NATURAL TOXIGENIC FUNGAL AND MYCOTOXIN OCCURRENCE IN MAIZE HYBRIDS

: The objective of the present study was to investigate the susceptibility of maize hybrids to the natural occurrence of toxigenic fungal species, in particular toxigenic Aspergillus and Fusarium species, and mycotoxins (aflatoxin B 1 (AFB 1 ), deoxynivalenol (DON) and total fumonisins B 1 , B 2 and B 3 (FBs)). Grain samples of six commercial maize hybrids (MAS 34.B – FAO 300, MAS 40.F, MAS 48.L, KWS Konfites and ZP 427 – FAO 400, and MAS 56.A – FAO 500) were collected at harvest in 2018. A total of seven fungal genera, Acremonium, Alternaria, Epicoccum, Fusarium, Nigrospora, Penicillium and Rhizopus , were identified of which only species from the genus Fusarium were present on maize grains of all hybrids tested. The incidence of Fusarium spp. was higher in the hybrids MAS 48.L (54.6%), and MAS 56.A (53.3%), compared to MAS 40.F (37.3%), KWS Konfites The agro-ecological conditions in 2018 favored the development of Fusarium species on maize grains of the hybrids tested, especially fumonisin producing species.


Introduction
Maize is the main cereal crop in Serbia, grown on about one million hectares (Statistical Yearbook of the Republic of Serbia, 2019). It is used for human and animal nutrition and industrial processing. However, under stress abiotic and biotic factors, the maize grains can be infected by mycotoxigenic fungi. The most common mycotoxigenic fungi isolated from harvested and stored maize grains belong species from the genera Fusarium, Aspergillus and Penicillium (Krnjaja et al., 2015). These fungal species produce secondary metabolites (mycotoxins) which cause adverse effects on animal and human health, and economic losses. In particular, species from genera Aspergillus and Fusarium, causers of ear rot, cause serious risk of mycotoxin accumulation in maize (Masiello et al., 2019). Recently, aflatoxins, which are secondary metabolites produced by Aspergillus flavus Link and A. parasiticus Speare have been isolated in high levels on the maize grains in Serbia (Kos et al., 2018;Obradović et al., 2018). Also, mycotoxins such as type B trichothecenes (deoxynivalenol) and zearalenone produced primarily of Fusarium graminearum Schwabe and fumonisins produced mainly of F. verticillioides (Sacc.) Nirenberg and F. proliferatum (Matsush.) Nirenberg have been detected on maize grains in Serbia (Jajić et al., 2008;Krnjaja et al., 2015;Obradović et al., 2018;Jakšić et al., 2019).
Farm animals are sensitive to higher mycotoxin levels. Among the four main aflatoxins (B 1 , B 2 , G 1 , and G 2 ), aflatoxin B 1 (AFB 1 ) is the most toxic and causes liver damage in animals. The toxic effects of deoxynivalenol (DON) on pigs are feed rejection, vomiting, reproductive, and neurological disorders (Biagi, 2009;Reddy et al., 2017). Leukoencephalomalacia of horses and porcine pulmonary edema of pigs are diseases caused by fumonisins B 1 , B 2 and B 3 (FBs) (Dohnal et al., 2010;Leggieri et al., 2015).
Maize grains can be contaminated with toxigenic fungi and their mycotoxins before and after harvesting. High temperatures and levels of humidity from silking to maturity stages are favourable conditions for fungal ear colonization by Fusarium species and synthesize of Fusarium mycotoxins (Logrieco et al., 2002), while drier growing seasons suitable for the growth of Aspergillus spp. and aflatoxin accumulation in maize crops (Giorni et al., 2019). The occurrence of mycotoxins in maize grains is also dependent on hybrid susceptibility (Blandino et al., 2017).

Natural toxigenic fungal and mycotoxin … 77
The control strategy for the prevention of fungal and mycotoxin contamination of maize grains includes pre-and post-harvest measures. The most important preventive measures in the pre-harvest time are good agricultural crop practices and the utilization of tolerant maize hybrids (Blandino and Reyneri, 2008). The drying grains to below 15% moisture content, insect control, the application of detoxification methods are the most common post-harvest measures in storages (Di Gregorio et al., 2014;Kumar and Kalita, 2017).
Since the selection and sowing of the tolerant and less susceptibility maize hybrids as one of the success measures in reduce fungal contaminants, the research purpose was to determine the natural occurrence of toxigenic fungal species, in particular, toxigenic Aspergillus, and Fusarium species, and mycotoxins, AFB 1 , DON, and FBs, in five foreign and one domestic maize hybrids in agro-ecological climate conditions in Serbia.
Hybrids were grown in 2018 in the experimental field of the Institute for Animal Husbandry, Belgrade-Zemun. The sowing and harvesting date of the hybrids was consistent with the FAO maturity groups. The plot size was 440 m x 50.4 m, sub-plot was 440 m x 8.4 m. Each maize hybrid sown in 12 rows, with a 0.7 m inter-row spacing. Crop densities were in accordance with manufacturers recommendations.
Maize grain samples were collected at harvest time. The sub-plot divided into three parts. A total of 30 ears were randomly taken per each hybrid (sub-plot), 10 ears from each part, then put in the paper bags and transferred to the laboratory. The maize grains of 10 ears (sub-sample) manually removed. A total of 18 maize grain sub-samples, each sub-sample approximately of 500 g weight, were kept at 4°C until analyses.
The moisture content of maize sub-samples was determined using OHAUS MB35 (USA) moisture analyser. In mycological analyses, maize grains were disinfected in 1% NaOCl (sodium hypochlorite) for a few minutes, rinsed in distilled water, and dried on the filter paper. Per each hybrid, 300 grains were plated on potato dextrose salt agar, 100 grains per sub-sample, 5 grains per plate (Krnjaja et al., 2019). After14 days of keeping plates on the room temperature, fungal species were identified using fungal keys of Leslie and Summerell (2006) and Watanabe (2002). The incidence of fungal species on maize grains was calculated according to Lević et al. (2012).
Before the mycotoxicological analysis, ground maize sub-samples dried during 72 h at 60°C. Enzyme-Linked Immunosorbent Assay (ELISA) for determining AFB 1 , DON, and FBs levels was applied according to the manufacturer's instructions Celer Tecna® ELISA kits in three repetitions. The limit of detection for AFB 1 . DON and FBs were 1 μg kg -1 , 40 μg kg -1 and 750 μg kg -1 , respectively.
Effect of different maize hybrids on the mycotoxin levels was determined by one-way analysis of variance (One-Way ANOVA) using SPSS software (IBM,Statistic 20). Comparing means values with significant differences at P ≤ 0.05 was done using the Tukey's test. The Pearson correlation coefficient was used in correlation analyses.

Results and Discussion
In a Belgrade area, according to the meteorological data of the Republic Hydro Meteorological Services of Serbia, in 2018, the mean monthly temperatures (˃ 20°C), total monthly rainfall (> 40 mm) and mean monthly relative humidity (RH) (˃ 60%) at the flowering stage (July) and milk stage (August) were suitable for fungal maize colonization (Graphic 1).
The incidence of Fusarium species on the maize grains was similar to the reported data of De Curtis et al. (2011) andCovarelli et al. (2011). Fusarium species were isolated from the grains of two commercial maize hybrids grown in Serbia, of which F. verticilioides, F. graminearum, and F. proliferatum were predominant. Mycotoxin analyses showed that in the hybrids tested, the mean levels of AFB 1 , DON and FBs did not exceed maximum limits of 5, 1750, and 4000 μg kg -1 , respectively, prescribed by European Commission (EC, 2007; and Serbian Regulation (Službeni Glasnik RS, 2014) for unprocessed maize.
Similar to our results, Blandino et al. (2017) have also established that FBs and DON levels in maize grains influenced by the type of the hybrids from FAO 500 and 600 maturity groups and environmental conditions. In addition, Van Rensburg et al. (2016) concluded that seven South African maize genotypes were differed in susceptibility to natural fungal and FBs contamination. Leggieri et al. (2015) found that there were no significant differences in the aflatoxins level between maize hybrids from different FAO maturity groups in two-year trials (2009)(2010)(2011). Also, in Serbia, in 2013, Krnjaja et al. (2016 have demonstrated that there were no significant effects of hybrids and the interaction effect of hybrids and location on the level of AFB 1 in the maize hybrids from FAO 300, 400, 500, and 600 maturity groups. Means followed by the same letter within a column are not significantly different according to Tukey's multiple comparison test (P ≤ 0.05); ** -significant at the 0.01 level of probability, *significant at the 0.05 level of probability, nsnot statistically significant Using the Pearson correlation analyses, a statistically significant positive correlation (P<0.05) was established among the incidence of F. verticillioides and F. proliferatum (r = 0.56) (data not presented). No significant positive correlations were established among the incidence of F. graminearum and F. subglutinans (r = 0.15), the incidence of F. verticilioides and F. proliferatum and the FBs level (r = 0.22 and r = 0.08, respectively), and the incidence of F. graminearum and the DON level (r = 0.40). The incidence of F. verticillioides and F. proliferatum was in no significant positive correlations with the moisture grain content (r = 0.22 and r = 0.24, respectively). The coefficients of correlation indicate that FBs and DON levels were influenced by the incidence of their Fusarium producers. In the studies of Balconi et al. (2014), it was also confirmed that the FBs level was depended on the incidence of F. verticillioides.

Conclusion
In this study, fungal species of the genus Fusarium, as economically important toxigenic species, were isolated from the grains of all maize hybrids, while Aspergillus species were not detected in any samples. Climatic factors in the growing season in 2018 were very favourable for the development of Fusarium species. Among Fusarium species, fumonisin producing species were dominant. The levels of mycotoxins, AFB 1 , DON, and FBs, were influenced by hybrids.
These results confirmed that in addition to climatic factors (temperature, rainfall, and RH), the susceptibility of hybrids was also one of the important risks for the appearance of toxigenic fungi and their mycotoxins. Therefore, investigation of susceptibility of hybrids should also be the focus of further studies with an aim for advancing integrated pest management control in the maize production.