PRESENCE OF DEOXYNIVALENOL IN WINTER WHEAT TREATED WITH FUNGICIDES

Natural occurrence of Fusarium spp. and concentrations of mycotoxin deoxynivalenol (DON) in the grain of the winter wheat moderately susceptible to Fusarium head blight (FHB) has been studied. Grain samples were collected from wheat crops intended mainly for human consumption. All wheat crops were treated with fungicides before (a.i. flutriafol – formulated as Fluoco, applied in dose of 0.5 l ha) and during the flowering phase of growing (a.i. thiophanate-methyl + epoxiconazole formulated as Eskorta plus and a.i. thiophanate-methyl formulated as Funomil, applied in doses of 0.75 and 0.5 l ha, respectivily). Among of Fusarium species only F. graminearum, as a well known producer of DON, was identified. This fungus was identified in 15 of 19 samples (78.9%) with incidence in positive samples of 2 to 28% (average, 14.0%). Presence of DON was established in 13 of a total 19 investigated wheat grain samples (68.4%). In positive samples DON was detected in concentrations from 69 to 918 μg kg (average, 478 μg kg). DON showed a significant and positive correlation at P≥0.05 with grain moisture content (r = 0.52*). Between the frequency of F. graminearum and concentration of DON and between the frequency of F. graminearum and grain moisture content, positive correlation was determined, but without statistical significance (r = 0.44 and r = 0.29, respectively).


Introduction
Wheat is one of the most important crop cultures grown in Serbia, on approximately 500,000 ha, with average yield of 3,700 kg/ha (Statistical Yearbook of Serbia, 2012).It has been used for thousands of years to provide food for humans.For livestock feeding wheat grain can be used as concentrated livestock feed, whereas whole plant can be used as fodder.
There are several toxigenic species of Fusarium that are also a major pathogens of cereal plants, causing Fusarium head blight (FHB) in wheat.It is the major wheat disease occurring worldwide, especially in temperate climate regions, causing reductions in yield and quality of wheat (Parry et al., 1995).In our agroclimatic condition, F. graminearum has been isolated as the most present species from Fusarium-infected grains (Lević et al., 2008;Krnjaja et al., 2011).This fungus produces different mycotoxins, mainly deoxynivalenol, which contaminate grain (Nakajima, 2007a;Stanković et al., 2012).
Fusarium species produce a wide range of mycotoxins of diverse structure and chemistry.Deoxynivalenol (DON) is a trichothecene mycotoxin with toxic effects on animals and human health.Testing of grains and animal feed on the occurrence of Fusarium mycotoxins attracts considerable attention and has been the subject of extensive investigations over the recent years.DON concentration in Fusarium-damaged grain generally increases with the percentage of damaged grain in a given sample.It was reported that the amount of DON produced by F. graminearum was positively correlated with fungal biomass (Wegulo, 2012).Semaškiené et al. (2006) have established a slight reduction in Fusarium infection in the plants treated with fungicides compared to untreated plants of spring cereals.Wegulo (2013) has been demonstrated the importance of applying of integrated management in cultivation of wheat, with an emphasis on application of fungicides and host resistance.
In this paper the results of the occurrence of F. graminearum and DON concentrations in samples of winter wheat grains collected from crops treated by fungicides have been presented.

Materials and Methods
The total of 19 samples of wheat grains of variety Takovčanka collected in 2013 from crops cultivated in Institute for Animal Husbandry, Belgrade, were used for mycological and mycotoxicological analysis.Wheat crops were treated twice with fungicides during the period of wheat growing.In the first half of April, the samples were treated with fungicide based on flutriafol (formulated as Fluoco) at a dose of 0.5 l ha -1 wheras the combination of fungicides based on epoxiconazole + thiophanate-methyl (formulated as Excorta Plus) and thiophanate-methyl (formulated as Funomil) at doses 0.75 and 0.5 l ha -1 , respectively, has been applied in the second half of May, in the flowering phase.Average weight of 1 kg per sample of wheat grains was taken immediately after the harvest in July 2013 using standard methods (European Commission, 2006).The moisture content of wheat grains was determined using a moisture analyzer (OHAUS MB35, USA).
For the mycological analysis the wheat grains, were first disinfected in 1% sodium hypochlorite solution (NaOCl) 3-5 minutes and rinsed twice in distilled water.After drying of grains on filter paper, 50 grains of each sample was distributed in 5 Petri plates (9 cm in diameter) containing water agar (WA) (10 grains per one plate) and kept in incubator (Memmert) at 25ºC during 7 days.Identification of colonies of fungi that overgrowth the wheat grains was done by microscopic examination of mycelium and spores, according to Burgess et al. (1994) and Watanabe (1994).The frequency and incidence of individual species was calculated per sample according to Lević et al. (2012).
The presence of DON was detected by enzyme-linked immunosorbent assay (ELISA).Five grams of sample 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) for 30 minutes.Homogenate was filtered through a Whatman filter paper 1.The filtrate was further analysed according to the manufacturer's instructions Celery Techna ® ELISA kits.Absorbance was measured at a wavelength of 450 nm on an ELISA reader spectrophotometer (Biotek EL x 800TM, USA).
Correlation between individual values obtained for grain moisture content, frequency of F. graminearum and concentration of DON was determined using Pearson's correlation coefficient.

Results
Moisture content of the samples of wheat grains ranged from 12.1 to 15.0%, with an average of 13.7%, for all tested samples (data not presented).
Based on mycological analysis F. graminearum, as well known producer of DON, was identified.This fungus was found in 15 out of 19 samples with incidence of 2 to 28% (average, 14.0%) in positive samples.In all investigated samples, species of genus Alternaria were identified, with an incidence of 84.8% (range 70-100%).Epicoccum spp.and Penicillium spp.were determined in 15.8% of samples, and A. flavus and Rhizopus in 5.3% of the samples, while in 10.5% of samples the isolated species have not been sporulated (sterile mycelia) (Table 1).Mycotoxicological analysis of wheat samples revealed the presence of DON in 68.4% of the tested samples.The concentration of DON in positive samples of wheat ranged from 69 to 918 μg kg -1 with an average concentration of 478 μg kg -1 (Table 2).The concentration of DON was exceeded the permissible limit (750 μg kg -1 ) prescribed by the Serbian Regulation (Official Gazzete, 2011) in only two samples (data not shown).A positive correlation was established between concentration of DON and the grain moisture content (r = 0.52*, P≥0.05), between concentration of DON and the frequency of F. graminearum (r = 0.44), as well as between the grain moisture content and the frequency of F. graminearum (r = 0.29).

Discussion
In the samples of wheat collected in 2013, the F. graminearum species was identified as a FHB disease-causing species, with an average incidence of 14.0%.In the study of Krnjaja et al. (2011b), it has been reported the incidence of F. graminearum of 82.50% in wheat grains collected in 2009, not treated with fungicides, whereas in untreated samples collected in 2010, the incidence of F. graminearum was 99.05% (Krnjaja et al., 2011a).Since in this study the grains were treated with fungicide, the lower incidence of F. graminearum was obtained, compared to the previous results of Krnjaja et al. (2011a,b).This finding confirmed the assumption that the application of fungicides could be a suitable preventive measure for the control of the occurrence of Fusarium spp.during the growing season of wheat.
In this study, 68.4% DON positive samples of wheat were determined with an average concentration of 478 μg kg -1 , whereas Krnjaja et al. (2011a,b) have established 100% DON positive samples with an average concentrations of 490 μg kg -1 in 2009 and 214 μg kg -1 in 2010.It could be assumed that these differences in an average concentration of DON has been the results of differences among the tested varieties of wheat, the differences in grain moisture content at harvest as well as climatic conditions, particularly in terms of temperature and rainfall during flowering and early stages of grain development.
By applying the fungicide tebuconazole and thiophanate-methyl, Wachowska et al. (2012), have effectively controlled FHB and isolated the lower level of Fusarium spp. in treated wheat grains in comparison with the control plants.By examining the efficiency of seven fungicides in order to reduce mycotoxins Nakajima (2007b) has found that preparations based on thiophanatemethyl have significantly reduced the content of DON and nivalenol (NIV) mycotoxin in wheat grain.Metcalfe et al. (2000) have found that the use of the sterol 14-α-demethylase inhibitors (DMI) fungicides (fluquinconazole, flutriafol and prochloraz) in full recommended doses had not caused resistance of pathogenic fungi of wheat to these fungicides.Nakajima (2007b) found that the efficacy of the control of DON and NIV was consistently lower than that of FHB severity and assumed that critical control point of DON and NIV might be different from that of FHB severity.The high levels of DON and NIV could be produced beyond 20 days after anthesis even by early infection and infection at a late stage.For this reason, the frequency of fungicide application has been important in the prevention of infection by Fusarium spp.and likewise for mycotoxin reduction.The general recommendation for timing of fungicide application is the beginning of flowering phase of growth, in which the plants are most susceptible to Fusarium infection.Likewise, developing control strategies that cover the late stage as well as the early stage would be desirable to reduce the risk of mycotoxin contamination in wheat (Nakajima, 2007b).
Based on the regulated maximum permissible concentration of mycotoxins in food and feed in Serbia (Official Gazette of RS, 2011), it could be concluded that the examined batches/lots of unprocessed wheat grains can be used as food and feed because its contained a concentration of DON under the safe limit (1250 μg kg -1 ).However, for direct human consumption only wheat containing DON less than 750 μg kg -1 can be used.

Conclusion
In conclusion, the use of fungicides in wheat crops significantly reduces the presence of the disease-causing species of FHB and thus prevents considerable DON production.These results may be of great importance especially in years when outbreaks of causal agents of wheat head fusariosis are expected.The use of fungicides in conjunction with other preventive measures to protect the wheat from the appearance of FHB is important to promote the development of integrated pest management strategy.