FUSARIUM INFECTION AND DEOXYNIVALENOL CONTAMINATION IN WINTER WHEAT

In this paper, the incidence of Fusarium-infected grain of winter wheat and the content of mycotoxin deoxynivalenol (DON) was studied in two Serbian cultivars Simonida and NS40S, both harvested in 2014. The level of Fusarium contamination of wheat grain was determined using phytopathological techniques based on the standard methodology while DON was detected by enzyme-linked immuno-sorbent assay (ELISA). The incidence of Fusarium-infected grain ranged from 12 to 19% for Simonida and NS40S, respectively. Fusarium graminearum, as well-known producer of DON mycotoxin, was identified among Fusarium species. In addition, Alternaria spp. was isolated in high percentage, with an average incidence of 53% (Simonida) to 63% (NS40S).The average content of DON ranged from 424 μg kg to 1101 μg kg for Simonida and NS40S cultivars, respectively. Statistically insignificant negative correlation (r = – 0.18) was determined between Fusariuminfected grain and DON in the cultivar Simonida and statistically insignificant positive correlation (r = 0.11) in the cultivar NS40S. The mean levels of DON in studied wheat samples of both tested cultivars were not higher than the maximum permitted limit (1250 μg kg) although the level of Fusarium-infected grain of both cultivars was relatively high. These results indicate that both wheat cultivars are susceptible to Fusarium infection and DON mycotoxin production in agro-ecological conditions of Serbia, but the cultivar NS40S being more susceptible compared to cultivar Simonida. In view of all stated above, regular health check of grains and developing strategies for integrated monitoring of incidence of Fusarium head blight are necessary preventive measures in protection of winter wheat.


Introduction
Wheat is one of the most important cereal crop grown in Serbia, on approximately 500,000 ha, with average yield of 3,700 kg/ha (Statistical Yearbook of Serbia, 2012).Besides maize, it is the main crop for human consumption, commonly used in the production of bread.It can also be applied in livestock nutrition, as an integral part of the feed mixture, while the straw is used as bedding in barns and stables.
DON belongs to a larger group of trichothechenes, the type Btrichothecene, with toxic effects on animals and human health.In livestock production, DON is the main cause of reproductive disorders in pigs (Biagi, 2009).In cattle, DON causes reduced food intake and lower milk production (Trenholm et al., 1985).DON concentrations significantly increase in Fusarium-damaged grain (Wegulo, 2012).
During the growing seasons in the field, the main factor responsible for FHB development and DON contamination is highly associated with weather conditions during the period of anthesis as the most susceptible growth stage for Fusarium infection.Intense rainfall during the anthesis disperses Fusarium inoculums from crop residues and promotes FHB infection (Landschoot et al., 2012).Likewise, crop residues and infested debris are reported such as the major sources of the primary inoculum for Fusarium spp.involved in FHB epidemics (Miedaner et al., 2008).According to Blandino et al. (2012) FHB infection and DON contamination of wheat grains caused by different factors, at first place climatic conditions, but also agronomic factors including previous crop residue management, cultivar susceptibility and fungicide applications.Similar to that, Eiblmeier and von Gleissenthall (2007) have stated that weather conditions at flowering, preceding crop, no or minimal tillage, susceptible cultivar, strobilurin as foliar fungicide (EC 31 -EC 59) and late harvest represent risk factors for increasing levels of DON in wheat grains.Therefore an integrated approach to the disease is appropriate to reduce the risk of high DON values in wheat grains.
Taking into consideration the importance of the harmful effect of Fusarium species and deoxinivalenol in wheat grains, the presence Fusarium spp.and deoxynivalenol (DON) in grains of two Serbian winter wheat cultivars, was analysed.

Materials and Methods
A total of 40 grain samples of two Serbian wheat cultivars, 20 samples of cultivar Simonida and 20 samples of cultivar NS40S were analysed in mycological and mycotoxicological tests.Grain samples were collected during harvest in 2014, from the crops production of the Institute for Animal Husbandry, Belgrade, Serbia.The tested wheat crops were sown in October 2013 in the field where maize was grown previously.During the growing season, the same basic cultural practices and crop protection measures were carried out for wheat crops of both cultivars, fertilization in late February was performed with ammonium nitrate fertilizer, crop protection measures against weeds, insects and pathogenic fungi were carried out at the wheat tillering stage, and treatment with fungicides against FHB was performed at the beginning of flowering.Samples of wheat grains were collected according to the method of the European Commission (2006a), and the moisture content of the grain was determined by moisture analyzer (OHAUS MB35, USA).
For the mycological analysis, grains were disinfected in 1% sodium hypochlorite (NaOCl) for 2-3 minutes, and rinsed twice in distilled water.After drying on filter paper, 100 of grains, of each sample, were distributed in Petri dishes with the nutrient medium (10 grains per Petri dish).Plates were incubated for 14 days at 20°C with alternating light and darkness.Two percent (2%) wateragar (WA) was used as nutrient medium, with NaCl (18g NaCl per 1 litre of medium) added to prevent the grain germination.Identification of colonies of fungi that have developed around the grains of wheat was done based on microscopic examination of mycelia and spores, as reported by Burgess et al. (1994) and Watanabe (1994).The incidence of fungal species were calculated according to Lević et al. (2012)

: I (%) = [Number of kernels in one sample in which a species occurred] / [Total number of kernels in the same sample] x 100.
For the mycotoxicological analysis, the wheat grain samples were ground to a fine powder with an analytical mill (IKA A11, Germany).All the samples were kept at 4ºC in the refrigerator before further analysis.Five grams of powder was mixed with 1 g of NaCl and homogenized in 25 ml of 70% (v/v) methanol in a 250 ml Erlenmeyer flask on the orbital shaker (GFL 3015, Germany).Homogenate was filtered through a Whatman filter paper 1.The level of DON was detected using the competitive ELISA method according to the manufacturer's instructions Celer Tecna ® ELISA kits.Absorbance was determined at a wavelength of 450 nm on an ELISA plate reader spectrophotometer (Biotek EL x 800TM, USA).The limit of detection (LOD) was 40 μg kg -1 and the limit of quantification (LOQ) was 125 μg kg -1 for wheat for DON.The mean recovery turned to be 104±12%.
The correlation between individual values for moisture content, the incidence of Fusarium spp.and DON concentration was determined using the Pearson correlation coefficient.

Results
According to the data of Republic Hydro-meteorological Service of Serbia, for 2014 for Belgrade area, from 1 to 31 May abundant rainfall (278.5 mm) was recorded, when wheat was in the pheno stage of flowering.These were very suitable climatic conditions for the development of Fusarium infection on spike wheat.
The average moisture content of the tested samples of wheat was 9.96% (ranged from 9.30 to 10.29%) for the cultivar Simonida and 10.38% (ranged from 9.56 to 11.16%) for the cultivar NS40S.According to mycological analysis the species from the genera Alternaria and Fusarium were determined with highest incidence.Based on morphological characteristics, F. graminearum was identified as only producer of DON mycotoxin.In case of Simonida cultivar, in the examined samples, incidence of F. graminearum was 12% (ranged from 7-19%) wheras in the cultivar NS40S it was 19% (ranged from 5-35%).The incidence of Alternaria spp. was 53% (ranged from 38-65%) in the cultivar Simonida, and 63% (range 46-72%) in the cultivar NS40S (Table 1).The presence of DON was detected in 100% of tested samples of both cultivars.The average concentration of DON was 424 µg kg -1 (ranged from 175 -610 µg kg -1 ) in the cultivar Simonida, and 1101 µg kg -1 (ranged from 214-1440 µg kg -1 ) in the cultivar NS40S (Table 2).The average concentrations of DON in unprocessed wheat grain of both tested cultivars were not above the maximum permitted limit (1250 µg kg -1 ) adopted by the European Commission (2006b).Statistically insignificant negative correlation (r = -0.18)was determined between the incidence of F. graminearum and concentrations of DON in the cultivar Simonida, and statistically insignificant positive correlation (r = 0.11) in the cultivar NS40S.The correlation between moisture content and incidence of F. graminearum evident in both cultivars was statistically insignificant negative, while between moisture content and concentration of DON statistically insignificant positive correlation was found in both tested cultivars.

Discussion
In this study, a high incidence of F. graminearum was established in the investigated samples of wheat grains of both studied cultivars, although, cultivar NS40S had higher average incidence of F. graminearum (19%) compared to the cultivar Simonida (12%).Similarly, the average concentration of DON in Simonida was lower (424 µg kg -1 ) in relation to the cultivar NS40S (1101 µg kg -1 ).These results are similar to those reported by Krnjaja et al. (2014) where the average incidence of F. graminearum was 14% and the average concentration of DON was 478 µg kg -1 in the 19 tested wheat samples of the Serbian cultivar Takovčanka, collected during the harvest 2013.It should be noted that in all these trials, grains sampled originated from the wheat crop treated with fungicide against FHB at the flowering stage of development.Based on earlier research of Krnjaja et al. (2011a, b), the average incidence of F. graminearum was higher than 80% in wheat grain samples originating from crops that were not treated with fungicide at the flowering stage of development.The average concentration of mycotoxin DON ranged from 214 to 490 µg kg -1 (Krnjaja et al., 2011a, b).These results pointed out that the agro-ecological and agro-climatic conditions in Serbia are very suitable for the occurrence of FHB and production of DON mycotoxin in wheat grain.Furthermore, maize as the previous crop and important host species for Fusarium spp.provided more inoculum especially in favourable weather conditions such as heavy precipitation during anthesis of wheat in 2014.In our study, differences were found between the cultivars in terms of the incidence of F. graminearum and the DON content.This indicated that the use of less susceptible cultivars to FHB can be an important factor in reducing the occurrence of harmful contaminants in wheat grain.
According to Mesterházy et al. (1999) resistance of cultivars has also an impact on the contents of DON.In most resistant cultivars, a low average level of DON was detected, and in some growing seasons, DON was not detected at all.In addition, great differences in the content of DON in the same cultivar of wheat have been also established, that involved a large diversity of pathogenic populations in the field (Mesterházy et al., 1999).According to recent research of Mesterházy et al. (2002), it was concluded that the level of resistance of given cultivar was more important in accumulation of DON than the aggressiveness of Fusarium isolates.This is confirmed by our research in which the differences among the cultivars in terms of accumulation of DON were determined, although the incidence of F. graminearum was high in both wheat cultivars.Blandino et al. (2012) reported that the main factors affecting the formation of DON in wheat grain were; susceptibility of a wheat cultivar, the preceding crop (especially maize and sorghum), soil tillage, fungicide application at anthesis of wheat.Contrary to the above, Müller et al. (2010) pointed out that the monitoring of FHB and DON content requires good knowledge of agricultural factors, primarily crop rotations and tillage practice, then climatic conditions especially annual precipitation and topographic factors (relief position, topographic wetness index TWI).In the research of these authors, the susceptibility ranking of wheat cultivars to Fusarium infection had no significant influence on DON accumulation.
According to data from other countries with similar geographical and climatic conditions, Mankevičiene et al. (2007) have established the incidence of DON in 62.5% to 100% of cereal samples from harvests 2004 and 2005 in Lithuania, with concentrations ranged up to 1121µg kg -1 .The high concentrations of DON were highly associated with abundant rainfall in both investigated years.Likewise, Pleadin et al. (2012) revealed the high mean concentrations of DON (up to 1454µg kg -1 ) in 103 feed samples from three regions of Croatia.

Conclusion
It can be concluded that both tested cultivars were susceptible to FHB, although, NS40S cultivar, showed higher incidence of F. graminearum and higher DON content compared to the cultivar Simonida.Although the average DON levels did not exceed the maximum permitted limit, it is necessary to develop and implement the methods for monitoring the risk of harmful contaminants in wheat grain.Therefore, it is essential to conclude that the individual methods such as the application of less sensitive or tolerant cultivars in the strategy of control of FHB and mycotoxins in wheat grain, have lower effect in preventing the risk of harmful contaminants in the food chain, compared to a combination of several measures of crop protection such as crop rotation, tillage practice and fungicide application at anthesis of wheat, especially under favourable climatic conditions, existing in Serbia.

Table 2 . Level of DON in tested grain samples of two Serbian wheat cultivars
a Number of positive samples/Number of total samples; b Mean concentration in positive samples