanins Two milliliters of leaf extract, two milliliters of HCl (2 N) and ammonia (2 ml) were mixed. The appearance of a pink red coloration that turned blue violet confirmed the presence of anthocyanins (Lerato et al., 2017). 2.2.3 Saponins Distilled water was added to the crude leaf extract in a test tube, followed by vigorous stirring. The formation of a persistent froth confirmed the presence of saponins in the extract (Lerato et al., 2017). 2.2.4 Coumarins A NaOH solution (10 %) was added to the leaf ex- tract. The formation of a yellow color confirmed the pres- ence of coumarins (Lerato et al., 2017). 2.2.5 Alkaloids The presence of alkaloids was assessed by adding three milliliters of HCl (1 %) to three milliliters of crude extract. The mixture was heated for twenty minutes. Subsequently, Mayer’s reagent was added in drips to the mixture. The formation of a cream precipitate or the oc- currence of a green coloration indicates the presence of alkaloids (Lerato et al., 2017). 2.2.6 Flavonoids The plant extract was treated with a NaOH solution (10 %). The appearance of an intense yellow color of the solution indicated the presence of flavonoids (Lerato et al., 2017). 2.2.7 Steroids Chloroform and H2O4 were added to the leaf ex- tract. The presence of steroids was indicated by a color change, from violet to blue or green, or the occurrence of a blue-green ring (Lerato et al., 2017). 2.2.8 Carbohydrates A boiled mixture of Fehling solutions A and B, with equal volumes, was added to the leaf extract. A red color- ed precipitate indicated the presence of reducing sugars (Jaradat et al., 2015). 2.3 DETERMINATION OF POLYPHENOLS COM- POUNDS CONTENT 2.3.1 Determination of total polyphenol content Total polyphenol content (TPC) was measured ac- cording to the colorimetric method (Singleton and Rossi, 1965), with some modification (using a UV spectropho- tometer). 200 µl of leaf extract were added to 1 ml of Folin-Ciocalteu reagent (diluted 10 times with distilled water) and 800 µl of Na2CO3 (7.5 %). The mixture was then incubated at 50 ºC for 30 minutes. Subsequently, the absorbance of the solution was measured at 765 nm. Gal- lic acid served as a control for the creation of a calibration curve to estimate the TPC (Cliffe et al., 1994). 2.3.2 Determination of total flavonoid content The total flavonoid content (TFC) was measured ac- cording to the Aluminium Chloride colorimetric meth- od (Djeridane et al., 2006), with some modification. In a test tube, 25 μl of the plant extract was mixed with 300 μl of NaNO2 and 300 μl of AlCl3 (10 %) and left for five minutes. 100 μl of NaOH (2 %) were added. The absorb- ance of this mixture was measured at 510 nm. Calibra- tion curve of standard quercetin solution was prepared to calculate TFC (Kim et al., 2003). 2.4 EXTRACTION OF PHENOLIC COMPOUNDS FOR LC-MS-MS ANALYSIS The crude extract was fractionated (liquid-liquid) by the addition of ethyl acetate to obtain ethyl acetate fraction. Extract fraction was dried and dissolved in 5 ml of methanol then stored at 4 °C until analysis. 2.5 LIQUID CHROMATOGRAPHY-MASS SPEC- TROMETRY ANALYSIS CONDITIONS (LC-MS- MS) The analysis of the Haloxylon scoparium leaf extract was carried out in the Technical Platform of Physico- Chemical Analysis (PTAPC-CRAPC)-Ouargla-Algeria, using a UPLC-ESI-MS-MS Shimadzu 8040 Ultra-High sensitivity with UFMS technology was employed and Acta agriculturae Slovenica, 119/4 – 20234 R. OTMANI et al. equipped with binary bump Nexera XR LC-20AD iden- tified the extracted phenolic compounds. For optimiza- tion of polyphones standards, we used direct injection without column. All standards were prepared in meth- anol with a 500 µg l-1 concentration. The ion trap mass spectrometer was used in both positive and negative ions with MRM mode (multiple reaction monitoring). The mobile phase was made of water, 0.1 % formic acid and 70 % methanol. The injection volume was six µl and the flow rate was 0.3 ml min-1. The samples were separated using an ultra-force C18 column (I.D. 2.5 mm × 100 mm, 1.8 µm particle size; Restek), the oven temperature was 25 °C. Isocratic elution was applied with 0.1 % formic acid and methanol. The injection volume was 10 ml and the flow rate was 0.30 ml min-1 (Ben amor et al., 2022). 2.6 BIOASSAY EXPERIMENT The bioassay experiments were arranged in a com- pletely randomized design, with four replications of each treatment. Seeds of Bromus rubens L., Phalaris minor L., Plantago lagopus L. and Ammi visnaga L. were placed on filter paper in sterile Petri dishes and treated with three ml of different concentrations of plant extract. The control was treated with three ml of distilled water. The petri dishes were kept under laboratory conditions with day temperature ranging from 19-24 °C and night temperature from 12-15 °C. The germination assessment was evaluated daily, for ten days, by counting the number of germinated seeds, measuring the shoot and the root lengths and determining fresh mass at the end of the ex- periment (Otmani et al., 2022). 2.7 EVALUATION OF ALLELOPATHIC EFFECTS The allelopathic effects can be defined as the inhibi- tion or the retardation of seed germination and reduc- tion or stimulation of root and shoot length and mass. 2.7.1 Calculation of inhibition percentage The inhibition percentage was calculated according to the equation proposed by Côme (1970). This param- eter explains the ability of a plant extract to inhibit seed germination. In the equation, mentioned below, N is the number of germinated seeds, and A is the total number of the sown seeds. Inhibition (%) = ((A-N)/A)*100 (1) 2.7.2 Average germination time The average germination time (AGT) was deter- mined through daily counting of germinated seed to the tenth day and calculated with the equation proposed by Labouriau (1983), being the expressed results in days. AGT = ∑ni*ti/∑ni = = (n1* t1+n2*t2+ ... +nn*tn)/n1+n2+……+nn (2) n1: number of germinated seeds at time t1. n2: number of germinated seeds at time t2. nn: number of germinated seeds at time tn. 2.7.3 Effects of extract on seedling’s growth After the germination test, the shoot and root lengths of the weed species were measured. Afterwards, the seedlings were separated into shoot and root parts in order to measure the fresh mass. 2.8 STATISTICAL ANALYSIS The results obtained from the various experimental tests were analyzed by one-way ANOVA with the “XL- STAT version 2014” software. Results were evaluated by the Fisher LSD test (p = 0.05), and presented as mean ± SD (Standard deviation). 3 RESULTS AND DISCUSSION 3.1 PHYTOCHEMICAL TESTS The results of the phytochemical screening, pre- sented in Table 1, clearly indicate the presence of dif- ferent secondary metabolites in Haloxylon scoparium leaf extract. These tests revealed the presence of phenols (tannins, saponins, coumarins, flavonoids), alkaloids and steroids. However, the absence of carbohydrates, antho- cyanin and betacyanin is noted. The phytochemical tests carried out on the leaf extract of Haloxylon scoparium allowed us to highlight the presence of several phytochemicals. These research results are in agreement with those obtained in previ- ous studies that indicated the richness of Haloxylon sco- parium in secondary metabolites (Haida et al., 2020). Ben kherara et al. (2021) confirmed the presence of six major compounds (alkaloids, flavonoids, saponins tan- Acta agriculturae Slovenica, 119/4 – 2023 5 Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara nins, anthocyanins, terpenes and sterols) and the absence of two other important compounds (leucoanthocyanins and cardinolids). A study done by Lachkar et al. (2021) showed that the aqueous and organic extracts of the aer- ial part of Haloxylon scoparium collected in Taza (Mar- roco) contains catechic tannins, flavonoids, saponins, alkaloids, anthracenosides, and free quinones. However, gallic tannins, sterols and anthraquinones were absent. Furthermore, Bourogaa and collaborators (2014) re- vealed the presence of flavonoids and alkaloids, while quinones and sterols are absent from the aqueous extract. In contrast, Zerriouh (2015) showed that the aqueous ex- tract of the aerial part of Hammada scoparia collected in Algeria is devoid of flavonoids but contains the alkaloids and saponins. These secondary metabolites possess allelopathic effects on the seed germination and seedling growth of weed species. These phytochemicals present in Haloxylon scoparium leaf extract might be controlling the observed allelopathic activity of the plant extract. alkaloids, flavo- noids, terpenoids, curcurbitacins, glycosides, coumarins, saponins and tannins are the plant components identi- fied as allelochemicals in the allelopathic effects of sever- al plant extracts on weeds and crops (Mseddi et al., 2018; Naz and Bano, 2013). 3.2 TOTAL POLYPHENOL AND FLAVONOIDS CONTENT The total polyphenol and total flavonoids contents obtained for leaf extract of Haloxylon scoparium are presented in Table 2. The total polyphenols content of the plant extract was determined in comparison to the standard gallic acid, expressed as mg GAE 100 g-1 of dry plant sample, whereas the total flavonoids content was measured in comparison to the standard quercetin, and expressed as mg QE 100 g-1 of dry plant sample. These interesting results of colorimetric analy- sis show a very high content of total polyphenols (588.33  ±  1.87 mg GAE g-1 DM) and total flavonoids (95.45 ± 1.21 mg QE 100 g-1 DM) in the Haloxylon scopar- ium leaf extract. These amounts were significantly better than those found by Allaoui et al. (2016) who obtained a high content of total polyphenol (397.743 mg GAE g-1 of extract) and flavonoid (82.835 mg QE g-1). The obtained results were three time higher than those quantified in the same studied plant species (Zeghada, 2009; Lachekar et al., 2021; Ben kherara et al., 2021) The extraction yields depend on the plant species, part of plant used, period of harvesting, climate and geographical position, drying conditions, plant material, nature and polarity of the solvent and the method and modality of extractions. The qualitative and quantitative analysis results show the superior biochemical quality of Haloxylon sco- parium. 3.3 LC-MS-MS ANALYSIS RESULTS The analysis results of Haloxylon scoparium leaf ex- tract by LC-MS-MS are shown in Table 3. This analysis revealed the presence of several secondary metabolites. Twenty-three phenolic compounds were detected based on the LC-MS-MS in which fifteen were identified by comparison with standards. The results of this charac- terization confirm the presence of vanillin, naringenin, folic acid, maleic acid, benzoic acid, myricetin, querce- tin, beta-carotene, butylhydroxy anisole (BHA), butyl- ated hydroxytoluene (BHT), rutin, cafeic acid, hydroxy- 4-coumarine, ascorbic acid, and gallic acid. However, keampferol, coumaric acid, picric acid, cinnamic acid, chlorogénic acid, chrysin, esculin, hesperetin were ab- sent. The phytochemical composition of Haloxylon sco- Table 1: Results of phytochemical tests of Haloxylon scoparium Pomel. leaf extract Constituents Leaf extract Tannins +++ Anthocyanin and Betacyanin - Saponins + Coumarins +++ Alkaloids +++ Flavonoids +++ Steroids +++ Carbohydrates - +++: Strong positive result, ++: Moderate positive result, +: Weak posi- tive result, - : Negative result Table 2: Quantitative results of Haloxylon scoparium Pomel. leaf extract Constituents Leaf extract Total Polyphenols Content (mg GAE/100 g DM) ± SD 588.33 ± 1.87 Total Flavonoids Content (mg QE / 100 g DM) ± SD 95.45 ± 1.21 GAE: Gallic acid equivalent; QE: Quercetin equivalent; DM: dry mass Acta agriculturae Slovenica, 119/4 – 20236 R. OTMANI et al. parium plant has not been the subject of many publi- cations. Few researchers have investigated its phenolic composition. Chemical characterization of Hammada scoparia essential oils confirmed the presence of carvac- rol, p-cymene, γ-terpinene and z-caryophyllene (Dri- oiche et al., 2019). In addition, Chao et al. (2013) showed the presence of some phenol acids such as Coumaric acid, Cinnamic acid and Caffeoylquinic acid, simple phenols (catechol and a chrysoeriol). However, Benkrief et al. (1990) identified isosalsoline dehydrosalsolidine, isosalsolidine (tetrahydroisoquinolines), N-methyl- corydaldine (isoquinolone), tryptamine and N-meth- yltryptamine (tryptamines) as minor alkaloids. Other studies have isolated and identified two principal alka- loids: carnegine and N-methylisosalsoline from Ham- mada scoparia leaf extract (Jarraya et al., 2008; Bouaziz et al., 2016). A new flavonol triglycoside has been isolated from the leaves of Hammada scoparia (Salah et al., 2002). 3.4 PERCENTAGE OF GERMINATION INHIBI- TION The allelopathic effect is expressed as the percentage of inhibition. The germination of target species, treated with Haloxylon scoparium leaf extract, decreased com- pared to the control. The degree of inhibition varies de- pending on the concentrations (Fig 1, 2, 3, and 4). A high inhibitory effect on germination was observed on all test- ed seeds, and the inhibition percentage increased with increasing concentrations of leaf extract. As illustrated in the graphs, at the 1 % and 2.5  % extract concentra- tions, the inhibition percentage values were, respectively, of 71.66-76.66 % for Bromus rubens , 63.33-93.33 % for Phalaris minor , 50.0-66.66 % for Plantago lagopus , 30.0- 43.33 % for Ammi visnaga The 5 % and 10 % extract concentrations significantly inhibited the germination of weed seeds, which corresponds to a 100% inhibition percentage. Table 3: LC-MS-MS-determined phenolic compounds of Haloxylon scoparium Pomel. leaf extract N° Compound Name Charge + / - Precursor m/z Product m/z Haloxylon scoparium 01 Keampferol [MH]+ 287.1 255.25 - 02 Vanillin [MH]+ 153.10 71.15 + 03 Naringenin [MH]+ 273.10 147.15 + 04 Coumaric Acid [MH]+ 165.10 59.10 - 05 Picric Acid [MH]- 227.8 198.05 - 06 Cinnamic Acid [MH]+ 149.1 77.2 - 07 Folic Acid [MH]+ 442.90 323.45 + 08 Maleic Acid [MH]+ 117.10 85.20 + 09 Benzoïc Acid [MH]+ 123.10 91.20 + 10 Chlorogénic Acid [MH]+ 355 73.15 - 11 Myricetin [MH]+ 336.25 72.15 + 12 Quercetin [MH]+ 303.10 85.05 + 13 Chrysin [MH]+ 255.10 223.30 - 14 Esculin [MH]+ 341.30 309.40 - 15 Hesperetin [MH]- 300.9 255.25 - 16 Beta-carotene [MH]+ 537.20 199.25 + 17 Butylhydroxyanisole (BHA) [MH]+ 181.10 140.15 + 18 Butylated hydroxytoluene (BHT) [MH]+ 221 161.30 + 19 Rutin [MH]+ 611.20 73.20 + 20 Cafeic Acid [MH]- 178.80 135.10 + 21 Hydroxy-4-Coumarine [MH]- 160.80 117.10 + 22 Ascorbic Acid [MH]- 174.90 131.10 + 23 Gallic Acid [MH]- 168.80 125.10 + + : present, - : not present Acta agriculturae Slovenica, 119/4 – 2023 7 Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara According to the present research, Haloxylon sco- parium leaf extract presents an allelopathic effect on the seed germination of weed species (Bromus rubens , Phalaris minor , Plantago lagopus , Ammi visnaga). These results are in agreement with those obtained by Karous et al. (2020); they demonstrate that the tested aqueous extract possessed an effective inhibitory activity against two weed species. Numerous studies have suggested that the presence of allelochemicals may cause a total or par- tial suppression of germination and a reduction in seed- ling growth (Qasem, 2002; Naz and Bano, 2013; Saadaoui et al., 2015; Mseddi et al., 2018). The presence of these secondary metabolites suggests that the plant might be of allelopathic and bioherbicidal importance. 3.5 AVERAGE GERMINATION TIME In the present study, it was observed that the average germination time of weed species treated with leaf ex- tract of Haloxylon scoparium increased in all treatments, from the lowest to the highest concentrations. The values show slower times compared to those of the control lot Figure 1: Inhibition percentage observed in control and treat- ed lots by various concentration of leaf extract of Haloxylon scoparium Pomel. on Bromus rubens L. For each concentra- tion, means (mean ± SD) followed by different letter (A, B, C) are significantly different at p < 0.05 level according to Tukey’s LSD test Figure 2: Inhibition percentage observed in control and treat- ed lots by various concentration of leaf extract of Haloxylon scoparium Pomel. on Phalaris minor L. . For each concentra- tion, means (mean ± SD) followed by different letter (A, B, C) are significantly different at p < 0.05 level according to Tukey’s LSD test Figure 3: Inhibition percentage observed in control and treat- ed lots by various concentration of leaf extract of Haloxylon scoparium Pomel. on Plantago lagopus L. For each concentra- tion, means (mean ± SD) followed by different letter (A, B, C, D) are significantly different at p < 0.05 level according to Tukey’s LSD test Figure 4: Inhibition percentage observed in control and treated lots by various concentration of leaf extract of Haloxy- lon scoparium Pomel. on Ammi visnaga L. For each concentra- tion, means (mean ± SD) followed by different letter (A, B, C, D) are significantly different at p < 0.05 level according to Tukey’s LSD test (Table 4). These results showed that the average germina- tion time varies between 6.14 and 8.75 days. The results of the current study show that Haloxylon scoparium leaf extract had an impact on average germi- nation time. Da Silva et al. (2016) reported that Ricinus communis leaf extract significantly affected the average germination time, which increased with concentrations. Allelochemicals can increase cell membrane permeabil- ity, which prevents plants from absorbing nutrients from their environment and affects their normal growth (Li et al., 2010). 3.6 EFFECT OF EXTRACT ON SEEDLING GROWTH The effect of Haloxylon scoparium leaf extract on the shoot and root growth of treated weed species (Bro- mus rubens , Phalaris minor , Plantago lagopus , Ammi visnaga) are shown in tables 5, 6, 7 and 8 respectively. In laboratory bioassay, all concentrations of leaf extract of Haloxylon scoparium decreased the seedling growth of weed species. They significantly decreased the shoot Acta agriculturae Slovenica, 119/4 – 20238 R. OTMANI et al. Table 4: Effect of Haloxylon scoparium Pomel. leaf extract on average germination time of Bromus rubens L., Phalaris minor L., Plantago lagopus L. and Ammi visnaga L Average Germination Time (AGT) (Days) Extract conc. (%) Bromus rubens Phalaris minor Plantago lagopus Ammi visnaga Mean ± SD Group Mean ± SD Group Mean ± SD Group Mean ± SD Group Control 6.33 ± 0.11 A 7.31 ± 0.08 A 6.19 ± 0.05 A 6.53 ± 0.03 A 1 % 6.14 ± 0.16 A 7.91 ± 0.16 A 6.68 ± 0.36 AB 7.33 ± 0.05 B 2.5 % 6.54 ± 0.66 A 8.75 ± 1.44 A 7.13 ± 0.25 B 7.64 ± 0.29 C 5 % - - - - 7.85 ± 0.76 C - - 10 % - - - - 8,63 ± 0.47 D - - LSD 0.72 1.51 0.72 0.30 -:100 % inhibition percentage Table 5: Effect of Haloxylon scoparium Pomel. leaf extract on shoot and root lengths and fresh mass of Bromus rubens L Bromus rubens Extract conc. ( % ) Shoot length (cm) Root length (cm) Shoot mass (g) Root mass (g) Mean ± SD Group Mean ± SD Group Mean ± SD Group Mean ± SD Group Control 12.61 ± 2.27 C 14.33 ± 1.49 C 0.0336 ± 0.0036 B 0.0262 ± 0.0078 B 1 % 5.45 ± 1.49 B 6.57 ± 1.05 B 0.0257 ± 0.0049 A 0.0096 ± 0.0024 A 2.5 % 2.12 ± 1.35 A 2.01 ± 1.05 A 0.0177 ± 0.0059 A 0.0058 ± 0.0020 A LSD 3.16 2.47 0.0085 0.0043 Table 6: Effect of Haloxylon scoparium Pomel. leaf extract on shoot and root lengths and fresh mass of Phalaris minor L Phalaris minor Extract conc. ( % ) Shoot length (cm) Root length (cm) Shoot mass (g) Root mass (g) Mean ± SD Group Mean ± SD Group Mean ± SD Group Mean ± SD Group Control 9.08 ± 0.65 B 9.26 ± 0.92 C 0.0155 ± 0.0019 B 0.0051 ± 0.0007 C 1 % 6.69 ± 0.87 A 3.97 ± 0.98 B 0.0096 ± 0.0018 AB 0.0018 ± 0.0009 B 2.5 % 5.45 ± 1.51 A 2.05 ± 1.41 A 0.0082 ± 0.0052 A 0.0005 ± 0.0001 A LSD 1.93 1.92 0.0061 0.0011 Table 7: Effect of Haloxylon scoparium Pomel. leaf extract on shoot and root lengths and fresh mass of Plantago lagopus L Plantago lagopus Extract conc. ( % ) Shoot length (cm) Root length (cm) Shoot mass (g) Root mass (g) Mean ± SD Group Mean ± SD Group Mean ± SD Group Mean ± SD Group Control 3.38 ± 0.49 D 4.03 ± 0.29 C 0.0084 ± 0.0015 C 0.0038 ± 0.0005 C 1 % 2.85 ± 0.29 C 1.53 ± 0.46 B 0.0058 ± 0.0024 B 0.0014 ± 0.0010 B 2.5 % 1.38 ± 0.25 B 0.28 ± 0.16 A 0.0026 ± 0.0021 A 0.0005 ± 0.0004 A 5 % ± 0.06 A 0.08 ± 0.03 A 0.0007 ± 0.0004 A 0.0001 ± 0.00005 A 10 % 0,35 ± 0.05 A 0,12 ± 0.05 A 0,0003 ± 0.0001 A 0,0001 ± 0.00005 A LSD 0.38 0.41 0.0026 0.0009 Acta agriculturae Slovenica, 119/4 – 2023 9 Phytochemical profile and allelopathic potential of Haloxylon scoparium Pomel (Chenopodiaceae) from Algerian Sahara length, root length, shoot fresh mass and root fresh mass of the test species compared to those of the control. This inhibitory effect on the root and shoot growth increased with increase of the concentrations. The present study indicate that Haloxylon scopari- um leaf extract presents allelopathic effect and contains allelochemicals responsible for the inhibitory activi- ties on the germination and the seedling growth of test weed species. Other authors in their studies on weeds and crops observed similar findings (Scavo et al., 2018; Bhowmik and Doll, 1984). Allelochemicals affect plant germination and growth (Salhi et al., 2013). The capac- ity to inhibit seed germination and seedling growth are a complex process, and several hypotheses about allelo- chemicals of plant extracts have been formulated. These hypotheses suggest that these compounds might affect enzymes responsible for plant hormone synthesis, to in- hibit the action of the amylase or inhibition of their tissue actions (Feeny, 1976). The alteration of the synthesis or activities of gibberellic acid in the seed could be due to the presence of phenolic compounds (Olofsdotter, 2001). Cell division and elongation are susceptible to the pres- ence of allelopathic compounds (Muller 1965), resulting in the reduction of root and shoot growth (Qasem, 2002). 4 CONCLUSION The results of the present research confirmed the strong allelopathic effects of Haloxylon scoparium leaf ex- tract, on the germination, shoot and root growth of test- ed weed species (Bromus rubens, Phalaris minor, Plan- tago lagopus, Ammi visnaga). The phenolic compounds present a great interest for the researchers due to their various biological activities. These findings encourages future research for identifying and characterizing ger- mination and growth inhibitors; it could be the source of these species’ significant allelopathic potential. These allelochemicals maight be used in the research and devel- opment of weed-controlling environmental herbicides. Table 8: Effect of Haloxylon scoparium Pomel. leaf extract on shoot and root lengths and fresh mass of Ammi visnaga L Ammi visnaga Extract conc. 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H., Vanderpas, J., Has- sani, B., Boutayeb, A., ... & Legssyer, A. (2014). Epidemiol- ogy of hypertension and its relationship with type 2 diabe- tes and obesity in eastern Morocco. Springerplus, 3(1), 1-7. https://doi.org/10.1186/2193-1801-3-644 Acta agriculturae Slovenica, 119/4, 1–9, Ljubljana 2023 doi:10.14720/aas.2023.119.4.15383 Original research article / izvirni znanstveni članek Effects of particle size on determination of the contents of grain and leg- ume dietary fibre and resistant starch Blaž FERJANČIČ 1, Mojca KOROŠEC 1, Saša PISKERNIK 1, Jasna BERTONCELJ 1, 2 Received Agugust 04, 2023; accepted November 14, 2023. Delo je prispelo 4. avgusta 2023, sprejeto 14. novembra 2023 1 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Ljubljana, Slovenia 2 Corresponding author, e-mail: jasna.bertoncelj@bf.uni-lj.si Effects of particle size on determination of the contents of grain and legume dietary fibre and resistant starch Abstract: Dietary fibre comprises non˗digestible car- bohydrates, including resistant starch, and lignin, and it is an important constituent of a healthy diet. The aim was to define the influence of particle size on contents determined for dietary fibre and resistant starch in unprocessed grain and canned le- gumes. Five samples of unprocessed and processed grains were analysed, as oatmeal, buckwheat, dehulled barley, wheat and spelt, and three canned legumes, as beans, chickpeas and peas, with and without their brine. Samples were initially milled un- screened, and then again through 500 μm or 350 μm screens. For unprocessed grain samples, there was generally no influ- ence of particle size, except for the 350-μm milling of dehu- lled barley, with significantly decreased contents determined for insoluble dietary fibre and resistant starch presumably due to damaging of starch granules and disrupting crystalline for- mation of starch. For canned legumes with and without their brine, particle size had little effect on contents determined for dietary fibre and resistant starch. Key words: dietary fibre, resistant starch, particle size, canned legumes, milling, grains Vpliv velikosti delcev na določitev vsebnosti prehranske vla- knine in rezistentnega škroba v žitih in stročnicah Izvleček: Prehranska vlaknina je sestavljena iz nepreba- vljivih ogljikovih hidratov, vključno z rezistentnim škrobom, in lignina ter je pomemben del uravnotežene prehrane. Cilj naše raziskave je bil preveriti vpliv velikosti delcev vzorca na določanje vsebnosti prehranske vlaknine in rezistentnega škro- ba v vzorcih žit in procesiranih žit ter konzerviranih stročnic. Analizirali smo pet vzorcev žit, in sicer ovsene kosmiče, ajdo, ješprenj, pšenico in piro ter tri vzorce konzerviranih stročnic, fižol, čičeriko in grah. Vzorci stročnic so bili analizirani z nali- vom in brez naliva. Vzorci so bili mleti na nedefinirano velikost ter na 500 μm ali 350 μm. V skupini žit ni bilo zaznati vpliva velikosti delcev, razen pri vzorcu ješprenja, mletega na velikost 350 μm, kjer sta bili določeni značilno manjši vsebnosti netopne prehranske vlaknine in rezistentnega škroba. Predvidevamo, da je to posledica poškodb škrobnih zrn in porušenja kristalinične formacije škroba. V vzorcih konzerviranih stročnic z ali brez naliva je imela velikost delcev zanemarljiv učinek na določeno vsebnost prehranske vlaknine in rezistentnega škroba. Ključne besede: prehranska vlaknina, rezistentni škrob, velikost delcev, konzervirane stročnice, mletje, žita Acta agriculturae Slovenica, 119/4 – 20232 B. FERJANČIČ et al. 1 INTRODUCTION Dietary fibre is recognized as an important compo- nent of the human diet, due to its beneficial effects on the gastrointestinal tract and its positive effects on reducing the risk of developing non˗communicable diseases. Di- etary fibre consists of carbohydrates that are resistant to hydrolysis by the human endogenous enzymes and that are not readily absorbed in the small intestine, as also for resistant starch. However, the fermentation processes by microbiota in the colon can partially use the dietary fibre for energy production, while metabolising it to bioactive compounds, such as short˗chain fatty acids (Fuller et al., 2016; Kendall et al., 2010). The definition of dietary fibre describes it as carbohydrate polymers with ten or more monomeric units that are not hydrolysed by the endog- enous enzymes in the small intestine of humans, and that belong to following four categories: (i) Edible carbohydrate polymers that occur natu- rally in the food consumed; (ii) Carbohydrate polymers that have been obtained from raw food material by physical, enzymatic or chemi- cal means, and that have been shown to have beneficial physiological effects on health, as demonstrated by the generally accepted scientific evidence available to com- petent authorities; (iii) Synthetic carbohydrate polymers that have been shown to have beneficial physiological effects on health, as demonstrated by generally accepted scientific evidence available to competent authorities. (iv) Carbohydrate polymers with three to nine monomeric units that belong to one of the previous cat- egories, if the national authorities decide to include them in these definitions (De Menezes et al., 2013). Dietary fibre can be classified based on its solubility in water. Insoluble dietary fibre (IDF) consists of mainly cellulose, lignin and some hemicelluloses, while (water)- soluble dietary fibre (SDF) consists of mainly pectin, gum and mucilage. The solubility of dietary fibre governs its physiochemical properties, as IDF has strong hygroscop- ic properties and can thus swell and absorb water. SDF, on the other hand, can form a gel network or a dense network under some physiochemical conditions, and can thus bind water in this way (Thebaudin et al., 1997). Increased intake of dietary fibre promotes more fre- quent defecation and lowers the risk of diabetes mellitus, obesity, coronary heart disease and various cancers; this increased intake also lowers cholesterol levels in the blood (Dahl & Stewart, 2015; Fernstrand et al., 2017; Perry & Wang, 2012; Tarcea et al., 2017). In terms of the physi- ological effects on blood sugar levels and lipid levels, SDF shows potential for great benefits through its regulatory effects (Kapoor et al., 2016). The particle size of the di- etary fibre itself can have an influence on the physiologi- cal properties of the colon. Smaller particles of bran have been shown to increase the microbiological production of short˗chain fatty acids, which appears to be due to the increased surface area of the smaller particles. Particle size of corn bran can also influence its swelling, with a consequent influence on faecal wet mass and faecal bulk- ing, with increases in liver cholesterol and butyrate levels (Ebihara & Nakamoto, 2001; Stewart & Slavin, 2009). The dietary fibre content of food is generally deter- mined by enzymatic–gravimetric methods, where the macro nutrients are digested in vitro or removed, and the remaining portion of the sample represents the dietary fibre. The most common of these methods are AOAC 985.29 and AOAC 991.43 (Westenbrink et al., 2013). Recently, new integrated methods that comply with the new definition of dietary fibre that also includes low mo- lecular weight SDF are gaining value in food composi- tion analyses (Zielinski & Rozema, 2013; Macagnan et al., 2016). The enzymatic breakdown of starch and pro- tein is very important for accurate determination of the dietary fibre content of a food. In the protocol for meth- ods AOAC 985.29 and AOAC 991.43, sample milling is defined, for particles to pass through a 500  µm screen (AOAC 991.43). Effects of sample particle size on the determination of dietary fibre content have been reported for animal feed samples, where it was shown that the particle size of the feed has effects on hemicellulose, cellulose and lignin determination. With decreasing particle size, the fibre contents determined also decreased. This was con- sistent regardless of forage cultivar, season of the animal feed preparation and annual variations in the animal feed (Ehle, 1984). Differences in dietary fibre content have also been reported for wheat bran that was milled to particle sizes of 50, 160, 400 and 750 μm, although here these differences were seen for SDF, while total dietary fibre (TDF) remained unchanged (Coda et al., 2014). The main concern about the accuracy of methods AOAC 985.29 and 991.43 is that these only quantify part of the resistant starch, while the proportion that is included in the dietary fibre determination is not known (Champ et al., 2003). The particle size of a sample can have effects on the enzyme activity of α˗amylase. For particles > 500 μm in size, starch granules can potentially remain entrapped and will not be reached by the enzyme. Then, for parti- cles < 350 μm in size, the α˗amylase activity approaches a constant value, which shows influence of particle size on releasing starch granules from plant cells, making them more susceptible to hydrolisation with α˗amylase (Al-Rabadi et al., 2009). Starch digestibility is related to particle size, due to the increase in surface area with the Acta agriculturae Slovenica, 119/4 – 2023 3 Effects of particle size on determination of the contents of grain and legume dietary fibre and resistant starch decrease in particle size. Here, this larger surface area of the finer ground particle allows for more rapid digestion of the starch and greater penetration of the amylase into the starch granules, therefore improving the starch di- gestibility (Mahasukhonthachat et al., 2010). As well as particle size, the food matrix and tech- nological processing are also important factors in enzy- matic breakdown of starch (Singh et al., 2010). Lipids and proteins can also have an influence on starch digestibility. In combination with lipids, starch can form amorphous structures that are similar to slowly digestible starch. A similar effect has been reported for starch interactions with protein, where the protein can form a structural ‘cage’ around starch granules, and thus prevent enzymat- ic digestion (Zhang et al., 2009). Changes in particle size, for dietary fibre analysis, can lead to disruption of cell wall, therefore freeing starch granules from the cell, also milling of the samples on smaller particle size can dis- rupt starch – protein complex or damage starch granules and not least milling can destroy crystalline formation of starch (Li et al., 2014). However, to the best of our knowledge, there are no studies on the influence of particle size of whole food samples on the dietary fibre content determined using enzymatic–gravimetric methods. The main aims of our study were thus to determine whether the particle size of five grains (i.e., oatmeal, buckwheat, dehulled barley, wheat, spelt) and three canned legumes (i.e., canned beans, chickpea, pea; with and without their brine) have an influence on the contents determined for dietary fibre, and resistant starch. Grain is frequently used as a model food for dietary fibre determination, while canned leg- umes are precooked food and therefore represent a dif- ferent analytical matrix compared to grain. Furthermore, the effects of the brine present in the cans on the legume content of dietary fibre, and resistant starch were deter- mined. 2 MATERIALS AND METHODS 2.1 SAMPLES AND SAMPLE PREPARATION For evaluation of the effects of particle size on the contents determined for dietary fibre and resistant and digestible starch, the grain (oatmeal, buckwheat, de- hulled barley, wheat, spelt) and canned legumes (beans, chickpea, pea; both with and without their brine) were air dried at 40 °C for 48 h. Their moisture contents were then determined gravimetrically from the difference in mass before and after drying at 105 °C for 5 h, along with the dry matter. Each sample was initially milled in a cyclone mill (AR100G31; Moulinex, Ecully, France) for 30 s, with no screening for particle size separation (i.e., unscreened). For 500  μm or 350  μm particle size the samples were milled as described, then passing of the samples through the mesh of desired size was ensured. If part of the sam- ple was unable to pass the mesh, the milling was contin- ued until all of the sample passed selected mesh. These milled samples were stored in plastic containers at –20 °C until analysis. 2.2 DETERMINATION OF DIETARY FIBRE The dietary fibre was determined according to en- zymatic–gravimetric method AOAC 991.43 (AOAC 991.43), in quadruplicate. The method was modified slightly: for the enzyme digestion, 50 ml centrifuge tubes were used instead of Erlenmeyer flasks, according to our previous study (Ferjančič et al., 2018). This method is based on three enzymes used under different conditions: heat stable α˗amylase, a protease, and an amyloglucosi- dase (all enzymes Cat N° 112979; Merck KGaA, Darm- stadt, Germany). The dietary fibre fractions were obtained as indi- gestible residues after this enzymatic digestion of the non-dietary fibre components. The IDF was obtained by filtration, and the SDF was precipitated from the fil- trate with 96 % ethanol. Determination of the residual ash content (ashed at 525 °C in a muffle furnace for 5 h, and weighed) and residual protein content from the nitrogen content after Kjeldahl, was carried out on the residues, for the corresponding data correction. The TDF was defined as the sum of IDF and SDF. All of these data are presented as fresh mass (FM). 2.3 DETERMINATION OF RESISTANT STARCH The resistant starch was determined according to AOAC 2002.02 method (McCleary et al., 2002), in quad- ruplicate, and with modifications for the sample mill- ing. The proposed particle size in the original method is given as <1 mm for dry samples, and < 4.5 mm for fresh samples. The samples in the present study were milled as described above. Resistant starch assay kit was used for resistant starch determination (K-RSTAR; Megazyme, Bray, Ire- land). Samples were incubated with pancreatic α–amyl- ase and amyloglucosidase for 16 h at 37 ° C, for hydro- lysation of digestible starch to glucose. Resistant starch is recovered in form of a pellet, obtained by centrifuga- tion of the sample. First centrifugation is followed by two suspensions of the pellet in 50 % ethanol (v/v) and Acta agriculturae Slovenica, 119/4 – 20234 B. FERJANČIČ et al. centrifugation. Resistant starch in the remaining pellet is dissolved in 2 M KOH and hydrolysed to glucose with amyloglucosidase. D-glucose is measured with glucose oxidase/peroxidase reagent (GOPOD), which develops a pink colour in presence of glucose that can be meas- ured by spectrophotometer. By obtaining concentration of glucose, content of resistant starch can be calculated. For the contents determined for resistant starch, the light absorbance was measured using a UV–Vis spectro- photometer (Carry 8458; Agilent, Victoria, Australia) at 505 nm (instead of 510 nm), following identification of the absorbance peak maximum at this wavelength. All of these data are presented as FM. 2.4 STATISTICAL ANALYSIS Statistical analyses were performed using the R com- mander software (version 3.3.3). To ensure the appropri- ateness of ANOVA, variances between the treatments were determined using Levene’s tests (α > 0.05). Further ANOVA was performed with post˗hoc Tukey’s tests. The threshold for statistical significance was p  ≤  0.05. For comparisons of the influence of the brine, F˗tests were performed to ensure the homogeneity of variance, fol- lowed by Student’s t˗tests. The threshold for statistical significance was p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 EFFECTS OF MILLING ON THE CONTENTS DETERMINED FOR DIETARY FIBRE AND RESISTANT STARCH OF THE GRAIN The data for the dietary fibre and resistant starch contents determined in grains are presented in Table 1. Across these five grain samples (i.e., oatmeal, buckwheat, dehulled barley, wheat, spelt) that were milled to the three different particle sizes (unscreened, or 500 μm, 350 μm screening), the highest TDF was determined for dehulled barley (10.15˗13.40 g 100 g-1 FM), followed by oatmeal (9.13˗10.01 g 100 g-1 FM) and wheat (8.96˗12.36 g 100 g-1 FM). These determined contents for TDF in the oatmeal grain are similar to those reported for nordic countries (10.8˗12.3 g 100 g-1 FM) (Rainakari et al., 2016). Similar- ly, Škrabanja et al. (2004) reported TDF of buckwheat as 2.7 to 21.3 g 100 g-1 FM across different milling fractions, which corresponds to TDF determined for buckwheat in the present study (3.92˗4.59 g 100 g-1 FM). TDF deter- mined for the dehulled barley was also similar to a previ- ous determination (10.8˗12.3 g 100 g-1 FM) (Yalcin et al., 2006), as also for wheat (9.2-20.0 g 100 g-1 FM) (Ciudad- Mulero et al., 2019) and spelt (8.8-14.9 g 100 g-1 dry mass [DM]) (Shewry & Hay, 2015). For dehulled barley, there was a significant decrease across the two specific particle sizes from 500 μm to 350 μm screening, respectively, for IDF and TDF determination. Similarly, for resistant starch determination, signifi- cant decreases across the unscreened to fine milling (i.e., 350 μm screening) of the samples were seen for dehulled barley, and also for wheat (Table 1). Such influences of the particle size on the contents determined for dietary fibre and resistant starch should be related to the enzyme ki- netics of the α˗amylase. Larger particles would be expect- ed to be less digested due to the slower penetration for larger particles, and therefore here it is possible that some starch in the samples with larger particle sizes remained undigested (Al-Rabadi et al., 2009). As well as considera- tion of the enzyme penetration for digestion of the starch in these samples, milling can also cause mechanical dam- age to starch granules, which can result in conversion of resistant starch to digestible starch (De La Hera et al., 2013). Differences in DF and resistant starch determina- tion can be explained by influence of milling on starch digestion kinetics. Dhital et al. (2011) reported influence of cryo-milling of starch granules on molecular structure of starch itself. Their results suggest influence of milling on disruption of helical and crystalline structures of the starch, without breaking the covalent bond of starch mol- ecules due to mechanical force. Furthermore, resistance of starch to hydrolysis is not purely molecular level effect but foremost inability of enzyme to digest starch due to mechanical obstacles, mainly absence of pores on starch granules, which are commonly present in rapid digested starch. Also, internal starch granules in the plant cell are commonly resistant to hydrolysis due to inaccessibility for enzymes. Milling however, can cause starch granule damage therefore facilitating starch hydrolysis (Dhital et al., 2010a). Also, an important note to starch digestion is resistance of starch to hydrolysis. Mechanisms behind the resistant starch are physical in nature (inaccessibility of starch to enzymes, recrystallization, physical entrap- ment and complexes with other macronutrients), there- fore it should be possible for mechanical manipulation of sample particle size to have an effect on starch digestion kinetics (Dhital et al., 2017). The absence of significant differences in other grain samples can be explained by differences in structural features (Dhital et al., 2010b). Acta agriculturae Slovenica, 119/4 – 2023 5 Effects of particle size on determination of the contents of grain and legume dietary fibre and resistant starch Ta bl e 1: U np ro ce ss ed g ra in d ry m at te r, di et ar y fib re co nt en t, an d re sis ta nt st ar ch co nt en t a cc or di ng to m ill ed sc re en se tti ng fo r p ar tic le si ze Sa m pl e M ill in g sc re en D ry m at te r D ie ta ry fi br e (g 1 00 g -1 F M ) Re sis ta nt st ar ch se tti ng (µ m ) (g 1 00 g -1 ) In so lu bl e So lu bl e To ta l (g 1 00 g -1 F M ) O at m ea l U ns cr ee ne d 96 .4 4. 35 ± 0 .4 9 a 4. 80 ± 0 .3 1 a 9. 15 ± 0 .3 3 a 0. 24 ± 0 .0 1 b 50 0 96 .4 4. 92 ± 0 .8 1 a 5. 09 ± 0 .4 6 a 10 .0 1 ± 1. 23 a 0. 15 ± 0 .0 3 a 35 0 96 .4 4. 42 ± 0 .3 5 a 4. 70 ± 0 .3 2 a 9. 13 ± 0 .3 3 a 0. 23 ± 0 .0 3 b Bu ck w he at U ns cr ee ne d 96 .8 2. 75 ± 0 .2 0 a 1. 17 ± 0 .1 4 a 3. 92 ± 0 .2 5 a 0. 72 ± 0 .2 0 a 50 0 96 .8 3. 32 ± 0 .3 5 b 1. 28 ± 0 .2 9 a 4. 59 ± 0 .3 2 b 0. 84 ± 0 .0 2 a 35 0 96 .8 3. 06 ± 0 .1 8 ab 1. 22 ± 0 .2 5 a 4. 28 ± 0 .3 5 ab 0. 62 ± 0 .1 6 a D eh ul le d ba rle y U ns cr ee ne d 93 .9 8. 23 ± 0 .4 4 c 5. 17 ± 0 .1 5 a 13 .4 0 ± 0. 33 c 2. 41 ± 0 .4 9 b 50 0 93 .9 5. 88 ± 0 .1 3 b 4. 95 ± 0 .4 4 a 10 .8 3 ± 0. 38 b 1. 52 ± 1 .6 4 ab 35 0 93 .9 4. 94 ± 0 .3 2 a 5. 21 ± 0 .1 3 a 10 .1 5 ± 0. 22 a 0. 19 ± 0 .1 2 a W he at U ns cr ee ne d 88 .6 7. 98 ± 0 .4 7 a 1. 25 ± 0 .2 7 a 9. 23 ± 0 .5 6 a 11 .9 4 ± 0. 84 b 50 0 88 .6 7. 64 ± 0 .4 2 a 1. 46 ± 0 .3 0 a 8. 96 ± 0 .3 5 a 10 .9 5 ± 0. 73 b 35 0 88 .6 10 .6 4 ±0 .1 0 b 3. 53 ± 0 .4 2 b 12 .3 6 ± 0. 28 b 6. 71 ± 0. 68 a Sp el t U ns cr ee ne d 89 .2 8. 02 ± 0 .7 8 b 1. 60 ± 0 .3 1 a 9. 62 ± 0 .9 9 b 12 .0 6 ± 0. 47 a 50 0 89 .2 7. 80 ± 0 .1 3 b 1. 70 ± 0 .1 4 a 9. 52 ± 0 .0 6 b 11 .6 0 ± 0. 37 a 35 0 89 .2 4. 16 ± 0 .3 6 a 1. 39 ± 0 .1 1 a 5. 55 ± 0 .2 0 a 12 .2 7 ± 1. 25 a FM , f re sh m as s D at a ar e m ea ns ± S D (n = 4) . M ea ns w ith d iff er en t l et te rs w ith in e ac h sa m pl e ar e sig ni fic an tly d iff er en t a cr os s t he p ar tic le si ze s ( p ≤ 0. 05 ; A N O VA w ith p os t-h oc T uk ey ’s te st s) Acta agriculturae Slovenica, 119/4 – 20236 B. FERJANČIČ et al. 3.2 EFFECTS OF MILLING ON THE CONTENTS DETERMINED FOR DIETARY FIBRE AND RESISTANT STARCH OF THE LEGUMES The data for dietary fibre and resistant starch for the legume samples (i.e., canned beans, chickpeas, peas; with and without their brine) are presented in Table 2. Across these three legume samples that were milled to the three different particle sizes (unscreened, or 500 μm, 350 μm screening), the highest TDF was determined for chickpeas (5.90-6.74 g 100 g-1 FM) and beans (6.09-6.65 g 100 g-1 FM), both without the brine. These relative levels for TDF were generally paralleled for IDF and SDF, with the exception of the legume samples without the brine. The dietary fibre data for these legumes are in agree- ment with other studies, where IDF and SDF were deter- mined for raw beans (11.4-19.9 g 100 g-1 DM; 2.42-3.40 g 100  g-1 DM; respectively) and raw peas (20.3  g 100  g-1 DM; 1.73  g 100  g-1 DM), and IDF for raw chickpeas (13.9 g 100 g-1 DM) (Li et al., 2002; De Almeida et al., 2006; Kleintop et al., 2013). Significant differences across the two specific parti- cle sizes from 500 μm to 350 μm screening were seen for TDF and IDF determined, as increases for beans without brine, and decreases for chickpeas with brine. Significant increases were also seen according to decreased particle size for SDF determined for beans with brine and chick- peas without brine. For resistant starch determination, again from 500 μm to 350 μm screening, significant difference was only seen as a decrease for peas without brine. Considering these data, generally the effects of the different milling processes of these foods for the deter- mination of the dietary fibre and resistant starch con- tents were not uniform. For example, for beans, this was seen for SDF determined with brine and for IDF, SDF and TDF determined without brine (lowest as 500  µm milling), and for chickpeas, numerically (but not sig- nificantly) for IDF and SDF determined without brine (lowest as 500  µm milling). On the other hand, across the unscreened to fine milling (i.e., 350 μm screening) of the samples, for chickpeas with brine this showed a sig- nificant increase in SDF and decrease in IDF, thus corre- sponding to decreased particle size. For the beans, chick- peas and peas, the uniform resistant starch determined would be a consequence of the food preparation, which transformed the resistant raw starch granules into digest- ible starch (Brouns et al., 2002). For the samples of grains and processed grains (previous chapter), in terms of the relatively uniform contents determined for resistant starch despite the different particle sizes might relate to starch gelatinisation. Starch gelatinisation occurs when starch granules receive enough energy to break their in- termolecular bonds, thus undergoing irreversible loss of the native structure. As a result of this gelatinisation, the starch granules become more readily digestible (Rooney &Pflugfelder, 1986). All legume samples were cooked be- forehand, due to the fact that they were canned, therefore starch in said samples should be gelatinised. Similar ef- fect could be induced in oatmeal, however the amount of water present in thermal treatment is different, thus not allowing for full gelatinisation of starch. The changes for the legume samples without the brine, as the canned beans, chickpeas and peas, were also examined for the dietary fibre and resistant starch con- tents determined (Table 2). Significant differences were observed without the brine compared to the samples with brine for TDF, IDF, SDF and resistant starch deter- mined. The TDF and IDF determined were significantly higher in all of these samples without the brine, while the SDF determined was significantly lower only for the beans and chickpeas without the brine. These data for TDF, IDF and resistant starch can be explained in terms of the differences in the dry matter contents. The sam- ples without the brine had higher dry matter contents in comparison to the corresponding samples with the brine (Table 2), and therefore there was an effect of dilution. At the same time, the differences in the SDF determined for these legume samples can be explained in terms of the solubility of this dietary fibre in the brine. A large pro- portion of SDF in the legumes in brine was dissolved in the brine, and this was thus lost when the brine was dis- carded prior to the analyses (i.e., some 50 %˗80 % of SDF lost in the brine). Also, the canned legumes used in the present study had been cooked and sterilised, with cook- ing previously shown to lower SDF (Martin-Cabrejas et al., 2006; Wang et al., 2008). At the same time, Shin et al. (2003) reported no changes in resistant starch contents in their samples with regard to the presence of brine, due to the low solubility of resistant starch in water. 4 CONCLUSIONS The particle sizes of the grain samples generally had non-uniform effects on the determined dietary fibre and resistant and digestible starch. Some particle size effects were seen for dehulled barley, where decreasing the parti- cle size from unscreened to 500 μm to 350 μm screening, the IDF determined significantly decreased, as also for the TDF determined in these samples. With the excep- tion of oatmeal, all of the grain showed some susceptibil- ity to these changes in particle size according to the IDF and TDF determined; however, overall, only the smallest particle size had any effect on dietary fibre determina- Acta agriculturae Slovenica, 119/4 – 2023 7 Effects of particle size on determination of the contents of grain and legume dietary fibre and resistant starch Ta bl e 2: L eg um e dr y m at te r, di et ar y fib re co nt en t, an d re sis ta nt st ar ch co nt en t a cc or di ng to m ill ed sc re en se tti ng fo r p ar tic le si ze Sa m pl e Br in e M ill in g sc re en D ry m at te r D ie ta ry fi br e (g 1 00 g -1 F M ) Re sis ta nt st ar ch se tti ng (µ m ) (g 1 00 g -1 ) In so lu bl e So lu bl e To ta l (g 1 00 g -1 F M ) Be an s W ith U ns cr ee ne d 17 .4 3. 02 ± 0 .1 8 a 1. 28 ± 0 .0 3 b 4. 30 ± 0 .2 1 a 0. 51 ± 0 .0 2 a 50 0 17 .1 2. 95 ± 0 .1 9 a 1. 15 ± 0 .0 4 a 4. 10 ± 0 .1 5 a 0. 47 ± 0 .0 7 a 35 0 17 .6 3. 06 ± 0 .1 2 a 1. 28 ± 0 .0 7 b 4. 34 ± 0 .1 5 a 0. 52 ± 0 .0 1 a W ith ou t U ns cr ee ne d 27 .8 5. 98 ± 0 .1 4 b, ** * 0. 67 ± 0 .0 5 a, ** * 6. 65 ± 0 .1 0 b, ** * 1. 34 ± 0 .0 3 a, ** * 50 0 27 .7 5. 39 ± 0 .3 0 a, ** * 0. 70 ± 0 .0 4 a, ** * 6. 09 ± 0 .2 7 a, * 1. 32 ± 0 .0 5 a, ** * 35 0 28 .5 5. 83 ± 0 .0 9 b, ** * 0. 68 ± 0 .0 9 a, ** * 6. 51 ± 0 .0 6 b, ** * 1. 32 ± 0 .0 4 a, ** * C hi ck pe as W ith U ns cr ee ne d 8. 8 1. 92 ± 0 .0 5 b 0. 38 ± 0 .0 2 a 2. 30 ± 0 .0 4 b 0. 40 ± 0 .0 3 a 50 0 9. 2 1. 86 ± 0 .0 6 b 0. 41 ± 0 .0 2 ab 2. 26 ± 0 .0 4 b 0. 40 ± 0 .0 6 a 35 0 9. 6 1. 50 ± 0 .1 8 a 0. 45 ± 0 .0 3 b 1. 95 ± 0 .1 5 a 0. 36 ± 0 .0 1 a W ith ou t U ns cr ee ne d 31 .4 6. 49 ± 0 .2 2 a, ** 0. 32 ± 0 .1 4 a, * 5. 90 ± 0 .6 9 a, * 1. 69 ± 0 .0 6 a, ** * 50 0 30 .9 5. 70 ± 0 .7 2 a, ** * 0. 20 ± 0 .0 6 a * 6. 74 ± 0 .1 9 a, ** * 1. 68 ± 0 .0 6 a, ** * 35 0 31 .5 6. 26 ± 0 .1 6 a, ** * 0. 40 ± 0 .0 5 b, 6. 65 ± 0 .1 6 a, ** * 1. 62 ± 0 .1 1 a, ** * Pe as W ith U ns cr ee ne d 13 .2 3. 44 ± 0 .1 4 a 0. 49 ± 0 .0 2 a 3. 92 ± 0 .1 3 a 1. 06 ± 0 .0 4 b 50 0 13 .0 3. 42 ± 0 .1 4 a 0. 48 ± 0 .0 4 a, 3. 89 ± 0 .1 2 a 1. 00 ± 0 .0 2 a 35 0 13 .5 3. 44 ± 0 .0 9 a 0. 48 ± 0 .0 2 a 3. 93 ± 0 .0 8 a 1. 01 ± 0 .0 4 ab W ith ou t U ns cr ee ne d 20 .2 5. 15 ± 0 .1 4 a, * 0. 39 ± 0 .0 3 a 5. 53 ± 0 .1 2 a, ** * 1. 21 ± 0 .0 3 b, ** * 50 0 20 .5 4. 83 ± 0 .1 2 a, ** * 0. 46 ± 0 .1 1 a, 5. 29 ± 0 .0 7 a, ** * 1. 22 ± 0 .0 1 b, ** * 35 0 20 .7 4. 87 ± 0 .3 0 a, ** * 0. 55 ± 0 .1 2 a 5. 42 ± 0 .2 0 a, ** * 1. 16 ± 0 .0 2 a, ** * FM , f re sh m as s D at a ar e m ea ns ± SD (n = 4 ). M ea ns w ith d iff er en t l et te rs w ith in e ac h sa m pl e ar e sig ni fic an tly d iff er en t a cr os s t he p ar tic le si ze s ( p ≤ 0. 05 ; A N O VA w ith p os t-h oc T uk ey ’s te st s) St ud en t’s t- te st * p ≤ 0. 05 , * * p ≤ 0 .0 1, ** * p ≤ 0 .0 01 , s ig ni fic an t d iff er en ce s b et w ee n sa m pl e w ith ou t v er su s w ith b rin e sa m pl e, w ith in e ac h pa rt ic le si ze Acta agriculturae Slovenica, 119/4 – 20238 B. FERJANČIČ et al. tion. The particle size in the present study had little or no systematic effect on resistant starch determined. This possibility of manipulation of dietary fibre de- termined was generally shown when these grain samples were milled according to the 350 µm screening. There- fore, for dietary fibre determined for grain samples, it is advisable to maintain the particle size at the level of the 500 µm screening, rather than for the 350 µm screening. In these canned (pre-cooked) legumes, particle size had little effect on the contents determined for dietary fibre, and also for resistant starch. However, overall, particle size can have some influence on the contents determined for dietary fibre and resistant determination, in terms of possible sources of error in such analyses, especially for grains. The additional part of the present study showed that consumption of these canned legumes with the brine increased the SDF intake, although due to the dilution effects seen in the DW analysis, less TDF would be con- sumed for the same quantity of food. 5 ACKNOWLEDGMENT The authors would like to thank the Slovenian Re- search Agency (ARRS) for financial support for the PhD of Blaž Ferjančič, and for Research Programme P4˗0234. 6 CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. 7 REFERENCES Al-Rabadi, G.J.S., Gilbert, R.G., Gidley, M.J. (2009). 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Delo je prispelo 23. avgusta 2023, sprejeto 29. novembra 2023 1 Visoka šola za upravljanje podeželja Grm Novo mesto, Novo mesto, Slovenija 2 Korespondenčni avtor, e-mail: joze.podgorsek@grm-nm.si The development of agricultural use in the area of the photo- voltaic power plant D3 next to the flow accumulation of the HE Brežice Abstract: Agrovoltaics as a method of dual use of agricul- tural land is the subject of many researches and projects of sci- entific research institutions and investors. The company Hidro- eletrarne na Spodnji Savi, d.o.o. wants to develop the concept of agricultural use of agricultural land between and under the existing panels of the solar power plant. The solar power plant is located in the municipality of Brežice, the plots of land are in the cadastral municipality of Krška vas. The total area suit- able for the development of agricultural use, on which the solar power plant is located, is 8.8 hectares. Through a case study, we reviewed the theoretical starting points for planning agricul- tural production in the area of the existing solar power plant. Through our study, we selected suitable species and varieties of plants that would be suitable for cultivation in this area. For the long-term financial sustainability of agricultural produc- tion, the study also checked the expected costs and revenues for the agricultural land use plan. Based on the case study, we conclude that this area could also be used for plant production, such as the production of asparagus, currants and raspberries. At the end of the research project, we proposed that in the fu- ture photovoltaics should be planned together with agricultural use, since there are significantly more possibilities for the dual use of agricultural land with the simultaneous planning of en- ergy and agriculture. Key words: agrovoltaics, dual use of agricultural land, flow accumulation of HE Brežice, photosynthesis light satura- tion point Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice Izvleček: Agrovoltaika kot način dvojne rabe kmetijskih zemljišč je predmet številnih raziskav in projektov znanstveno raziskovalnih inštitucij in investitorjev. Podjetje Hidroelektrar- ne na Spodnji Savi, d.o.o. želi na obstoječi deponiji s postavlje- no sončno elektrarno ob pretočni akumulaciji hidroelektrarne v Brežicah razviti koncept kmetijske rabe kmetijskih zemljišč med in pod obstoječimi paneli fotovoltaične elektrarne. Sončna elektrarna je postavljena v občini Brežice, parcele so v k.o. Kr- ška vas. Celotna površina, primerna za razvoj kmetijske rabe, na kateri v večjem delu stoji sončna elektrarna, je 8,8 hektarjev. S študijem primera smo pregledali teoretična izhodišča za načr- tovanje kmetijske pridelave na območju obstoječe sončne elek- trarne. S študijo smo izbrali primerne vrste in sorte rastlin, ki bi bile primerne za pridelavo na tem območju. Za dolgoročno finančno vzdržnost kmetijske pridelave smo v študiji preverili tudi pričakovane stroške in prihodke za načrtovano kmetijsko rabo zemljišč. Na podlagi študije primera ugotavljamo, da bi to površino lahko uporabili tudi za rastlinsko pridelavo, kot je pri- delovanje špargljev, ribeza in malin. Ob zaključku raziskovalne naloge smo predlagali, da se v bodoče načrtuje fotovoltaika sku- paj s kmetijsko rabo, saj je možnosti za dvojno rabo kmetijskih zemljišč ob sočasnem načrtovanju energetike s kmetijstvom bistveno več. Ključne besede: agrovoltaika, dvojna raba kmetijskih ze- mljišč, pretočna akumulacija HE Brežice, svetlobna saturacijska točka fotosinteze Acta agriculturae Slovenica, 119/4 – 20232 J. PODGORŠEK 1 UVOD Republika Slovenija se je v strateških dokumentih zavezala povečati delež obnovljivih virov energije v strukturi pridobljene energije. Obnovljive vire energije podpirata dve ključni strategiji, kot sta Nacionalni ener- getski in podnebni načrt (NEPN) (Ministrstvo za in- frastrukturo, 2022) in Strateški načrt Skupne kmetijske politike (SN SKP) (Ministrstvo za kmetijstvo, gozdarstvo in prehrano, 2022). NEPN med drugim predvideva vsaj 27 % obnovljivih virov energije v bruto končni rabi ener- gije. Prav tako predvideva do leta 2030 zmanjšanje emisij toplogrednih plinov v sektorju kmetijstvo za 1 % glede na leto 2005. Enako tudi SN SKP naslavlja obnovljive vire energije kot pomemben korak k zmanjšanju emisij toplogrednih plinov na področju kmetijstva in gozdar- stva. Poleg vezave CO2 intenzivno naslavlja investicije za pridobivanje energije iz obnovljivih virov, med drugimi tudi iz sonca. V tem delu se sklicuje na NEPN in v kl- jučnih delih, povezanih z energetiko, povzema njegovo strategijo. Po drugi strani pa SN SKP izrazito poudarja zaščito proizvodnega potenciala kmetijskih zemljišč za pridelovanje hrane. Številne raziskovalne inštitucije v Evropi in Svetu so pričele z intenzivnim raziskovanjem kombinirane rabe kmetijskih zemljišč za namene pridelovanja hrane in pridobivanja električne energije iz sonca. Nastaja nova skupna raba kmetijskih zemljišč, agrovoltaika. Številni raziskovalci že raziskujejo to novo tehnološko področje in spremljajo možnosti za razvoj agrovoltaike na kmetij- skih zemljiščih. Predvsem iščejo priložnosti in prednosti za oba načina rabe kmetijskih zemljišč. Tako 1Gorjian s sod. (2022) ugotavlja, da implementacija agrovoltaike izjemno hitro narašča. Trenutno se za proizvodnjo elek- trične energije še vedno postavljajo klasični neprosojni paneli. Takšni so postavljeni tudi na proučevani fotovol- taični elektrarni D3 ob pretočni akumulaciji HE Brežice. Pri tem tipu agrovoltaike je največja, kritična omejitev za rastlinsko pridelavo pod paneli senčenje, kar v svo- jih raziskavah navajajo tudi drugi raziskovalci (Tromm- sdorff s sod., 2022; Takashi in Nagashima , 2019; Tani s sod., 2014; Herbert, 2018). Zato se v raziskavah vse bolj uporabljajo delno prosojni paneli, zasnovani na podlagi kristalnega silicija, tankoplastnih panelov, luminisenčnih solarnih koncentratorjev in panelov z na sončno sevanje občutljivim barvilom. Rezultati poskusov kažejo, da so delno prosojni paneli, zasnovani na podlagi kristalnega silicija, najbolj razširjeni v raziskovalnih projektih. Ti moduli imajo namreč v primerjavi z ostalimi številne prednosti, kot so nizki stroški, velika stabilnost in od- pornost na vremenske vplive ter velika učinkovitost. Z razvojem novih tehnologij zlasti pri tankoplastnih pane- lih, ki uporabljajo tehnologijo organskih panelov ali pa- nelov z na svetlobo občutljivim barvilom, se povečujejo možnosti agrovoltaike, saj ti paneli prepuščajo fotosintet- sko aktivno sončno sevanje, ostali spekter svetlobe pa se uporabi za proizvodnjo električne energije. Nekatere štu- dije dokazujejo povečanje ekonomske vrednosti kmetij z uvajanjem agrovoltaike. Harshavardhan s sod. (2016) ocenjuje, da posta- vitev sončnih elektrarn v kombinaciji z rastlinsko proi- zvodnjo z na senčenje tolerantnimi vrstami poveča eko- nomsko vrednost kmetij za preko 30 %. Še več, nekateri raziskovalci (2Gorjian s sod., 2022) vidijo priložnosti agrovoltaike tudi v lastni proizvodnji elektrike za po- trebe električnih kmetijskih strojev in sodobne opreme, vključno z roboti v kmetijstvu. Kot navajajo raziskoval- ci, sta pred nami dva glavna izziva, ki trenutno zavirata široko uporabo sodobnih električnih kmetijskih strojev. To sta visoki začetni stroški v povezavi s postavitvijo sončne elektrarne in pomanjkljivosti v tehnologijah za shranjevanje električne energije. Poleg pridobivanja električne energije pravilno na- črtovana in rastlinski pridelavi prilagojena agrovoltaika tudi izboljšuje nekatere razmere za rast gojenih rastlin v tej kombinaciji. Trommsdorff s sod. (2022) navaja pred- nosti agrovoltaike za gojene rastline, kot so zmanjšanje potreb po namakanju za 20 % v sušnih obdobjih, možnost zbiranja vode za potrebe namakanja, zmanjševanje vetr- ne erozije, uporaba konstrukcije fotovoltaike za zaščitne mreže ali folijo in optimizacija razpoložljive svetlobe za gojene rastline. Raziskovalci navajajo tudi prednosti za proizvodnjo električne energije zaradi povečanja učin- kovitosti fotovoltaičnih modulov zaradi konvekcijskega hlajenja in povečanje učinkovitosti bifacialnih panelov, ki uporabljajo osvetlitev za proizvodnjo elektrike na obeh straneh, saj je večja razdalja med paneli, tlemi in sosedn- jimi vrstami panelov. Za določitev vrst rastlin, ki bi uspešno uspevale v tej kombinaciji dvojne rabe kmetijskih zemljišč, je potrebno upoštevati svetlobno saturacijsko točko fotosinteze po- sameznih rastlin. Optimizacijo fotosinteze in nadzorni model je možno vzpostaviti ravno na podlagi te lastnosti rastlin (Xin s sod., 2019). Svetlobna saturacijska točka fo- tosinteze kaže na vpliv spreminjanja jakosti fotosintetsko aktivnega sevanja na učinkovitost fotosinteze. Nekatere rastlinske vrste namreč dosežejo za njih značilen največji učinek fotosinteze pri manjši jakosti svetlobe kot druge rastlinske vrste (Thrommsdorff s sod., 2022). Princip svetlobne saturacijske fotosinteze točke pri rastlinah, ki so ali niso tolerantne na senčenje, je prikazan na sliki 1 Nekatere rastlinske vrste imajo svetlobno saturacij- sko točko blizu 1.000 µmol s-1 m-2 ali celo pod 1.000 µmol s-1 m-2 , spet druge bistveno višje. In višja, kot je svetlobna saturacijska točka, večjo jakost fotosintetsko aktivnega Acta agriculturae Slovenica, 119/4 – 2023 3 Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice sevanja potrebujejo te vrste. Saturacijske točke fotosinte- ze nekaterih rastlinskih vrst so navedene v preglednici 1. Številni raziskovalci (Trommsdorff s sod., 2022; Ta- kashi in Nagashima , 2019; Tani s sod., 2014; Herbert, 2018) navajajo vpliv agrovoltaike na kmetijsko proi- zvodnjo. Sončni paneli senčijo površino pod sabo. In- tenzivnost senčenja je v veliki meri odvisna od načina postavitve panelov. Pravilno načrtovana agrovoltaika je zasnovana tako, da kolikor je mogoče čim manj ovira kmetijsko pridelavo, zato so običajno sončnimi paneli visoko nad rastlinami. Pri takšnih postavitvah pride do razpršenega difuznega sevanja fotosintetsko aktivnega sevanja, kar omogoča rastlinsko pridelavo neposredno pod sončnimi paneli. Glede na postavitev sončnih pa- nelov je potrebno za rastlinsko pridelavo načrtovati tudi ustrezno namakanje. Ne glede na to so v običajnih letih brez temperaturnih ekstremov pridelki gojenih rastlin pod sončnimi paneli nekoliko manjši kot pri vzgoji brez kombinacije s sončnimi paneli. Pri 30 do 35 % senčenju zaradi sončnih panelov so pridelki manjši za okoli 10 % pri solati, 30 % pri brokoliju, 20 % pri ozimni pšenici, 15 % pri krompirju, 20 % pri zeleni. V letih, ko je bilo poletje nadpovprečno toplo s temperaturnimi ekstremi blizu 40 oC pa so bili pridelki pri pridelavi pod sončnimi paneli celo večji. Tako so bili pridelki večji pri zeleni za okoli 12 %, krompirju za okoli 11 %, vinski trti za okoli 25 %, koruzi okoli 5 %, brokoliju okoli 40 %, papriki 40 %. Stallknecht E. J. s sod. (2023) navaja, da je za kvalite- ten pridelek tržne pridelave bazilike zadosten že indeks dnevne svetlobe malo nad 12 mol m-2 dan-1, kar se doseže z uporabo selektivnih modulov fotovoltaične elektrarne. Podatki teh raziskovalcev kažejo tudi, da pa je pridelek paradižnika manjši že pri minimalnem zmanjšanju foto- sintetsko aktivnega sevanja. 2 MATERIALI IN METODE Agrovoltaika postaja zanimiva tudi za nekatere in- vestitorje v Sloveniji. Eden od resnih investitorjev se po- javlja podjetje Hidroelektrarne na Spodnji Savi, d.o.o. z vizijo po razvoju kmetijske rabe na območju fotovolta- ične elektrarne D3 ob pretočni akumulaciji HE Brežice. Za preverjanje potenciala kmetijske rabe smo uporabili metodo študije primera. Tako smo na konkretnem pri- meru preverili potencial in investicijsko vrednotili razvoj kmetijske rabe. 2.1 OBMOČJE RAZISKAVE Fotovoltaična elektrarna D3 ob pretočni akumula- ciji HE Brežice leži v občini Brežice, k.o. Krška vas na parcelah 6741, 6676/2, 6746, 6749, 6784, 6779, 6321/2, 6775, 6790, 6770, 6331/2, 6680, 6752, 6766, 6763, 6755. Velikost celotne parcele je okoli 88.000 m2. Na tej parceli so v smeri vzhod-zahod postavljeni klasični neprosojni fotovoltaični moduli. Postavitev fotovoltaične elektrarne D3 je predstavljena na sliki 2. Kmetijsko pridelavo naj bi umestili med posame- zne linije fotovoltaičnih panelov glede na natančen na- črt postavitve modulov, razmakov med njimi in višinami fotovoltaičnih panelov. Prav tako smo upoštevali višino Slika 1: Svetlobna saturacijska točka fotosinteze pri različni intenzivnosti sončnega sevanja in stopnja fotosinteze (Thrommsdorff s sod., 2022) Figure 1: Light saturation point of photosynthesis at dif- ferent intensities of solar radiation and photosynthesis rate (Thrommsdorff et al., 2022) Preglednica 1: Svetlobne saturacijske točke fotosinteze neka- terih rastlinskih vrst (Thrommsdorff s sod., 2022; Hidaka s sod., 2023) Table 1: Light saturation points of photosynthesis of certain crop species (Thrommsdorff et al., 2022; Hidaka et al., 2023) Rastlinska vrsta Svetlobna saturacijska točka (µmol s-1 m-2) Paradižnik 1985 Kumare 1421 Jajčevci 1682 Zelje 1324 Cvetača 1095 Solata 857 Špinača 889 Jagode 800 – 1200 Borovnice 600 - 800 Jagodičevje 700 – 1000 Šparglji 900 – 1100 Acta agriculturae Slovenica, 119/4 – 20234 J. PODGORŠEK rastlin predlagane rastlinske pridelave. Rastlinsko pride- lavo smo umeščali z vidika minimalnega senčenja foto- voltaičnih panelov glede na razvojno fazo rastlin in viši- no sonca v posameznih mesecih leta. 2.2 KONCEPT POSTAVLJENE FOTOVOLTAIČNE ELEKTRARNE D3 Fotovoltaični moduli fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice so postavljeni v 52 vrst v smeri vzhod – zahod, s čimer se doseže največja učinkovitost pridobivanja sončne energije iz sonca, saj so posamezni paneli orientirani proti jugu. Dolžine posa- meznih vrst fotovoltaičnih modulov so od 30 m do 150 m. Postavitev modulov glede na osončenost je predsta- vljena na sliki 3. Paneli fotovoltaične elektrarne so postavljeni na predhodno pripravljeno konstrukcijo. Konstrukcija je zasnovana po nizih, ki se lahko poljubno sestavljajo v različne dolžine posamezne linije, glede na prostorske možnosti. Konstrukcija za 1 niz je zasnovana tako, da se nanjo lahko postavi 17 panelov v dveh vrstah, skupno 34 panelov. Konstrukcija niza je sestavljena iz 5 okvirjev na razdalji 4,15 m, medsebojno povezanih z vzdolžnimi no- silci, na katere se pritrjujejo moduli. Konstrukcija je po- stavljena pod naklonom 30 o. Višina okvirjev je določena tako, da je spodnji rob panelov 1 m od temelja oziroma 1,1 m od tal. Podrobnejši podatki konstrukcije so pred- stavljeni na sliki 4. V študiji smo pripravili načrt kmetijske rabe prosto- ra med posameznimi vrstami panelov. 2.3 METODA IZBIRE PRIMERNE KMETIJSKE RABE Poseben izziv v raziskavi je bila izbira primerne kmetijske rabe na lokaciji že postavljene fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice. In- vestitorji na že postavljeni in delujoči elektrarni želijo vzpostaviti kmetijsko rabo zemljišč predvsem okoli sa- mih panelov, v primeru reje manjših živali pa tudi pod paneli. Glede na dejstvo, da je elektrarna že postavljena, smo v študiji primera morali kmetijsko rabo povsem pri- lagoditi obstoječemu stanju. Zato smo pri načrtovanju kmetijske rabe sladili usmeritvi investitorja, da kmetijska raba ne sme senčiti ali kako drugače ovirati delovanja fo- tovoltaične elektrarne. Prav tako smo upoštevali dejstvo, da investitor želi izvajati ekološko pridelavo hrane in da želi kombinirati rejo živali z rastlinsko pridelavo. Izbor rastlin smo naredili na podlagi sledečih para- metrov: - primernost rastlin za gojenje pri manj močni svet lobi (nižja svetlobna saturacijska točka fotosin teze), Slika 2: Postavitev fotovoltaičnih modulov na fotovoltaični elektrarni D3 ob pretočni akumulaciji HE Brežice (HESS, 2023) Figure 2: Installation of photovoltaic modules on the photovoltaic power plant D3 next to flow accumulation of HE Brežice (HESS, 2023) Acta agriculturae Slovenica, 119/4 – 2023 5 Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice - habitus rastline v polni rodnosti, ki ne sme senčiti fotovoltaične elektrarne, - možnost ekološke pridelave, - želje lastnika fotovoltaične elektrarne. Pri izboru primernih živalskih vrst pa smo upošte- vali sledeče parametre: - višina živali, - agresivnost živali, - sposobnost živali za pašo, - uporabnost gnoja za gnojenje ekološke pridelave rastlin, - želje lastnika fotovoltaične elektrarne. V nadaljevanju smo naredili podrobnejšo analizo po principu študije primera zgolj za predlagano rastlin- sko pridelavo, rejo živali v analizo nismo vključili. 2.4 METODA EKONOMSKE ANALIZE Za investitorja smo pripravili tudi kratko ekonom- sko analizo načrtovane rastlinske pridelave. Osredoto- čili smo se predvsem na investicijski strošek, izračunali predviden letni donos in potrebo po delovni sili za vzdr- ževanje v enem letu polne rodnosti zasnovane rastlin- ske pridelave. Investicijske stroške smo ocenili glede na potrebne materiale za postavitev rastlinske pridelave in aktualne cene na trgu, na podlagi prejetih ponudb v letu 2023. Predviden donos smo izračunali glede na priča- kovan pridelek in aktualne cene na trgu v sezoni 2023. Podatke o potrebnem času za vzdrževanje rastlinske pri- delave pa smo črpali iz Kataloga kalkulacij za načrtovanje gospodarjenja na kmetijah v Sloveniji (Jerič s sod., 2001). Slika 3: Postavitev fotovoltaičnih modulov na fotovoltaični elektrarni D3 ob pretočni akumulaciji HE Brežice (HESS, 2023) Figure 3: Installation of photovoltaic modules on the photovoltaic power plant D3 next to flow accumulation of HE Brežice (HESS, 2023) Slika 4: Podrobnejša predstavitev načrta konstrukcije za mon- tažo panelov fotovoltaične elektrarne D3 (HESS, 2023) Figure 4: Detailed presentation of the construction plan for the installation of D3 photovoltaic power plant panels (HESS, 2023) Acta agriculturae Slovenica, 119/4 – 20236 J. PODGORŠEK 3 REZULTATI IN DISKUSIJA Na podlagi parametrov izbire rastlinskih vrst smo se odločili za sledeče rastlinske vrste: - maline (Rubus idaeus L.), - ribez (Ribes), - šparglji (Asparagus officinalis L.). Na obstoječi fotovoltaični elektrarni D3 smo pre- dlagali sledečo razporeditev posameznih rastlinskih vrst, kar prikazuje tudi slika 5: - od vrste 15 do vrste 28: pridelovanje malin, - od vrste 28 do vrste 40: pridelovanje ribeza, - od vrste 40 do vrste 52: pridelovanje špargljev. Na površini fotovoltaične elektrarne D3 od vrste 1 do vrste 15 smo predvideli rejo kokoši nesnic oziroma drobnice z namenom pridobivanja ekološkega gnoja za gnojenje predlagane rastlinske pridelave. Rejo živali v študijo nismo vključili, zato je tudi nismo podrobneje analizirali. 3.1 POTREBNA OPREMA ZA VZPOSTAVITEV KMETIJSKE RABE ZEMLJIŠČ 3.1.1 Maline Za pridelovanje malin smo predlagali površino od vrste 15 do vrste 28 fotovoltaičnih panelov. Glede na na- črtovano višino malin v času obiranja smo predlagali eno vrsto malin v medvrstni prostor fotovoltaičnih modulov s sajenjem čim bližje najvišjemu delu fotovoltaičnih mo- dulov. Priporočena sadilna razdalja v vrsti je pri malinah 40 cm, zato je za predlagano površino potrebnih 6.300 sadik. Za pridelovanje malin predlagamo sorte, ki omogo- čajo ekološko pridelavo in imajo manjši habitus, torej ne senčijo panelov fotovoltaične elektrarne. Predlagali smo sledeče sorte: Clarita, Enrosadira in Polka. Za namakanje malin smo predlagali kapljično na- makanje. Za postavitev kapljičnega namakanja predlaga- ne površine malin so potrebni sledeči materiali: - kapljična cev: 1.900 m, - gibka cev za sekundarni razvod namakanja: 105 m, - priključne spojke: 13 kosov. Maline se sadijo na grebene, ki jih je potrebno pre- kriti s tkanino proti razvoju plevela. Predlagali smo tka- nino teže 100 g m-2, širine 1,25 m. Potrebno je okoli 1.900 dolžinskih metrov te tkanine. Za pripravo opore predlaganega nasada malin se potrebujejo sledeči materiali: - betonski stebri 7 x 7,5 cm, višine 2,8 m: 450 kosov - kovinska sidra, 0,85 m x 130 mm: 80 kosov - napenjalna žica 2,5 mm: 100 kg. Po postavitvi nasada malin bo potrebno postaviti še oporo za prekrivanje malin s folijo v času rodnosti, pri čemer se priporoča uporaba obstoječe konstrukcije elek- trarne kot pomemben konstrukcijski element. 3.1.2 Ribez Za pridelovanje ribeza smo predlagali površino od vrste 28 do vrste 40 fotovoltaičnih panelov. Glede na na- črtovano višino grmov smo predlagali eno vrsto ribeza v medvrstni prostor fotovoltaičnih panelov. Priporočena sadilna razdalja v vrsti je 1 m, zato je za predlagano povr- šino potrebnih 1.800 sadik. Izbor sort ribeza je temeljil na možnosti ekološke pridelave in višini posameznih grmov z vidika morebit- nega senčenja panelov fotovoltaične elektrarne. Predla- gamo sledeče sorte ribeza: Jonkher van Tets in Rovada (rdeči riberz) ter Ben Nevis in Titania (črni ribez). Tudi ribez se namaka kapljično. Za postavitev kapl- jičnega namakanja predlagane površine ribeza so potreb- ni sledeči materiali: - kapljična cev: 1.900 m, - gibka cev za sekundarni razvod namakanja: 105 m, - priključne spojke: 12 kosov. Ribez se sadi v vrste, ki jih je potrebno prekriti s tka- nino proti razvoju plevela. Predlagali smo tkanino teže 100 g m-2, širine 1,25 m. Potrebno je okoli 1.800 dolžin- skih metrov te tkanine. 3.1.3 Šparglji Za pridelovanje špargljev smo predlagali površino od vrste 40 do vrste 52 fotovoltaičnih panelov. Priporoče- na sadilna razdalja v vrsti je 30 cm, za načrtovano površi- no je potrebnih 4.350 sadik. Glede na načrtovano višino zelenja po končanju pobiranja špargljev predlagamo eno vrsto špargljev v medvrstni prostor fotovoltaičnih pane- lov. Pri izboru sort špargljev smo sledili izhodišču, da bo pridelava potekala na ekološki način in prilagojenosti sort na težja zemljišča. Predlagamo sledeče sorte: Eros, Marte in Giove. Šparglje je priporočljivo namakati. Za pripravo kapljičnega namakanja predlagane površine špargljev so potrebni sledeči materiali: - kapljična cev: 1.500 m, - gibka cev za sekundarni razvod namakanja: 115 m, - priključne spojke: 12 kosov. Material, ki je potreben za zasnovo predlagane ra- stlinske pridelave po posamezni rastlinski vrsti predsta- vljamo v preglednici 2. Acta agriculturae Slovenica, 119/4 – 2023 7 Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice 3.2 OKVIRNI STROŠKI VZPOSTAVITVE IN POTREBA PO DELOVNI SILI 3.2.1 Maline Ocenjena vrednost sadik in materiala za postavitev nasada malin iz preglednice 3 je po prejetih ponudbah iz marca 2023 okoli 14.000 EUR (brez DDV). Za napravo predlaganega nasada malin se ocenju- je porabo okoli 400 delovnih ur. Za spravilo pridelka in vzdrževanje nasada pa okoli 2.500 ur letno. Izrazita de- lovna konica je v času spravila pridelka. Samo za spravilo pridelka se predvideva porabo okoli 2.200 ur časa, kar pomeni okoli 3,1 FTE v času od sredine junija do konca oktobra (Jerič s sod., 2001). Pri bruto urni postavki 6,92 EUR (Odredba o uskladitvi najnižje bruto urne postav- ke za opravljeno začasno ali občasno delo v kmetijstvu, 2023) je ocenjen strošek dela 20.068 EUR. Pričakovan pridelek malin na predlagani površini je okoli 14.000 kg. Pri prodajni ceni 5,00 EUR kg-1 je priča- kovan prihodek 70.000 EUR. 3.2.2 Ribez Ocenjena vrednost sadik in materiala za postavitev nasada ribeza iz preglednice 3 je po prejetih ponudbah iz marca 2023 okoli 6.500 EUR (brez DDV). Za napravo predlaganega nasada ribeza se ocenju- je porabo okoli 100 delovnih ur. Za spravilo pridelka in vzdrževanje nasada pa okoli 1.200 ur letno. Izrazita de- lovna konica je v času spravila pridelka (konec junija do začetka avgusta) (Jerič s sod. 2001). Samo za spravilo pri- delka ocenjujemo porabo okoli 1.000 ur časa, kar pomeni okoli 3,8 FTE v času od konca junija do začetka avgusta. Pri bruto urni postavki 6,92 EUR (Odredba o uskladitvi najnižje bruto urne postavke za opravljeno začasno ali Slika 5: Razporeditev izbranih rastlinskih vrst na obstoječi fotovoltaični elektrarni D3 Figure 5: Distribution of selected crop species at the existing photovoltaic power plant D3 Acta agriculturae Slovenica, 119/4 – 20238 J. PODGORŠEK občasno delo v kmetijstvu, 2023) je ocenjen strošek dela 8.996 EUR. Pričakovan pridelek ribeza na predlagani površini je okoli 7.000 kg. Pri prodajni ceni 4,00 EUR kg-1 je priča- kovan prihodek 28.000 EUR. 3.2.3 Šparglji Ocenjena vrednost sadik in materiala za postavitev nasada špargljev iz preglednice 3 je po prejetih ponudbah iz marca 2023 okoli 3.600 EUR (brez DDV). Za napravo predlaganega nasada špargljev se oce- njuje porabo okoli 120 delovnih ur. Za spravilo pridelka in vzdrževanje nasada pa okoli 600 ur letno (Jerič s sod., 2001). Izrazita delovna konica je v času spravila pridelka, ki v času polne rodnosti traja 8 tednov (prvo leto spravila pobiramo poganjke 3 tedne in drugo leto 5 tednov). Za spravilo pridelka ocenjujemo porabo okoli 550 ur časa, kar pomeni 1,5 FTE v času obiranja. Pri bruto urni po- stavki 6,92 EUR (Odredba o uskladitvi najnižje bruto urne postavke za opravljeno začasno ali občasno delo v kmetijstvu, 2023) je ocenjen strošek dela 4.982 EUR. Pričakovan pridelek špargljev v času polne rodno- sti je na predlagani površini okoli 2.400 kg špargljev. Pri prodajni ceni 5,00 EUR kg-1 je pričakovan prihodek 12.000 EUR. Predvideni stroški zasnove predlagane rastlinske pridelave na fotovoltaični elektrarni D3 in stroški enega leta vzdrževanja nasada, vključno z obiranjem, ter pred- viden pridelek in prihodek v polnem letu rodnosti so po posameznih rastlinskih vrstah predstavljeni v pregledni- ci 3. Preglednica 2: Vrste in količine potrebnega materiala za zasnovo predlagane rastlinske pridelave na fotovoltaični elektrarni D3 Table 2: Types and quantities of material needed for the design of the proposed crop production at the D3 photovoltaic power plant Rastlinska vrsta Material Maline Ribez Šparglji število sadik (kos) 6.300 1.800 4.350 kapljična cev (m) 1.900 1.900 1.500 gibka cev - sekundarni razvod namakanja (m) 105 105 115 priključne spojke (kos) 13 12 12 tkanina (m) 1.900 1.800 betonski stebri (kos) 450 kovinska sidra (kos) 80 napenjalna žica (kg) 100 Preglednica 3: Predvideni stroški in prihodki za postavitev in vzdrževanja nasada v enem letu polne rodnosti po posameznih rast- linskih vrstah predlagane rastlinske pridelave na fotovoltaični elektrarni D3 Table 3: Estimated costs and revenues for the installation and maintenance of the plantation in one year of full fertility by crop species of the proposed crop production at photovoltaic power plant D3 Rastlinska vrsta Predvideni stroški Maline Ribez Šparglji material iz preglednice 2 (EUR) 14.000 6.500 3.600 delo za postavitev nasada (ure) 400 100 120 delo za postavitev nasada (EUR) 2.768 692 830 delo za vzdrževanje in obiranje (ure) 2.500 1.200 600 delo za vzdrževanje in obiranje (EUR) 17.300 8.304 4.152 Predviden prihodek pričakovan pridelek (kg) 14.000 7.000 2.400 pričakovana prodajna cena (EUR/kg) 5,00 € 4,00 € 5,00 € pričakovan prihodek 70.000,00 € 28.000,00 € 12.000,00 € Acta agriculturae Slovenica, 119/4 – 2023 9 Razvoj kmetijske rabe na območju fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice 3.3 DISKUSIJA Študija primera, ki smo jo izvedli na konkretnem primeru, kaže na potencialno možnost rastlinske pridelave na območju Fotovoltaične elektrarne D3 ob pretočni akumulaciji Brežice. Pri načrtovanju rastlinske pridelave smo se povsem prilagodili obstoječi fotovoltaični elektrarni, ki je že postavljena. Paneli so postavljeni na primernih razdaljah in se ne senčijo med sabo. Prav tako razdalje med paneli omogočajo nekatere vrste rastlinske pridelave. Pri tem gojene rastline ne senčijo panelov. Zasnova nasada malin in ribeza je predvidena z zastirno folijo, ki preprečuje rast in razvoj plevelov in hkrati omogoča vsakoletno obnavljanje nasada z novimi poganjki. Zastirna folija je široka 1,25 m, kar pomeni, da bo pokrivala le grebene, kjer bodo rastle predlagane kulture. Ostali del površine bo zatravljen (trenutno je celotna površina zatravljena). Zato bo potrebno stalno vzdrževanje travne površine. Glede na prostorsko stisko glede na sajene rastline med dvema vrstama panelov bo traktorsko mulčenje trave onemogočeno, zato bo potreb- na košnja z manjšo samohodno kosilnico. Načrtovana rastlinska pridelava okoli panelov fo- tovoltaične elektrarne bo omogočala stalen dostop do elektrarne. Rastlinska pridelava ne bo posegala v komu- nikacijske in logistične poti, ki so potrebne za vzdrževan- je in spremljanje fotovoltaične elektrarne. Prav tako bo v vsakem trenutku omogočen dostop do katerega koli panela oziroma električne napeljave v primeru potrebe po odpravi okvar na katerem koli delu fotovoltaične elek- trarne. Investitor fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice je študijo naročil z namenom, da se po metodi študije primera ugotovijo realne možnos- ti za kmetijsko pridelavo na lokaciji fotovoltaične elek- trarne. Investitorji naj se ne bi ukvarjali s kmetijsko pri- delavo, temveč bodo študijo uporabili za potencialnega najemnika zemljišč. Zainteresiran pridelovalec bo lahko na podlagi študije dokaj predvidljivo zasnoval rastlin- sko pridelavo na podlagi pogodbenega razmerja. Glede na ekonomske izračune, ki sicer ne vključujejo potrebne strojne opreme in stroškov za pripravo pridelka za trg (pakirnice, hladilnice, embalaža ipd.) predlagamo, da se pogodbeno razmerje sklepa za čas amortizacijske dobe posamezne rastlinske vrste in konstrukcije pri postavitvi nasada malin, vsekakor pa ne krajši od 10 let. 4 SKLEPI Študija primera na primeru fotovoltaične elektrarne D3 je pokazala, da je rastlinska pridelava mogoča in eko- nomsko zanimiva. Stroški zasnove rastlinske pridelave so ocenjeni na 24.100 EUR. Za zasnovo rastlinske pridelave je potrebnih 620 delovnih ur, zato je strošek dela za za- snovo rastlinske pridelave ocenjen na 4.290 EUR. Torej je skupen strošek zasnove rastlinske pridelave ocenjen na 28.390 EUR. Rastlinska pridelava zahteva vsakoletno oskrbo in spravilo pridelka. Ocenjujemo, da je v enem letu polne rodnosti zasnovane rastlinske pridelave za ta dela sku- paj potrebnih 4.300 delovnih ur, kar predstavlja strošek dela v višini 29.756 EUR. Največ dela je potrebnega za spravilo pridelka. Za nemoteno opravljanje dela in redno oskrbo rastlinske pridelave je letno potrebnih okoli 550 delovnih ur, medtem ko je za spravila pridelka letno po- trebnih okoli 3.750 ur. Predlagamo, da se za pokrivanje tega izrazito sezonskega dela najame sezonska delovna sila za opravljanje začasnega dela v kmetijstvu. Predvideni prihodki od predlagane rastlinske pride- lave so v skupni vrednosti 110.000 EUR. Zato ocenjuje- mo, da je vrednost rastlinske pridelave ekonomsko zani- miva. Prav tako ocenjujemo, da razlika med ocenjenimi stroški in prihodki omogoča izvajanje vseh tehnoloških ukrepov, ki jih ekološko pridelovanje zahteva, ter opreme in embalažo za spravilo pridelka. Za še učinkovitejšo rabo zemljišč za kmetijsko rabo je potrebno skupno načrtovanje fotovoltaike s kmetijsko pridelavo. Pri tem je potrebno uskladiti višino, na kateri se postavljajo paneli fotovoltaične elektrarne glede načr- tovane kmetijske rabe zemljišč in tip panelov, ki zagotavl- jajo zadostno prepustnost fotosintetsko aktivnega sevan- ja za zmanjševanje vpliva senčenja na višino in kvaliteto pridelka. Raziskava po metodi študije primera, ki smo jo izvedli na konkretni lokaciji fotovoltaične elektrarne D3 ob pretočni akumulaciji HE Brežice je ena prvih takšnih raziskav v Sloveniji. Rezultati raziskave, ki nakazujejo na možnost rastlinske pridelave na obstoječi fotovoltaični elektrarni, bodo v nadaljevanju lahko podlaga za načr- tovanje novih fotovoltaičnih elektrarn, kjer bi se z bolj- šim načrtovanjem prostor še bolje izkoristil za rastlinsko pridelavo. Kot smo že omenili, študija nakazuje na pri- ložnosti pri sočasnem načrtovanju rastlinske pridelave in fotovoltaične elektrarne, pri čemer se vidik kmetijske rabe in vidik energetske rabe prostora prilagajata drug drugemu s ciljem optimizacije obeh načinov rabe. Študija nakazuje priložnost za optimizacijo rabe kmetijskih površin. Z vidika razvoja dvojne rabe prostora bo potrebno uskladiti zakonodajo na način, da bodo dvojne rabe kmetijskih zemljišč dopustne in izvedljive. Trenutna zakonodaja omejuje investicije v dvojno rabo kmetijskih zemljišč, ki so po namenski rabi kmetijska zemljišča. Navedena dejstva nam nakazujejo, da bodo za bo- Acta agriculturae Slovenica, 119/4 – 202310 J. PODGORŠEK doče urejanje agrovoltaike v Sloveniji potrebne dodatne študije in raziskave z namenom optimizacije dvojne rabe kmetijskih zemljišč. Na podlagi pridobljenih rezultatov bo zakonodajalec lahko pripravil zakonodajni okvir, ki bo omogočal investicije. Z vidika kmetijskih gospodarstev je agrovoltaika lahko priložnost za povečanje prihodkov in s tem stabilizacijo delovnih mest, kar bo utrdilo kmetij- ska gospodarstva in jim povečalo odpornost ob vremen- skih, tržnih ali drugih odmikih v kmetijski pridelavi. 5 VIRI Gorjian S., Bousi E., Ozdemir O. E., Trommsdorff M., Kumar N. M., Anand A., Kant K., Chopra S. S. (2022). Progress and Challenges of Crop Production and Electricity Genera- tion in Agrivoltaic Systems Using Semi-transparent Photo- voltaic Technology. Renewable and Sustainable Energy Re- views, 158, New York: Pergamon. https://doi.org/10.1016/j. rser.2022.112126 Gorjian S., Ebadi H., Trommsdorff M., Sharon H., Demant M., Schindele S. (2021). The advent of modern solar-powered electric agricultural machinery: A solution for sustainable farm operations. Journal of Cleaner Production, 292. https:// doi.org/10.1016/j.jclepro.2021.126030 Harshavardhan D., Joshua P. (2016). The potential of agrivol- taic systems. 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(2014) Improve- ment in lettuce growth by light diffusion under solar pa- nels. Agricultural Meteorology, 70, 139-49. https://doi. org/10.2480/agrmet.D-14-00005 Trommsdorff M., Dhak I.S., Ozdemir O.E., Ketzer D., Wein- berger N., Rosch C. (2022). Agrivoltaics: solar power ge- neration and food production. Science Direct; Solar Energy Advancements in Agriculture and Food Production Systems, 159–210. https://doi.org/10.1016/B978-0-323-89866- 9.00012-2 Trommsdorf M., Gruber S., Keinaht T., Hopf M., Hermann C., Schonberger F., Hogy P., Zikeli S., Ehmann A., Weselek A., Bodmer U., Rosch C., Ketzer D., Weinberger N., Schindele S., Vollprecht J. (2022). Agrivoltaics: Opportunities for Ag- ri-culture and the Energy Transition. Fraunhofer Institute for Solar Energy Systems ISE, Freiburg, Germany, 72 str. Xin P., Li B., Zhang H., Hu J. (2019). Optimization and control use of the light environment for greenhouse crop produ- ction, Scientifiv Reports, 9, 8650. https://doi.org/10.1038/ s41598-019-44980-z Acta agriculturae Slovenica, 119/4, 1–16, Ljubljana 2023 doi:10.14720/aas.2023.119.4.15748 Original research article / izvirni znanstveni članek Influence of mulches on soil moisture and water infiltration in the toma- to crop Imerina TANKEUOO KOPA 1, 2, Mohamed MEDDI 1, Abdelhadi AMMARI 1 Received August 23, 2023; accepted November 22, 2023. Delo je prispelo 23. avgusta 2023, sprejeto 22. novembra 2023 1 National Higher School of Hydraulics (ENSH), Water and Environmental Engineering Laboratory (GEE), Blida, Algeria 2 Corresponding author, e-mail: i.tankeuookopa@ensh.dz Influence of mulches on soil moisture and water infiltration in the tomato crop Abstract: Soil moisture is a key parameter of soil monitor- ing for observation of vegetation growth, predicting crop pro- duction, and improving water resource management. In this study, the objective is to compare the evolution of soil mois- ture in different mulches to determine the best mulch and its characteristics of infiltration in the soil. The experiment was conducted during the summer season in July-September 2022 on four different mulches (wood chips, sawdust, straw, mix- ture), and control at the experimental plot of Blida. The results showed that silt is the main matrix of the soil. The analysis of infiltration data identified modified Kostiakov as the best mod- el of the study site, whose period of plant growth represents the phase during which we have a better infiltration under the mixture. The application of the mulch changes the moisture mainly at 15 cm and the conductivity at 10 cm. In addition, the mixture is the best mulch to conserve moisture in the soil while reducing the frequency of irrigation. The correlation between soil moisture and conductivity was overall very good. This was due to the effect of mulch, soil texture, plant root development, and capillary rise. Key words: tomatoes, mulching, soil moisture, infiltra- tion, soil electrical conductivity Vpliv mulčenja na vlažnost tal in infiltracijo vode v nasadu paradižnika Izvleček: Vlažnost tal je glavni parameter pri spremljanju lastnosti tal pri opazovanju rasti, napovedovanju pridelka in pri izboljševanju upravljanja z vodnimi viri. Predmet te raziskave je bil primerjati razvoj vlažnosti tal pri različnih načinih mulče- nja z namenom določiti najboljši način mulčenja za infiltracijo vode v tla. Poskus je potekal v poletni sezoni od julija do sep- tembra 2022 s štirimi načini mulčenja (lesni sekanci, žagovina, slama, mešanica) in kontrolo na poskusnem polju v Blidi, Al- žirija. Rezultati so pokazali, da je bil kremenčev drobir glavna sestavina anorganskega dela tal. Analiza podatkov o infiltraciji vode je pokazala, da se je izkazal spremenjen Kostiakov model kot najboljši za preučevanje tal, v katerem predstavlja obdobje rasti rastlin fazo, v kateri je infiltracija vode v tla najboljša pri mulčenju z mešanico. Uporaba mulčenja spreminja vlažnost tal v glavnem na globini 15 cm in prevodnost tal na globini 10 cm. Dodatno je mešanica materialov za mulčenje najboljša, ker ohranja vlažnost tal in hkrati zmanjšuje pogostost namakanja. Korelacija med vlažnostjo in prevodnostjo tal je bila nasplošno zelo dobra kar je bilo posledica mulčenja, teksture tal, razvoja korenin in kapilarnega dviga. Ključne besede: paradižnik, mulčenje, vlažnost tal, infil- tracija, električna prevodnost tal Acta agriculturae Slovenica, 119/4 – 20232 I. TANKEUOO KOPA et al. 1 INTRODUCTION One of the fundamental soil parameters affecting the life of plants, animals, and microorganisms is soil moisture (Safari et al., 2021). Commonly defined as the amount of water present in the unsaturated zone, it is a key element for predicting agricultural production and improving water resource management. Its measurement in precision agriculture is an essential agronomic com- ponent for monitoring crop growth (Goel et al., 2020). Due to the importance of the role played by soil mois- ture, some scientists have developed different instrumen- tation observation methods, including the dialectical method by time domain reflectometry (TDR). Indeed, it is a method that cannot be ignored for the quality, ease of use, and accuracy of its measurements (Freire et al., 2020). Other researchers have instead focused on the is- sue of water conservation in the soil, establishing differ- ent techniques, including mulching (Stelli et al., 2018). An old practice (Jacks et al., 1955), consists of covering the soil surface with other materials, called “mulch” (Al- metwally et al., 2019; Telkar et al., 2017). They can be applied to perennial or seasonal crops to: conserve wa- ter (Zegada-Lizarazu and Berliner, 2011), improve crop performance and wind control (Sharma et al., 2023; Sim- sek et al., 2017), increase plant health and vitality (Stelli et al., 2018), improve the action of microorganisms in the soil, and increase soil organic matter (Sharma et al., 2023; Simsek et al., 2017). According to an experiment conducted on different organic and inorganic mulches (Safari et al., 2021), the ideal depth is 15 cm with an in- crease of 2 to 5 % compared to bare soil (control). Or- ganic materials are the most recommended as they can actively promote soil desalination and assist in the deg- radation of pesticides and other pollutants (Telkar et al., 2017). Several studies have been conducted in semi-arid and arid areas on the effects of mulching in conserving soil moisture (Almetwally et al., 2019; Mkhabela et al., 2019; Stelli et al., 2018; Simsek et al., 2017; Telkar et al., 2017) with interesting results. Some have also tested the effects of mulching on specific crops, such as tomatoes. It’s a crop that is widely cultivated for its fruits that are consumed fresh or processed (Chaux and Foury, 1994). Currently, it represents the most cultivated and processed crop in the world (Sharma et al., 2023). Tomatoes play a major role in Algeria’s agricultural economy. According to the Ministry of Agriculture and Rural Development (MADR), nearly 33,000 hectares of land are devoted to this crop (horticultural and industrial), with an aver- age production of 11 million quintals and average yields of around 311 Qx ha-1 (MADR, 2009 in Tarchag, 2020, MADR, 2011 in Amichi et al., 2015). However, according to Sharma et al. (2023), water is one of the elements that directly affects tomato productivity. In addition to effi- cient use of available soil moisture, weed control, spacing, timing of planting, and judicious application of manure and fertilizer are all aspects that influence the success of tomato production (Lamont, 2005). The Mitidja, which is heavily dominated by vegetable crops and arboriculture, is one of the most fertile plains in Algeria (Meddi et al., 2013). However, in recent years, this plain is facing a de- cline in the water table of at least 40 m in some areas. This phenomenon is mainly due to poor water management, overexploitation of the water table by various industries, and drought episodes combined with the importance of irrigated areas (Djouda-Hallah, 2014). Another impor- tant process to consider in agriculture is infiltration, as it is one of the important components of the soil water balance in semi-arid areas (Liao et al., 2021). Further- more, researchers (Oku and Aiyelari, 2011) have shown that soil infiltration properties can be quantified by fit- ting field infiltration data to model infiltration. Similarly, detailed knowledge of soil infiltration rates and charac- teristics can increase irrigation water use efficiency and reduce water losses (Haghiabi et al., 2011; Xing et al., 2017). To this end, scientists (Liao et al., 2021; Farid et al., 2019; Vand et al., 2018, Furman et al., 2006; Mishra et al., 2003) have developed several models to determine the infiltration rate and its characteristics. The most commonly used are Philips, Kostiakov, modified Kostiakov, and Horton because of their perfor- mance and efficiency. However, despite all the advantages mentioned, mulching in general and organic mulching in particular is not a common practice in agricultural crops in Algeria. However, this technique can be a de- sign solution to the great theme that is the conservation of moisture to the maximum and with efficiency in the soil, without destructuring and impoverishing it. That is why we studied the effects of mulching on soil moisture and infiltration of the soil in the cultivation of tomatoes. The specific objectives were to (1) characterize the soil of the site where tomato plants were planted; (2) determine the best infiltration model that best fits the study area; (3) study the effects of mulching on soil electrical conductiv- ity, soil moisture retention and identify the best mulch among those studied as well as its ideal depth. 2 MATERIALS AND METHODS 2.1 EXPERIMENTAL SITE The experiments were carried out in the Mitidja plain, at the experimental site of the National Higher School of Hydraulics (ENSH) of Blida, Algeria (36°30’31’’ N, 2°53’15’’ E, 110 m) (Fig. 1), during the period from Acta agriculturae Slovenica, 119/4 – 2023 3 Influence of mulches on soil moisture and water infiltration in the tomato crop July to September 2022. According to Laribi et al. (2023), this plain extends today over four Wilayas (Blida, Tipaza, Bourmedès and Algiers), with an average annual tem- perature of 18 °C and a rainfall between 600 mm and 900 mm/year. According to this author, this plain is located in the bioclimatic Mediterranean subhumid area with mild winters. An agro-meteorological station was installed in the plot to obtain data on rainfall, air and soil surface temperature, wind speed, solar radiation and humidity. The data processing for the period July-September 2022 shows that the range of air temperatures varies from 25.25 °C (September) to 50.77 °C (August). On the other hand, on the surface of the ground, the range tempera- ture is about 28.00  °C (September)–54.45 °C (August). The average relative humidity is 64.71 % with a minimum of 33.13 % and a maximum of 75.41 %; solar radiation is 247.70 W m-², wind speed is 2.54 m s-1 and evapotran- spiration is 129.95 mm/day. There was no precipitation during this period. 2.2 SITE DESIGN At this site, five plots of 4 m² were built (Fig. 2) (i.e. a total area of 40 m² with the respective additions of coarse (wood chips) and fine (sawdust) white wood bark mulch, wheat straw (cut to about 5 cm), the mixture of the first three mulches (with the same quantity of each mulch), and bare soil (here referred to as the control). The choice of mulches was based on the availability of inexpensive, abundant, and effective local materials. To monitor moisture until fruiting, 3-week-old ‘Bahadja F1’ hybrid tomato plants were planted in these plots. The dis- tance between the ramps was 50 cm and 40 cm for the to- mato plants and drippers, respectively. Each dripper was placed at the base of each plant and was self-regulating. In total, there were 12 tomato plants and 12 drippers per plot. The plants were irrigated with the surface drip sys- tem according to their needs at one-hour (1h) intervals in the evening. For the irrigation control, in addition to the Figure 1: Location map of Mitidja plain Figure 2: Design of the experimental setup and installation of mulch on tomato plants Acta agriculturae Slovenica, 119/4 – 20234 I. TANKEUOO KOPA et al. literature, we used the template provided by the company responsible for the TDR 150 device, which visualizes the delineation of the moisture at the wilting point (WP) and the field capacity (FC) for each soil type. During this ir- rigation period, the average flow rate used was 7.72 l h-1. 2.3 SOIL SAMPLING The soils on which the tomato plants were planted were first physically analyzed to determine soil texture and bulk density. Five samples of disturbed soil, one per plot, were taken with a hand auger from 0 to 30 cm deep. Then five more undisturbed samples (always at the same points and depth) were taken with special samplers (cylinders). The entire sample collection was sent to the Agroecology Laboratory of the National Higher School of Hydraulics (ENSH) for analysis. The international method of Robinson (1949) was used to determine the different proportions of sands, clays, and silts of the re- worked samples. Also, the texture of each soil was de- termined through its texture diagram. To determine the bulk density of each sample, a balance was used for wet and dry weighing, and an oven at 105 °C was used to dry the samples. 2.4 INFILTRATION MEASUREMENT The double ring infiltrometer with an outer diam- eter of 32 cm and an inner diameter of 11 cm was in- stalled in the ground to measure the infiltration rate in the field. During our fieldwork, this device was driven into the ground at a depth of 5 cm on a flat surface. The float was placed inside the inner ring to read the amount of water infiltrating into the soil. Measurements were taken at one-minute intervals. Infiltration measurements were taken before sowing tomato plants on bare soil in each plot, during the period of tomato plant growth un- der mulch, and at the end of crop growth at each soil sampling point. The rest of the experiment continued until the equilibrium infiltration rate was reached. After reaching this equilibrium infiltration rate (mm min-1) in the soil, the experiment was stopped. This experiment was then repeated three times per plot. 2.5 DETERMINATION OF PARAMETERS OF DIF- FERENT INFILTRATION MODELS Philip, Kostiakov, and modified Kostiakov are the three models used in the study due to their popularity, effectiveness, and the type of soil present in the study area (Kostiakov, 1932; Philip, 1957). Experimental field data were used to evaluate these infiltration models and to obtain numerical values of the model hydraulic pa- rameters. The hydraulic parameters of each model were determined using EXCEL software. The equations used for each model are described below. - Philips model This model is one of the physical models com- monly used to estimate infiltration. Based on Darcy’s law and the law of conservation of mass (Vand et al., 2018, Philips, 1957), it has the equation: (2.1) Where: f(t) is the infiltration rate at time t, S is the soil sorptivity, K is the hydraulic conductivity of the soil at saturation, and t is the time since infiltration began. In this study, to estimate the values of the parameters S and K, the infiltration rate data (mm min-1) were fitted as a function of time transformed by least squares regression for all data obtained before sowing, during growth and at the end of fruiting at the five points. - Kostiakov model Kostiakov is a model based on the collection of ex- perimental data obtained in the field, as well as in the laboratory. As an empirical model, it allows estimating the infiltration rate according to the equation: (2.2) Where f(t) is the infiltration rate at time t; A and B are the parameters of the unknown equation represent- ing the infiltration characteristics of the soil, with A the initial measurement of infiltration rate and soil structural condition, and B the stability index of the soil structure, t the time. To determine its parameters A and B, the loga- rithms (ln) of the infiltration measurements f(t) and time (t) were taken. - Modified Kostiakov model Also known as Kostiakov-Lewis or Mezencev it is commonly used in the infiltration function for surface irrigation applications (Haverkamp et al., 1988, Furman et al., 2006). Its equation is as follows: (2.3) Where F(t) is the cumulative infiltration rate as a function of time; A and B are the hydraulic parameters of the equation, t is time, and fc is the stable infiltration rate. As with Kostiakov, the logarithms (ln) of the infiltration f(t) and time (t) measurements were taken to determine the A and B parameters. Acta agriculturae Slovenica, 119/4 – 2023 5 Influence of mulches on soil moisture and water infiltration in the tomato crop 2.6 SELECTION OF THE INFILTRATION MODEL ACCORDING TO PERFORMANCE CRITERIA The comparison between the simulated and meas- ured field data was performed according to the selected performance criteria to determine the best - fitting mod- el in the range. The different criteria selected were based on their popularity and efficiency. These include, among others : - Determination Coefficient (R²) (2.4) - The Mean Square Error (RMSE) (2.5) - Nash and Sutcliffe efficiency coefficient (NASH) (2.6) - Error Index (PBIAS) (2.7) where n is the number of observations, xi is the ob- served infiltration depth, yi is the simulated infiltration depth and is the mean of observed data The best-fitting model was selected based on the maximum of the coefficient of determination R², Nash; the minimum of the root mean square error (RMSE) and the prediction error index (PBIAS), as established by Mo- riasi et al. (2007). 2.7 MEASUREMENT OF MOISTURE AND ELEC- TRICAL CONDUCTIVITY IN THE SOIL The measurement of the amount of water in the soil was carried out throughout the period from the sowing of the tomato plants to the fruiting stage in each of the plots. It was carried out from July to September 2022. In our study, the TDR 150 was used because of its afford- ability, reliability, accuracy and ease of use. What’s spe- cial about this device is that it can measure soil moisture, soil electrical conductivity and soil temperature at the same depth and on the same time scale. For this study, we considered only the first two parameters, because of their importance in irrigation and nutrient management. In the field, its spikes were inserted at 5 cm, 10 cm, 15 cm and 20 cm depths next to the tomato plants in each plot. Measurements were taken daily, three repetitions per plot between 4–8 p.m. and reported on data sheets. The data obtained was then processed in Excel software to produce graphs of the evolution of humidity and elec- trical conductivity at different depths. 3 RESULTS 3.1 PHYSICAL CHARACTERIZATION OF SOIL The granulometric analysis by sedimentometry car- ried out according to the method of Robinson (1949) on five (05) samples, made it possible to obtain the percent- ages of sand (% S), silt (% L), and clay (% A) particles at a depth of 30 cm (Tab. 1). Observation of this table shows that silt is the main matrix of these soils, whose main texture is fine silty. The values of apparent density are between 1.24 and 1.67 g cm-3 with an average of 1.33 g cm-3. The water holding capacity varies from 32 to 38 % depending on soil type, wilting point varies from 14 to 17 %, and field capacity varies from 32 to 38 %. Table 1: Physical analysis of soil samples at 30 cm depth Samples Sand % Clay % Silt % Textural Class Bulk density WP (%) FC (%) SWC (%) Wood chips 41.7 10.3 49 Loam-Silty Loam-Silty sand 1.24 14 35 35 Sawdust 19.9 8.5 71.5 Silty loam 1.3 17 38 38 Wheat straw 38.1 11 52 Silty loam-loam 1.24 15 36 36 Mixture 21.9 7 70.5 Silty loam 1.18 17 38 38 Control 17.5 6 76.3 Silty loam 1.67 17 38 38 Note: WP = wilting point, FC = field capacity and SWC = Soil water capacity. The value of WP, FC and SWC were given by the company responsible for the TDR 150 device and the granulometric analysis was performed at the ENSH pedology laboratory Acta agriculturae Slovenica, 119/4 – 20236 I. TANKEUOO KOPA et al. 3.2 COMPARISON OF INFILTRATION RATES Figure 3 shows the evolution of the infiltration rate observed in the field in each mulch and in the control before sowing, during the growth and after the ripening of the tomato plants. The general appreciation of these graphs highlights the rapidity of water infiltration into the soil during plant growth in all mulches and bare soil (control). However, a closer look at the sawdust graph shows that the infiltration rate is slightly higher after the fruiting phase. In short, it evolves through the different phases. Based on this observation, we can deduce that this mulch improves the humidity of the soil over time, unlike other mulches. In fact, after the growth and fruiting phase, the soil water infiltration behavior of other mulches tends to be similar to that obtained during the pre-sowing phase of tomato plants. This behavior is best illustrated in the mixture. With this in mind, factors such as soil plow- ing, mulch decomposition and root system development need to be taken into consideration to explain the evolu- tion of the humidity. 3.3 SELECTION OF THE BEST MODEL FOR THE STUDY SITE The identification of the best infiltration model (Table 2) followed the determination of the hydraulic parameters (Table 2a) and the evaluation of the perfor- mance criteria (Table 2b). 3.3.1 Determination of the hydraulic parameters of each model in each mulch before sowing, dur- ing growth and after fruiting of tomato plants Table 2a shows the hydraulic parameters of each model for each mulch before sowing, during growth, and after maturation of the tomato crop. From the annota- tion of these dashboards, it can be seen that high simu- lated hydraulic conductivity values are recorded for each model in all three phases during the plant development phase. The mixture also records very high values during this phase. 3.3.2 Evaluation of performance criteria for Philip, Kostiakov and modified Kostiakov models Table 2b shows the different performance criteria used to determine the best model under each mulch in the study area. Looking at the values obtained from the different performance criteria for different models in each mulch, we note that the modified Kostiakov is the best model in the study area. We also note that the Philip model has the lowest PBIAS value, but unlike Kostiakov, very high RMSE values were found. Overall, these three models are applicable in the area, although modified Ko- stiakov is the best of the three. 3.4 EFFECT OF MULCH ON SOIL ELECTRICAL CONDUCTIVITY Daily, soil electrical conductivity data were re- corded in each mulch at different depths (Fig. 4). From the general shape of these curves, it can be seen that the highest conductivity values are recorded at 10 cm depth and lower at 5 cm depth at each mulch. However, a dif- ference is observed first in wood chip and sawdust be- tween 08/22/2022 - 01/09/2022, then in control between 16/09/2022 - 26/09/2022, where there is a high peak at 5 cm depth, unlike the rest of the depths. The individual observation of each figure shows that high conductivity rates are recorded within: - treatments (wood chips, sawdust, straw, and mix- ture) between 08/22/2022-01/09/2022 and between 11/09/2022-21/09/2022 - bare soil (control) between 27/08/2022-06/09/2022 and between 16/09/2022-26/09/2022 The comparative study of the variation of the elec- trical conductivity of the soil shows overall very high rates of conductivity between 17/08/2022-06/09/2022. The overview of Table 3 notices a slightly more pro- nounced increase in conductivity at 10 cm depth (0.44 µS cm-1). The following classification can be made: 10 cm (0.44 µS cm-1) > 15 cm (0.42 µS cm-1) > 20 cm (0.39 µS cm-1) > 5 cm (0.27 µS cm-1. Also, a more pronounced in- crease in conductivity at the level of wood chips (0.41 µS cm-1), followed by sawdust (0.37 µS cm-1), straw (0.37 µS cm-1), control (0.38 µS cm-1), mixture (0.36 µS cm-1). The difference between the mulches and the control varied from 0.1 to 0.3 µS cm-1. The standard deviation values for the different mulches at each depth are less than 3 %. The very low deviations indicate that the data points are close to the mean. Showing that the data are homogeneous. 3.5 EFFECT OF MULCH ON SOIL MOISTURE An overview of the various graphs at 5 cm, 10 cm, 15 cm and 20 cm depth (Fig.5) in each mulch shows that sawdust has a high water content at almost all soil depths, Acta agriculturae Slovenica, 119/4 – 2023 7 Influence of mulches on soil moisture and water infiltration in the tomato crop Figure 3: Comparison of infiltration rate (mm h-1) observed before seeding tomato plants (blue color), during plant growth (or- ange color) and after fruiting tomatoes (green color) in the wood chip, sawdust, wheat straw, mixture and control Table 2a: Hydraulic parameters of different models before sowing tomato plants, during tomato plant growth and after maturation of tomato plants Plot Period P K KM     S (mm h-1) K (mm h-1) M A (mm h-1) B M A (mm h-1) B M Wood chips Before 9.27 80.07 87.65 82.47 0.07 87.65 0.63 0.85 1.46 During 61.73 87.12 146.03 106.87 0.26 146.03 4.19 0.64 2.43 After 21.35 102.16 120.77 108.34 0.11 120.77 1.41 0.75 2.01 Sawdust Before 53.13 105.47 153.45 124.22 0.2 153.45 3.68 0.64 2.55 During 53.54 96.93 155.34 111.34 0.23 155.34 3.59 0.63 2.58 After 60.81 136.7 204.57 148.65 0.21 204.57 3.98 0.66 3.4 Wheat straw Before 47.9 68.78 108.4 87.94 0.24 108.4 3.39 0.62 1.8 During 54.62 103.57 155.69 122.01 0.21 155.69 3.81 0.62 2.59 After 28.73 97.16 122.38 105.96 0.15 122.38 1.93 0.7 2.03 Mixture Before 41.03 64.75 104.57 82.83 0.19 104.57 3.18 0.48 1.74 During 44.02 164.69 214.4 173.55 0.14 214.4 2.95 0.67 3.57 After 25.05 75.54 98.52 85.52 0.13 98.52 1.84 0.57 1.64 Control Before 59.07 69.5 122.46 93.11 0.26 122.46 4.23 0.58 2.04 During 59.07 69.5 122.46 93.11 0.26 122.46 4.23 0.58 2.04 After 24.83 18.38 34.56 31.05 0.27 34.56 2.11 0.5 0.57 Min   0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 Max   0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 Mean   42.94 89.35 130.08 103.80 0.20 130.08 3.01 0.63 2.16 Note. S= Sorptivity (mm h-1); K= parameter related to saturated hydraulic conductivity (mm h-1); A and B = hydraulics parameters with A measure- ment initial infiltration (mm h-1) and B is index evaluated stability; P= Philip model; K= Kostiakov model; KM= Modified Kostiakov model Acta agriculturae Slovenica, 119/4 – 20238 I. TANKEUOO KOPA et al. Ta bl e 2b : P er fo rm an ce cr ite ria a nd se le ct io n of th e be st m od el in e ac h m ul ch (c hi p, sa w du st , w he at st ra w, m ix tu re ) a nd co nt ro l Pl ot So il Te xt ur e Pe rio d R² N A SH RM SE PB IA IS Be st m od el       P K K M P K K M P K K M P K K M   W oo d ch ip s L- Ls Be fo re 0. 48 0. 70 0. 83 0. 48 0. 69 0. 99 5. 27 4. 03 1. 03 0. 00 8. 33 -1 3. 33 M od ifi ed Ko st ia ko v D ur in g 0. 90 0. 92 0. 96 0. 90 0. 94 0. 99 12 .2 7 8. 96 2. 21 0. 00 3. 92 -1 9. 35 A fte r 0. 70 0. 81 0. 94 0. 70 0. 82 0. 99 7. 82 6. 07 1. 24 0. 00 12 .4 7 -9 .5 7 Sa w du st lf Be fo re 0. 90 0. 91 0. 98 0. 90 0. 94 0. 99 9. 83 7. 82 1. 61 0. 00 15 .4 2 -1 2. 91 M od ifi ed Ko st ia ko v D ur in g 0. 91 0. 90 0. 98 0. 91 0. 93 0. 99 10 .5 5 9. 36 1. 11 0. 00 23 .2 8 -7 .2 3 A fte r 0. 87 0. 91 0. 96 0. 87 0. 92 0. 99 15 .0 5 11 .6 3 1. 75 0. 00 17 .5 3 -1 3. 29 W he at st ra w lf Be fo re 0. 91 0. 94 0. 97 0. 91 0. 96 0. 99 7. 88 5. 39 1. 93 0. 00 3. 36 -1 7. 24 M od ifi ed Ko st ia ko v D ur in g 0. 92 0. 92 0. 98 0. 92 0. 94 0. 99 9. 61 7. 85 1. 37 0. 00 17 .3 5 -1 2. 69 A fte r 0. 81 0. 91 0. 94 0. 81 0. 92 0. 99 7. 95 5. 01 1. 45 0. 00 5. 80 -1 6. 70 M ix tu re lf Be fo re 0. 94 0. 86 0. 99 0. 94 0. 84 0. 99 5. 72 10 .1 3 0. 28 0. 00 45 .4 5 -2 .0 1 M od ifi ed Ko st ia ko v D ur in g 0. 79 0. 79 0. 98 0. 79 0. 79 0. 99 14 .4 2 14 .5 1 0. 82 0. 00 42 .2 3 -0 .3 0 A fte r 0. 95 0. 90 0. 99 0. 95 0. 87 0. 99 3. 24 5. 27 0. 30 0. 00 12 .6 6 -2 .0 4 C on tr ol lf Be fo re 0. 95 0. 95 0. 98 0. 95 0. 96 0. 99 7. 69 6. 96 1. 63 0. 00 12 .2 2 -1 2. 06 M od ifi ed Ko st ia ko v D ur in g 0. 95 0. 95 0. 98 0. 95 0. 96 0. 99 7. 69 6. 96 1. 63 0. 00 12 .2 2 -1 2. 06 A fte r 0. 96 0. 97 0. 98 0. 96 0. 94 0. 99 2. 17 2. 88 1. 06 0. 00 7. 27 -6 .6 0 M ea n     0. 86 0. 89 0. 96 0. 86 0. 89 0. 99 8. 48 7. 52 1. 30 0. 00 15 .9 7 -1 0. 49   M in     0. 48 0. 70 0. 83 0. 48 0. 69 0. 99 2. 17 2. 88 0. 28 0. 00 3. 36 -1 9. 35   M ax     0. 96 0. 97 0. 99 0. 96 0. 96 0. 99 15 .0 5 14 .5 1 2. 21 0. 00 45 .4 5 -0 .3 0   N ot e. P = Ph ili p m od el ; K = K os tia ko v m od el ; K M = M od ifi ed K os tia ko v m od el Acta agriculturae Slovenica, 119/4 – 2023 9 Influence of mulches on soil moisture and water infiltration in the tomato crop while wood chips have a lower water content. However, at a depth of 20 cm, the opposite phenomenon is ob- served. Wood chips retain more moisture than sawdust. The moisture data (Fig. 5) obtained on each mulch before and after irrigation allowed us to establish an existing relationship between the parameters: irriga- tion, moisture, mulch and actual evapotranspiration. The evapotranspiration calculated with the Pennan- Montheih formula was corrected with the crop coef- ficients of tomato to have plot - specific values. After irrigation under different mulches, the observation is mentioned in the level: - of the wood chips of great moisture conserva- tion in the soil between the end of August - beginning of September (25/08/2022-09/09/2022), that is 15 days of maximum retention. The observation of the graph shows a particular retention of only three days, justified by a strong increase in real evapotranspiration during this pe- riod. In addition, nine irrigation cycles were carried out during the crop. - of sawdust of a great retention of moisture in the soil of maximum 13 days at the beginning of the growth and flowering stage of the plant (11/07/2022-04/08/2022). Observation of this graph also identifies eight rounds of irrigation during the development of the plant. - of the straw, the great retention was identified Table 3: Averages and standard deviations of soil conductivity retained for different mulches (wood chips, sawdust, wheat straw, mixture) and control at 5 cm, 10 cm, 15 cm and 20 cm depths Mulches Averages and standard deviations of soil conductivity (µS cm-1) 5 cm 10 cm 15 cm 20 cm Mean (%) SD (%) Wood chips 0.24 0.5 0.46 0.43 0.41 0.10 Sawdust 0.32 0.41 0.38 0.37 0.37 0.03 Wheat straw 0.26 0.48 0.41 0.34 0.37 0.08 Mixture 0.2 0.39 0.43 0.43 0.36 0.10 Control 0.31 0.44 0.44 0.35 0.38 0.06 Mean (%) 0.27 0.44 0.42 0.39 SD (%) 0.04 0.04 0.03 0.04 Note: SD = standard deviation, Mean = average of soil conductivity in different mulches and depths Figure 4: Variation in soil conductivity (µS cm-1) each daily of wood chips (blue color), sawdust (orange color), wheat straw (gray color), mixture (yellow color) and control (green color) at 5 cm, 10 cm, 15 cm and 20 cm depth Acta agriculturae Slovenica, 119/4 – 202310 I. TANKEUOO KOPA et al. in the second cycle of the plant between 23/07/2022- 01/08/2022 which is a maximum of 13 days of conserva- tion. During the growth of the plant, the observation of the graph shows eight rounds of irrigation as the sawdust. - of the mixture, conservation of humidity in the soil between 20/07/2022-10/08/2022 that is to say a max- imum of 22 days. On the graph, we can also see that dur- ing the period of growth of the plant, seven rounds of irrigation were carried out. - of the control, a maximum of 13 days between 07/29/2022-10/08/2022. Also on the graph, we see that during the growth the plants were irrigated 10 times. Considering the average moisture content data in Table 4, the following classification by the average mois- ture content obtained in percentages is sawdust (35.6 %) > straw (34.3 %) > mixture (33.9 %) > control (33.8 %) > wood chips (33.7 %). We also note that 15 cm (39.7 %) is the depth at which we record a very high rate of water storage in the soil. In the same table, we observe a slightly significant difference in low water retention in wheat Figure 5: Effects of wood chips, sawdust, wheat straw, mixture, control on moisture at 5 cm; 10 cm; 15 cm and 20 cm depth each daily; relationship between soil moisture (%), irrigation (mm) and reel evapotranspiration (ETR) in mm/day to different mulches and control. The vertical bars represent plant growth stages; the horizontal bars represent the number of days (d) of moisture re- tention in the soil after each irrigation round and the small histograms represents the number of irrigated turns for each treatment Table 4: Averages, and standard deviations soil moisture retained for different treatments (wood chips, sawdust, wheat straw, mixture and control) at 5cm, 10cm, 15cm and 20cm depths Mulches Averages and standard deviation of soil moisture (%) Depths 5 cm 10 cm 15 cm 20 cm Mean (%) SD (%) Wood chips 29.6 31.6 39 34.5 33.7 3.5 Sawdust 34.3 34.5 40.4 33.1 35.6 2.8 Wheat straw 32.4 32.5 40.3 32.1 34.3 3.5 Mixture 29.3 31.5 39.6 34.9 33.9 3.9 Control 31.6 31.4 39.4 33 33.8 3.3 Mean (%) 31.4 32.3 39.7 33.5 SD (%) 1.8 1.2 0.5 1.0 Note: SD = standard deviation, Mean = average of soil moisture in different mulches and depths Acta agriculturae Slovenica, 119/4 – 2023 11 Influence of mulches on soil moisture and water infiltration in the tomato crop straw and the control; being of the order of 0.3 % (be- tween 20 cm and 5 cm depth) and 0.2 % (between 10 cm and 5 cm depth), respectively. Similarly, indicating the homogeneity of the data, the standard deviation values are low (less than 8 %). If we make a correlation between the averages of the values of electrical conductivity of the soil and humidity at different depths in different mulches (Tab.5), we obtain good results of coefficient of determination going from 0.70 to 0.95 in almost all the depths. 4 DISCUSSION The results of the particle size analysis showed that silt is the main matrix of the soils of the different plots, whose main texture is fine silt. These results are consist- ent with those obtained by Ecrement and Seghir (1971) in the region. According to Skhiri (2019), the average bulk density within the silty soils is 1.3 g cm-3. This result is consistent with that obtained in the study area, which is 1.33 g cm-3. The general observation of the graphs showing the evolution of the infiltration rate observed in the field in each mulch and bare soil, before sowing, during the growth and after the ripening of the tomato plants, allows us to see that the infiltration of water into the soil is more rapid during the period of plant growth. This phenomenon is observed in all the mulches and in the bare soil (control). This can be explained, on the one hand, by the mulching of the soil surface and, on the other hand, by the plowing of the soil before sowing, in addition to the root development of the plant during this period. Several researchers (Liao et al., 2021, Farid et al., 2019) indicate that mulching would facilitate infiltration after irrigation or very heavy rainfall. The hydraulic pa- rameters of each model observed in each mulch before sowing, during growth and after ripening of the tomato crop show that high values of simulated hydraulic con- ductivity are recorded for each model during the three phases of plant development. The mixture, which is the mulch, records the highest values. Some researchers at- tribute this increase to the mulching of the soil surface. They consider that mulching improves, if not increases, the rate of hydraulic conductivity in the soil (Mkhabela et al., 2019; Stelli et al., 2018, Simsek et al., 2017). As for the high values of the mixture, according to Bear (1972), they are justified by the very high ability of the soil under this mulch to let water through, unlike other mulches. Based on the evaluation of the selected performance criteria, the modified Kostiakov emerged as the best model in the study area. According to several researchers (Niyazi et al., 2022; Vand et al., 2018; Yuemei et al., 2008), the best model is the one with minimum RMSE and PBIAS, maximum R² and NASH. In our study, modified Kostia- kov is the best because it has: R² equal to 0.996; Nash equal to 0.99997; RMSE equal to 0.279 and PBIAS equal to -19.35. Overall, these three models are applicable in the field, with the best being the modified Kostiakov. This result is similar to that obtained by Smerdon et al. (1988). These researchers have shown that modified Kostiakov is one of the best models to apply in surface irrigation. Thus, from these performance criteria, we can establish the following ranking: modified Kostiakov > Kostiakov≥ Philip. From the research conducted by Zolfaghari et al. (2012) on seven infiltration models, it is found that mod- ified Kostiakov and Kostiakov are the two models with better ranking among all models. Mirzaee et al., (2014) on the one hand state that modified Kostiakov is the best model for fine silty loam soils and on the other hand it is among the best models suitable for loam, silty clay loam and clay loam soils. About the graphs illustrated con- cerning the simulation of the infiltration rate and the cu- mulative infiltration, we note that the best simulation is within the soil mulched with the mixture. Likewise, fol- lowing the values of NASH and R² obtained at the level of the mixture, after the stage of maturation of the tomato, we identify very good values and therefore very good simulations, contrary to the first two stages. The plausible explanation is the presence of the mulch, because accord- ing to some researchers (Simsek et al., 2017; Zhang et al., 2014), mulching improves infiltration into the soil. As well, as the colored presence of roots in the soil coupled with the soil texture. The average electrical conductivity values obtained in the soil are, on the whole, very low. Let us remember that electrical conductivity is a very important parameter in agriculture. It is mainly used to determine soil salinity, but it can also be used to estimate other soil properties (soil water content, soil temperature, soil pH, soil tex- ture, organic matter, solution ion concentration, etc.) at the non-saline soil level (Cornwin and Lesh, 2005). Ac- Table 5: Correlation between average soil electrical conduc- tivity and soil moisture values at 5cm, 10 cm, 15 cm, 20 cm depths for different treatments 5 cm 10 cm 15 cm 20 cm Average humidity (%) 31.40 32.30 39.70 33.50 Average conductivity (µS cm-1) 0.27 0.44 0.42 0.39 R² Conductivity - Humidity 0.70 0.73 0.91 0.95 Note: R² = coefficient of determination Acta agriculturae Slovenica, 119/4 – 202312 I. TANKEUOO KOPA et al. cording to the range suggested by the NRCS Soil Survey Handbook, we begin to have saline soil when the con- ductivity value is higher than 2 dS m-1 and non-saline soil when the conductivity value is lower than 2 dS m-1. The values obtained in our study are well below the range sug- gested by the latter for non-saline soils. This is an assur- ance that the soil quality is ideal for agriculture. Also, a more pronounced increase in conductivity was observed at the level of wood chips (0.41 µS cm-1), followed by saw- dust (0.37 µS cm-1), straw (0.37 µS cm-1), control (0.38 µS cm-1), mixture (0.36 µS cm-1). Some authors (Simsek et al., 2017) have shown that mulching does not affect pH, electrical conductivity, bulk density and carbonate content in the soil. On the other hand, others have shown that all types of mulches have a much more positive effect because they maximally reduce the electrical conductiv- ity within the soil properties (Kumar et al., 2012). Ac- cording to our results, we notice that the electrical con- ductivity increases in almost all mulches, except at the level of the mixture, where it decreases compared to the control. The low value recorded at the level of the mix- ture compared to the control is indeed consistent with the results of the authors mentioned above. On the other hand, the opposite phenomenon (increase in conductiv- ity) observed in the other mulches may be due, accord- ing to some researchers (Sadek et al., 2019, Pakdel et al., 2013, Chalker-Scott, 2007), to the decomposition of the organic mulch under the effect of appropriate nutrients released in the soil, which become available to plants. But also to the texture and other properties of the soil, which unfortunately we could not study. For example, the level of phosphorus in the soil, as pointed out by Donogemma et al. (2008). The peaks in conductivity observed between 17/08/2022-06/09/2022 could be due to soil moisture. According to the work of (Costa et al., 2014), electrical conductivity is strongly influenced by soil moisture. This is similar to the result obtained in our study during this period. We found during the same period, an increase in soil moisture. The increase in conductivity that is slightly more pronounced at 10 cm depth may be due to the root development of the plants and the texture of the soil. Synthesizing the moisture data on graphs of each mulch at different depths, it is very clear that the mixture retains more moisture than the rest of the mulch and that the ideal retention depth is 15 cm. According to Dinu- shika et al. (2019) and Adams (1996), in arid or semi-arid zones, mulching would increase water retention in the soil from 0-40 cm depth. However, Stagnari et al. (2014) state that in a Mediterranean environment, mulching in general and straw in particular would increase the water retention in the soil from 5-15cm depth. According to an experiment conducted on different organic and inorgan- ic mulches (Safari et al., 2021), the ideal depth is 15 cm with an increase varying from 2 to 5 % compared to the control on bare soil. The ideal depth chosen in our study is within the range suggested by the above authors. How- ever, the rate recorded by Safari et al. (2021) is mainly consistent with sawdust. Kumar and Dey, (2011) remind us that the use of mulches on the soil surface would in- crease water diffusion under the vapor gradient during the growing season. They added by saying that this fac- tor would increase the maximum water absorption under mulch. Next, we note that the mixture was irrigated less than the other mulches. This justifies its low percent- age difference in soil water storage (2.3 %) compared to sawdust. Finally, we see that the mixture conserved a lot of soil moisture in the first two cycles. Over time, how- ever, it tends to have the same characteristics as straw in particular. The plausible explanation for this is that the wood chips and sawdust mulch decomposed faster over time than the straw. The work of Kaboneka et al. (2021) and Boyer (2021) emphasizes the fact that these mulches have in common a very slow decomposition. In fact, according to the studies carried out in Burundi by Kaboneka et al. (2021) on the decomposition of wheat straw, it appears that this mulch is characterized by a predominance of substances resistant to decomposition (cellulose, hemicellulose, lignin) and by a very high ratio of carbon to nitrogen (C/N) (76.4), that is, three times higher than the standard (25) ideal for the mineralization of nitrogen and the rapid release of nutrients contained in organic materials. Likewise, the study by Boyer (2021), conducted in Quebec on the bibliographic research of the potential use of organic mulches, showed that the C/N ratio of wood chips is about 39.2; that is, slightly high- er than the standard set. Furthermore, Nicolardot et al. (2001) point out that this ratio is considered to be the simplest biochemical indicator of the quality of organic matter, its decomposability and nutrient release poten- tial. According to Kaboneka et al. (2021), its value indi- cates whether an organic substrate is rapidly or slowly decomposable. Thus, comparing the ratios of the values obtained by their authors shows that wheat straw does indeed take longer to start decomposing compared to wood. This result supports the one obtained in the study area. Furthermore, the straw was the mulch that retained the least amount of water in the soil during the period 13/08/2022-16/08/2022. This can be explained by the peak of water demand observed during the same period. Thus, at the level of mulches, the effective classification is as follows: mixture (07), sawdust (08), straw (08), wood chips (09) and control (10). The fact that the number of irrigated turns is lower for the mulch than for the bare soil control allows us to say that the mulch used allows to reduce the irrigation frequency. This result is in line with the researches carried out by some scientists (Ahmad et Acta agriculturae Slovenica, 119/4 – 2023 13 Influence of mulches on soil moisture and water infiltration in the tomato crop al., 2020, Chalker-Scott, 2007, Rasmussen, 1999). The lat- ter emphasize the fact that the application of mulch can significantly reduce the frequency of irrigation and even eliminate the need for irrigation. The choice of mulch is crucial in that some will reduce irrigation more than oth- ers, as we observed with the mulches used in the study. During the period from 10/08/2022 to 12/09/2022, when real evapotranspiration was high (marked by two peaks, one on 13/08/2022 and the other on 09/09/2022), we find that the mulched soils were more resistant than the bare soil control because they were less irrigated. This result is consistent with that of Duchaufour et al. (2017), who mention that under the effect of mulching, soil evapo- transpiration is reduced. Overall, it is noted that the mulches used retain more moisture in the soil than the control. This result is consistent with the research of Mkhabela et al. (2019) and Telkar et al. (2017), who in- dicated that soil moisture is retained under mulch. They also indicated that the percentage of water stored in the soil is higher under mulch than under bare soil. However, in our study, this result is not entirely true for the wood chips, which is an exception to the rule. In fact, the differ- ence between the control and the wood chips is slightly higher than that of the wood chips, i.e. 0.1 %. This differ- ence can be explained by the short duration of the study, but also by the nature of the soil. In fact, the wood chips, which are more enriched with sand, retain less moisture due to their texture. Also, since the difference between the two soils is very small, we can take into account the time of the study. Considering the differences that ex- ist between the wood chips and the sawdust in terms of moisture retention and the number of watering cycles, we can explain them by the grinding of the wood. The finer the wood is ground, the more water it retains in the soil. About the availability of the mulches used in the study, it should be noted that wood waste is widely avail- able in Algeria. Indeed, (Irinslimane, 2007), points out that its recycled waste amounts to 7 to 8 million tons per year, and comes mainly from forestry operations and sawmills. As for (Chachoua, 2015), cereal growing ac- counts for more than 50 % of useful agricultural areas. The figures given by these researchers reassure us that sufficient water is being used for large areas of irrigation fields in Algeria. The correlation between the average values of soil electrical conductivity and soil moisture at different depths in different mulches gave good results with co- efficients of determination ranging from 0.70 to 0.95 in almost all depths. The strong correlation observed re- flects the influence of soil moisture on conductivity. This result is in agreement with the one obtained by Costa et al. (2014) in Brazil. 5 CONCLUSION In addition to climate change, the overuse of water, chemical fertilizers and pesticides in the cultivation of to- matoes in Algeria has become almost commonplace, in the quest for extreme profitability. Our study is intended to be useful in the sense that, in addition to conserving soil moisture, it can contribute to soil restructuring, as well as enrich the soil with the organic matter and nutri- ents plants need to grow and produce good yields. In ad- dition, knowledge of the soil’s infiltration rate and char- acteristics enables better irrigation management. For scientists and researchers, this study contrib- utes to a better understanding of organic mulch and soil moisture in crops. As for legislators, we encourage them to set up a policy to raise awareness among individuals, farmers and water managers of the advantages of organic mulch in agriculture. Through this study, which aims to be simple in its application, economical and save time and energy, we en- courage the above stakeholders to increasingly practice the use of organic mulch in their various crops. This lim- its weeds, preserves the environment, reduces irrigation requirements, improves water infiltration and conserves soil moisture. What’s more, once decomposed, they form compost, ideal for plant growth. This reduces the need for nitrogen fertilizers. However, if the organic mulch chosen has a positive effect on the various aspects mentioned above, it should be noted that the questions of application density, effect on the growth of tomato crops, and agricultural yield de- pending on the mulch chosen remain unknown. As is the question of soil temperature and organic matter content. We are currently working on these questions to gain a better understanding them. 6 ACKNOWLEDGMENTS The study was carried out at the Water and Envi- ronmental Engineering Laboratory (GEE) of the High- er National School of Hydraulics (ENSH) of Blida. 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Acta agriculturae Slovenica, 119/4, 1–7, Ljubljana 2023 doi:10.14720/aas.2023.119.4.15939 Original research article / izvirni znanstveni članek Effect of the alcohol content on sensory perception of the fruit spirits Arman ŠEKERIĆ 1, Milenko BLESIĆ 1, Pakeza DRKENDA 1, Nermina SPAHO 1, 2 Received August 31, 2023; accepted November 22, 2023. Delo je prispelo 31. avgusta 2023, sprejeto 22. novembra 2023 1 University of Sarajevo, Faculty of Agriculture and Food Sciences, Sarajevo, Bosnia and Herzegovina 2 Corresponding author, e-mail: n.spaho@ppf.unsa.ba Effect of the alcohol content on sensory perception of the fruit spirits Abstract: Fruit spirits must have an aroma of the raw ma- terial, which is balanced by ethanol. Since many aroma com- pounds are more soluble in ethanol than in water, ethanol is the most important carrier of aroma compounds. The alcohol con- centration seems to be crucial for the sensory profile of spirits. Alcohol content of 40% vol is the standard alcoholic strength of fruit spirits. Regulations specify a minimum alcohol content of 37.5% vol. However, ethanol reduction can result in change in sensory profile of spirits. The aim of this research is to deter- mine whether lowering the alcohol content of spirits may make them less acceptable to customers. On this occasion, 5 pairs of fruit spirits were sensory tested: pear, plum, apple, raspberry, and grape spirits, each with a commercial and reduced alcohol concentration to 37.5% vol. The results showed that customers can recognize the difference in alcohol content of fruit spirits and dilution to lower alcohol content led to decreasing aroma for all tastes fruit spirits. However, typicality and intensity of fruit odour and the overall note of the spirits, were very similar perceived for Williams, plum and grape spirits whereas apple and raspberry spirits showed better characteristic at higher al- cohol content. Key words: fruit spirits, alcohol content, aroma, sensory perception Učinek vsebnosti alkohola na senzorično zaznavanje žganih pijač Izvleček: Čeprav sta voda in etanol glavni sestavini sa- dnih žganih pijač, ne določata njihove narave in zvrsti, niti nji- hove senzorične kakovosti. Vzrok za to je prisotnost številnih aromatskih sestavin. Sadne žgane pijače morajo imeti aromo njihovih izvirnih surovin, ki je uravnavana z etanolom. Glede na to, da je veliko aromatičnih sestavin bolj topnih v etanolu kot v vodi, je etanol najbolj pomemben nosilec aromatskih spojin. Koncentracija alkohola je odločilna za senzorični pro- fil žganih pijač. Za konzumiranje je standardna alkoholna moč žganih sadnih pijač 40 volumskih odstotkov. Predpisi določa- jo minimalno vsebnost etanola kot 37,5 volumskih odstotkov, kar uporabljajo mnoge distilerije pri proizvodnji žganih pijač. Namen te raziskave je bil ugotoviti, če lahko takšno zmanjša- nje v vsebnosti alkohola potencialno zmanjša sprejemljivost žganih pijač pri potrošnikih. V ta namen je bilo preiskušenih 5 parov žganih sadnih pijač in sicer hruškovo, slivovo, jabolčno, malinovo in grozdno žganje, v vseh primerih s komercialno in na 37,5 % vol. zmanjšano koncentracijo alkohola. Rezultati so pokazali, da uživalci zaznavajo razliko v vsebnosti alkohola v žganih sadnih pijačah, ker razredčenje na manjšo vsebnost al- kohola zmanjša aromo in okus vseh preiskušenih žganih pijač. Kljub temu so značilen vonj, okus kot splošne značilnosti bili podobno zaznani pri žganju iz hrušk viljamovk, sliv in grozdja, med tem, ko sta žganji iz jabolk in malin imeli boljše lastnosti pri večjih vsebnostih alkohola. Ključne besede: sadne žgane pijače, vsebnost alkohola, aroma, senzorično zaznavanje Acta agriculturae Slovenica, 119/4 – 20232 A. ŠEKERIĆ et al. 1 INTRODUCTION Spirits are alcoholic beverages produced by three successive processes: fermentation, distillation and alter- native ageing in wood barrel. During the production of fruit spirits, numerous volatile substances are produced. Fruits are the source of the so-called primary aroma of spirits, which are the components that give the beverage identity and uniqueness for a particular fruit species or variety (Januszek et al., 2020; Spaho et al., 2023). Most volatiles are produced during alcoholic fermentation (St- acner et al., 2023), creating a fermentative or secondary aroma of spirits. Distillation is a process that controls the alcohol concentration and the composition of volatile compounds in distillates. It is enabled by fraction cutting (Spaho et al., 2013; Xiang et al., 2020), thermal energy input and reflux rates (Heller & Einfalt, 2022), and is strongly influenced by the type and design of distillation apparatus used (Balcerek et al., 2017; Rodríguez-Solana et al., 2018; Hodel et al., 2021). During distillation, alco- hol and water are the actual carriers of hundreds of vola- tile compounds contained in the initial fermented mash. The quantity and quality of these volatile compounds in the vapor depend on their boiling point, their better solubility in water or ethanol, and the variation of etha- nol content during distillation. The ethanol is enhanced and refined during the distillation process. Balcerek et al. (2017) and Xiang et al. (2020) demonstrated that increas- ing the final alcohol concentration in the heart fraction resulted in lower amounts of main volatile components in the distillates. Wie et al. (2018) showed that ester spe- cies and amounts increased significantly with increasing alcohol concentration in the heart fraction, while acid- ity decreased. During distillation heart fraction separate from head and tail fractions because these fractions are responsible for negative aroma attributes. With head fraction the majority of acetaldehyde, ethyl acetate, ac- etone are removed. Those compounds give sharp and unpleasant flavour. The tail fraction contains, acetic ac- ids and fusel oils, such as propyl, butyl and amyl alcohols and their isomers, that are associated with unpleasant aroma attributes (Bohn et al., 2022).The maximum alco- hol content in the middle (heart) cut of fruit distillates af- ter distillation might be 86% vol, although in practice it is usually around 65-75% vol, depending on the distillation apparatus used (Durr, 2010; Lukić et al., 2011; Esteban- Decloux, et al., 2021; Tian-Tian et al., 2022; Lončarić et al., 2022). After distillation is completed, the fresh high- proof distillate has to storage for a period of time to har- monize. The concentration of alcohol in the heart frac- tion is especially important for the aging process, since the extraction of wood components, the clarity of the distillates, and the volatile compounds strongly depend on the alcohol content of the distillates (Różański et al., 2020; Valcarcel-Munoz et al., 2022; Butron et al., 2023). A spirit straight from the still is not palatable, so it must rest for at least three months. The fresh distillates have a high alcohol concentration, and the sharpness of the alcohol affects the sensory perception of the usual fruit aromas. They also contain a large number of alde- hydes, even if they have been properly separated before- hand, which, due to their stale and pungent smell and taste, lead to an inharmonious, unripe overall impres- sion. The aroma-determining esters, which mostly form during storage of the distillate, are also missing (Scholt- ten, 1999). Consequently, the distillate must be diluted un- til bottling. The greater the dilution of the alcohol with distilled or demineralized water, the fewer odour com- ponents a spirit has. Today, alcohol concentrations of 40 to 45% vol. are common. This is the alcohol content to which consumers are accustomed. However, the EU Regulation (No. 2019/787) for fruit and wine spirits stip- ulates a minimum alcohol content of 37.5% vol. These regulations allow distilleries to offer spirits with a lower alcohol content than usual. For the industry, this means an increase in sales value, as the addition of water to di- lute spirits is commonly regarded as a means of stretch- ing production volumes. If finished spirit is diluted from 40 to 37.5% alcohol by volume, this means that 6.7 litres of distilling water were added to 100 litres of 40% vol al- cohol distillates. It means 6.7 more litres of beverages for the industry. This is added value for industry, but the question is: Is it acceptable to consumer? Do consumers perceive the alcohol reduction in fruit spirits and do they welcome the sensory changes caused by this reduction? Currently, there is limited data on the effects of alcohol reduction on the perceived sensory quality of these spirits and their appeal to consumer. Therefore, this study examined the impact of alcohol reduction in fruit spirits on consumer′ perceptions and potentially reduction of their acceptabil- ity of spirits with low alcohol content. 2 MATERIALS AND METHODS 2.1 MATERIALS This study evaluates five spirits produced from pear Williams, plum, apple, raspberry, and grape spirits. The spirits were purchased from various producers on the market. With the exception of raspberry spirits, these spirits were selected for their distinct aroma and popu- larity among customer from the West Balkan (Mrvcic et al., 2021). Each original bottled spirit with declared alco- Acta agriculturae Slovenica, 119/4 – 2023 3 Effect of the alcohol content on sensory perception of the fruit spirits hol content served as a control, and the corresponding sample was prepared by reducing the alcohol concentra- tion to 37.5% vol. Table 1 shows the alcohol content of the samples. 2.2 METHODS 2.2.1 Sensory analysis Sensory analysis was performed by a consumer panel. Consumer panel members were recruited through online and in-person surveys. After 72 people were surveyed, a group of 30 individuals was selected based on their past experience with consuming spirits. They claimed to be moderate drinkers who believed they un- derstand the range of quality of spirits. The panel con- sisted of 80% men and 20% women between the ages of 20 and 60. All samples were sensory analysed using three sen- sory tests: paired comparison difference test, paired pref- erence test and descriptive test (Stone and Sidel, 2004). Sensory analysis was performed in two separate sessions. In the first session, assessors are used the paired compari- son difference test and the paired preference test, and in the second session, the descriptive test. The two coded products of each fruit spirit (con- trol and reduced alc. sample) are served for the test of differences. In the directional test, the two presentation orders are AA, BB, AB, BA, where A is the control and B is the sample with reduced alcohol concentration. The paired samples are served simultaneously, and the indi- vidual is asked if “there is a difference.” Each assessors received a set of five pairs of samples (Fig. 1). They have taken a break between evaluation of each single paired. The order of the spirit series was randomised. The asses- sors were asked if “there is a difference”. If they notice a difference, they must choose more desired (preferred) samples. After a one-hour break, the assessors evaluate the samples by a descriptive test. Prior to the analysis, the as- sessors received a brief training in the evaluation of spir- its as well as insight into the sensory attributes of spirits. The five sensory attributes were evaluated: typicality of odour and intensity of fruit odour, aroma, mouthfeel and overall note. Typicality of odour and intensity of fruit odour were evaluated by ortonazal while the aroma and mouthfeel were evaluated by retronazal. The overall sen- sation was evaluated as general impression of the spirit quality. Each sensory attribute was evaluated using a 5-point intensity scale (1-very weak, 5-very strong). 2.2.2 Statistical analysis Analysis of paired comparison difference test based on the binominal distribution of answers. The binomial Table1: Alcohol content of commercial fruit spirits and their reduced value Spirits from Declared alcohol content in %vol Reduced alcohol content in %vol Wiliams 40 37.5 Plum 42 37.5 Apple 40 37.5 Raspberry 43 37.5 Grape 43 37.5 Figure 1: Series of five pairs of spirit samples that served to assessor, where A is the commercial alcohol content and B is the reduced alcohol content Acta agriculturae Slovenica, 119/4 – 20234 A. ŠEKERIĆ et al. test is used to determine the probability of selecting the correct answer. Based on the total number of traces, the number of correct choices is taken from the table of bino- mial numbers to determine significance at the 0.05 prob- ability level (O′ Mahony, 1986). The Chi-square test was used to test whether the testers showed a significant pref- erence for one of the samples (Meyners, 2007). The mean scores for the sensory atribbutes of the spirits were tested with a t-test using the Microsoft Excel software program. 3 RESULTS AND DISCUSSION The assessor evaluated each pair of spirits and asked, „Are the samples different?“ The paired condense responses for all samples tested are shown in Table 2. Sensory analysis of all spirits revealed significant differences between commercial and reduced alcohol content (Table 2). The assessors found a significant dif- ference in the alcohol content of the tested spirit samples. This means that the sensory perception of beverages is significantly influenced by the alcohol concentration. This was evident in all fruit species (varieties) tested in this experiment. Raspberry spirits had the fewest incor- rect responses in the evaluation, possibly due to the sig- nificant difference in alcohol content between the com- mercial and reduced versions of the spirits. Although the difference in alcohol content between the commer- cial (40 % vol) and light (37.5 % vol) versions of apple spirits was not as great, most incorrect responses were observed. However, the distribution of responses shows that respondents perceived a difference between apple spirits with high and reduced alcohol content. In the statistical analysis of the preference test, only the responses of the assessors who correctly identified the differences were considered. The results of the prefer- ence test are shown in Figure 2. Regarding the preference test, many of the assessors indicated that they preferred the beverages with higher alcohol content. However, a statistically significant dif- ference is observed between pairs of the commercial and light versions of Williams pear, apple, and raspberry spirits. Although more assessors indicated that a stronger sample of grape and plum spirits was more acceptable to them, there was no statistically significant difference in the distribution of responses between these pairs. In a descriptive test, assessors were asked to rate the sensory attributes of each pair of spirits. Figure 3 shows the average scores for each sensory attribute of the spirits along with the results of the t-test. Sensory perception of fruit spirits has been shown to be influenced by ethanol concentration, consistent with the findings of Ickes and Cadwallader (2017; 2018). Spirits with higher alcohol content were mostly evaluated favourably by the assessors compared to their “light” versions. According to average ratings, Williams pear spirits with 40 % vol were rated significantly better in aroma than Williams pear spirits with 37.5 % vol. All other sensory attributes of Williams pear spirits were rat- ed about the same. The difference in alcohol content was not sufficient to clearly distinguish all individual sensory properties except aroma. Nikičević (2005) states that a higher alcohol content is the ultimate for Williams pear spirits, as flavour and pleasure aroma are favoured at an alcohol content of more than 40 % vol. Similar to Williams pear spirits, plum spirit with 42 % vol of alcohol was perceived significantly superior in aroma, while differences in smell attributes between stronger and lighter versions of spirits were not per- ceived. Mouthfeel (warming sensation) was also more intense for stronger plum spirits than for lighter version of plum spirits. This is not surprising, as ethanol causes a warming sensation in the mouth (Demiglio and Pick- ering, 2008; Longo et al., 2017; Ickes and Cadwallader, 2017). However, mouthfeel is rated significantly better for apple spirits with lower alcohol concentration. Other studies have found that increasing the alcohol percent- age causes a higher assessment of hotness or a burning mouthfeel experience (Le Berre et al.,2007; Jones et al 2008). The dilution of the samples, with distilled wa- ter coused significant changes to the sensory profiles of apple and raspberry spirits. All sensory attributes of Table 2: Number of responses of the assessors in the test of paired differences - commercial and reduced alcohol concentrations in the tested spirits where * indicates significance Samples of spirits Correctly noted difference Incorrectly noted difference Significance at p < 0.05 Williams pear 27 3 * Plum 26 4 * Apple 23 7 * Raspberry 28 2 * Grape 26 4 * Acta agriculturae Slovenica, 119/4 – 2023 5 Effect of the alcohol content on sensory perception of the fruit spirits stronger apple and raspberry spirits are significantly bet- ter scored. In the study of Wei et al. (2018) it was shown that the concentration of esters, terpenes, and alkanes increased with increasing alcohol concentration. These authors also reported that spirtis with higher content of alcohol were more fragrant than the spirits with lower alcohol. Also, Durr et al. (2010) pointed out that in fin- ished spirits, some primary aroma compounds become more prominent at higher alcohol concentrations. The most important primary aroma compounds are terpenes, phenol compounds, aromatic ethyl esters of short-chain fatty, but also aldehyde compounds and alcohols (Spaho et al., 2021; Wang et al., 2022). Because apple and raspberry spirits in this study, are significantly better scored in aroma and overall note in stronger versions of spirts it appears that these spirits are more characterized by primary aroma components where higher concentration of alcohol affects the release of apple and raspberry aromas. Many factors, as stated by Lyu et al. (2021), can influence the results of this sen- sory analysis: physical and chemical properties of volatile aroma components, low detection threshold, so that ev- ery dilution of spirits leads to a decrease in the aromatic value of aroma components, or physiological factors dur- ing tasting. More exact claims cannot be made unless the aromatic components in the tested spirits are identified and quantified analytically. The influence of alcohol content was not as pro- nounced in distinguishing the sensory characteristics of grape-derived spirits. Grape spirit with 43 % vol alcohol was evaluated better in terms of aroma perception and less intense mouthfeel, while there were no differences between grape spirits with 43 and 37.5 % vol alcohol in the perception of other sensory properties. Although grape spirits with higher alcohol content (between 43 and 45 % vol) are frequently offered on the Balkan mar- ket, this study found that consumers did not perceive any Figure 2: The results of the test preferences between fruit spir- its with commercial alcohol concentration (higher % vol) and reduced alcohol concentration of alcohol (37.5 %vol), where * indicates significance at p < 0.05 for the Chi-square test Figure 3: Sensory evaluation of Williams’s pear, plum, apple, raspberry and grape spirits with higher alcohol content (40 % vol, 42 % vol, 40 %vol, 43 %vol and 43 % vol, respectively) and their „light“ version with 37.5 % vol of alcohol. The attributes with different letter are statistically different according to the results of t-test and p < 0.05; ns-no significance Acta agriculturae Slovenica, 119/4 – 20236 A. ŠEKERIĆ et al. changes in sensory quality when alcohol was diluted to 37.5 % vol. Our results are consistent with the findings of Scholten (1999), according to which the lowest possible alcohol content is preferred for spirits with sensitive, fine aromas, e.g., grape spirits, so that the fruit-typical odour and aroma can be better perceived. 4 CONCLUSIONS According to the results of the paired comparison difference test, and the descriptive test, the consumers noticed the difference between the »strong and laight« version of spirits much more easily, although it was much more difficult to determine what this difference was ma- nifested in. In other words, they know what they like but are unsure why. Consumer panels are susceptible to vari- ous biases, including response bias, and the sample size of the consumer panel used in this study may limit the generalizability of the results. Nonetheless, the outcomes of this study have repeatedly shown that consumers can detect a difference in the alcohol content of fruit spirits and, as a result, prefer Williams pear, apple, and rasp- berry spirit with alcohol content higher than 37.5 % vol. The aroma of all fruit spirits with a higher alcohol content than 37.5% vol significantly better. In other sen- sory attributes our findings showed that the fruit spirits with commercial alcohol content and their dilutions ver- sion were more similar to one another. Reduction in eth- anol concentration can affect consumers’ perception of grape, plum and Wiliams pear spirits in terms of aroma and mouthfeel but in terms of fruit odour and intensity and overall note cannot. The apple and raspberry spirits, had better sensory quality in “stronger” versions of the spirits and these results indicate that it is better to bottle apple and raspberry spirits with high alcohol concentra- tion. 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Characterization of odor-active com- pounds in the head, heart, and tail fractions of freshly distilled spirit from spine grape (Vitis davidii Foex) wine by gas chromatography-olfactometry and gas chromato- graphy-mass spectrometry. Food Research International, 137, 109388. https://doi.org/10.1016/j.foodres.2020.109388 Acta agriculturae Slovenica, 119/4, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.4.16094 Original research article / izvirni znanstveni članek A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay Elham R. S. SOLIMAN 1, 2 Received September 09, 2023; accepted November 27, 2023. Delo je prispelo 9. septembra 2023, sprejeto 27. novembra 2023 1 Cytogenetics and Molecular Genetics Unit, Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt 2 Corresponding author, e-mail: Elham_soliman@science.helwan.edu.eg A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay Abstract: Ricinus communis seeds harbor high oil and polyphenolics contents that hinder DNA extraction. Here, a rapid and efficient protocol for isolating total DNA from R. communis seeds was developed. The current method implies the use of repeated cycles of freeze/heat shock for the seed tis- sue to lyse the cells. DNA isolated with this protocol was suc- cessfully used as a template for PCR amplification of the inter- nal transcribed spacer (ITS) region of the rRNA encoding gene that widely used for molecular identification of different plant species. As far to our knowledge, this study is the first one that report the efficient use of freeze/heat shock repeated cycles for isolation of a high-quality DNA from plant cells. The current protocol would support the subsequent analysis for seed lot pu- rity analysis. Key words: castor bean seed, cell lysis, heat shock, ITS, PCR, sequencing Hitra in učinkovita metoda DNK ekstrakcije iz semen klo- ščevca (Ricinus communis L.), bogatih na oljih, primerna za PCR analizo Izvleček: Semena kloščevca so bogata na oljih in poli- fenolih kar ovira ekstrakcijo DNK. V tej raziskavi je bil razvit hiter in učinkovit protokol za izolacijo celokupne DNK iz se- men kloščevca. Metoda uporablja ponavljajoče se cikle zmr- zovanja in segrevanja, kar povzroči lizijo celic v tkivu semena. DNK izolirana po tem protokolu je bila uspešno uporabljena kot osnova za PCR namnoževanje ITS regij v rRNK, ki kodirajo gene, na širokouporabljene pri molekularni identifikaciji raz- ličnih rastlinskih vrst. Kolikor nam je znano, je to prva raziska- va, ki poroča o učinkoviti rabi ponavljajočih se cikličnih šokov zmrzovanja in segrevanja za izolacijo visoko kakovostne DNK iz rastlinskih celic. Ta protocol bo pripomogel k hitri analizi čistosti semen. Ključne besede: kloščevec, lizija celic, vročinski šok, ITS, PCR, sekvenciranje Acta agriculturae Slovenica, 119/4 – 20232 E. R. S. SOLIMAN 1 INTRODUCTION Castor bean (Ricinus communis L.) seed is one of the most common oil seed plants that is widely used eco- nomically in biodiesel production, cosmetics industry, lubricants, biomedical applications and as a rich animal fodder (Patel et al., 2016; Sturtevant et al., 2019; Sánchez et al., 2019; Awais et al., 2020). The seeds are externally protected by hard, brittle, mottled brown and shinning testa. The outermost layer of the seeds coat is the waxy cuticle, which represents the first barrier to water im- bibition. The mature desiccated seed coat is rich in the oxidative products of polyphenolic compounds includ- ing phenolic acids, tannins, and flavonoids which are the source of the brown color of the mature seed coat, and play a significant role in plant disease resistance. Also, peroxidases and other antioxidant scavenging enzymes are commonly found in seeds coats (Moïse et al., 2005). Employing genomics and molecular technologies promise to accelerate our knowledge of seeds and thus open new potentials for uses and conservation. The low concentration of DNA present in seed tissues makes it difficult to isolate intact DNA. Additionally, the DNA in seeds can be tightly bound to various proteins, polyphe- nols, and sugars. These biochemical interactions further complicate the process of extracting high quality DNA from seeds (Roy Davies, 1977; Sliwinska, 2006; Lee et al., 2020). The isolation of a good quality DNA is a pre- requisite for any molecular biology work because pro- teins, polyphenols and polysaccharides impurities that co-precipitate with the DNA may hinder any enzymatic action, such Taq DNA polymerase in Polymerase Chain Reaction (PCR) and subsequent sequencing (Lakay et al., 2007; Wnuk et al., 2020) and endonucleases in genotyp- ing and blotting techniques (Brown, 2001; Harju et al., 2004; Shiraishi and Iwai, 2020). PCR based technique is successfully used to detect R. communis candidate genomic loci that are associated with important agronomic traits (Fan et al., 2019). PCR is essential for specific gene detection that prevailed in molecular identification and characterization (Manju- nath and Sannappa, 2014). Enhancing the current seed traits to add value or to overcome an existing problem may be achieved by the generation of genetically modi- fied plants. This wouldn’t be achieved without isolating a good quality DNA that would serve as a template for a PCR reaction. The production of transgenic R. communis plants expressing the Bacillus thuringiensis cry1Aa gene help in lepidopteran insect pest management that were responsible for 30–50 % of yield losses (Muddanuru et al., 2019). CTAB (Cetyl trimethylammonium bromide) DNA based extraction protocol and tailored modification ver- sions of it, is widely used for isolating DNA from different plant tissues. However efficient, the CTAB is harsh and toxic in nature for human health and should be followed by phenol purification to ensure sufficient DNA purity. The phenol is volatile and can burn the skin (Doyle and Doyle, 1990; Shukla et al., 2018; Aboul-Maaty and Oraby, 2019). The used CTAB based protocol is time consuming as it requires long incubation time reached up to 60 min at 65 0C (Novaes et al., 2009). Alternatively, the commer- cial DNA extraction kits is an efficient substitute, despite the associated cost burden. So, a rapid efficient isolation method is of demand to facilitate any genomics and mo- lecular biology work. Therefore, the current study was designed for devel- oping a rapid and efficient seed DNA isolation method that implies repeated cycles of freeze/heat shock to lyse the seed cells. Subsequent removal of the coagulated pro- teins and lipids by chloroform extraction was made and finally, high quality DNA was precipitated by ethanol. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS Ten different samples of R. communis seeds were collected from their wild habitat in Egypt. Their DNA was extracted according to the newly developed protocol and used as a template for PCR amplification to ampli- fy the ITS region of the corresponding rRNA encoding gene (Cheng et al., 2016). 2.2 MATERIALS AND EQUIPMENTS - 1.5 ml Eppendorf tubes - Mortar and pestle - Tips - Cooling centrifuge (Hettich MIKRO 22) - Crushed ice - UV-transilluminator (Vilber Lourmat-Germany) - Digital balance (RADWAG Wagi Elektroniczne, AS 220/C/2) - Micropipettes - Vortex (VELP SCIENTIFICA) - Water bath (MLW W21) - Gel electrophoresis unit - Thermal cycler (Biometra, Germany) 2.3 REAGENTS - Lysis buffer: 2 % Triton X-100 (ADVENT), 100 Acta agriculturae Slovenica, 119/4 – 2023 3 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay mM NaCl (POWER CHEMAL), 1 % SDS (Sodium Dodecyl Sulfate), 10 mM Tris-HCl (pH 8.0) (OX FORD) and 1 mM EDTA (pH 8.0) (Hoffman and Winston, 1987, Harju et al., 2004) - Ice cold 99 % ethanol (POWER CHEMICAL) or isopropyl alcohol - 70 % ethanol - 1 × TAE buffer - Ethidium bromide (ALPHA CHEMIKA) - Chloroform (SIGMA-ALDRICH) - 3 M sodium acetate pH 5.2 - TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0, autoclaved) - Agarose (molecular grade, Cleaver Scientific Ltdm CAS 9012-36-6) 2.4 THE DETAILED PROTOCOL 1. In 1.5 ml Eppendorf tubes, 0.1 g crushed de- coated R. communis seeds were combined with 400 μl of the DNA lysis buffer. And then vortexed vigorously in a bench top vortex for up to one minute, followed by incu- bation on ice. a. TIP: Avoid using a large amount of seed sample because the polysaccharides, polyphenols, and their de- rivatives in the seed would increase concurrently. These substances could interfere negatively with proper DNA isolation, reducing the isolated amount. b. TIP: Rather than using a mortar and pestle, the sample could be crushed inside a round end 2 ml Eppen- dorf tube using a glass rod. c. TIP: Grinding in liquid nitrogen would facilitiate the grinding process and increase DNA integrity. 2. After two minutes on crushed ice, the tubes were immersed abruptly in a 95 °C water bath for 5 minutes with interval vortexing. 3. After repeating the freeze/heat shock procedure exactly as described above, the tubes were vigorously vortexed for a continuous 30 seconds. TIP: The solution takes on the appearance of a milky emulsion. 4. 400 μl of chloroform was added and vigorously vortexed for one minute, followed by centrifugation at 4 °C, 5 min, 10,000 rpm (RCF: 17,507). TIP: Chloroform and isoamyl-alcohol can be mixed in a ratio of (24:1 v/v). 5. The aqueous layer was transferred to a new tube and the chloroform purification was repeated twice. TIP: The chloroform purification should be repeat- ed whenever necessary till the aqueous fraction is clear. 6. To allow DNA precipitation, the clear aqueous layer was transferred to a new tube containing 1 ml of ice-cold 99 % absolute ethanol and 40 µl of 3 M sodium acetate pH 5.2. After 15 minutes on ice, the tubes were centrifuged for 10 minutes at 4 °C and 10,000 rpm. TIP: Instead of absolute ethanol, isopropyl alcohol may be used. 7. Following the removal of supernatants, DNA pel- lets were washed with 0.5 ml of 70 % ethanol and quickly centrifuged at 4  °C for 2 minutes at 10,000 rpm. The pellet was then air dried for 5 minutes. TIP: To expedite the drying of the DNA pellet, in- vert the Eppendorf on a piece of clean. tissue. 8. Resuspend the DNA in 30 μl TE buffer [10 mM Tris, 1 mM EDTA (pH 8.0)]. The samples were stored at -20 °C until they were used. TIP: It is recommended to dissolve the DNA pellet in a small volume of TE buffer to ensure that the DNA is adequately concentrated. If the sample was dissolved in a larger volume, it resulted in a low concentration of DNA. Reprecipitate your DNA and resuspend it in a reduced volume of TE buffer. 2.5 QUANTIFICATION AND VISUALIZATION OF DNA The concentration of isolated DNA was determined using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Scientific-USA). The optical density (OD) at A260 and A280 was used to determine the purity. Elec- trophoresis of samples was performed on a 1 % agarose gel in 1× TAE (Tris-Acetate- Ethylenediaminetetraacetic acid) buffer containing 0.5 µg ml-1 ethidium bromide. The DNA was visualized and photographed with the aid of a UV-transilluminator embedded in Gel Documenta- tion system (Virballurmate-Germany). 2.6 PCR AMPLIFICATION AND SEQUENCING The PCR was carried out according to the manufac- turer’s instructions in a 50 μl reaction volume using (BI- OLINE cat # BIO-21108) Mytaq Red DNA polymerase master mix. ITS1 (5’ TCCGTAGGTGAACCTGCGG 3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC 3’) primers were used. The reaction mixture contains 1 × mytaq Red DNA polymerase master mix, 2.0 μl of each primer at a concentration of 10 pm l-1, 1.0 μl of DNA template, and 0.25 μl of MyTaqTM DNA polymerase (5U μl-1). The fol- lowing PCR cycles were run in a Thermal Cycler (Biom- etra, Germany): Initial denaturation was carried out for 3 minutes at 95 °C, followed by 35 cycles of 20 seconds at 95 °C (denaturation), 20 seconds at 55 °C (annealing), 30 seconds at 72 °C (extension), and then a final extension Acta agriculturae Slovenica, 119/4 – 20234 E. R. S. SOLIMAN for 10 minutes at 72 °C. On a 1 % agarose gel, the PCR products were separated. The amplified fragments were excised and gel pu- rified according to the manufacturer’s protocol using the PCR-M clean up system (VIOGENE cat# PF1001). Following that, their sequences were determined at GATC Company using an ABI 3730xl DNA sequencer and an ITS4 primer. The obtained nucleotide sequenc- es were validated against the NCBI database using the MEGA blast tool (https://blast.ncbi.nlm.nih.gov/Blast. cgi). Geneious 11.1.5 software was used to align all se- quences. The following sequences were deposited in the NCBI GenBank: MN880879, MN880880, MN880881, MN880882, MN880885, MN880886, MN880887, MN880888, MN880889, and MN880890. 3 RESULTS The following is a description of a rapid DNA isola- tion protocol called the “ES DNA isolation method” (Fig. 1). The method is based on the use of repeated freeze- and-heat shock cycles to disrupt the cell wall and release genomic DNA from crushed de-coated R. communis seeds in lysis buffer. Following that, two rounds of chlo- roform purification were performed to remove co-bound proteins and lipids to the DNA following cell lysis. After transferring the aqueous phase to a clean tube, the DNA is easily precipitated using absolute (Abs.) ethanol and 3 M sodium acetate (pH 5.2). Following a 70 % ethanol wash, the air-dried pellet was dissolved in TE buffer to ensure the extracted DNA was preserved for the long term. The isolated DNA’s purity and lack of degradation are demonstrated by gel electrophoresis (Fig. 2). Figure 2: Gel electrophoresis of DNA isolated from the de- coated R. communis seeds used in the current protocol; “ES DNA isolation method”. Lanes 1 to 10 refers to the ten samples used. λ DNA refers to serial dilution of undigested λ DNA for quality control and quantification of isolated DNA. The flourecent bands towards the bottom of the gel likely represent degraded RNA, which commonly co-purifies during DNA extraction protocols Table 1: Quantitative estimates of DNA purity and concentra- tion revealed by Nanodrop Spectrophotometer Sample ID Concentration ng µl-1 A260/A280 readings (DNA Purity) Rc1 95.1 2 Rc 2 81.4 2.57 Rc 3 49.5 2.3 Rc 4 55.6 2.28 Rc 5 79.7 1.99 Rc 6 52.8 2.11 Rc 7 56.9 2.3 Rc 8 60.1 2.5 Rc 9 66.3 1.99 Rc 10 55.7 2.44 Figure 1: Flow chart representation of the current R. com- munis seed DNA isolation protocol termed “ES DNA isolation method” Acta agriculturae Slovenica, 119/4 – 2023 5 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay The A260/280 ratio, which ranges between 1.99 and 2.57 for all R. communis seed DNA samples examined, indicates the quality of the isolated DNA (Table 1). The isolated DNA concentration ranges between 49.5 and 95.1 ng µl-1. The yield and purity of the isolated DNA were sufficient for performing polymerase chain reaction amplification (Fig. 3). All samples were amplified to the expected product size of 700 bp. All PCR products were sequenced and the nucleo- tide sequences verified using the National Center for Bio- technology Information’s (NCBI) website. Alignment of the ITS sequences was performed to demonstrate their homology (Fig.4). The matched sequences revealed that all samples shared a significant degree of homology. The results demonstrates that the sequenceable PCR products obtained are of a high grade. Figure 3: PCR-amplification of the ITS-rRNA encoding gene of the different R. communis seeds using the DNA extracted by the current method “ES DNA isolation method” as a template. Lanes 1 to 10 represent the ten R. communis samples. 100 bp ladder refers to the DNA ladder. NTC refers to non-DNA template of the PCR, no amplification means no PCR contamination Figure 4: Partial sequence alignments of the ITS-rRNA encoding gene sequences obtained from the 10 R. communis seeds using Geneious 11.1.5 software. The data confirms the quality of the obtained PCR products that could be sequenced Acta agriculturae Slovenica, 119/4 – 20236 E. R. S. SOLIMAN 4 DISCUSSION While a PCR-based assay requires adequate quality genomic DNA, a rapid isolation methodology is required to permit the processing of large numbers of samples. Be- cause the majority of polyphenolic chemicals in seeds are localized in their seed coats, de-coating the seeds reduces their interference with DNA isolation (Moïse et al., 2005). Repeated freeze-heat shock cycles are employed to shat- ter the cell wall and release genomic DNA from crushed de-coated R. communis seeds in lysis buffer, obviating the necessity for enzymatic or mechanical degradation (Har- ju et al., 2004). The lysis buffer contains mild detergents such as Triton X-100 and SDS and is frequently used to lyse cells, extract proteins, extract oils, and permeabilize live cell membranes by dissolving protein-lipid and lipid- lipid interactions without denaturing proteins (Johnson, 2013). However, freezing and heat shock may affect cell wall permeability and denature proteins, thereby expe- diting their removal, without the need for hazardous pol- yvinyl pyrrolidone (PVP), phenol, or β-mercaptoethanol (John, 1992; Shukla et al., 2018). Seed polysaccharides, polyphenols, and their deriv- atives may impair the integrity of isolated DNA from R. communis seed (Porebski et al., 1997). When cells are ly- sed, these compounds covalently link to DNA, impairing DNA integrity and impeding PCR amplification (Wnuk et al., 2020). Therefore, the 60 °C CTAB incubation may be particularly important to dissociate polysaccharides from DNA, allows efficient and thorough lysis of cells and breakdown of proteolytic enzymes that could otherwise degrade DNA. This contributes to high DNA yields with CTAB and improve purity of the extracted DNA. The current lysis protocol using repeated freeze-heat shock cycles may achieve comparable degree of enzymatic deg- radation and release of DNA from cellular components without prolonged optimum heated incubation (Carey et al., 2023). Chloroform can be used to remove these com- pounds during DNA extraction. Additional to removing denatured proteins, it aids in the removal of other col- ouring chemicals such as pigments and dyes. Chloroform facilitates the separation of lipids, proteins, and cellular detritus into the organic phase, while the recoverable DNA is dissolved in the aqueous phase and then easily precipitated with absolute (Abs.) ethanol. Because the low seed DNA content necessitates efficient DNA pre- cipitation, 3 M sodium acetate (pH 5.2) was added to the absolute ethanol during the precipitation process (Júnior et al., 2016; Heikrujam et al., 2020). Gel electrophoresis was used to determine the feasi- bility of the current DNA isolation approach. No degra- dation was seen on the gel. Additionally, this was also ob- served with the nanodrop measurements. The A260/280 ratio of isolated DNA varied between 1.99 to 2.57 for all R. communis seed DNA samples examined, indicating that the DNA was of acceptable quality (Nzilibili et al., 2018). The isolated DNA concentration ranged between 49.5 and 95.1 ng µl-1, which was sufficient for down- stream applications such as PCR. Although the A260/280 ratio for high-quality DNA should be between 1.8 and 2, values greater than 2 have been observed previously for DNA samples isolated from various plant tissues us- ing a modified CTAB-based approach (Aboul-Maaty and Oraby, 2019). This could be attributed to ionic strength and altered pH of the solutions used in the extraction process (Wilfinger et al., 1997; Boesenberg-Smith et al., 2012). Despite the fact that the DNA isolated using the current approach had a slightly higher A260/280 ratio, the DNA isolated was effectively employed for the down- stream application, PCR. The extracted DNA was used as a template for PCR amplification of the ITS region of the rRNA encoding gene, which is commonly used for molecular identification and assessment of the molecular diversity of eukaryotic cells (Cheng et al., 2016; Yang et al., 2018; Ghareb et al., 2020; Soliman and A., 2021). PCR amplification of the extracted DNA samples yielded the expected product size of approximately 700 bp, as shown in Figure 3. The amplified PCR products were successful- ly sequenced, and the obtained sequences were deposited in the NCBI database (Fig. 4) (Soliman and A., 2021). These results demonstrate that the isolated DNA was of suitable quality for PCR amplification and downstream sequencing applications. 5 CONCLUSION This rapid DNA extraction protocol demonstrates the ability to isolate DNA from seed tissues even with- out relying on specific chemical reagents typically used for extraction, such as CTAB. By utilizing simple, readily accessible lysis buffer components and efficient physi- cal disruption of cells through freeze-heat cycles. This method provides an alternative approach to extract PCR- suitable DNA without requiring specialized chemicals. While CTAB is considered a staple reagent for high- quality DNA purification, this work shows that effective extraction is still achievable using very basic buffers and lysing techniques. The simplicity and accessibility of the reagents needed could make this rapid protocol easily adoptable, especially in resource-limited settings where procuring chemicals like CTAB may be difficult or cost- prohibitive. The proposed approach would facilitate the molecular investigation of seed lot quality and the char- acterization of germplasm. Acta agriculturae Slovenica, 119/4 – 2023 7 A rapid and efficient DNA extraction method from high oily content seeds: Ricinus communis L.- apt for PCR based assay 5.1 DECLARATIONS 5.1.1 List of abbreviations PVP, polyvinyl pyrrolidone. ITS-rRNA, Internal transcribed spacer of ribosomal RNA encoding gene. 5.1.2 Ethics approval and consent to participate “Not applicable” 5.1.3 Consent for publication “Not applicable” 5.1.4 Availability of data and material All data generated or analyzed during this study are included in this article. 5.1.5 Competing interests “The author declare that she has no competing in- terests” 5.1.6 Funding This study received no funding grant 5.1.7 Authors’ contributions ERSS: generates the idea, performs all experimental work, analyze the data, submit the sequences to the data- base, wrote the manuscript, revised it, and corresponding the publication. 5.1.8 Acknowledgements “I am very gratefully thanking Prof. Soliman M. S. A, Cyto-genetics and Molecular Genetics section, Bot- any and Microbiology department, Faculty of Science, Helwan University, (Egypt) for providing R. communis seeds.” 6 REFERENCES Aboul-Maaty, N.A.F. and Oraby, H.A.S. (2019). 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BioTechniques, 22, 474–481. https://doi.org/10.2144/97223ST01 Wnuk, E., Waśko, A., Walkiewicz, A., Bartmiński, P., Bejger, R., Mielnik, L. and Bieganowski, A. (2020) The effects of humic substances on DNA isolation from soils. PeerJ, 8. https:// doi.org/10.7717/peerj.9378 Yang, R.-H., Su, J.-H., Shang, J.-J., Wu, Y.-Y., Li, Y., Bao, D.-P. and Yao, Y.-J. (2018) Evaluation of the ribosomal DNA in- ternal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing.(ed D Cullen). PLOS ONE, 13, e0206428. https://doi.org/10.1371/ journal.pone.0206428 Acta agriculturae Slovenica, 119/4, 1–11, Ljubljana 2023 doi:10.14720/aas.2023.119.4.16395 Original research article / izvirni znanstveni članek Effect of sowing time and fertilizer on the protein content, seed- and pro- tein yield of dry beans (Phaseolus vulgaris L.) Andrea GYÖRGYINÉ KOVÁCS 1, 2, Gabriella TÓTH 1, Tamás SIPOS 1, Béla SZABÓ 3, István HENZSEL 1 Received October 17, 2023; accepted November 24, 2023. Delo je prispelo 17. oktobra 2023, sprejeto 24. novembra 2023 1 University of Debrecen, IAREF, Research Institute of Nyíregyháza, Nyíregyháza 2 Corresponding author, e-mail: gyorgyine@agr.unideb.hu 3 University of Nyíregyháza, Institute of Engineering and Agricultural Sciences, Department of Agricultural Sciences and Environmental Management,Nyíregyháza Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) Abstract: The beans are an important protein source. In our three-year experiment, we examined the effect of sowing times and fertilizer doses on the protein content of the seed and the protein yield per hectare. We set up the trial on sandy soil, without irrigation, in four replications with 300,000 plant per hectare in Nyíregyháza (Hungary). 10 m2 plots were in randomized blocks, with three sowing times (in normal time, earlier and later) and three fertilizer doses (0  %; 100  % and 150 %). We measured the nitrogen content of the seed with a Vario-Max CNS analyzer. The protein contents were counted from that value. The data were evaluated with Excel and SPSS 22.0. In examined 3 years the largest protein contents were in the third sowing time with 150 % fertilizer dose. In 2016-17 the yield and protein yield of the third sowing time were larger than the value of earlier sowing times for all three fertilizer doses. This was because of favourable weather. The significant effect of increased fertilizer doses was not proved at a given sowing time. If the weather was favourable, then the significant relationship among the sowing time, protein content, yield, and protein yield was positive and strong. Key words: bean, protein, yield, sowing time, fertilizer Učinek časa setve in gnojenja na vsebnost beljakovin v seme- nu in pridelku beljakovin v suhem fižolu (Phaseolus vulgaris L.) Izvleček: Fižol je pomben vir beljakovin. V triletnem poskusu smo preučevali učinek časa setve in odmerka gnojil na vsebnost beljakovin v semenu in na pridelek beljakovin na hektar. Poskus je bil zasnovan na peščenih tleh, brez namaka- nja, v štirih ponovitvah, s 300,000 semeni na hektar, v Nyíre- gyházi (Madžarska). 10 m2 velike ploskve so bile razporejene v naključnih blokih, s tremi časovi setve (normalen čas, zgodnja in pozna setev) in s tremi odmerki gnojil (0 %; 100 % in 150 %). Vsebnost dušika v semenih je bila izmerjena z Vario-Max CNS analizatorjem. Vsebnosti beljakovin so bile izračunane iz teh vrednosti. Podatki so bili ovrednoteni z Excelom in SPSS 22.0. V treh letih poskusa je bila ugotovljena največja vsebnost belja- kovin v tretjem času setve in pri odmerku gnojil 150 %. V letih 2016-17 sta bila pridelek in pridelek beljakovin v tretjem obdo- bju setve večja kot pri zgodnji setvi, pri vseh odmerkih gnojil. Vzrok za to je bilo ugodno vreme. Značilen učinek povečanja odmerka gnojil ni bil ugotovljen pri nobenem času setve. V pri- meru ugodnega vremena je bila značilna in močna povezava med časom setve, vsebnostjo beljakovin, pridelkom in pridel- kom beljakovin. Ključne besede: fižol, beljakovine, pridelek, čas setve, gnojila Acta agriculturae Slovenica, 119/4 – 20232 A. GYÖRGYINÉ KOVÁCS et al. 1 INTRODUCTION Ensuring the protein requirement is an important aspect of both human nutrition and animal feeding. Leg- umes have 2-3 x more protein than cereals (Siddiq et al., 2010). The protein content of beans seed is 24-28 % (Chávez-Mendoza et al., 2019; Kahraman & Onder, 2013; Bildirici & Oral 2020). The ratio of protein and fiber is also important (Brick et al., 2022). Bean flour is excellent for supplementing lysine-poor wheat flours, improving the ratio of essential amino acids in bread while reduc- ing carbohydrate content, thus improving the nutritional value of the bread produced (Hoxha, 2020; Singh, 2017). Its flour can also play a major role in the production of gluten-free products (Siddiq, 2010). In addition, the ethi- cal question of the use of animal proteins and their effect on health is gaining more and more space. Bean protein is also used in the production of vegetable meats, muf- fins, mayonnaise and yogurt (Ferreira, 2022). Grown of dry beans are strongly affected by climate change. In our country this appears with extremely high summer temperatures occurring, as well as the rather ex- treme distribution and intensity of precipitation. In terms of consumption, it is important how much protein a unit of food contains, and in terms of produc- tion, how much protein we can produce from a unit of area. This gives one aspect of the economy. Based on the role of beans in nutrition, we consid- ered it important to investigate the effect of fertilizer dose and sowing time on the protein content of seed beans and the protein yield per hectare. Beans are an ecologically sensitive crop. With this experiment, we get an answer, to how we can reduce the effects of the extreme weather conditions experienced nowadays with the technological elements mentioned above. In this publication, we present the response of seed protein content and protein yield of the Start pearl bean variety to sowing time and fertilizer treatments. 1.1 LITERATURE REVIEW Factors affecting the protein content are the variety and the weather, soil, irrigation and through these, ferti- lization and sowing time (Singh, 2017; Bildirici & Oral, 2020). Barampama & Simards (1993) in their experiment statistically confirmed that the variety and locality affect the nutrient content of the plant. The effect of sowing time and fertilization can be lead back to favourable cul- tivation conditions of the variety. The beneficial effect of fertilization can be only en- forced under favourable weather conditions, especially the amount of precipitation (Ermolaev & Radkov, 1975; Unk, 1984; Kádár, 2005; Togay & Anlarsal, 2008; Bel- laloui et al., 2011b, Bellaloui et al., 2013; Kawaka et al., 2018; Celmeli et al., 2018). Islam et al. (2016) and Shehata et al. (2011) proved in their experiment that the amount of protein was af- fected to a different degree by the use of different types of manure. Nitrogen is involved in protein formation (Marschner, 1995). Several researchers have shown the effect of nitrogen (Kádár, 2005; Morshed et al., 2008; Bal- láné Kovács, 2011; Gulmezoglu & Kayan, 2011; Soratto et al., 2017; Varfolomeyeva et al., 2021), phosphorus (Yin et al., 2016; Kahraman & Onder, 2013; Bildirici & Oral, 2020) and potassium (Bellaloui et al., 2013) fertilizers to increase the protein content. Miya & Modi (2015) also statistically confirmed the effect of NPK fertilization on increasing total crude protein. When organic manure is applied with NPK fertilizer, it increases the protein con- tent more than if they were applied separately (Dikshit & Khatik, 2002; Jagannath et al., 2002; Hegazi et al., 2011; Chaturvedi et al. 2012; Tomar et al., 2016; Saikia, 2018). In Singh’s (2002) experiment different organic fertilizers and pesticides were used to influence yield and protein content. However, there is also literature where N fertili- zation did not increase the NPK content of the seed (Nas- cente, 2017; Ovacikli & Tolay, 2020). The effect of the vintage is reflected in the higher pro- tein content of the dryer year (Somos, 1983; Nemeskéri & Nagy, 2003; Asemanrafat & Honar, 2017), respectively the control had a higher protein concentration in non- irrigated conditions than the fertilized one (Bellaloui et al., 2011b). According to Celmeli et al. (2018), the pro- tein content of cereal crops decreased as the crop grows. Asemanrafat & Honar (2017) confirmed this in the case of beans. The effect of sowing time on the protein content of soybeans was shown by Bellaloui et al. (2011a). Based on their studies, they found that early-sowed soybeans had a higher protein content. Singh et al. (2012) investigated the protein content of wheat semolina at different sow- ing times under irrigated and non-irrigated cultivation conditions. It was established that the temperature dur- ing the period of grain saturation had an effect on the amount of protein quantity. In non-irrigated conditions, the early-sown wheat had a higher protein content due to the lower temperature. However, in irrigated cultivation, the protein content was higher in the late sowing period. The role of weather is important because strongly influences utilization of fertiliser and crop yield. Beans are a water-intensive plant, one of the yield limiting fac- tors is the lack of rainfall (Nagy, 2006). Its water require- ment during vegetation is 300-400 mm, the most critical is the period of flowerbud formation, when it is very sen- sitive to drought. Its water-demanding period lasts from Acta agriculturae Slovenica, 119/4 – 2023 3 Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) budding to 12-16 days after the initial set. During this period, a single irrigation of 30 mm results in a 50 % yield increase of green pods (Tóth, 1979; Hadnagy, 1981). It needs a relative humidity above 65 % for pod set (Géczi, 2003). The effect of temperatures above 25-30 °C during flowering and atmospheric drought is unfavourable for the yield (Kádár, 2005). The yield and element content were more influenced by weather conditions than row spacing and fertilizer treatments (Russo, 2006). At the legumes, the increase in yield often entails a decrease in the protein content of the seed (Varfolomeyeva et al., 2021). There are differences in protein content between varieties (Már & Juhász, 2003; Köse et al., 2019). Cultiva- tion of the short growing season (60-90 days) varieties is safer in drought-prone climates (CGIAR, 2016). The ex- amined Start variety is also like this, in our country it can be safely sown until May 20, because it can ripen its crop. 2 MATERIAL AND METHODS We set up the experiment with the institute’s three dry bean (Phaseolus vulgaris L.) cultivars (‘Start’, ‘Hópe- hely’ and ‘Diana’) in Nyíregyháza (Hungary) in 2015-17. We examined the effects of sowing times, plant densities and nutrient supply on yield and yield characteristics. We describe in this publication the protein content test of the Phaseolus vulgaris ‘Start’ at a plant density of 300,000 plant ha-1 was examined over a 3-year period. Phaseolus vulgaris ‘Start’ is a white, pearl-shaped and small-seeded dry bean cultivar. The experiment was set up on sandy soil (Table 1.) without irrigated conditions with randomized blocks in 4 replications on 10 m2 plots. The first sowing was when the soil temperature has permanently risen to 14 °C. The second sowing was at the generally accepted beginning of May (~ May 8-10), and the third sowing happened until May 20. The plant densities were set 200 000; 300 000 and 400 000 germs ha-1. Treatment of nutrient replenishment included the control (0 %), 100 % and 150 % NPK dose based on Antal (1983) and Velich (1994) recommended 95 kg N, 40 kg P and 80 kg K to achieve 1 ton grain yield. Used fertilizers: ammónium-nitrát (N 34 %), szuperfosz fát (P 18 %) and kálium-szulfát (K 50 %). We calculated the protein content by the nitrogen values of the sample which was taken from 50 g minced dry seed crop. We measured the nitrogen values with a Vario-Max CNS analyzer and multiplied them with a conversion factor of 6.25. The study was in 3 replicates which were taken from the crop of field replicates. We used the SPSS software package for the evaluation. At the one-way analysis of variance, the homogeneous sample was tested by Tukey’s-b, and the non-homogeneous sam- ple was tested by the Games-Howel test at a 5 % SD level. Spearman’s and Pearson’s correlation analysis were used for the quantify relationship between the factors. The weather factors were analyzed with an Excel program. The weather is discussed in more depth because the rainfall and temperature conditions during flowering have a significant impact on the yield of beans (Table 2). In 2015 in the third sowing time, the proportion of hours with relative humidity above 65 % was the lowest and the temperature also was very high strongly decreas- ing the yield. During the flowering of the 2nd sowing season, temperatures above 30 °C did not occur and the relative humidity was also favourable. 1st sowing time re- ceived the most rainfall during its flowering period. In 2016 more rainfall fell (24 mm) during the flow- ering of the 1st sowing time than during the flowering of the other two sowing times. The number of hours of favourable relative humidity, which is necessary for gen- erative processes, was the highest here. However, the number of hours of critical temperature values was the highest in this sowing time, and the yield was the lowest. Table 1: Soil characteristics and GPS position of the experiment 2015 2016 2017 GPS position 47.978401, 21.675888 47.974961, 21.691528 47.975930, 21.697846 pH (KCl) 6,00 5,58 7,12 Plasticity index according to Arany 27 35 38 Water-soluble total salt (m m-1) % 0,02 0,04 0,04 CaCO3 (m m -1) % < 0,100 < 0,100 4,34 Organic carbon in humus (m m-1) % 0,842 1,98 2,07 AL-soluble P2O5 (mg kg -1) 96,4 123 142 AL-soluble K2O (mg kg -1) 247 211 328 KCl-soluble NO3- + NO2- - N (mg kg -1) 10,1 51,6 35,8 Acta agriculturae Slovenica, 119/4 – 20234 A. GYÖRGYINÉ KOVÁCS et al. During the flowering of the 1st sowing season, the tem- perature was above 30 °C twice, through several days (5 and 3 days). In the 2nd sowing season, the temperature was above 30 °C through 3 days on two occasions. In the 3rd sowing season it happened only once, which lasted for 3 days. In the 3rd sowing season, which had the largest yield, 15 mm of rainfall fell during flowering. This favoured crop formation. The number of hours of critical tempera- ture values and the values of relative humidity were the lowest in the 3rd sowing times among the 3 sowing times. Lower temperatures also supported crop formation. The percent of critical values within the flowering period is an important indicator. The relative humidity did not vary greatly between sowing times. However, in the first two sowing seasons, the proportion of hours above 30 and 25 °C during the flowering period was very high, which resulted in a low yield. In 2017, there was no big difference in the flowering weather of the 1-2. sowing times. The rainfall amounts also developed similarly. However, the 3rd sowing time had a much lower proportion of values above the critical temperature during the flowering time, which was shown in a significant increase in yield. The value of the 3rd sow- ing time was also more favourable in the proportion of hours with relative humidity above 65 %. The big differences between crop results could also be attributed to the weather. 3 RESULTS AND DISCUSSION 3.1 DIFFERENCES BETWEEN SOWING TIMES IN THE AVERAGE OF FERTILIZER TREATMENTS IN THE CASE OF THE Phaseolus vulgaris ‘Start’ In 2015-16-17, the seed yield of the 3rd sowing time had the highest seed protein content. Its significant dif- ference was different from the other two sowing times in a given year. In 2016 we did not prove any difference between them using the GH method. The protein con- tent of the 1st sowing time was significantly lower than that of the 3rd sowing time in 2015 and 2017. We verified a statistical difference between the seed protein content of the 1st and 2nd sowing times in 2015. In contrast to the other two years, the value of the 2nd sowing time was sig- nificantly lower than the protein content of the 1st sowing time (Table 3). There was a significant difference between seed yields in all three years between each sowing time. In 2016-17, when the soil and weather conditions were more ideal, the yield increased as the sowing times were postponed. In 2015 acidic soil with little humus content, extremely high temperatures during the flowering period and a growing season with poor rainfall were unfavour- able for beans, so the 3rd sowing time produced the least and the 2nd sowing time the most yield. Table 2: Weather characteristics during flowering 2015 2016 2017 Sowing time 1 2 3 1 2 3 1 2 3 Length of flowering (day) 20 10 14 17 15 13 20 17 15 65 % < relative humidity (h) 379 195 220 334 272 235 397 336 317 Average of affected days (h) 19 20 16 20 18 18 20 20 21 In percentage of flowering hours 79 81 65 82 76 75 83 82 88 30 °C < number of hours 17 0 71 61 46 20 18 16 5 Average of affected days (h) 9 0 8 8 8 7 3 3 5 In percentage of flowering hours 4 0 21 15 13 6 4 4 1 25 °C < number of hours 66 42 152 156 129 87 144 122 58 Average of affected days (h) 7 6 12 10 11 9 10 9 7 In percentage of flowering hours 14 18 45 38 36 28 30 30 16 Rainfall amount 24 14 13 24 9 15 50 50 44 Acta agriculturae Slovenica, 119/4 – 2023 5 Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) We also confirmed significant differences in pro- tein yield between all three sowing times. In the average of the treatments, the 3rd sowing time had significantly the highest protein yield and the 1st sowing time had the lowest in 2016-17. In 2015, due to the extreme weather, different results were obtained. Despite significantly the highest protein content of the 3rd sowing time, its protein yield was the lowest because of the very low yield. 3.2 DIFFERENCES BETWEEN THE RESULTS OF FERTILIZER DOSES IN THE AVERAGE OF THE SOWING TIMES IN THE CASE OF THE Phaseolus vulgaris ‘Start’ We verified a significantly difference in protein con- tent in 2016. The value of the treatment without fertilizers was statistically proven to be lower than the treatments with fertilizers (Figure 1-2). In 2015 and 2017, it was ob- served that the value of the treatment without fertilizers was the smallest and the value of the treatment with the most fertilizers was the highest. In yield per hectare and protein yield, we did not show any statistically proven differences between the treatments and the differences were also small. 3.3 DIFFERENCES BETWEEN FERTILIZER TREATMENTS AT A GIVEN SOWING TIME IN THE CASE OF THE Phaseolus vulgaris ‘Start’ 3.3.1 Seed protein content The unfertilized treatment had the lowest seed pro- tein content in all 3 sowing times and during all 3 years. The significantly deviation of this was different per year and per sowing time. In 2016, we significantly verified that the protein content of the seeds of fertilized plots was higher than that of non-fertilized plots (Table 4). We established the same thing in 2017, with the clarification that in the 2nd sowing season, the protein content of the two fertilized treatments was also significantly different from each oth- er: the treatment receiving 150 % had the highest protein content. This year, in the 3rd sowing time, the difference between the protein contents was minimal, we did not verify a significant difference between the fertilizer treat- ments. In 2015, the protein content of the treatment with a 150 % fertilizer dose was significantly higher than that of the non-fertilized treatment in the first two sowing sea- sons. In the 3rd sowing season, the protein content of the 100 % fertilizer dose differs significantly from that pro- tein of the unfertilized. 3.3.2 Yield (kg ha-1) In 2015-16, when the fertilizer treatments were ex- amined by sowing time, we did not prove a significant Table 3: Protein content, seed yield, and protein yield data of 3 years per sowing times (2015-2017) Protein content (%) Seed yield (kg ha-1) Protein yield (kg ha-1) 2015 2016 2017 2015 2016 2017 2015 2016 2017 1. sowing time 27.9 a 23.4 21.6 a 200.9 a 98.8 a 248.5 a 55.8 a 22.3 a 53.9 a 2. sowing time 26.1 b 25.6 22.8 a 336.3 b 224.2 b 712.0 b 87.5 b 56.4 b 162.1 b 3. sowing time 32.1 c 26.4 28.8 b 19.7 c 751.7 c 1490.3 c 6.3 c 197.2 c 428.8 c Figure 1: Effects of fertilizer doses in the average of the sowing times on protein content Figure 2: Effects of fertilizer doses in the average of the sowing times on seed yield and protein yield Acta agriculturae Slovenica, 119/4 – 20236 A. GYÖRGYINÉ KOVÁCS et al. difference using the Tukey-b method between the ferti- lizer treatments. In 83 % of cases, the without fertilizers had the most yield. In 2015, at the 1st sowing time, at the LSD 5  % level, the 150  % dose fertilizer treatment had yield significantly less than the treatment that received the 100 % dose. In 2016, in the case of the 3rd sowing time, at the LSD 5 % level, we verified a significant difference between the without fertilizer treatment with the highest yield and the treatment that received the 100 % dose. In 2017, the difference in the yield of the earlier and later sowing times was minimal, so we did not detect a significant difference. In the 2nd sowing season, the yield per hectare of the treatment with a 100 % fertilizer dose was significantly higher than the yield of the treatment without fertilizer and treatment with a dose of 150 %. In the 2nd sowing season of 2017, we statistically ver- ified that the protein yield of the treatment with a 100 % dose of fertilizer was significantly higher than the yield of the treatment without fertilizer and with a dose of 150 %. This can be explained by its significant surplus of yield, which was able to compensate for its protein content, which was located in the average protein content of the other two fertilizer doses. In 2015-16, except for the 1st sowing time, the treat- ment without fertilizers always had the highest protein yield, despite the lowest protein content. However, this difference in protein yield was not significant. We showed a significant difference at the LSD 5 % level between the lowest and the highest protein yield in 2015. In 2015, in the 1st and 3rd sowing times, the protein yield of the treat- ment with the highest fertilizer dose was significantly the lowest, but in the 2nd sowing time, the protein yield of the treatment with 100 % fertilizer dose was the lowest. In the 1st sowing seasons, the protein yield of treatments with the highest fertilizer dose was the lowest, which was also statistically confirmed in 2015. This trend was also observed in 2017. It was likely that the early sowing was unfavourable for the small-eyed, white-seeded Start va- riety, which was only amplified by the higher fertilizer dose. In the case of a lower seed protein content, the pro- tein yield may be higher due to the higher yield. For ex- ample: in 2017, in the case of 100 % treatment of the 2nd sowing time, and in 2015-16, regardless of the sowing times, in the treatments without fertilizers. 3.4 DIFFERENCES BETWEEN THE RESULTS OF SOWING TIMES AT A GIVEN FERTILIZER TREATMENT IN THE CASE OF THE Phaseolus vulgaris ‘Start’ 3.4.1 Protein content In 2015 and 2017, regardless of the fertilizer dose, the seed protein content of the 3rd sowing time had sig- nificantly higher than that of the previous sowing times Table 4: Effect of treatment combinations on protein content, yield and protein yield   2015 2016 2017 Fertilizer dose 1. sowing time 2. sowing time 3. sowing time 1. sowing time 2. sowing time 3. sowing time 1. sowing time 2. sowing time 3. sowing time Protein content (%) 0 27,2 A a 24,9 A a 30,8 A b 19,0 A a 23,6 A b 25,1 A c 20,6 A a 20,8 A a 28,7 b 100% 27,6 A a 25,7 AB b 33,1 B c 25,1B 26,3B 26,7 B 22,3 B a 22,7 B a 28,1 b 150% 28,9 B a 27,7 B a 32,4 AB b 26,2B 26,9B 27,4B 22,0 B a 24,8 C b 29,5 c Yield (kg ha-1) 0 219,1 AB a 372,4 b 30,1 c 134,0 a 302,1 a 895,6 A b 274,4 a 531,1 A b 1482,0 c 100% 280,3 B a 312,6 a 23,0 b 110,1 a 171,3 a 647,8 B b 279,2 a 1039,8 B b 1473,0 c 150% 103,4 A a 323,9 b 6,1 c 52,2 a 199,1 a 711,8 AB b 191,8 a 565,2 A b 1516,0 c Protein yield (kg ha-1) 0 59,6 A a 92,4 A b 9,4 A c 25,3 a 71,4 b 224,3 c 56,9 a 110,8 A b 424,6 c 100% 77,8 B a 80,2 B a 7,7 A b 27,8 a 44,9 ab 172 b 62,4 a 236,1 B b 415,5 c 150% 29,9 C a 89,8 A b 1,9 B c 13,8 a 53 a 195,2 b 42,4 a 139,2 A b 446,2 c Note on the figures: Capital letter: indicates a significant effect of fertilizer doses within the same sowing time and the same year Lower case: indicates a significant effect of sowing times within the same fertilizer dose and same year Acta agriculturae Slovenica, 119/4 – 2023 7 Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) (Table 4). The 1st and 2nd sowing times were significantly different at the 100 % fertilizer treatment in 2015 and in the 150 % dose treatment in 2017. In 2016, on the protein content of the fertilized treatments had no significant effect by sowing times. In the treatment without artificial fertilizers, the protein content of the 3rd sowing time was significantly higher than the that of 1st sowing time. 3.4.2 Yield (kg ha-1) In 2017, regardless of the fertilizer doses, the yield increased significantly in the 2nd and 3rd sowing times. In 2016, the 3rd sowing time also had significantly the high- est yield, however, the yields of the 1st and 2nd sowing times did not differ significantly from each other. In 2015, regardless of the fertilizer doses, due to the extreme weather, the 2nd sowing time had significantly the highest yield. This was also significantly different from the result of the 3rd sowing time, except for the treatment with a 100 % fertilizer dose, where the yield of the 1st sowing season was not significantly lower. 3.4.3 Protein yield (kg ha-1) The differences in protein yield (kg ha-1) formed in accordance with the yield in 2015 and 2017. We observed a deviation from this in 2016, where the protein yield of the 3rd sowing time was significantly higher in all ferti- lizer treatments. In the case of those without artificial fertilizers, the protein yield of each sowing time differed significantly. In the treatment with a 100  % fertilizer dose, despite the large difference, the 2nd sowing time did not differ significantly from the protein content of the 3rd sowing time, even though the 2nd sowing time had 74 % less protein yield compared to the 3rd sowing time. 3.5 RESULTS OF TREATMENT COMBINATIONS IN THE CASE OF THE Phaseolus vulgaris ‘Start’ 3.5.1 Protein content In 2015 and 2017, the highest seed protein content was measured in the 3rd sowing time treatment combina- tions. In 2016, the protein content of without fertilizer treatments were the least. The largest protein content was measured in 150 % fertilizer dose of 2nd and 3rd sowing times. 3.5.2 Yield and protein yield (kg ha-1) In 2015, treatments of 3rd sowing time produced sig- nificantly less than other treatments. The highest fertiliz- er dose treatment of 1st sowing time also produced little, so did not differ significantly from the yield of 3rd sowing times. The treatment without fertilizer of 2nd sowing time yielded the highest that did not significantly differ from the yield of treatment with fertilizer of 2nd sowing time and from the yield of treatment with 100 % dose of 1st sowing time. In 2016-2017, the treatment combinations of 3rd sowing time were significantly more productive than treatments of earlier-sowing times. Accordingly, its pro- tein yields also were significantly higher than those of earlier sowing times. The treatments with fertilizer were not significantly more productive than the control. 3.6 CORRELATIONS BETWEEN THE EXAMINED ELEMENTS IN THE CASE OF THE Phaseolus vulgaris ‘Start’ The yield volume, protein content and protein yield were correlated with each other at the 1 % significance level. In all three years, we showed a very strong, positive relationship between yield and protein yield (Table 5). Only the significant relationships are shown in the table. Between the protein content and protein yield, we verified a very strong, positive relationship in 2017. The high protein content resulted in a high protein yield (also due to the increased yield per sowing time). However, we showed a strong, negative relationship between them in 2015. The reason for this was that the highest protein content of the 3rd sowing time was combined with the lowest protein yield because of the low yield. In 2017, the increasing yield was coupled with higher protein content, and in 2015, a lower yield was coupled with higher pro- tein content. In all three years, in most cases, the sowing time was related with protein content, yield per hectare and pro- tein yield at the 1 % significance level. In 2016-17, we ver- ified a very strong positive relationship with the protein yield, in 2017 we showed a very strong relationship with the yield per hectare, and in 2016 a strong significant re- lationship. We verified a medium significant relationship with the protein content in all three years. In 2015, the sowing time had a negative, medium relationship with yield per hectare and protein yield be- cause of the extreme growing conditions. Acta agriculturae Slovenica, 119/4 – 20238 A. GYÖRGYINÉ KOVÁCS et al. 3.7 DISCUSSION IN THE CASE OF THE Phaseolus vulgaris ‘Start’ The yield of beans is strongly defined by the weather. This was also reflected in the yield as Kádár (2005) and Russo (2006) established during their experiment that the temperature during flowering and the lack of precipi- tation greatly influence the yield. In 2016-17 the fertilizer was utilized by the effect of favourable weather, thereby the protein content of seed, yield, and yield protein were increased. 3.7.1 Effect of sowing times in the average of fertilizer treatments The seed protein content of the 3rd sowing season was the highest in each of the 3 investigated years. In the case of Start beans variety, this was different from what was found in the literature in the case of soy and wheat. Bellaloui et al. (2011a) found that early-sowed soybeans had a higher protein content. Singh et al. (2012) deter- mined that in non-irrigated conditions, the early-sown wheat had a higher protein content due to the lower tem- perature. Early sowing of the Start bean variety is unfa- vourable due to the small, white seed. In case of favourable weather, the 2nd and 3rd yield of sowing times was greater than that of the 1st (early) sow- ing and their protein yield also increased. 3.7.2 The effect of fertilizer treatments at a given sow- ing time The non-fertilized treatment had the lowest seed protein content in all 3 sowing times during 3 years, which is understandable, since many literatures support the protein-increasing effect of fertilization. The protein yield of the treatment that received a 100 % fertilizer dose in the 2nd sowing season of 2017 was significantly higher. In the early sowing times, the protein yield of treatments with the highest fertilizer dose was the lowest, that differ- ence was significant in 2015. The reason for this was that the Start variety produced very little in the early sowings, because early sowing with a 150 % fertilizer dose was un- favourable for it. 3.7.3 Effect of fertilizer treatments in the average of sowing times In 2016 the protein content of the treatment without fertilizer was significantly lower than that of the treat- ment with fertilizer. We did not verify a significant dif- ference in protein yield per hectare between the fertilizer treatments. 3.7.4 The effect of sowing times in a given fertilizer treatment In 2015 and 2017 independently of the fertilizer dose, the seed protein content of the later sowing was significantly higher than the values of the earlier sowing times. In a favourable weather, the later sowing times had significantly the highest yield and protein yield. 3.7.5 Results of treatment combinations In examined 3 years the largest protein contents were in the third sowing time with 150 % fertilizer dose. In 2016-17 the yield and protein yield of the third sowing time were larger than the value of earlier sowing times. This was because of favourable weather. The significant effect of increased fertilizer doses was not proved. If the weather was favourable, then the significant relationship among the sowing time, protein content, yield, and protein yield was positive and strong. 4 CONCLUSIONS The observations of Ermolaev & Radkov (1975) and Table 5: Significant correlation values of 3 years between the examined factors Protein content (%) Seed yield (kg ha-1) Protein yield (kg ha-1) 2015 2016 2017 2015 2016 2017 2015 2016 2017 Protein content (%) 1 1 1 -,845** ,862** -,811** ,909** Seed yield (kg ha-1) 1 1 1 ,995** ,996** ,992** Sowing time ,536** ,425* ,798** -,518** ,885** ,938** -,530** ,920** ,938** Fertilizer ,780** ,387* Acta agriculturae Slovenica, 119/4 – 2023 9 Effect of sowing time and fertilizer on the protein content, seed- and protein yield of dry beans (Phaseolus vulgaris L.) Unk (1984) are still valid today, the yield-increased ef- fect of fertilizers only takes effect in case of favourable weather. We verified significantly different on one time of 9 variations. Examining the effect of fertilizers by sowing times, we already showed significantly different in more cases. The protein content of treatments with fertilizer was higher than that of without fertilizer. In 2015, we verified significantly that the protein yield of treatment with 150 % fertilizer dose was least at the 1st and 3rd sowing times, because its yield was very little by the unfavourable weather. In 2017, at the 2nd sowing time, the protein yield of treatment with 100 % fertilizer dose was significantly more than that of other fertilizer treatments. Examining the sowing times, with the favourable weather, the 3rd sowing time was the more favourable at the protein content, yield and protein yield for the grow- ing of the white and small-seed Start variety. 5 REFERENCES Antal, J. (1983): Növénytermesztők zsebkönyve,. Mezőgazdasági Kiadó, Budapest, 189. Asemanrafat, M., & Honar, T. 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(2021): The effect of mineral fertilizers on the yield of vegetable beans, depending on the competition be- tween plants in crops. E3S Web of Conferences 273, 01005 INTERAGROMASH 2021 https://doi.org/10.1051/e3s- conf/202127301005 Velich, I. (1994): Bokor- és karósbab. In: S. Balázs (Ed.), Zöldségtermesztők Kézikönyve (pp. 381-395). Mezőgazda Kiadó, Budapest. Acta agriculturae Slovenica, 119/4, 1–6, Ljubljana 2023 doi:10.14720/aas.2023.119.4.16424 Original research article / izvirni znanstveni članek Effectiveness of local biopesticides in the control of Diamondback Moth (Plutella xylostella L.) in cabbage production in Zanzibar, Tanzania Ahmed Gharib KHAMIS 1, 2, Ali Abdulla ALI 1, Mwanamanga Haji ALI 1, Ali Seif NASSOR 1, Mohammed Omar HAMAD 1, Salma Omar MOHAMMED 1, Mohammed Dhamir KOMBO 1 Received October 21, 2023; accepted November 27, 2023. Delo je prispelo 21. oktobra 2023, sprejeto 27. novembra 2023 1 Department of Agricultural Research, Zanzibar Agricultural Research Institute, Zanzibar, Tanzania 2 Corresponding author, e-mail: ahmadboycd@gmail.com Effectiveness of local biopesticides in the control of Dia- mondback Moth (Plutella xylostella L.) in cabbage production in Zanzibar, Tanzania Abstract: This experiment was conducted to determine the effectiveness of different types of local biopesticides to control diamondback moth (DBM) in cabbage production in Zanzibar. The experiment was conducted in horticulture farms at Zanzibar Agricultural Research Institute. The experiment was conducted in a randomized complete block design, with 6 treatments and a control, each of which was replicated 3 times. The following plants were used as biopesticide which are garlic (T1), hot pepper (chilli) (T2), clove (T3), mixture of garlic and pepper (T4), mixture of garlic and clove (T5), synthetic insecti- cide (T6) and a control (T0). The parameters were evaluated by conducting initial data collection (before treatment) and final data collection (after treatment). The average number of leaves affected by DBM and yield in terms of average mass (kg) of cab- bage were recorded. Results showed that T6 was significantly associated with a lowest average number of affected leaves and higher yield followed by T4 and T5. Overall, garlic-treated plots had recorded higher yield compared to the non-garlic treated plots. Therefore, garlic extract as local biopesticide can effec- tively repel DBM. This study recommends that garlic can be used as an alternative to using synthetic chemicals to control DBM in cabbage crop. Key words: cabbage, biopesticide, agriculture, diamond- back moth, Zanzibar, Tanzania Učinkovitost lokalnih biopesticidov za uravnavanje kapusne sovke (Plutella xylostella L.) pri gojenju zelja v Zanzibarju, Tanzanija Izvleček: Poskus je bil izveden za določitev učinkovito- sti različnih lokalnih biopesticidov za nadzor kapusne sovke pri gojenju zelja v Zanzibarju. Poskus je potekal na vrtnarskih kmetijah v Zanzibarju, na Zanzibar Agricultural Research Institute. Poskus je bil izpeljan kot popolni naključni bločni poskus s šestimi obravnavanji v treh ponovitvah in kontrolo. Obravnavanja so bila sledeča: česen (T1), čili (T2), klinčki (T3), mešanica česna in paprike (T4), mešanica česna in klinčkov (T5), sintetični insekticid (T6) in kontrola (T0). Obravnavanja so bila ovrednotena z zbiranjem podatkov pred in po obravna- vanjih. Pridobljeni podatki so obsegali število po sovki napa- denih listov in pridelek zelja, izražen kot poprečna masa zelja (kg). Rezultati so pokazali, da je imelo obravnavanje T6 zna- čilno najmanjše število napadenih listov in večji pridelek, temu sta sledili obravnavanji T4 in T5. Nasplošno je bil pridelk na ploskvah, kjer so sovko zatirali s česnom večji, v primerjavi s ploskvami brez česna. Iz tega lahko sklepamo, da bi lahko bili izvlečki česna uporabljeni kot učinkovit lokalni biopesticid za zatiranje kapusne sovke. Izsledki raziskave kažejo, da bi lahko uporabili česen kot alternativo sintetičnim kemičnim priprav- kom za uravnavanje zeljne sovke pri pridelavi zelja. Ključne besede: zelje, biopesticidi, kmetijstvo, kapusna sovka, Zanzibar, Tanzanija Acta agriculturae Slovenica, 119/4 – 20232 A. G. KHAMIS et al. 1 INTRODUCTION The agriculture sector contributed an average of 25 % of the total GDP and employing the majority of the country’s worksforce in Zanzibar. On average, 70  % of the population depends directly or indirectly in the agri- culture sector for their livelihood. However, diseases and pests such as insect infestation in agricultural production is among a key causes of quantitative damage of the crops which finally leads to degraded in quality and quantity of food (Stathas et al., 2023). Cabbage, Brassica oleracea L. ssp. oleracea convar. capitata (L.) Alef. is a green leafy vegetable with very high nutritional value and it is infested by varieties of insects. This crop is cultivated mainly by small-scale farmers in Zanzibar. The Diamondback Moth, Plutella xylostella (L., 1758) is one of the greatest threats to cabbage production in many parts of the world (Hill & Foster, 2000). It is a major cabbage pest that can cause a 100 % loss in yield if not well controlled and managed. In recent years, crop production in various tropical and sub-tropical countries has been largely affected by developed resistance to a wide range of synthetic chemical to control infestations. The introduction of these chemical products into the nat- ural environment resulted in the disruption of biological balance, and poses a great threat to human, animals and environmental health (Magierowiczet al., 2020). In ad- dition, they are known to cause cancers and abnormali- ties, and they remain in the environment for many years (Scholtz et al., 2002). On the other hand, biopesticides are naturally oc- curing compounds or agents that are obtained from animals, plants, or microorganisms such as bacteria, and are used to control wide range of agricultural pests and pathogens (Feniboet al., 2021). The use of biopesticides to protect crops against pests has a long history in in- sect pest management (Moshi & Matoju, 2017). Previous literature shows that the use of biopesticides is more ad- vantageous than the use of their conventional chemical pesticides, as they are eco-friendly and poses little threat to food safety (Khursheed et al., 2022). These opportuni- ties coupled with increasing costs of synthetic pesticides as well as increased consumer demand of organic prod- ucts have created the impetus to search for potential bi- opesticides. In spite of the large number of researches conduct- ed on effectiveness of biopesticides (Rusdi & Rusaldy, 2023), hardly any evidence, especially in Zanzibar have been conducted and published. Zanzibar has always been renowned for producing exceptionally good spices and herbs such as cloves, cinnamon, cardamon and black- pepper (Mahenya et al., 2014). Most of these products are good sources of bioactive compounds which may provide inhibitory activity against pests. In the absence of effective management options to tackle pests, there would be extensive dependence on synthetic pesticides for their management on crop, with significant negative impacts human, animal and the environment (Akutseet al., 2020). Therefore, there was a need for a research to identify specific biopesticides in the control of pests such as diamondback moth in Zanzibar. The main objective of this study was to determine the effectiveness of local biopesticides in the control of diamondback moth in cabbage production. The information obtained from this research is expected to contribute in finding solutions for for a better cabbage production among small holder farmers, especially in reducing the level of pests attack. 2 MATERIALS AND METHODS 2.1 STUDY SITE The research was conducted in Zanzibar, Tanzania. Zanzibar is a one of the two partner states that form the United Republic of Tanzania, comprised of two main islands – Unguja and Pemba. The experiment was con- ducted at the horticulture farms of Zanzibar Agricultural Research Institute (ZARI) which is located at Kizimbani area. The area is about 5 kilometers from Zanzibar Town. The institute is situated at latitude 60 south, longitude 390 east and 20 m above sea level. The area receives an average rainfall of 1564 mm/annum and annual average temperature of 25.7 0C. The experiment was conducted from June to September 2021. 2.2 CABBAGE PLANTING The chinese cabbage seeds (Michihili type) were bought from local agro-dealers directly to the field of 1000 m2. The land was ploughed followed by harrow- ing on 24th June, 2021. Field measured and laid out was conducted using measuring tape, rope, a hammer and pegs to mark the planting area. On 06th July; total 63 raised planting beds of 6 x 1.5 m (length x width) were made. Each bed was separated from one another by 50 cm apart. Seeds were sown directly to the prepared seed beds on 23rd July; at a spacing of 60 cm x 30 cm. Before sowing, soils were mixed and incorporated with about 45 kg of cow dung manure per bed, whereby each plant was estimated to take 0.75 kg of manure, this was done on 14th July, 2021. Transplanting and gap filled was done on after weeding which was done three days before. Acta agriculturae Slovenica, 119/4 – 2023 3 Effectiveness of local biopesticides in the control of Diamondback Moth (Plutella xylostella L.) ... 2.3 TREATMENT APPLICATIONS There were 6 treatments which are garlic (local cul- tivar) water extract (T1), hot pepper (Habanero type) water extract (T2), clove (local type) water extract (T3), mixture of garlic and pepper water extract (T4), mixture of garlic and clove water extract (T5), synthetic insecti- cide (T6) and a control (T0). These treatments were allo- cated randomly in an experimental area of 33m2 each. The number of treatments were allocated in a Randomized Complete Blocked Design (RCBD) in three replications (Rusdi & Rusaldy, 2023). 1 litre of each botanical spice water extracts was applied in 3 beds of a respective treat- ment for all replications in the morning before 10 o’clock using a 7 litre sprayer. Garlic, chilli pepper and cloves were separately blended and grounded respectively then mixed with water and soap to make stock solutions as previously explained (Hardiansyah & Al Ridho, 2020). In summary, 1 kg of plant materials (pepper, cloves, garlic ) were diluted in 5 litres of water. For mixed biopesticides ( e. g garlic and cloves/ pepper), the ratio was 1:1. During application, 1 litre of stock solution was then diluted in 5 litres of water. Biopesticide application was done after 48 hours from the time bio-pesticides prepared and were applied once for both biopesticides and synthetic. Data collection was done before application of pesticides and after application on 20th August and 30th August respec- tively. 2.4 DATA COLLECTION Presence of diamondback moth attack and yields were collected before and after treatments. The data col- lection continued untill at harvest stage (maturity). At harvest, marketable yield (mass in kg) data were record- ed for each cabbage plant. The mass (kg) of cabbage was measured obtained from a randomly selected plants in each plots. The parameters for data collection were num- ber of total leaves, number of affected leaves per plant and yield (mass) of harvested cabbage. The level of ef- fectiveness of the studied biopesticide treatments was calculated using the formula as follows:- EI = Description: EI = Insecticidal (biopesticide) efficacy (%) Ca = Number of infested cabbage leaves in the con- trol (without treatment biopesticide) Ta = Number of infested cabbage leaves in the bi- opesticide treatment. 2.5 DATA ANALYSIS All statistical results were considered significant if p < 0.05. One-Way analysis of variance (ANOVA) was per- formed to determine the significant differences in num- ber of affected leaves. All analyses were performed using STATA software version 16. 3 RESULTS AND DISCUSSION 3.1 DBM INFESTATION AND EFFECTIVENESS OF BIOPESTICIDE The intensity of DMB attacks is presented in Table 1 and Figure 1 below. This study shows that T6 (synthetic insecticide) was significantly associated with the lowest average number of affected leaves among all set of treat- ments followed by T4 (mixture of garlic and pepper) and T5 (Mixture of garlic and clove). With reference to the control, T3 (clove only) was significantly associated with greater average number of affected leaves in cabbage plants. As T4 and T5 are local biopesticides associated with a lower attack intensity of DBM, the significant re- duction of the DBM attack on cabbage after their appli- cation was indicative of the potency of garlic extract in controlling this pests. This significant finding highlights that garlic extract can be used to control DBM and other plant pests affecting cabbages such as red flour beetle, aphids, and whiteflies (Batool Syeda & Butt, 2021). It can be seen that all treatments provided very low level of effectiveness in controlling DBM. In comparison between treatments, T4, T5 and T6 had relatively higher efficiency compared to the rest. Meanwhile, T2 and T3 exhibited negative efficiency in controlling DMB which may implies that they are not very effective. The results reported in this study are in line with the findings re- ported previously using garlic extract to control plant pests (Batool Syeda & Butt, 2021; Hardiansyah & Al Ridho, 2020). For-example, garlic was intercropped with cabbage and found to have a strong repellant property against DMB (Karavina et al., 2014). Treatment of garlic is known to be a potential mean than other chemical be- cause it provide pungent smell which plant pests do not like (Batool Syeda & Butt, 2021). Garlic belongs to same family as onion and it is known to contain similar repel- lent properties to insects (Elmadawyet al., 2023). Also, garlic is known to contain sulphur compounds which deter insects from feeding on plants. The use of garlic to control pests was reported to be cheaper, safer and en- vironmentally friendly. Futher research needs to exam- ine how much and how long the level of effectiveness of Acta agriculturae Slovenica, 119/4 – 20234 A. G. KHAMIS et al. garlic is related to the function of DBM and other pests during dry and rainy season. 3.2 YIELD OF CABBAGE The findings concerning average mass (kg) of har- vested cabbage is presented in Table 2. Result shows that the highest yield was found in T6 followed by T1 and T4. Even though the garlic extracts reduced DBM attacks, their yield performance was not comparable to the syn- thetic insecticide. This may be due to the fact that the ac- tive ingredient of garlic, allicin is known to degrade very fast compared to chemicals (Baidoo & Mochiah, 2016). This study showed that there was no statistically signifi- cant variations in yield among treatments (p > 0.05). As shown earlier, T1, T4 and T6 had significantly lower in- tesity of DBM attack, which may influence good growth and development of cabbages. However, data shows that the lowest yield were in T0 and T5. Further analysis re- vealed that there was significant difference in yield be- tween T0 with T1 and T6, which may be attributed by the differences in the intensity of DBM infestation (Table 1). In comparison, Baidoo and Mochiah (2016) found that yield of plots sprayed with garlic were significantly high- er compared to the control (Baidoo & Mochiah, 2016). 4 CONCLUSION It can be concluded that using garlic extract as lo- cal biopesticide can effectively repel diamondback moth. Therefore, this study recommends that it can be used as an alternative to using synthetic chemicals to control Table 1: Average number of affected leaves per plants before and after treatments and efficiency of biopesticides in cabbage Types of treatments Average number of leaves  Average number of affected leaves Efficacy (%)Before After New leaves after treatment  Before After Net affected leaves P-value T0 10.03 14.87 4.83 2.67 4.13 1.47 Reff Reff T1 10.47 15.20 4.73 2.47 3.70 1.23 0.382 15.9 T2 9.87 15.00 5.13 2.00 3.77 1.77 0.154 -20.5 T3 8.63 13.60 4.97 1.47 3.03 1.57 0.002 -6.8 T4 9.70 14.57 4.87 1.77 2.87 1.10 0.003 25.0 T5 9.10 14.37 5.27 1.30 2.60 1.30 < 0.001 11.4 T6 9.47 14.93 5.47 1.73 2.57 0.83 0.001 43.2 Figure 1: Average number of affected leaves per plant before and after treatment Acta agriculturae Slovenica, 119/4 – 2023 5 Effectiveness of local biopesticides in the control of Diamondback Moth (Plutella xylostella L.) ... diamondback moth in cabbage crop. Further studies are needed to confirm the effectiveness of biopesticides to control plant pests for other horticultural products in different planting seasons. 4.1 FUNDING Funding for this study was obtained under Zanzibar Agricultural Research Institute (ZARI). 4.2 DATA AVAILABILITY The data to support this findings are available from authors upon special request. 4.3 CONSENT FOR PUBLICATION Not applicable. 4.4 COMPETING INTERESTS The authors declare no conflict of interest. 4.5 ACKNOWLEDGEMENTS We would like to acknowledge the research staff, field workers and the data collectors without whom the present study would have not been possible. 5 REFERENCES Akutse, K., Subramanian, S., Maniania, N., Dubois, T., & Eke- si, S. (2020). Biopesticide Research and Product Develop- ment in Africa for Sustainable Agriculture and Food Secu- rity – Experiences From the International Centre of Insect Physiology and Ecology (icipe). 4. https://doi.org/10.3389/ fsufs.2020.563016 Baidoo, P., & Mochiah, M. (2016). Comparing the effective- ness of garlic (Allium sativum L.) and hot pepper (Capsi- cum frutescens L.) in the management of the major pests of cabbage (Brassica oleracea (L.). Sustainable Agriculture Research, 5, 83. https://doi.org/10.5539/sar.v5n2p83 Batool Syeda, H., & Butt, S. (2021). Repellent activity of extracts of black pepper, black seeds, garlic and white cumin against red flour beetle. American Scientific Research Journal for En- gineering, Technology, and Sciences, 84(1), 153-161. Elmadawy, A. A., Omar, A. F., & Ismail, T. (2023). Bags impreg- nated with garlic (Allium sativum L.) and parsley (Petrose- linum crispum (Mill.) Fuss) essential oils as a new biope- sticide tool for Trogoderma granarium Everts, 1898 pest control. Acta agriculturae Slovenica, 119(1), 1–13. https:// doi.org/10.14720/aas.2023.119.1.2707 Fenibo, E. O., Ijoma, G. N., & Matambo, T. (2021). Biopesticides in Sustainable Agriculture: A Critical Sustainable Develop- ment Driver Governed by Green Chemistry Principles. 5. https://doi.org/10.3389/fsufs.2021.619058 Hardiansyah, M. Y., & Al Ridho, A. F. J. I. J. o. A. R. (2020). The effect of garlic (Allium sativum) extract pesticides in repelling rice eating bird pests. 3(3), 145-152. https://doi. org/10.32734/injar.v3i3.3947 Hill, T. A., & Foster, R. E. (2000). Effect of insecticides on the diamondback moth (Lepidoptera: Plutellidae) and its para- sitoid Diadegma insulare (Hymenoptera: Ichneumonidae). Journal of Economic Entomology, 93(3), 763-768. https:// doi.org/10.1603/0022-0493-93.3.763 Karavina, C., Mandumbu, R., Zivenge, E., & Munetsi, T. J. J. A. R. (2014). Use of garlic (Allium sativum) as a repellent crop to control diamondback moth (Plutella xylostella) in cab- bage (Brassica oleraceae var. capitata). 52(4). Khursheed, A., Rather, M. A., Jain, V., Wani, A. R., Rasool, S., Nazir, R., . . . Majid, S. A. (2022). Plant based natural products as potential ecofriendly and safer biopesticides: A comprehensive overview of their advantages over con- ventional pesticides, limitations and regulatory aspects. Mi- crobial Pathogenesis, 173, 105854. https://doi.org/10.1016/j. micpath.2022.105854 Magierowicz, K., Górska-Drabik, E., & Golan, K. (2020). Effects of plant extracts and essential oils on the behavior of Ac- robasis advenella (Zinck.) caterpillars and females. Journal of Plant Diseases and Protection, 127(1), 63-71. https://doi. org/10.1007/s41348-019-00275-z Mahenya, O., Aslam, M. J. S., Tourism: Destinations, A., & Cui- sines. (2014). Rediscovering Spice Farms as Tourism At- tractions in Zanzibar, a Spice Archipelago. 38, 97. https:// doi.org/10.21832/9781845414443-008 Moshi, A. P., & Matoju, I. (2017). The status of research on and application of biopesticides in Tanzania. Review. Crop Protection, 92, 16-28. https://doi.org/10.1016/j.cro- pro.2016.10.008 Rusdi, & Rusaldy, A. (2023). The effectiveness of fragrant bi- opesticide of lemon cigarette biopesticide to control fruit flies on large chilli plants. IOP Conference Series: Earth and Environmental Science, 1153, 012030. https://doi. org/10.1088/1755-1315/1153/1/012030 Table 2: Average mass of cabbage after harvest Types of treatments Mass (Kg) T0 0.46 T1 0.63 T2 0.54 T3 0.51 T4 0.58 T5 0.47 T6 0.64 Acta agriculturae Slovenica, 119/4 – 20236 A. G. KHAMIS et al. Scholtz, M., Voldner, E., McMillan, A., & Van Heyst, B. J. A. E. (2002). A pesticide emission model (PEM) Part I: model development. 36(32), 5005-5013. https://doi.org/10.1016/ S1352-2310(02)00570-8 Stathas, I. G., Sakellaridis, A. C., Papadelli, M., Kapolos, J., Papadimitriou, K., & Stathas, G. J. (2023). The effects of insect infestation on stored agricultural products and the quality of food. 12(10), 2046. https://doi.org/10.3390/ foods12102046 Acta agriculturae Slovenica, 119/4, 1–11, Ljubljana 2023 doi:10.14720/aas.2023.119.4.12497 Review article / pregledni znanstveni članek Herbicides weed management in changing environmental conditions Zvonko PACANOSKI 1, 2, Arben MEHMETI 3 Received February 23, 2023; accepted November 11, 2023. Delo je prispelo 23. februarja 2023, sprejeto 11. novembra 2023 1 Ss. Cyril and Methodius University, Faculty for Agricultural Sciences and Food, Department of Plant production, Skopje, R. North Macedonia 2 Corresponding author, e-mail: zvonkop@zf.ukim.edu.mk 3 University of Prishtina, Faculty of Agriculture and Veterinary, Department of Plant Protection, Prishtina, R. Kosovo Herbicides weed management in changing environmental conditions Abstract: Elevate CO2 levels in the atmosphere might have prominent effects on weed phenology, consequently chang- ing herbicide performance on weeds. Increased atmospheric CO2 concentration increase leaf thickness and reduce stomatal number and conductance potentially reducing the absorption of POST–emergence applied herbicides. From the other side, higher temperature stimulates stomata conductance, reduce the viscosity of epicuticle waxes, thus increasing the penetra- tion and diffusion of herbicides as a result of changes in the composition and the permeability of the cuticle. However, in some circumstances higher temperatures might cause hastened metabolism, which consequently decreases herbicide activity on target plants. In conditions of higher RH, cuticle hydration and stomatal conductance increases, consequently increases the permeability and translocation particularly of hydrophilic herbicides into the leaves. Similar, under higher irradiance, stomata stay open, photosynthetic rate increases consequently increasing absorption, penetration and subsequent phloem translocation of POST–em systemic herbicides in weed tissue. Drought might cause increased cuticle thickness and increased leaf pubescence, with consequent reductions in herbicide ab- sorption into the leaves. Rainfall after POST–emergence herbi- cides application might reduce their efficiency through washing out. Increased frequency and intensity of precipitation will have a negative effect on absorption, translocation, and activity of PRE–emergence herbicides. Key words: environmental conditions, weeds, control, herbicides Uravnavanje plevelov s herbicidi v razmerah spreminajočega se okolja Izvleček: Povečane koncentracije CO2 v ozračju bi lahko imele znatne učinke na fenologijo plevelov kar bi posledično lahko spremenilo učinkovanje herbicidov nanje. Povečane kon- centracije CO2 v ozračju povečujejo debelino listov in zmanjšu- jejo število rež, kar potencialno zmanšuje njihovo prevodnost in potencialno zmanjšuje absorbcijo POST–em nanešenih her- bicidov. Po drugi strani višje temperature pospešujejo prevo- dnost rež in zmanjšujejo viskoznost epikutikularnih voskov in s tem povečujejo penetracijo in difuzijo herbicidov kot posledico sprememb v sestavi in prevodnosti kutikule. V nekaterih razme- rah lahko višje temperature pospešijo presnovo, ki posledično lahko zmanjša aktivnost herbicidov na tarčnih rastlinah. V raz- merah večje relativne zračne vlažnosti se povečata hidratacija kutikule in stomatarna prevodnost kar posledično poveča per- meabilnost in translokacijo, še posebej hidrofilnih herbicidov v liste. Podobno v razmerah večjega obsevanja ostajajo reže dalj časa odprte, povečana fotosinteza posledično poveča absorbci- jo, penetracijo in translokacijo sistemskih POST-em herbici- dov po floemu v tkiva plevelov. Suša lahko povzroči povečanje debeline kutikule in dlakavosti listov kar posledično zmanjša absorbcijo herbicidov vanje. Dež lahko po nanosu POST–em herbicidov zmanjša njihovo učinkovitost zaradi izpiranja. Po- večana pogostost in jakost padavin imata negativni učinek na absorbcijo, translokacijo in aktivnost PRE–em herbicidov. Ključne besede: okoljske razmere, pleveli, nadzor, herbi- cidi Acta agriculturae Slovenica, 119/4 – 20232 Z. PACANOSKI and A. MEHMETI 1 INTRODUCTION Agriculture production in terms of quantity and quality, as well as agronomic practices, including weed management, may be affected significantly in conditions of climate change (Varanasi et al., 2016). Elevating CO2 levels associated with changes in temperature and pre- cipitation are important concerns for upcoming weed management and crop production. Taking into account the greater physiological flexibility (Ziska et al., 2010; Davidson et al., 2011; Billore, 2019) and their greater in- tra specific genetic variation (Dukes & Mooney, 1999), weeds are expected to show greater competitiveness and better accommodation regarding increasing CO2 con- centrations and temperature in comparison with crops (Singh et al., 2011; Varanasi et al., 2016). Considering its positive effect on weed growth, shifting environmen- tal conditions will impact directly or indirectly on the weed control methods by reducing their effectiveness on weeds and making them a considerable issue for sustain- able agriculture production as well as costlier in same time (Ziska et al., 1999; Karl et al., 2009). Climatic vari- ation factors are estimated to have significant effects on the growth and physiological processes of weedy plants, like growing rate, stomatal conductance, and photosyn- thetic efficiency (Fuhrer, 2003; Manisankar & Ramesh, 2019). Elevate CO2 and temperature, sunlight intensity, relative humidity, rainfall, and drought influence the cov- erage, penetration, translocation, persistence and activ- ity of herbicides (Muzik, 1976; Hatzios & Penner, 1982; Bailey, 2003; Bailey, 2004; Malarkodi et al., 2017). Addi- tionally, interactions among these environmental factors may have uncertain consequences on herbicide efficacy (Sutherland et al., 2017). Numerous studies confirmed that shifting climate conditions might also decrease the susceptibility of weeds to some herbicides (Varanasi et al., 2016; Ziska, 2016; Fernando et al., 2016; Matzrafi et al., 2018). For example, elevated CO2 reduced the ef- ficacy of glyphosate and glufosinate against Cirsium ar- vense (L.) Scop., and Elytrigia repens (L.) Desv.ex Nevski (Ziska & Teasdale, 2000). Similarly, Manea et al. (2011) reported that glyphosate efficacy at increased CO2 con- centrations is diminished in C4 weeds such as Eragostis curvula (Schrad.) Nees, Paspalum dilatatum Poir., and Chloris gayana Kunth, as a result of increased leaf area and total plant biomass. Higher temperatures may worse the consistency of cuticular lipids, thereby increasing the absorptivity and penetration of herbicides through the cuticle (Price, 1983; Patterson et al., 1999); for ex- ample, uptake and translocation of 14C-glyphosate was found to be higher at 22 °C than at 16 °C in Desmodium tortuosum (Sv.) DC. (Sharma & Singh, 2001). Although tendency of elevated air temperatures is to increase ab- sorption and translocation of most POST-em applied herbicides (Patterson et al., 1999), in some cases higher temperatures also might encourage rapid metabolism, which consequently decreases herbicide efficacy on tar- get plants (Kells et al., 1984; Madafiglio et al., 2000; Medd et al., 2001; Johnson & Young, 2002). Increased CO2 and temperature might change weed growth phenology, with shortened the period spent in the seedling stage, i.e. the stage of greatest POST-em herbicide efficacy (Ziska et al., 1999). Also, changes in these factors caused alteration in leaf morphology, leaf surface characteristics or variation in root-to-shoot ratio which affect herbicide absorption, distribution and efficacy (Olesen & Bindi, 2002; Poorter & Navas, 2003; Ziska et al., 2004; Dukes et al., 2009). Ad- ditionally, enhance in tuber and rhizome growth, joined with enhance in biomass, particular in perennial weeds (Oechel & Strain, 1985), would induce a dilution effect on any herbicide treatment (Patterson, 1995), making their control more complicated (Patterson et al., 1999). Modifications in environmental factors, such as drought spells or prolonged rainy periods, might restrict the field conditions necessary for optimal herbicide applications (Amare, 2016). Generally, dry soil conditions decrease the activityof PRE-em herbicides, affect their behavior in the soil and the herbicide effectiveness “windows” due to strong herbicide adsorption (Bailey, 2004; Howden et al., 2007), whereas severe or frequent rainfall after the appli- cation may cause herbicide leaching (Soukup et al., 2004; Pacanoski & Mehmeti, 2021) and dilution (Kanampiu et al., 2003). 2 INTERACTION CO2 – HERBICIDE EFFI- CACY The importance of interaction CO2 concentra- tion - herbicide efficacy has occupied research attention in recent decades as a result of the constant increase in concentrations of atmospheric CO2. Elevate CO2 lev- els in the atmosphere might have prominent effects on weed phenology (Anwar et al., 2021), consequently al- tering herbicide effectiveness on weeds (Ziska, et al., 1999; Ziska & Teasdale, 2000; Ziska et al., 2004; Ziska & Runion, 2007). One of the most pronounced effects of increased CO2 concentrations is the minimizing of sto- matal conductance, which could increase up to 50 % in some weeds (Bunce, 1993). Minimized number and sto- matal conductance with increasing CO2 could decrease transpiration resulting in decreased herbicide absorption and efficacy, particularly of POST-em applied herbicides (Bunce & Ziska, 2000; Ziska & McClung, 2008; Ziska, 2008). Additionally, Nowak et al. (2004) and Ainsworth & Long (2005) indicated that C3 and C4 weeds grown Acta agriculturae Slovenica, 119/4 – 2023 3 Herbicides weed management in changing environmental conditions in condition of increased CO2 concentrations have in- creased leaf pubescence and developed thicker cuticle. Apart from increasing leaf thickness, increased CO2 con- centrations might also generate partially stomatal closure (Ziska, 2008; Jackson et al., 2011). These characteristics might minimize up take and efficacy of POST-em applied herbicides. Manea et al. (2011) found that in three of four C4 grass species tolerance to glyphosate in conditions of raised CO2 is significantly increased. Similar results were obtained by Ziska & Goins (2006). The explanations for the minimized efficacy of the herbicides could be that el- evating CO2 increase leaf consistency and reduce stoma- tal number and their conductivity potentially reducing the absorption of POST-em applied herbicides. Further- more, an increase in the apparent photosynthesis rates as a result of increased CO2 concentrations, mainly in C3 weeds, might cause rapid seedling growth, the most susceptible stage for optimal weed control, which could modify the efficacy of POST-em herbicides. For example, Chenopodium album L., a C3 weed, demonstrated higher tolerance to glyphosate as a result of increased growth and plant biomass at raised CO2 concentration (Ziska et al., 1999). In addition, perennial weeds may become even more troublesome, if vegetative growth is stimulated as a result of increased photosynthesis in relation to elevated CO2. This could be due to less herbicide translocation as the root system becomes more vigorous. In this context, Elymus repens (L.) Gould (Ziska & Teasdale, 2000) and Cirsium arvense (Ziska et al., 2004) showed prominent tolerance to glyphosate due to elevated CO2 levels, which caused large stimulation of belowground growth. Elevate CO2 levels increases concentration of starch in leaf tis- sue (Patterson, 1995), particularly in C3 weeds (Wong, 1990), but reduce protein concentration (Bowes, 1996; Taub et al., 2008; Loladze, 2014). Reduction of protein content results to diminished demand for aromatic and branched-chain amino acids synthesis, which may re- duce the efficacy of many herbicides, including ALS and EPSPS inhibitors (Patterson et al., 1999; Varanasi et al., 2016). Changed environmental conditions, particularly rising of CO2 concentration and temperatures, stimulate weed growth through modification of photosynthesis, pigment production, as well as overall metabolic activ- ity. Because of that, herbicides photosystem I and II and pigment inhibitors may become more effective. However, the effects of rising CO2 on herbicide efficacy is species determined. Namely, at double atmospheric CO2 con- centrations, efficacy of metsulfuron on Amaranthus ret- roflexus L. decreased by 4.6 %, efficacy of imazethapyr in control of Stellaria media (L.) Vill. was unchanged, whereas efficacy of imazamethabenz-methyl over Avena fatua L. improved by 15.7 % (Archambault et al., 2001). According same authors, in the same conditions, efficacy of linuron in control of Polygonum convolvulus L. was re- duced by 15 %, whereas status quo in the efficacy was reported for metribuzin on Chenopodium album L. and bromoxynil on Kochia scoparia (L.) Schrad., respectively. The effects of rising CO2 on ACCase inhibitors varied and depend on weed species. At double–environment CO2 concentrations clodinafop efficacy in control of Ave- na fatua increased by 8.6 %, while Avena fatua L. control was not affected by sethoxydim. No change in the efficacy was reported for control of Avena fatua and Setaria vir- idis (L.) P.Beauv.by fluazifop (Archambault et al., 2001). Further, decreasing of clopyralid efficacy for 8.9 % was noted in control of Senecio vulgaris L., whereas increas- ing of efficacy of 2,4-D for 26.9 % was obtained in control of Polygonum convolvulus L. (Archambault et al., 2001). Increased frequency of herbicide applications might ex- ceed CO2 caused declines in efficacy, but might bring additional risks for human and animal health because it might increase the occurrence and concentration of these chemicals in the environment (Ziska et al., 2004). 3 INTERACTION TEMPERATURE – HERBI- CIDE EFFICACY Temperature has multiple impacts on weed growth and development as well as herbicide efficacy. Alterations in the apparent photosynthesis rate, respiration, phloem translocation, and protoplasmic flux, as well as rate of water up take and transpiration, leaves formation, cu- ticle compactness and hydration, number and aperture of stomata will affect uptake, diffusion, and metabolism of herbicides (Bailey, 2004; Zanatta et al., 2008; Roden- burg, et al., 2011). Higher temperature encouraged sto- mata conductivity, reduced the viscosity of cuticle waxes, thus increasing the uptake and diffusion of herbicides as a result of modifications in the structure and the perme- ability of the cuticle (Price, 1983; Chandrasena, 2009). Abutilon theophrasti Medik. plants treated with acifluor- fen at lower (20/15 oC) day/night temperature regime showed 70 % higher production of epicuticular wax on the leaf surface than plants in condition of higher (32/22 oC) day/night temperature. Decreasing of the wax pro- duction was connected with better efficacy of herbicides when temperature increased, corroborating the assump- tion of higher herbicide efficiency as cuticle structure al- tered. This study also confirmed that when temperature increased from 20/15 oC to 32/22 oC, there was a 25 % increase in the acifluorfen absorption applied alone and 99 % increase in acifluorfen absorption applied with oil– based surfactant (Hatterman-Valenti et al., 2011). Ganie et al. (2017) noted that the efficacy of 2,4-D and glypho- sate in control of Ambrosia artemisiifolia L. and Ambrosia Acta agriculturae Slovenica, 119/4 – 20234 Z. PACANOSKI and A. MEHMETI trifida L. might be enhanced if applied at higher (29/17 oC) day/night temperature regime, because of improved absorption and translocation compared with applications during lower (20/11 oC) day/night temperatures. Study in greenhouse conditions using different night/day temper- atures (5/10 °C, 15/20 °C, and 20/25 °C) demonstrated that Raphanus raphanistrum L. grown in conditions of lower (5/10 °C) temperatures was poorly controlled with 1,200 g ai ha–1 of glufosinate. Contrary, 100 % mortal- ity was achieved under higher temperatures15/20 °C and 20/25 °C, respectively for the same dose (Kumaratilake & Preston, 2005), indicating increased efficacy of glufosi- nate under increased air temperature. Flumiclorac exhib- ited higher activity on Amaranthus retroflexus (threefold) and Chenopodium album (sevenfold) with increasing of temperatures from 10 °C to 40 °C (Fausey & Renner, 2001). Johnson & Young (2002) reported for threefold increase in mesotrione efficacy in control of Abutilon theophrasti and Xanthium strumarium L. with increasing of temperatures from 18 °C to 32 °C. Similarly, at higher temperature, fluthiacet was twice and three times more effective in control of Amaranthus retroflexus and Che- nopodium album, respectively, compared to the efficacy observed at 10 oC (Fausey & Renner, 2001). Atrazine ap- plied at 15:00 h, when the air temperature was the high- est, provided the greatest control of Ambrosia artemisii- folia L. and Abutilon theophrasti (Stewart et al., 2009). Stopps et al. (2013) confirmed that glyphosate efficacy in control of Ambrosia artemisiifolia L. and Abutilon theo- phrasti, Amaranthus spp., increased when herbicide was applied between noon and 6 pm, which coincides to the higher air temperatures during the day. Contrary, the ef- ficacy of bromoxynil on Abutilon theophrasti declined by up to 45 % when applied at 24:00 h, when the air tem- perature was the lowest (Stewart et al., 2009). Irrespective of temperature increase, dicamba/diflufenzopyr provid- ed > 95 % control of Amaranthus retroflexus, and Am- brosia artemisiifolia, Chenopodium album. On the other hand, lower temperatures reduced control of Abutilon theophrasti by 7 % to 15 % (Stewart et al., 2009). Similar, in research of Ziska et al. (1999) glyphosate efficacy was reduced in control Ambrosia trifida and Ambrosia arte- misiifolia at low temperatures. Although the tendency of higher atmospheric tem- peratures is to enhance absorption and translocation of most POST-em applied herbicides, in some circumstanc- es higher temperatures might cause hastened metabo- lism, which consequently decreases herbicide activity on target weeds (Johnson & Young, 2002). Enhanced me- tabolism rate was the reason for reduction of pinoxaden efficacy on Brachypodium hybridum (L.) P. Beauv. con- trol and other grasses in conditions of higher tempera- ture (Matzrafi et al., 2016). Ou et al. (2018) tested effects of temperature on Kochia scoparia (L.) Schrad. growth treated with glyphosate and dicamba under three day/ night temperatures: 17.5/7.5 °C; 25/15 °C; and 32.5/22.5 °C. Visual above‐ground dry biomass, injury and mortal- ity data indicated greater sensitivity to both glyphosate and dicamba when Kochia scoparia was grown in con- ditions the two cooler day/night temperature regimes. Similar trend was noted in investigation of Kleinman et al. (2016) when Conyza bonariensis (L.) Cronq., Co- nyza canadensis (L.) Cronq., and Kochia scoparia were treated with glyphosate. A significant variation in con- trol of Amaranthus palmeri S. Watson with mesotrione was obtained when the weed was grown in conditions of low and high day/night temperature regimes (25/15 °C and 40/30 °C, respectively) compared to optimum day/ night temperature (32.5/22.5 °C). Related to weed height, injury, and mortality, Amaranthus palmeri S. Watson was more susceptible to mesotrione at 25/15 °C and less susceptible at 40/30 °C compared to 32.5/22.5 °C (Godar et al., 2015). Pyrithiobac provided higher efficacy in con- trol of Amaranthus palmeri at 18 oC (25 % dry mass ac- cumulation) than at 40 oC (70 % dry mass accumulation), although the highest efficacy was recorded at 27 oC (only 2.5 % dry weight accumulation) (Mahan et al., 2004). Mesotrione efficacy in control of Digitaria sanguinalis (L.) Scop. and Amaranthus rudis J.D.Sauer decreased by six and seven times when temperature increased from 18 °C to 32 °C (Johnson & Young, 2002). Increased tem- peratures as well as increased metabolic activity of the weeds nullify increased herbicide translocation, because herbicide metabolisation increases at higher tempera- ture, as well (Martini et al., 2015; Matzrafi et al., 2016). Higher temperatures also might generate diminishing of herbicide absorption due to quick drying of spray drop- lets to solid deposits (Devine et al., 1993) and volatility of some herbicides, such as growth regulators herbicides causing in vapor drift and possible injury on non target broadleaf crops (van Rensburg & Breeze, 1990; Strachan et al., 2010). Further, soil temperature has an effect on the ab- sorption and translocation of PRE-em herbicides within the weed plant, as well as their persistence in the soil (Ro- denburg et al., 2011). Warmer soil temperatures might reduce efficacy of PRE-em herbicides through rising volatility and degradation by soil microorganisms. For example, higher temperature had a great impact on the volatilization of the triallate from the soils. According Atienza et al. (2001) triallate losses increased from 7 % to 41 % in loamy soil and 14 % to 60 % in sandy soil, respectively with rising temperatures from 5 °C to 25 °C. Opposite, in the controlled trial conditions, low soil tem- peratures (around 10 °C) decreased the efficacy of ala- chlor and EPTC (Mulder & Nalewaja, 1978). Acta agriculturae Slovenica, 119/4 – 2023 5 Herbicides weed management in changing environmental conditions 4 INTERACTION RELATIVE HUMIDITY – HERBICIDE EFFICACY Relative humidity (RH) is mainly important for the activity of POST-em herbicides through its effects on herbicide absorption, including interactions between the herbicide droplets, leaf cuticle, and accessibility of water in or round droplets (Devine et al., 1993). In conditions of higher RH, cuticle hydrating and stomatal conductiv- ity increases, consequently increases the penetrability and translocation particularly of hydrophilic herbicides into the leaf surface (Kudsk et al., 1990; Wichert et al., 1992; Shaw et al., 2000; Hatterman-Valenti et al., 2011). Penetration as well as efficacy of most POST-em her- bicides is usually higher when weeds were exposed to higher RH after spraying than before, concluding that slowly droplets drying might be the reason for higher efficacy at higher RH levels rather than cuticle hydrat- ing (Ramsey et al., 2002). The susceptability of Digitaria sanguinalis and Amaranthus rudis to mesotrione was two and four–times higher at 85 % RH compared with 30 %, respectively (Johnson & Young, 2002). Glufosinate am- monium efficacy in control of Avena fatua significantly increased (> 95 %) at higher RH compared with its effica- cy at lower (40 %) RH. Additionally, penetration of glu- fosinate ammonium was higher when Avena fatua plants were exposed to higher RH for 30 min before and after application compared with those left at constantly lower RH (Ramsey et al., 2002). Efficacy of acifluorfen on Am- brosia artemisiifolia and Xanthium strumarium was 30 % higher when it was applied at 85 % RH compared with its efficacy at 50 % RH (Ritter & Coble, 1981). Likewise, acifluorfen, fomesafen, and lactofen provided higher ef- ficacy in control of Ipomoea lacunosa L., Ipomoea hedera- cea Jacq. var. integriuscula, Sida spinosa L., and Xanthium strumarium at 85 % RH, compared to the condition of 50 % RH (Wichert et al., 1992). Similarly, when the efficacy of acifluorfen was estimated on trials carried–out for two consecutive years, it was concluded that there was higher control of Xanthium strumarium obtained in the year of higher RH condition (Shaw et al., 2000). Casley & Cou- pland (1985) stated that higher RH increased glyphosate performance due to slower evaporation from the plant surface, while Mathiessen & Kudsk (1996) claimed that higher RH had no significant influence on glyphosate ef- ficacy. 5 INTERACTION SUNLIGHT INTENSITY – HERBICIDE EFFICACY Alterations in sunlight intensities influence on the plants anatomy, morphology, and physiology, which con- sequently have an effect on herbicide performance in the plants. Stomatal conductivity and formation of leaf cuticle are positively correlated with sunlight intensity (Hull et al., 1975; Raschke et al., 1978). Under conditions of higher irradiation, stomata stay open, photosynthetic rate increases consequently increasing uptake, penetra- tion and subsequent phloem translocation of POST-em applied herbicides in weed plant tissue (Fausey & Renner, 2001; Hwang et al., 2004; Camargo et al., 2012). Efficacy of clethodim, talkoxydim and bentazon proportionally increased with increasing of sunlight intensity (McMul- lan, 1996, Hatterman-Valenti et al., 2011). In study of Fausey & Renner (2001) flumiclorac provided nine times higher control of Chenopodium album at light intensity of 1,000 μmol m–2 s–2 than at 4 μmol m–2 s–2. Control of Amaranthus retroflexus was 15 times more effective with the same herbicide under higher light intensity compared to the lower one. In same study, fluthiacet was also more effective in control of these two species at irradiance condition of 1000 μmol m–2 s–2, as compared to the ef- ficacy obtained at 4 μmol m–2 s–2. Similar, oxadiazon and oxadiargyl reduced the growth of Echinochloa crus–galli (L.) P.Beauv. in the presence of light, but were completely ineffective in the dark (Hwang et al., 2004). UV light re- duced the efficacy of tralkoxydim and clethodim, which indicates that application of these graminicides when sunlight intensity is higher during the day might increase their efficacy. Filtering UV light for 4 h after application improved efficacy of these herbicides between 13 and 55 % (McMullan, 1996). UV light is obviously significant to cyclohexanedione herbicide efficacy because these her- bicides are unstable in UV light (Campbell & Penner, 1985; Falb at al., 1990; McInnes et al., 1992). Similar, 14C- paraquat penetration and efficacy in control of Abutilon theophrasti, Chloris virgata Sw. and Digitaria sanguinalis was reduced during the UV-B treatment because of in- creasing leaf epicuticular wax deposition (Wang et al., 2006). On the other hand, in lower irradiance conditions, tendency of plants is to form thinner leaves with greater specific leaf surface and plant height to catch accessible sunlight required for photosynthesis. These adjustments Acta agriculturae Slovenica, 119/4 – 20236 Z. PACANOSKI and A. MEHMETI in weed growth and leaf mofphology determinate the herbicide amount that is received and retained by the weed (Upasani & Barla, 2018). For example, surface cov- erage as well as absorption of POST-em herbicides is en- hanced in weed with higher branching, whereas leaves with thicker structure retard herbicides penetration causing decreased herbicide efficacy (Riederer & Schon- herr, 1985). 6 INTERACTION DROUGHT AND RAIN- FALL PATTERN – HERBICIDE EFFICACY Herbicides might become less effective because of alteration of the external environment (drier and warm- er conditions) or alterations in anatomy, physiology, and phenology of the weed flora (Clements et al., 2014; Chau- han et al., 2014; Ziska & McConnell, 2015). In this con- text, POST-em herbicide efficacy might be significantly influenced by drought. Drought might cause enlarged cuticle thickness and intensify grow of leaf pubescence, with consequent reductions in herbicide penetration into the leaves (Patterson, 1995). For example, the weed cuti- cle under arid conditions was 50-80 % thicker relative to optimal available water situations (Hatterman-Valentiet al., 2011). Increasing aridity and drought might reduce herbicide penetration, intensify herbicide volatilization, and consequently reduce its effectiveness. Drought influ- enced weeds are more challenge for control with POST- em herbicides than weeds that are actively growing in conditions without environmental stress. For example, for systemic POST-em applied herbicides is necessary active weed growth to be effective. In that context, in conditions of drought spells efficacy of glyphosate in control of Abuthilon theophrasti was reduced two and eight–fold when it was applied in two and six leaves weed growth stages, respectively (Zhou et al., 2007). Survival of glyphosate-resistant biotype of Echinochloa colona (L.) Link treated with double glyphosate rate (1440 g ha-1) in condition of no water deficiency was only 19 %, but un- der water deficiency this value increased by 62 % (Mol- laee et al., 2020). Likewise, under dry soil conditions usually activity of PRE-em herbicides is reduced the due to strong herbicide soil adsorption (Arıkan et al., 2015). These herbicides are highly dependent on accessible wa- ter for relocation into the zone of weed seed germination (Olson et al., 2000). Herbicide photodecomposition is common process which takes place on the soil surface, and if optimal moisture does not become accessible in period of few days after application, weed control is of- ten inadequate. Even for considerably persistent herbi- cides, inability to penetrate into the soil surface because of the moisture shortage give weeds opportunity to ger- minate without any herbicide injuries. Jursık et al. (2013) claimed a reduced pethoxamid efficacy under dry soil conditions. Contrary, increased soil moisture promotes the efficacy of many, PRE-em herbicides, including PRO- TOX inhibitors (Hatterman-Valenti et al., 2011). Rainfall after POST-em herbicides application might reduce their efficiency through washing out. In- creased frequency and intensity of precipitation will have a negative effect on penetration, translocation, and activ- ity of PRE-em herbicides (Bailey, 2004; Rodenburg et al., 2011). An unusual increase in precipitation might cause leaching of PRE-em herbicides (Soukup et al., 2004; Pa- canoski & Mehmeti, 2021), and consequent crop injury (Pacanoski et al., 2020) and under soil water contamina- tion (Froud-Williams, 1996). From the other side, scarce rainfall amounts during the season might cause water– deficit conditions that impact herbicide efficacy (Zanatta et al., 2008; Keikotlhaile, 2011). For example, situations of water deficit reduced the absorption of acifluorfen (Hatterman-Valenti et al., 2011). Pereira et al. (2011) reported that Eleusine indica (L.) Gaertn. grown under water–stress conditions was not effectively controlled by sethoxydim. Similarly, control of Eleusine indica with fenoxaprop-p-ethyl, topramezone, foramsulfuron, 2,4-D + dicamba + MCPP + carfentrazone, and thiencarba- zone-methyl + foramsulfuron + halosulfuron-methyl at soil moisture contents < 12 % was unsatisfactory (Shekoo- fa et al., 2020). Urochloa plantaginea (Link) R.D.Webster grown under water–deficit stress was less susceptible to ACCase–inhibiting herbicides when applied during the later growth stages (Pereira, 2010). 7 MULTIPLE INTERACTIONS Atmospheric CO2 and air temperature elevate simultaneously. The result can be completely differ- ent when both factors are taken into account together in comparison when only one factor is considering. In weeds, alteration in temperatures and CO2 concentra- tions might modify net photosynthesis rates result- ing with modification in carbohydrate accessibility and stability causing in altered weed physiological and bio- chemical capabilities. Increased CO2 and atmospheric temperature might decrease herbicide efficacy by chang- ing herbicide penetration, translocation and metabolism, subsequently increasing herbicide decomposition in weeds and decreasing herbicide availability for the target weed (Matzrafi, 2019). For example, reduced glyphosate susceptibility was observed in Chenopodium album and Conyza canadensis in response to elevated temperature, (32/26 °C) combined with raised CO2 (720 ppm). Ac- cording obtained results by Matzrafi et al. (2019), 61.1 Acta agriculturae Slovenica, 119/4 – 2023 7 Herbicides weed management in changing environmental conditions %, 69.0 % and 64.0 %, respectively of the plants tested survived in conditions of mutual effects of higher tem- perature/elevated CO2 concentration. Further, the ef- ficacy of cyhalofop-butyl was reduced about 50  % in multiple–resistant Echinochloa colona plants grown un- der higher CO2 concentration (700 ± 50 ppm) or high (35/23 oC) day/night temperature regime compared to multiple–resistant plants at ambient conditions. Higher CO2 and temperatures increased the level of resistance to multiple–resistant E. colona to cyhalofop–butyl, as well (Refatti et al., 2019). Opposite, mutual effects of ambient CO2 concentration (400-450 ppm) and day/night tem- perature (20/10 °C) and increased CO2 concentrations (400-450 ppm, 800–900 ppm) and day/night tempera- ture (25/15 °C), did not reduce efficacy of glyphosate in control of Lactuca serriola L., Hordeum murinum L., and Bromus tectorum L. (Jabran & Doğan, 2018). Interaction between CO2 concentrations and water deficiency was studied by Weller et al. (2019). According their results, efficacy of glyphosate in control of glyphosate resistant and susceptible Chloris truncata R.Br. biotypes in condi- tion of moisture stress (50 % field capacity) and increased CO2 level (750 ppm) was significantly reduced. Few stud- ies have examined correlation between temperatures and RH. When higher temperatures are related with higher RH levels, there is increased cuticle hydrating, which consequently increases herbicides penetration and effi- cacy (Price, 1983). With simultaneous temperature and RH increasing, the efficacy of metribuzin also increased, while at lower temperatures (10 °C and 20 °C) caused no significant decreasing in its efficacy (Gealy & Buman, 1989). Opposite, glufosinate ammonium provided high- er efficacy on Setaria faberi Herrm. at higher RH as well as higher temperature (Anderson et al., 1993). 8 CONCLUSION The successfulness of weed management is pre- dicted to change together with the changing of environ- mental conditions. In conditions of rising CO2 and air temperature, and unpredictable drought spells and pro- longed rainfall forecasts, the possibility of herbicides ei- ther to generate crop injure or being ineffective at weed control is expected, as well. Elevated CO2 and tempera- tures might cause anatomical, morphological and physio- logical changes in weeds, their growth and development, all that could impact on absorption, translocation, and metabolism of herbicides and on the entire efficacy of herbicides. Conditions of soil water deficiency decrease the activity of PRE-em herbicides, affect their persistence in the soil and the “windows” for herbicide effectiveness due to strong herbicide adsorption, while severe or fre- quent rainfall after the application may cause herbicide leaching and dilution. One–sided and repeated herbicide use is estimated to result in appearance of resistant weed biotypes. Changes of environmental conditions might hasten this. In these circumstances, additional herbicide applications at higher rates might be needed to control such weeds, but additional activities increase the cost of control. Modification strategies are existing, but the expenditures of realizing such strategies (e.g. herbicide with new active ingredient, higher herbicide rates) are uncertain. Specific national legislation regulated herbi- cides use. In case of changing environmental conditions, which encourage weed species spreading out of their ge- ographical boundaries, new herbicidal active ingredient might be essential to control them effectively. Commonly it takes a lot of time to obtain state agreement for a new herbicide active ingredient or an active ingredient that is not been previously used locally. 9 REFERENCES Ainsworth, E. A., & Long, S. P. (2005). What have we learned from 15 years of free– air CO2 enrichment (FACE)? A meta–analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytology, 165, 351-371. https://doi.org/10.1111/j.1469- 8137.2004.01224.x Amare, T. (2016). Review on impact of climate change on weed and their management. Journal of Agricultural, Bio- logicaland Environmental Statistics, 2, 21–27. https://doi. org/10.11648/j.ajbes.20160203.12 Anderson, D.M., Swanton, C. J., Hall, J. C., Mersey, B. G. (1993). 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Weed Science, 47, 608-615. https://doi.org/10.1017/ S0043174500092341 Acta agriculturae Slovenica, 119/4, 1–12, Ljubljana 2023 doi:10.14720/aas.2023.119.4.15212 Review article / pregledni znanstveni članek Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphinema index Thorne & Allen, 1950 Julija POLANŠEK 1, 2, 3, Franci Aco CELAR 2, Saša ŠIRCA 1 Received July 26, 2023; accepted November 10, 2023. Delo je prispelo 26. julija 2023, sprejeto 10. novembra 2023 1 Kmetijski inštitut Slovenije, Ljubljana, Slovenija 2 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za agronomijo, Ljubljana, Slovenija 3 Korespondenčni avtor, e-naslov: julija.polansek@kis.si Non-chemical control options against the virus vector nema- todes Xiphinema index Thorne & Allen, 1950 Abstract: Nematodes are widespread organisms that ex- hibit remarkable ubiquity, biodiversity, and adaptability in di- verse ecosystems. While most nematodes are beneficial, there are also some parasitic species that have harmful effects. Among these plant-parasitic nematodes is the species Xiphinema index. It primarily colonizes the root environment of grapevines (Vitis vinifera L.), as these vines serve as its primary host. Although the nematode’s direct effects on roots are not particularly prob- lematic, it poses a significant threat to grapevine because it can transmit and introduce Grapevine Fanleaf Virus (GFLV), a member of the genus Nepovirus. Infection with GFLV can result in yield losses of over 80 %. Therefore, it is imperative to take preventive measures to contain the uncontrolled spread of nematodes and the resulting infections in vineyards. In ad- dition to transmission through planting material, agricultural machinery and implements in vineyards are also important vectors. Traditional chemical methods of controlling X. index have proven ineffective due to the nematodes’ resilience and widespread distribution in the soil. Future efforts should there- fore focus on pursuing alternative, more effective approaches. In addition to intercropping, the efficacy of bacterial and fungal preparations has also been tested and offers great potential for further research. Key words: virus-transmitting nematodes, Xiphinema in- dex, nepoviruses, Vitis vinifera, GFLV, nematicides, biological control Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphi- nema index Thorne & Allen, 1950 Izvleček: Ogorčice so organizmi, ki jih zaradi njihove številčnosti, raznolikosti in prilagodljivosti najdemo praktično povsod. Medtem ko je večina ogorčic koristnih, pa poznamo tudi take, ki s svojim parazitiranjem povzročajo škodo. Med škodljive ogorčice uvrščamo tudi rastlinsko-parazitsko vrsto Xiphinema index. Najdemo jo lahko v bližini korenin žlahtne vinske trte (Vitis vinifera L.), saj je prav ta njena glavna gosti- teljica. Ogorčica ni tako problematična z vidika neposrednega napada korenin, ampak vinski trti predstavlja grožnjo zaradi prenosa in vnosa virusa pahljačavosti listov vinske trte (GFLV) iz rodu Nepovirus. Virus namreč na vinski trti povzroči bolezen kužne izrojenosti vinske trte, kar vodi v ekonomsko nekonku- renčnost vinogradov. Okužba lahko privede tudi do več kot 80 % izpada pridelka. V izogib nenadzorovanemu širjenju ogorčic in posledično okužbam v vinogradih je pomembna preventiva, saj poleg prenosa s sadilnim materialom, pomembnega prena- šalca predstavljata tudi kmetijska mehanizacija in fizični pre- nos z orodjem. Ker se je kemično zatiranje ogorčice X. index zaradi njene trdoživosti in razporeditve v tleh izkazalo za neu- činkovito, je potrebno v prihodnje stremeti k alternativnim in predvsem učinkovitejšim pristopom. Poleg vmesnih posevkov so preverjali delovanje pripravkov na podlagi nekaterih bakterij in gliv, ki predstavljajo velik potencial za nadaljnja raziskovanja. Ključne besede: virusonosne ogorčice, Xiphinema index, nepovirusi, vinska trta, GFLV, nematocidi, biotično varstvo Acta agriculturae Slovenica, 119/4 – 20232 J. POLANŠEK et al. 1 UVOD Ogorčice so najbolj raznolike in najštevilčnejše predstavnice večceličnih organizmov, ki so prisotne tako v kopenskih kot tudi vodnih ekosistemih, in lahko pa- razitirajo večino rastlinskih in živalskih vrst (Smythe in sod., 2019; Lazarova in sod., 2021). Prav zaradi velike raznolikosti in fenotipske prilagodljivosti ocenjujejo, da se je tekom evolucije razvilo do približno milijona različ- nih vrst (Hugot in sod., 2001; Smythe in sod., 2019). Naj- številčnejše so v tleh živeče ogorčice (Lazarova in sod., 2021).Večina vrst talnih ogorčic je v kmetijstvu koristnih, saj prispevajo k razgradnji organske snovi v tleh in so pomemben člen v prehranjevalni verigi (Smiley, 2005). Vsaj polovica vseh ogorčic je prostoživečih (Baldwin in sod., 2000; Hugot in sod., 2001; Smythe in sod., 2019), pomembne pa so predvsem ogorčice, ki parazitirajo ži- vali in rastline (Archidona-Yuste in sod., 2020). Največji poudarek in večina raziskav je namenjenih prav parazit- skim ogorčicam, predvsem v povezavi z zdravstvom in kmetijstvom (Smythe in sod., 2019). Rastlinsko-parazit- ske ogorčice predstavljajo približno 15 % vseh trenutno znanih ogorčic (Wyss, 1997; Decreamer in Hunt, 2006; Wick, 2012; Archidona-Yuste in sod., 2020). V tleh živeče ogorčice povzročajo škodo na števil- nih vrtninah, okrasnih rastlinah, sadnem drevju in vinski trti, kar povzroča velike izgube na ravni svetovnega gos- podarstva. Ocenjujejo, da rastlinsko-parazitske ogorčice zmanjšajo količino kmetijskih pridelkov za 8,8–15 %, kar se odraža z izgubami od 90 do 160 milijard evrov po ce- lem svetu (Abad in sod., 2008; Nicol in sod., 2011; Jones in sod., 2013; Singh in sod., 2013; Singh in sod., 2015; Andret-Link in sod., 2017; Coyne in sod., 2018; Wernet in Fischer, 2023). Poleg fizičnih poškodb, ki jih ogorčice povzročajo na rastlinah, so lahko tudi prenašalke viru- sov ter zaradi povzročenih poškodb na rastlinskem tkivu vplivajo tudi na intenzivnejši vdor fitopatogenih bakterij in gliv (Garcia in sod., 2019; Hajji-Hedfi in sod., 2019; Wernet in Fischer, 2023). Ena pomembnejših virusnih bolezni vinske trte je kompleks kužne izrojenosti vinske trte. To bolezen po- vzroča 15 virusov iz družine Secoviridae, ki jih prenašajo talne ektoparazitske vrste ogorčic iz rodov Longidorus, Paralongidorus in Xiphinema, ki so uvrščeni v družino Longidoridae (Andret-Link in sod., 2004; Martelli in Boudon-Padieu, 2006). Med temi ogorčicami ima naj- večji vpliv na pridelavo vinske trte vrsta Xiphinema in- dex Thorne & Allen, 1950, ki je prenašalka virusa pahl- jačavosti listov vinske trte (GFLV) (Andret-Link in sod., 2004). Med več kot 4.100 znanimi vrstami rastlinsko-pa- razitskih ogorčic je vrsta X. index uvrščena na 8. mesto na seznamu 10 najpomembnejših ogorčic glede na njihov znanstveni in gospodarski pomen (Jones in sod., 2013; Andret-Link in sod., 2017). Tako ogorčica X. index, kot virus GFLV sta od leta 2019 na seznamu nadzorovanih nekarantenskih škodl- jivih organizmov v Evropski uniji (EPPO, 2023). V Slo- veniji je bila prisotnost ogorčice X. index prvič potrjena leta 1978 (Hržič, 1978), kasneje je bila najdena in preu- čevana v obdobju med letoma 2002 in 2004, ko so njeno najdbo potrdili v Vipavski dolini in na Krasu (Urek in Širca, 2005). Prisotnost X. index je zaenkrat omejena le na Primorski vinorodni okoliš, številčnost populacije pa je v nekaterih vinogradih zelo visoka, tudi več kot 1000 osebkov na 1 kg vinogradniške zemlje (Širca in Theuer- schuh, 2020). 2 RASTLINSKO PARAZITSKE OGORČICE Ogorčice uvrščamo v deblo Nematoda Cobb, 1932. Na podlagi morfoloških raznolikosti so med ogorčicami določili dve glavni skupini, kopenske ogorčice – Secer- nentea in vodne ogorčice – Adenophorea (Smythe in sod., 2019). Sledile so molekularne analize 18S rDNA, ki so filogenetsko drevo oblikovale v tri glavne skupine oz. podrazrede: Chromadoria, Dorylaimia in Enoplia (De Ley, 2006; Blaxter in sod., 2011; Smythe in sod., 2019). Več kot 4.100 vrst je prepoznanih in identificiranih kot rastlinsko-parazitskih ogorčic, kar predstavlja nekje 15 % vseh trenutno znanih ogorčic (Wyss, 1997; Decreamer in Hunt, 2006; Wick, 2012; Archidona-Yuste in sod., 2020). Kot navaja Smythe s sodelavci (2019), fitoparazitske oz. rastlinsko-parazitske ogorčice uvrščamo v dva reda – Rhabditida (podred Tylenchina) in Dorylaimida (po- dred Dorylaimina), medtem ko De Ley (2006) dodaja še red Triplonchida (podred Diphtherophorina). Ogorčice, ki se prehranjujejo kot fitofagi oz. rastlin- ski paraziti, se lahko hranijo na različne načine (Slika 1) (Urek in Hržič, 1998; Vieira in Gleason, 2019): - ektoparazitsko (črpanje rastlinskih sokov z vbo- dom v rastlinsko tkivo od zunaj), - semiendoparazitsko (sesanje rastlinskih sokov z delnim prodorom v rastlinsko tkivo) ali - endoparazitsko (izčrpavanje rastline celotno ži- vljenjsko obdobje s popolnim vdorom v rastlinsko tkivo). Rastlinsko-parazitske ogorčice hrano pridobivajo iz gostiteljskih rastlin. Kljub temu, da so organizmi zelo majhni (v dolžino običajno merijo 1 mm), lahko povzro- čijo velike izgube pridelka, kar predstavlja znatno eko- nomsko škodo. Rastlinsko parazitske ogorčice se med sabo precej razlikujejo, lahko jih najdemo na koreninah, steblih kot tudi listih. V kmetijstvu je največji poudarek namenjen endoparazitskim ogorčicam, ki napadajo ko- reninski sistem in podzemna rastlinska tkiva (Vieira in Acta agriculturae Slovenica, 119/4 – 2023 3 Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphinema index ... Gleason, 2019). Medtem ko se ektoparazitske ogorčice prosto gibljejo okrog rastlin in se hranijo z rastlinskimi celicami (primer vrste rodu Longidorus in Xiphinema – ogorčice prenašalke virusov), endoparazitske ogorčice prodrejo v tkivo rastline in eno ali več svojih življenjskih obdobij preživijo v rastlinskem tkivu. Ločimo migrator- ne in sedentorne endoparazite. Migratorne endoparazit- ske ogorčice prodrejo v rastlinsko tkivo in se nato znotraj tkiva hranijo in premikajo (primer vrste rodu Pratylen- chus – ogorčice koreninske pegavosti). Sedentorne endo- parazitske ogorčice pa prav tako vstopijo v rastlinsko tki- vo, potujejo do mesta prehranjevanja in se tam ustalijo, se ne gibljejo več po rastlini (primer vrste rodu Meloido- gyne – ogorčice koreninskih šišk in vrste rodu Globodera – cistotvorne ogorčice) (Urek in Hržič, 1998; Vieira in Gleason, 2019). Ob pojavu bolezenskih znamenj na rast- linah je za odkrivanje in identifikacijo ogorčic potrebna morfološka (pregled organizmov pod mikroskopom) in molekularna analiza, saj je zgolj na podlagi bolezenskih znamenj nemogoče natančno določiti, za kateri organi- zem gre (Jackson, 2020). Ogorčice se v grobem med sabo ločijo predvsem po ustnem aparatu in načinu prehranje- vanja (Bilgrami in Brey, 2005). 2.1 OGORČICE, KI PARAZITIRAJO VINSKO TRTO Številne vrste ogorčic lahko napadejo korenine žlahtne vinske trte (Vitis vinifera L.), vendar le nekate- re izmed njih povzročijo znatno škodo (Urek in Hržič, 1998; Jackson, 2020). Ogorčice, ki napadajo vinsko trto, so omejene na prehranjevanje na koreninah. Prehran- jujejo se s srkanjem citoplazemske tekočine iz korenin- skih celic. To jim omogoča suličasto bodalo – stilet, ki predre v gostiteljske celice. Hranjenje je lahko omejeno na površje korenin, v kolikor pa ogorčice prodrejo v rast- lino, se zadržujejo predvsem v koreninski skorji. Poleg neposredne škode, ki jo povzroči hranjenje, in posledič- ne motnje delovanja koreninskega sistema, kar drugim patogenom lajša okužbo rastlin, lahko nekatere ogorčice prenašajo tudi viruse (Jackson, 2020). Rastlinam so najbolj nevarne ogorčice koreninskih šišk iz rodu Meloidogyne in virusonosne ogorčice iz rodu Xiphinema. Medtem ko Meloidogyne spp. povzroča- jo znatne poškodbe na koreninah s tvorbo zadebeli- tev (šišk), ogorčice iz rodu Xiphinema prenašajo viruse vinske trte. Druge ogorčice, ki se občasno prehranjujejo na koreninah vinske trte, vključujejo vrste iz rodov Pra- tylenchus (ogorčice koreninske pegavosti), Tylenchulus (citrusove ogorčice) in Criconemella (obročaste ogorčice) (Urek in Hržič, 1998; Jackson, 2020). Gospodarsko ško- do na vinski trti povzročajo rastlinsko-parazitske ogor- čice več različnih vrst; najpogosteje zastopane ogorčice v vinogradih so: Xiphinema index, Meloidogyne ethiopica Whitehead, 1968, Mesocriconema xenoplax Raski, 1952 in Tylenchulus semipenetrans Cobb, 1913 (Aballay in sod., 2009; Baginsky in sod., 2013). Kot je že omenjeno, lahko nekatere ogorčice, ki se hranijo z rastlinskim sokom preko ustnega aparata, pre- našajo iz okuženih na zdrave rastline fitopatogene viru- se, so njihovi prenašalci (Urek in Hržič, 1998). Ogorčice prenašalke virusov uvrščamo v dve družini (Taylor in Brown, 1997; Urek in Hržič, 1998): - družina Longidoridae: rod Longidorus, Paralongi- dorus in Xiphinema (razred Dorylaimea) in - družina Trichodoridae: rod Paratrichodorus, Tri- chodorus (razred Enoplea). Ogorčice prenašalke virusov so ektoparaziti, ki živi- jo prosto v tleh in se hranijo na koreninah rastlin (Urek in Hržič, 1998). Kot navajata Taylor in Brown (1997) so ogorčice iz rodov Longidorus, Paralongidorus in Xiphi- nema prenašalke nepovirusov (virusi iz rodu Nepovi- rus), medtem ko ogorčice iz rodov Paratrichodorus in Trichodorus prenašajo tobraviruse (virusi iz rodu Tobra- virus). Najštevilčnejši rod ogorčic, prenašalk virusov je rod Xiphinema, sledi rod Longidorus s 183 vrstami, rod Trichodorus zajema 52 vrst, sledita pa rodova Paralongi- dorus s 34 vrstami in Paratrichodorus s 30 vrstami (Hod- da, 2022). Rod Xiphinema je ena najbolj raznolikih sku- pinskih vrst virusonosnih ogorčic z več kot 280 vrstami (Coomans, 2000; Ye in sod., 2004; Decraemer in Hunt, 2006; Gutiérrez-Gutiérrez in sod., 2010; Hodda, 2022). Devet od približno 280 poznanih vrst Xiphinema so po- trjeno prenašalke nepovirusov (Decraemer in Robbins, 2007; Gutiérrez-Gutiérrez in sod., 2013). Glede na veliko Slika 1: Ilustracija različnih načinov prehranjevanja rastlin- sko-parazitskih ogorčic na prerezu korenine (Vir: Vieira in Gleason, 2019) Figure 1: Illustration of a cross-section of a root with different feeding habits of plant-parasitic nematodes (Figure: Vieira and Gleason, 2019) Acta agriculturae Slovenica, 119/4 – 20234 J. POLANŠEK et al. morfološko raznolikost te skupine razdelimo rod Xiphi- nema v dve različni skupini (Decraemer in Hunt, 2006; Gutiérrez-Gutiérrez in sod., 2013; Archidona-Yuste in sod., 2016): - skupina Xiphinema americanum sensu lato (ame- riška skupina), ki obsega kompleks približno 60 vrst (Ye in sod., 2004; Gutiérrez-Gutiérrez in sod., 2010; Gutiér- rez-Gutiérrez in sod., 2013; EFSA in sod., 2018; Archido- na-Yuste in sod., 2020) in - skupina Xiphinema non-americanum (neameriška skupina), ki obsega kompleks več kot 220 vrst (Thorne, 1935; Coomans, 2000; Decraemer in Hunt, 2006; Archi- dona-Yuste in sod., 2020). Xiphinema index je uvrščena v neameriško skupino ogorčic – Xiphinema non-americanum group (Taylor in Brown, 1997; EFSA in sod., 2018). 2.1.1 Ogorčice vrste Xiphinema index Prvi dokaz o prenosu rastlinskega virusa z rastlin- sko-parazitsko ogorčico sega v pozna petdeseta leta 20. stoletja. Dokazali so, da talna ektoparazitska vrsta X. index, prenaša virus pahljačavosti listov vinske trte (GFLV), ki je eden glavnih povzročiteljev bolezni kužne izrojenosti vinske trte (Hewitt in sod., 1958). Bolj kot direktne poškodbe ogorčic X. index so problematične zaradi prenosa virusa GFLV (Andret-Link in sod., 2004; Van Ghelder in sod., 2015). Bolezen, ki jo povzroča virus GFLV se na rastlinah odraža s skrajšanimi internodiji, slabša je kakovost plodov, izpad pridelka lahko presega 80 % (Andret-Link in sod., 2004; Van Zyl in sod., 2012; Rubio in sod., 2020). Evropska populacija ogorčic iz rodu Xiphinema je tolerantnejša za nizke temperature in jih lahko v tleh naj- demo praktično celotno koledarsko leto (Flegg, 1968a, b; Taylor in Brown, 1997). Vrsta X. index preživi med –11 in 35 °C, vendar je konstantna temperatura 45 °C ali –22 °C 10 dni zapored zanje smrtonosna (Van Zyl in sod., 2012). Populacija X. index ima raje težka tla, ki so manj iz- postavljena suši, njihovo prisotnost pa so potrdili tudi v vlažnih peščenih tleh (Esmenjaud in sod., 1992). Takšne rastne razmere so ugodne tudi za gojenje vinske trte, ki je njihova glavna naravna gostiteljica (Andret-Link in sod., 2017). Gibanje ogorčic je omejeno na nekaj centimetrov na leto (Pitcher, 1975; Taylor in sod., 1994). Ogorčice se v tleh premikajo vodoravno in navpično, večinoma v smeri rasti korenin, na katerih se hranijo (Thomas, 1981; Esmenjaud in sod., 1988). Na območju korenin je tudi gostota populacije največja (Feil in sod., 1997). Običaj- no jih je manj v plitvih slojih tal. V vinogradniških tleh se X. index najpogosteje nahajajo na globini 0,3–1,5 m, tam kjer je večina mladih korenin/koreninskih laskov (Esmenjaud in sod., 1992; Feil in sod., 1997; Villate in sod., 2008). Zadržujejo se lahko tudi na globini 3,6 m (Raski in sod., 1965). Širijo se s kontaminirano opremo, sajenjem okuže- nih sadik in prenosom zemlje. Da bi preprečili širjenje ogorčic iz vinograda v vinograd je treba opremo, kot so traktorji, sadilniki, grebenarji in delovni škornji, po upo- rabi v vsakem vinogradu očistiti (Bileva in sod., 2009; Esmenjaud in Bouquet, 2009). Tudi voda povzroča aktiv- no migracijo ogorčic v tleh, saj se ogorčice lahko pasivno širijo s potoki, poplavnimi vodami in pronicajočo vodo v vinogradih (Roccuzzo in Cianco, 1991). Razvojni krog ogorčic se razlikuje od vrste do vrs- te, nanj pa močno vplivajo okoljske razmere (Weischer, 1975; Taylor in Brown, 1997). Jajčeca odložijo v tla, bli- zu mesta hranjenja, ta pa se izležejo spomladi ali zgo- daj poleti, ko odženejo nove korenine. X. index zaključi svoj življenjski krog na vinski trti v 7–9 mesecih. Ta čas pa je krajši, v kolikor so ogorčice prisotne v rastlinjaku (Andret-Link in sod., 2017). Med ogorčicami iz rodu Xiphinema so samci zelo redki, izjema je vrsta X. diversi- caudatum Micoletzky, 1927. Razmnožujejo se nespolno – partenogenetsko. Posamezna ličinka vrste X. index lah- ko ustvari populacijo, kljub temu da zelo redko pride do spolnega razmnoževanja (Villate in sod., 2010). Glavni morfološki parametri so (Andret-Link in sod., 2017): - dolžina telesa, - oblika in velikost glave in ustnega aparata, - struktura in dolžina bodala, - struktura in položaj vodilnega obroča, - položaj in tip genitalnih organov samice. Dodatne morfološke značilnosti na ravni vrste so razvoj in struktura ženskega reproduktivnega organa, oblika repa v vseh razvojnih stadijih ter prisotnost oz. odsotnost samcev (Andret-Link, 2017). Nepovirus GFLV se pri ogorčicah vrste X. index zadržuje v požiralniški cevi in v notranji plasti kutikule odontoforja (Slika 2) (Taylor in Robertson, 1970; Brown in sod., 1995; Wang in sod., 2002; Širca in Urek, 2016; Sanfaçon, 2020). 3 MOŽNOSTI ZATIRANJA OGORČIC VRSTE X. index Pomembno je, da sadimo le neokužen sadilni ma- terial, ki je predhodno testiran na prisotnost virusov (Demangeat in sod., 2004; Pompe-Novak in sod., 2005). Predvsem je potrebna pazljivost pri možnostih prenosa ogorčic s prenašalci, saj na ta način lahko preprečimo po- javnost napadov in okužb v nasadih (Djennane in sod., 2021; Schurig in sod., 2021a; Schurig in sod., 2021b). Acta agriculturae Slovenica, 119/4 – 2023 5 Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphinema index ... lematično, saj njihova jajčeca v mirujočem stanju v tleh preživijo tudi več let. Vprašljivo je tudi prodiranje ne- matocidov v globino do 1,5 m in več, saj se tam nahaja večina koreninskega sistema. Predvsem fumiganti so veljali za potencial pri omejevanju širjenja in pojavnosti ogorčic, a se slabo aplicirajo do večjih globin, prav tako pa so slabo učinkoviti v ilovnatih tleh (Jackson, 2020). Na podlagi poskusov z uporabo kemičnih sredstev, kar- bamatov in organofosfatov, z nanosom na vinogradniška tla enkrat ali dvakrat letno so ugotovili, da ti niso dovolj učinkoviti. Neučinkovitost so pripisali vplivu namakanja, uporabi organskih dodatkov in načinu nanosa (Baginsky in sod., 2013). Uporaba nematocidov je po svetu še vedno precej razširjena, a je njihovo delovanje vprašljivo tudi z vidi- ka neciljnega delovanja, saj poleg ciljnih organizmov – ogorčic, v tleh negativno učinkujejo tudi na talne mik- roorganizme in druge koristne organizme. Z njihovim uničenjem pa posledično siromašimo tla (Chitwood, 2003; Dong in Zhang, 2006; Atreya, 2008; Brun in sod., 2008; Pires in sod., 2022). 3.2 BIOTIČNO ZATIRANJE Ker je uporaba kemičnih nematocidov precej ome- jena in neučinkovita, so potrebe po alternativnih, trajno- stnih in okolju prijaznih pristopih vse večje (Wernet in Fischer, 2023). Kot možen pristop v boju proti vrsti X. in- dex v vinogradih se je izkazala kombinacija kolobarjenja in strniščnih dosevkov (Aballay in sod., 2004). Prav tako lahko na populacijo ogorčic v vinogradniških tleh vpliva- jo antagonistične rastline s svojimi koreninskimi izločki (Bello in sod., 1998), hkrati pa lahko pozitivno vplivajo na vinsko trto in spodbujajo rast korenin (Birch in sod., 1993; Baginsky in sod., 2013). Biotično varstvo rastlin Zaradi vse večjih omejitev pri uporabi kemičnih ne- matocidov in njihove neučinkovitosti se je trenutno kot najučinkovitejša metoda za iztrebljanje X. index v tleh izkazala vsaj 7 letna praha oz. zemljišče v mirovanju, saj lahko ogorčice v vinogradniških tleh preživijo najmanj 4 leta brez prisotnosti gostiteljske rastline. Do prenosa virusa pa lahko pride tudi po predhodni 5 letni prahi (Demangeat in sod., 2005; Villate in sod., 2008; Meng in sod., 2017; Nguyen in sod., 2019; Wernet in Fischer, 2023), saj se lahko ogorčice v tleh ohranijo zaradi pri- sotnosti koreninskih ostankov po izkrčitvi trt (McKenry in Buzo, 1996; Jackson, 2020). Tako dolgotrajni postopki iztrebljanja ogorčic iz tal predstavljajo ekonomsko nepri- vlačno metodo (Demangeat in sod., 2004). 3.1 KEMIČNO ZATIRANJE – NEMATOCIDI V Sloveniji so na seznamu registriranih fitofarma- cevtskih sredstev – nematocidov navedeni štirje priprav- ki (Tabela 1) (FURS, 2023). Učinkovitost nematocidov je slaba in ne vodi do želenih učinkov. Zatiranje ogorčic vrste X. index je prob- Slika 2: Morfologija sprednjega dela ogorčic rodu Xiphinema in zadrževalno mesto virusnih delcev (obarvano rdeče): v notranji plasti kutikule odontoforja (odf) in požiralniške cevi (prirejeno po Andret-Link in sod., 2017) Figure 2: Morphology of the anterior region of nematodes genus Xiphinema and the viral particles retention site (red coloured): inner layer of the odontophore‘s cuticule (odf) and esophageal bulb (Figure: Andret-Link et al., 2017) Tabela 1: Seznam registriranih nematocidov v Sloveniji (Vir: FURS, 2023) Table 1: List of registered nematicides in Slovenia (Reference: FURS, 2023) Pripravek Aktivna snov Formulacija Basamid granulat dazomet mikrozrnina Profume insecticide sulfuril fluorid plin Velum prime fluopiram koncentrirana suspenzija Votivo FS 240 Bacillus firmus sev I-1582 koncentrirana suspenzija za tretiranje semena Acta agriculturae Slovenica, 119/4 – 20236 J. POLANŠEK et al. zajema tudi biotične agense različnega taksonomskega izvora: entomopatogene ogorčice, parazitoidne žuželke, patogene bakterije, glive, viruse ter plenilce (Mesa-Valle, 2020; Topalović, 2020; Pires in sod., 2022). 3.2.1 Vmesni posevki V interakciji z ogorčico X. index je bilo preučevanih več posevkov rastlin iz družine nebinovk (Asteraceae) (Tsay in sod., 2004) in posevkov iz družine križnic (Bra- sicaceae) (Halbrendt, 1996). Večina jih je bila učinkovitih proti vrsti X. index (Insunza in sod., 2001; Aballay in sod., 2004). Učinek teh vmesnih posevkov je odvisen od vrste izbranih rastlin, tipa tal, ogorčice, ki je v tleh prisotna, in načina gojenja. V primeru neprimerne izbire vmesnega posevka lahko to vodi do škodljivih učinkov za glavni posevek (McLeod, 1994; Baginsky in sod., 2013). V ra- ziskavi Baginskya in sodelavcev (2013) so v vinogradih preizkušali učinkovitost vmesnih posevkov oljne ogršči- ce (Brassica napus var. napus L.) in krmne repe (B. rapa var. rapa L.). Oba posevka sta tako kot nematocidi zman- jšala populacijo vrste X. index v vinogradniških tleh. Ne- katere raziskave poljskih poskusov v vinogradu poročajo o obetavnih rezultati s posevki krmne grašice (Vicia villo- sa Roth) in žametnice (Tagetes minuta L.). Pod nadzoro- vanimi razmerami v rastlinjaku so se najbolj izkazali bela lupina (Lupinus albus L.), niger (Guizotia abyssinica (L. f.) Cass.) in žametnica (Tagetes minuta L.), saj so znatno zmanjšali populacijo vrste X. index v primerjavi z golimi tlemi, takimi brez posevka (Villate in sod., 2012). Abal- lay in Insunza (2002) poročata o rezultatih pridobljenih z gojenjem in vključevanjem oljne ogrščice (Brassica na- pus) v vinograde. Populacija vrste X. index se je v tleh ob prisotnosti oljne ogrščice znatno zmanjšala v primerjavi s kemičnim zatiranjem (fenamifos). Mulč pokrovnih po- sevkov, kot so kapusnice (Brassica spp.) lahko služi kot biofumigant, ki lahko zmanjša populacijo ogorčic v tleh (McLeod in Steel, 1999; Rahman in Somers, 2005). Ka- pusnice namreč tvorijo glukozinolate, ki so v živi rastlini neaktivni. Po mulčenju te spojine hidrolizira encim mi- rozinaza, pri čemer se sproščajo hlapni izotiocianati. Ti sodelujejo pri biofumigaciji vinogradniških tal (Kruger in sod., 2013). V poskusu Pensec in sod. (2013) pa se je sadrenka oz. šlajer (Gypsophila paniculata L.) izkazala z bionematocidnim delovanjem in ob tem ni vplivala na delovanje mikorize. 3.2.2 Glive Alternativo kemičnemu zatiranju lahko predstavlja- jo tudi koristni talni mikroorganizmi. Eden od učinko- vitih primerov je arbuskularna mikoriza (AM), ki omili škodo povzročeno s strani rastlinsko parazitskih ogor- čic (Pozo in Azcon-Aguilar, 2007; Schouteden in sod., 2015, v Hao in sod., 2018). V poskusu Hao in sodelavcev (2012) so dokazali, da je izolat arbuskularne mikorizne (AM) glive Rhizoglomus irregularis Walker & Schüsler, 2010 imenovan BEG141 zmanjšal tvorbo šišk na koreni- nah, ki so jih povzročile brezvirusne ogorčice X. index in zmanjšal namnožitev ogorčic v tleh, ki so obdajale vinsko trto cepljeno na podlago SO4 (Vitis berlandieri × V. riparia). V kasnejši študiji je Hao s sodelavci (2018) preučeval učinke glive R. irregularis na razvoj virusonos- ne ogorčice X. index in okužbe z GFLV. Ugotovili so, da je prisotnost AM glive na koreninah vinske trte cepljene na podlago SO4 zmanjšala nastanek šišk na koreninah povzročenih s strani ogorčice X. index, zmanjšana pa je bila tudi namnožitev ogorčic v tleh. V primeru vnosa 10 osebkov vrste X. index v lonec, kjer je bila AM gliva, viru- sa GFLV pri vinski trti po 90 dneh niso potrdili, medtem ko so v primeru vnosa 100 osebkov potrdili GFLV tako pri rastlinah v loncih brez AM kot z AM glivo. Torej bi se z manjšanjem populacije vrste X. index in njihovega prodiranja v korenine zmanjšala tudi okuženost vinske trte z GFLV. V poskusu Boosalis in Mankau (1965) so ugotovili, da je endoparazitska gliva Catenaria anguillulae Sorokin, 1876 parazitirala ogorčico vrste X. index. Kot parazit se je izkazala tudi pri ogorčicah Panagrellus redivivus Linnae- us, 1767 in samicah ogorčice Heterodera schachtii Sch- midt, 1871, kar kaže na njihov velik potencial za biotično zatiranje ogorčic (Voss in Yyss, 1990). Galper s sodelavci (1991) so ugotovili, da je ekstrakt glive Cunninghamella elegans Lendner, 1907, gojene na kolagenu kot viru ogl- jika in dušika, zmanjšal gibljivost ogorčic X. index v in vitro razmerah. Prav tako se je potencial te glive pri bio- tičnem zatiranje ogorčic povečal, ko so kolagen dodajali v rastni substrat (0,1 % m/m). Kot ekološko in stroškovno učinkovito alternativo varstvu pred ogorčicami so z različnimi raziskavami po- trdili glivo Arthrobotrys flagrans Sidorova, Gorlenko & Nalepina, 1964 (prej Duddingtonia flagrans). Izkazala se je, da ima velik potencial za uporabo kot biotični nema- tocid (Araújo in sod., 2008; Vilela in sod., 2012; da Silva in sod., 2013; Braga in de Araújo, 2014; Mostafanezhad in sod., 2014). To sta potrdila tudi Wernet in Fischer (2023), saj se je gliva Arthrobotrys flagrans (Duddingtonia flagrans) v njuni raziskavi izkazala za učinkovit mikroorganizem za zatiranje ogorčice X. index. V lončnem poskusu s figo (Ficus carica L.) je prisotnost glive A. flagrans zmanjšala število ličink vrste X. index. Figa ni gostitelj virusa GFLV, je pa dober gostitelj ogorčice X. index, zato je mogoče zagotoviti, da brezvirusne ogorčice ostanejo neokužene Acta agriculturae Slovenica, 119/4 – 2023 7 Možnosti nekemičnega zatiranja virusonosnih ogorčic Xiphinema index ... z virusom tekom poskusa. Monteiro s sodelavci (2018) pa je potrdil pozitivno delovanje glive A. flagrans na sadike paradižnika, saj se je ob prisotnosti le-te v tleh povečala absorpcija elementov in mineralov, kar je po- zitivno vplivalo na rast in razvoj sadik. Prav s poskusi na paradižnikih so dokazali nematocidno delovanje glive A. flagrans, saj se je po vnosu klamidiospor v rastni substrat zmanjšalo število ličink vrste Meloidogyne javanica Tre- ub, 1885 za 73 % (Monteiro in sod., 2020; v Wernet in Fischer, 2023). Wernet in Fischer (2023) sta s poskusi preverila in- terakcije gliv A. conoides Drechsler, 1937, A. flagrans, A. oligospora Fresen., 1850, A. musiformis Drechsler, 1937, Drechslerella stenobrocha Drechsler, 1950 in Dactylelina haptoyla Drechsler, 1950 z bakterijami iz rodov Delftia, Bacillus, Pseudomonas, Enterobacter in Serratia. Ugoto- vila sta, da so bakterije Bacillus subtilis Ehrenberg, 1835, Pseudomonas stutzeri Lehmann in Neumann, 1896, Ente- robacter cloacae Jordan, 1890, Serratia marcescens Bizio, 1823 in Delftia po 48 urah na hifah gliv vrste A. conoides in A. oligospora tvorile »pasti« oz. zanke, ki ogorčice uja- mejo in jih nato prebavijo. Ta interakcija gliv in bakterij, ter tvorba pasti na hifah gliv bi lahko v prihodnje pred- stavljala velik potencial biotičnega zatiranja patogenih ogorčic. Darago in sodelavci (2013) pa so preverili nemato- cidno delovanje gliv iz rodu Trichoderma. Poskus so za- snovali v in vitro razmerah s šestnajstimi sevi šestih vrst: T. atroviride Bissett, 1984, T. harzianum Rifai, 1969, T. rossicum Bissett, Kubicek & Szakacs, 2003, T. tomentosum Bissett, 1991, T. virens Arx, 1987 in T. asperellum Samu- els, Lieckf. & Nirenberg, 1999. Ocena testov smrtnosti ogorčic X. index je pokazala, da so glive Trichoderma spp. sposobne zmanjšati populacijo vrste X. index v in vitro razmerah. Najboljše rezultate so dosegli z glivo T. harzianum. 3.2.3 Bakterije Eno od možnosti v boju proti ogorčicam X. index pa so preizkušali v raziskavi Aballaya in sod. (2011; 2012). Iz korenin vinskih trt so pridobili več kot 400 izolatov bakterij iz 25 rodov. Izolata bakterij Brevibacillus brevis Migula, 1900 in Bacillus megaterium de Bary, 1884 sta pokazala nematocidno delovanje tako v in vitro razme- rah, kot pri lončnem poskusu. Rizobakterije, izolirane iz žlahtne vinske trte so zmanjšale škodo na mladih ko- reninah, ki jo povzroči vrsta X. index in so potencialni biotični agensi za nadaljnje raziskave o možnostih zati- ranja oz. omejevanja pojavnosti ogorčice v vinogradih. Preprečitev tveganja za okužbo vinske trte z GFLV sicer ni dosežena, saj tudi pri majhni populaciji ogorčic lahko pride do okužbe, vendar prisotne bakterije pripomorejo k večji zaščiti korenin pred neposrednimi poškodbami ogorčic (Aballay in sod., 2012). Potencial pri varstvu rastlin pred ogorčico X. index so ugotovili tudi pri bakteriji Pasteuria penetrans Thorne, 1940 (Sturhan, 1985). Prav tako se je pozitivno izkazala bakterija Paenibacillus sp. (sev B2), izolirana iz navadne- ga sirka (Sorghum bicolor L. Moench), saj izloča peptid penimiksin. Prisotnost bakterije je znatno zmanjšala po- pulacijo vrste X. index v tleh in tvorbo šišk na koreninah vinske trte. Penimiksin je v in vitro razmerah zmanjšal aktivnost ogorčic X. index (Hao in sod., 2017). Delovanje nekaterih rizobakterij na vrsto X. in- dex so preverjali Aballay in sodelavci (2020). Korenine vinske trte v rastlinjaku so inokulirali z različnimi for- mulacijami rizobakterij (tekočina, prašek in izotonična raztopina). Ugotovili so, da so bili izolati rizobakterij Brevibacterium frigoritolerans Delaporte in Sasson, 1967, Bacillus amyloliquefaciens Fukomoto, 1943, Bacillus me- gaterium, Bacillus thuringiensis Berliner, 1915, Bacillus weihenstephanensis Lechner in sod., 1998 in Pseudomo- nas fluorescens Migula 1895 učinkoviti proti ogorčicam X. index, saj je bila njihova učinkovitost primerljiva z učinkovitostjo kemičnega nematocida Rugby®200 CS (kadusafos). Formulacija (prašek, tekočina, izotonična raztopina) na samo učinkovitost ni imela vpliva. 4 ZAKLJUČEK Virusonosne ogorčice vrste Xiphinema index so v tleh sposobne preživeti več let. Kljubujejo različnim okoljskim in talnim razmeram, ob tem pa v svojem or- ganizmu vseskozi ohranjajo virus GFLV, ter na ta na- čin predstavljajo nevarnost za vinsko trto, ki je njihova glavna gostiteljica. Nepovirus GFLV, ki ga vrsta X. index prenaša, namreč povzroča virusno bolezen kužne izro- jenosti vinske trte, ki postopno vodi do velikega izpada pridelka in kasneje v propad trt. Za zdaj učinkovite- ga trajnostnega pristopa za obvladovanje te bolezni ni. Najučinkovitejši pristop v boju z boleznijo predstavlja zmanjšanje populacije prenašalk virusa v tleh. Ker pa je značilna porazdelitev teh ogorčic v tleh tudi do globine 3 in več metrov, nakazuje na izjemno težavnost pri varstvu rastlin in predstavlja v »okuženih« vinogradih velik izziv. Priporočljiva je 10 letna praha pred ponovno zasaditvi- jo vinograda, kar pa je iz ekonomskega stališča nespre- jemljivo. Prav zaradi obstojnosti, globinske porazdelitve in zmožnostjo preživetja v neugodnih razmerah je učin- kovitost kemičnih pripravkov, nematocidov, v tleh, kjer so ogorčice prisotne, precej slaba. Vprašljivo pa je tudi njihovo neciljno delovanje in vpliv na ostale mikroorga- nizme v tleh. Vse več raziskav se nagiba k uporabi okolju Acta agriculturae Slovenica, 119/4 – 20238 J. POLANŠEK et al. prijaznejših in učinkovitejših strategij. Kot učinkoviti so se izkazali različni vmesni posevki, ki s svojimi korenin- skimi izločki vplivajo na zmanjšanje populacije ogorčic v tleh. Prav tako se vse več raziskav nagiba k uporabi raz- ličnih mikroorganizmov, ki bi lahko v prihodnosti učin- koviteje nadomestili kemične pripravke. Preučevanje in- terakcij med vrsto X. index, virusom GFLV in vinsko trto predstavlja vsekakor velik izziv. Področje nekemičnega zatiranja virusonosnih ogorčic pa bo tudi v prihodnje deležno raziskovanja, saj bi z razvojem novih strategij za- tiranja ogorčic ali kakršne koli drugačne prekinitve pre- nosa virusa na vinsko trto omogočili zmanjšanje izgube pridelka in dohodka na vinogradniških kmetijah. 5 ZAHVALA Aktivnosti mlade raziskovalke potekajo v sklopu programske skupine Agrobiodiverziteta (P4-0072), ki jo financira Javna agencija za raziskovalno dejavnost Repu- blike Slovenije (ARRS). 6 VIRI Abad, P., Gouzy, J., Aury, J. M. in sod. (2008). Genome sequen- ce of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology, 26, 909–915. https://doi. org/10.1038/nbt.1482 Aballay, E., Insunza, B. (2002). Evaluation of plants with ne- maticidal properties in the control of Xiphinema index on table grapes cv. Thompson Seedless in the central zone of Chile. 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