Trichothecene chemotypes of Fusarium graminearum species complex in Serbia

The species Fusarium graminearum represents one of the most economically important factor that threaten agricultural production in the world. In addition to affecting yield reduction and grain quality, this species is of great importance due to its ability to synthesize mycotoxins. Knowing the toxicological profile of F. graminearum species is essential for agriculture and food industry, because trichothecene chemotypes (NIV, 3ADON, 15ADON) have different toxicological properties that directly affect human and animal health. In this study, 20 isolates of F. graminearum originating from maize and wheat were examined. DNA isolation was performed from the mycelium of the fungus using the commercial kit Dneasy Plant Mini Kit according to the manufacturer’s instructions. Chemotype detection was performed using two sets of specific primers (TRI3 and TRI12) designed for trichothecenes by Ward et al. in 2002. After the polymerase chain reaction was completed, the products were electrophoretically separated on a 1% agarose gel, and visualization was performed by staining with ethidium bromide and observing under UV light. Amplified fragments were read visually, and a positive reaction indicated the presence of DNA fragment of the expected size. All isolates of F. graminearum examined in this study had products of size 610bp at TRI3 and 670bp at TRI12, which indicates that isolates originating from Serbia belonged to 15ADON chemotype

Model prediction of ruminal dry matter digestibility of serbian maize genotypes

The focus of this study is on the physical quality traits, the carbohydrates and in vitro dry matter digestibility (IVDMD) of various maize kernel genotypes produced in Serbia. Furthermore, the aim was to determine the relationship among these quality traits, as well as, their effects on the IVDMD. Ten maize genotypes with different endosperm type and kernel color have been studied (2018 growing season). All kernel traits significantly varied among selected maize genotypes. IVDMD ranged from 83.1-91.2%. In vitro regression model of IVDMD in ruminants was obtained. The physical quality traits such as test weight (TWt), 1000-kernel weight (KWt), density (Den) and hard endosperm portion (HE) had significant role in predicting the digestibility of maize kernel. The IVDMD was mostly affected by test weight and density in the FOP model (p<0.01 level). The determined in vitro digestibility model can serve for screening various maize kernel genotypes due to estimate their utility value for feed industry

Combined effect of cover crops and bio-fertilizer on sustainable popcorn maize production

Cover crops play an important role in low-input cropping systems, increasing the use of agro-ecosystem services. Due to the lack of information about the impact of cover crops and bio-fertilizers on popcorn maize (Zea mays everta Sturt.) growth and yield quality, especially the popping volume and nutritive quality, such as concentrations of protein and mineral elements, this research aimed to provide essential information. The interrelation between popcorn maize productivity and quality with important groups of soil microorganisms presents additional novelty. The results demonstrated that field pea is a beneficial cover crop, especially when combined with a bio-fertilizer, supporting the accumulation of maize biomass, chlorophyll, yield potential, and the concentrations of protein, Ca, Mg, Fe, and Zn. In addition, field pea residues promoted N-fixing bacteria, and the number of total microorganisms, especially actinomycetes and decomposing bacteria, which could promote nutrient uptake and grain quality. Residues of cover crop mixtures, common vetch + winter oats and field pea + winter oats, promoted the total number of microorganisms in the soil, and up to the end of vegetation, a greater number of decomposition and ammonification microorganisms were found, especially when the bio-fertilizer was applied, which consequently could support greater maize biomass. Popping volume, as a main trait of popcorn maize, had the highest value in the common vetch + winter oats variant, supporting again the statement that quality traits could be enhanced in sustainable production. Unlike living cover crops, mulch mainly affected soil microbial communities and promoted the development of actinomycetes and cellulolytic microorganisms during the growing season. The results of this research could contribute to the development of sustainable popcorn maize production for improved grain quality. They could also serve as a basis for isolating beneficial soil microorganisms to develop new bio-fertilizers that could improve maize production in synergy with cover crop

Genetic variability in tocopherols content in maize landraces

Improvement of maize grain composition is one of the most important breeding goals. Maize is generally characterized by low levels of vitamin E in its kernels. Still, its biofortification through breeding could provide sufficient vitamin E as a staple crop or seed oil. Tocopherols are particularly important due to their role in protecting the plant from oxidative stress and contributing to the nutritional quality of the grain. The highest biological activity is found in α-tocopherol and therefore is most interesting for maize breeding. This work aimed to evaluate the whole kernel tocopherols composition of 54 maize landraces with different kernel colours and kernel types. High-Performance Liquid Chromatography (HPLC) was employed to quantify and determine the levels of α-tocopherol, γ+ß -tocopherol, and δ-tocopherol within these landraces. The detected ranges for tocopherols were 0.83 to 3.65 μg g−1 for α-tocopherol, 17.59 to 38.52 μg g−1 for γ+ß -tocopherol, and 1.52 to 11.43 μg g−1 for δ-tocopherol. The highest value of α-tocopherol was observed in the landrace with yellow flint kernels, while the highest γ+ß –tocopherol was measured in the landrace with orange semi-dent kernels. At the same time, landrace with yellow dent kernels had the highest δ-tocopherol. On the contrary, landraces with white dent and semi-dent kernels had the smallest values for all tocopherol types. Genetic diversity of landraces has an important role in maize breeding and could significantly impact the improvement of new varieties as a valuable source of useful traits. The presence of a wide genetic diversity of landraces based on tocopherol content makes them more than suitable to be used for further maize breeding improvement programs

Impact of deficit irrigation and planting density on grain yield and water productivity of maize grown under temperate continental climatic conditions

Variable frequent drought periods in Vojvodina region (the southeastern part of the Pannonian Plain, Serbia), severely limit maize (Zea mays L.) production under rainfed conditions. A four-year field experiment was conducted to investigate the effects of sprinkler deficit irrigation and plant density on yield and water use efficiency of maize grown on silty clay loam soil under temperate continental climate. The experiment included fully irrigated crop (I1), three deficit irrigation treatments (I2, I3 and I4 corresponding to 80, 50 and 40 % of crop evapontranspiration, respectively) and non-irrigated treatment (I0), and three plant densities (LPD: 54,900 plants ha–1 ; MPD: 64,900 plants ha–1 ; HPD: 75,200 plants ha–1 ) in four replicates. The results showed that grain yield, CWP (crop water productivity) and IWP (irrigation water productivity) varied significantly with irrigation amounts, plant densities and seasons. The irrigation rates and plant density interact significantly. MPD and HPD differed significantly from LPD in almost all seasons. With increasing irrigation and plant density, yield and CWP showed an increasing trend. The relative values of IWP increased with the rise of plant density and decreased with the amount of irrigation. The highest four-year average yield (15.03 t ha–1 ) was obtained in the I1-HPD treatment, while the lowest (9.30 t ha–1 ) was obtained in the I0-HPD treatment. The highest average values of CWP and IWP were recorded at I3-HPD or/and I2-HPD. The lowest CWP and IWP values were determined for I4- HPD and I4-MPD, respectively. In the pedo-climatic conditions of Vojvodina and similar regions, we recommend growing maize under the I1-HPDtreatment to achieve high yields. Under the water shortage conditions, the application of I2-HPD treatment is a favorable strategy saving 37 % of irrigation water, while reducing maize grain yield by ~10 %

Genetic characterization of soybean genotypes using microsatellite markers

Soybean (Glycine max (L.) Merr.) is one of the most economically important grain crops because of its nutrient richness and well-balanced seed composition. The genetic base of soybean is highly narrow because it is largely a self-pollinated species. Molecular characterization of 21 cultivars and eight experimental lines of soybean originating from Serbia and Croatia was performed using 25 microsatellite (SSR) markers. A large proportion of SSR markers was polymorphic (92%) and highly informative (PIC=0.525). Five unique alleles were observed, while the average number of alleles per locus was three. The average value of genetic similarity was relatively high (GS=0.654), which can be explained by the fact that the studied varieties originated from a narrow geographical area whose genetic basis is predominantly composed of elite genotypes of North American germplasm. Cluster analysis of soybean genotypes using the UPGMA method and the NTSYSpc statistical program showed a significant correlation with the pedigree data of the genotypes. Two numerically equal clusters with two subclusters were clearly observed. Individual genotypes of groups showed minor deviations in clustering, taking into account their origin. For some of the examined genotypes, pedigree information was missing, which led to the formation of seemingly less related groups

Fusarium spp. infection, mycotoxin contamination, and some agronomic traits in winter barley as affected by N fertilization under Serbia conditions

Fusarium head blight (FHB), caused by different Fusarium species, is the most devastating disease of small cereal grains, including barley (Hordeum vulgare L.) This study aimed to investigate the influence of N fertilization rates (0 kg N ha-1 - N0, 50 kg N ha-1 - N50, and 100 kg N ha-1 - N100) on Fusarium and mycotoxin (deoxynivalenol - DON and zearalenone - ZEA) contamination and some agronomic traits (plant height - PH, spike length - SL and thousand kernel weight - TKW) in two barley cultivars, NS 565 and Etincel, harvested in 2019 and 2020. Climatic conditions during two successive seasons were favourable for Fusarium infection, providing a high incidence of FHB-causing species, of which F. graminearum species complex (FGSC) strains were the most frequent (on average > 34% per treatment). The N rates and barley cultivars had nonsignificant effects on the incidence of FGSC strains. However, N rates significantly influenced mycotoxin levels in 2019, with the highest DON at N100 (5209.67 μg kg-1) and ZEA levels at N50 (47.11 μg kg-1). In 2020, there were nonsignificant differences between N rates for DON levels, while ZEA was not detected. In both years, the sixrow barley ‘Etincel’ had significantly higher DON and ZEA levels than the two-row barley ‘NS 565’. Agronomic traits, PH and SL, were affected by barley cultivars, and TKW was affected by N rates and barley cultivars. The highest TKW was at N100, followed by N50 and N0

Modelling of Genotype × Environment interaction for grain yield of late maturity maize hybrids in Serbia by climate variables

With global climate change, including unpredictable geographic and temporal weather patterns causing significant Genotype × Environment interaction (GEI), modelling by climate variables can reveal their influence on phenophases of maize (Zea mays L.) development. The objectives of this study were dissection of the phenotypic variation of grain yield of late maturity maize hybrids grown in multi-environment trial, and quantification of the influence of climatic factors on the GEI for each vegetative and reproductive phenophase. Eight FAO 700 maize hybrids were evaluated across five locations in Serbia during 2020 and 2021 years. The hierarchy of sources of variation according to three-way ANOVA were: Year (Y) > Location (L) > Location×Genotype (G) > L×Y×G > G > L×Y > Y×G. The average maximum temperature (mxt, 22.1%), average minimum temperature (mnt, 19.2%), average mean temperature (mt, 18.2%) and relative humidity (rh, 15.1%) in April significantly influenced emergence stage. The mxt (21.1%) and mt (15.7%) in May influenced significantly vegetative phases V1-V9. June contributed the largest percentage of the sum of squares of the GEI with mxt (25.2%), mnt (20.9%), mt (16.1%) influencing vegetative phases V10-V18 and tassel emergence. In July mxt (17%), mt (15.6%), precipitation sum (15.2%), and sunshine hours sum (15.1%), influenced R1, R2, R3, and R4 reproductive phases. In August mxt (23.2%), mnt (20.8%), mt (15.7%), rh (17.1%) influenced R5 reproductive phase. The extreme heat as a stressor had a more critical role for late maturity maize hybrids production than drought in crucial phenophases of maize development

New toxigenic species of the genus Aspergillus on maize kernels

Species of the genus Aspergillus are distributed throughout the world but are primarily represented in subtropical and warm temperate climates. In warm, humid subtropical or tropical climates or even in temperate climates in which the growing season is unusually hot and dry, species of the genus Aspergillus can infect seeds in the fields. The movement of fungal species threatens food security and creates global public concern worldwide. Climate change is altering the distribution, incidence and intensity of plant pests that is moving north into more temperate zones of Europe. In recent years, Serbia has been facing climate changes accompanied by a warming trend, which is a cause for concern. A more frequent occurrence of Aspergillus species was observed in the years in which the weather conditions were characterized by high temperatures, lack of precipitation and pronounced drought conditions (2012, 2015, 2017). The intensive outbreaks of Aspergillus species were in 2012. In addition to the previously known species Aspergillus flavus, which was a storage pathogen, it also occurs in field conditions. We discovered a new species of A. parasiticus in Serbia, for the first time. During the 2015 and 2017 we identified new species of the genus Aspergillus section Nigri. In order to adapt to climate change, priority is given to the cultivation of drought-resistant maize hybrids. The introduced challenges require additional climate change adaptation strategies, including various changes in production systems, in order to maintain long-term productivity

First report of Aspergillus welwitschiae causing maize ear rot in Serbia

In recent years, countries in Southeast Europe are facing climate changes characterized by extreme hot weather, which contribute to the increased frequency of Aspergillus species. Because of these changes, Aspergillus parasiticus was isolated, for the first time, from maize grain in Serbia (Nikolic et al, 2018). The presence of black powdery mycelia on maize ears indicated occurrence of species of the genus Aspergillus section Nigri, which led to the need for detailed identification of these fungi. Disease incidence ranged from 10 and 15% in August 2013. Maize ears with black powdery symptoms were collected from field in Zemun Polje, Serbia. Symptomatic kernels were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed three times with sterilized water, then incubated at 25°C in the dark for 7 days on potato dextrose agar (PDA). Twenty isolates were identified as genus Aspergillus section Nigri. Monospore cultures formed black cottony colonies with a yellowish border on PDA. The average colony diameter was 50 mm. In order to reliably identify, isolates were transferred to Malt Extract agar (MEA) and Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. On MEA fungal colonies were dense, black, with yellowish border. The reverse side was colorless to pale yellow, with a yellow ring in the middle. The average size of conidia was 4.3 μm. The conidia were globose to sub-globose, smooth to roughened, which coincides with previous research (Silva et al, 2020). Given that the fungi Aspergillus niger and Aspergillus welwitschiae are morphologically indistinguishable (Susca et al, 2016), species level identification was completed by analysis of a partial sequence of the internal Page 1 of 5transcribed spacer (ITS) region (ITS1/ITS4 primers) and calmodulin gene (CMD5/CMD6 primers) (Samson et al., 2014). The sequences were compared with the sequences of A. welwitschiae strains registered in the GenBank database based on nucleotide similarity, and results showed 99,64 and 100% similarity with ITS (OL711714) and calmodulin (KX894585), respectively. The sequence was deposited in GenBank with accession numbers OQ456471 (ITS) and OQ426518 (calmodulin). We also confirmed the presence of this species with specific primers (AWEL1/AWEL2) designed by Susca et al. 2020. Pathogenicity test was performed in Zemun Polje on the same maize hybrid from which the fungal species was isolated. Using artificial inoculations by the injecting conidial suspension into the silk channel, three days after 50% of plants reached the silking stage. Twenty ears were inoculated with each isolate, in four replicates (Reid et al, 1996). Inoculum was prepared from 7-day-old colonies on PDA, and 2 ml of a conidial suspension (1×106 spores/ml) was used. Control plants were inoculated with sterile water. All inoculated ears showed symptoms, similar to those from field infections. Control ears were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch’s postulates. Based on molecular, morphological and pathogenic properties, the isolates were identified as A. welwitschiae. This is the first report of A. welwitschiae as the causal agent of black maize ear rot not only in Serbia, but also in the other countries of the Western Balkans. Given that the fungus A. welwitschiae synthesizes both ochratoxin A (OTA) (Battilani et al, 2006) and fumonisin (FB) (Frisvad et al, 2011), further studies should be focused on assessment its aggressiveness and toxicological profile