Colonisation of winter wheat grain by Fusarium spp. and mycotoxin content as dependent on a wheat variety, crop rotation, a crop management system and weather conditions.

Field experiments were conducted during three consecutive growing seasons (2007/08, 2008/09 and 2009/10) with four winter wheat (Triticum aestivum L.) cultivars - 'Bogatka', 'Kris', 'Satyna' and 'Tonacja' - grown on fields with a three-field crop rotation (winter triticale, spring barley, winter wheat) and in a four-field crop rotation experiment (spring wheat, spring cereals, winter rapeseed, winter wheat). After the harvest, kernels were surface disinfected with 2% NaOCl and then analysed for the internal infection by different species of Fusarium. Fusaria were isolated on Czapek-Dox iprodione dichloran agar medium and identified on the basis of macro- and micro-morphology on potato dextrose agar and synthetic nutrient agar media. The total wheat grain infection by Fusarium depended mainly on relative humidity (RH) and a rainfall during the flowering stage. Intensive rainfall and high RH in 2009 and 2010 in the period meant the proportions of infected kernels by the fungi were much higher than those in 2008 (lack of precipitation during anthesis). Weather conditions during the post-anthesis period changed the species composition of Fusarium communities internally colonising winter wheat grain. The cultivars significantly varied in the proportion of infected kernels by Fusarium spp. The growing season and type of crop rotation had a distinct effect on species composition of Fusarium communities colonising the grain inside. A trend of a higher percentage of the colonised kernels by the fungi in the grain from the systems using more fertilisers and pesticides as well as the buried straw could be perceived. The most frequent species in the grain were F. avenaceum, F. tricinctum and F. poae in 2008, and F. avenaceum, F. graminearum, F. tricinctum and F. poae in 2009 and 2010. The contents of deoxynivalenol and zearalenon in the grain were correlated with the percentage of kernels colonised by F. graminearum and were the highest in 2009 in the grain from the four-field crop rotation. The content of T-2/HT-2 toxins was the highest in 2010 in grain from the three-field crop rotation and it was correlated with the isolation frequency of F. langsethiae.

A simple, direct plating method, alternative to dilution plating, for estimation of the abundance of Penicillium verrucosum on incubated cereal grain.

Because dilution plating is more labor intensive than direct plating, we would like to propose the use of a direct plating technique for estimation of Penicillium verrucosum abundance in cereal grain in incubation studies, with use of very selective and indicative for the fungus agar DYSG medium. The proposed method is based on the measurement of the diameter of P. verrucosum colonies grown around cereal kernels placed on DYSG medium. In three different experiments wheat grain contained a great range of P. verrucosum CFU number (from < 25 to 77 x 10(6) per 1 g). When P. verrucosum CFU number was at least as high as 10(2) per 1 g of the grain, 100% of the wheat kernels, placed on the surface of DYSG medium, were surrounded by colonies of P. verrucosum. The diameter (x, mm) of P. verrucosum colonies surrounding wheat kernels on DYSG medium was correlated with the fungal CFU number (y) on the wheat grain. The relationship is described by the exponential regression equation (y = 0.1258 e (0.9309x), R2 = 0.96). The relationship became linear (y = 0.404 x - 0.901, R2 = 0.96) after transformation of P. verrucosum CFU numbers to logarithms to base 10.

Growth of Penicillium verrucosum and production of ochratoxin A on nonsterilized wheat grain incubated at different temperatures and water content.

The results of two experiments with wheat grain inoculated with Penicillium verrucosum are reported. In Experiment I, wheat grain, containing 10, 20 and 30% water, was incubated for 2 weeks at 10, 15, 21 and 28 degrees C. In Experiment II, wheat grain, containing 14, 16, 18, 20 and 22% water, was incubated for 2 weeks at 10, 15, and 20 degrees C. At initial moisture content (IMC) of the wheat grain up to 16% neither P. verrucosum growth nor ochratoxin A (OTA) formation were observed. In the range of IMC from 18% to 22% both the fungal growth and OTA synthesis were distinct, and the parameters were higher at higher temperature in the range 10-21 degrees C. A temperature of 28 degrees C was probably too high for proper metabolism of the fungus, including OTA formation. OTA formation was distinctly related to P. verrucosum abundance in the temperature range 10-21 degrees C, expressed both as the counts of fungal colony forming units (CFU) on agar DYSG medium and diameters of the fungal colonies growing around the wheat kernels placed on the surface of DYSG medium. OTA formation and abundance of P. verrucosum were negatively correlated with the percentage of wheat kernels, placed on DYSG medium, with growing colonies of fungi different from P. verrucosum. CFU counts of P. verrucosum on the wheat grain were significantly related to the diameter of the fungal colonies growing around the wheat kernels placed on DYSG medium. The relationship is described by an exponential regression equation.

An attempt to protect winter wheat against Gaeumannomyces graminis var. tritici by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides.

Pseudomonas fluorescens strains III107 and II21 and Bacillus mycoides strains JC192 and K184, stimulating growth of winter wheat, were chosen for the studies. The bacterial strains inhibited on agar nutrient medium the growth of Gaeumannomyces graminis var. tritici (Ggt)--the pathogenic fungus causing take-all on wheat. Both strains of pseudomonads synthesized relatively high amounts of Fe3+ chelators. The strains of bacilli were characterized by the very fast spreading on agar media. Furthermore, strain II21 was highly cyanogenic, and strain JC192 highly chitinolytic. Bacterization of winter wheat seeds (especially with strains III107 and JC192) significantly reduced the percentage of the plants infested with the pathogen in the 28 day glasshouse pot experiment. In the plot experiment, the winter wheat seeds were inoculated with a mixture of strains III107, II21 and JC192. Due to the bacterization the yield of wheat grain and straw was higher in comparison to the series with Ggt alone by 122% and 75%, respectively, but it amounted only to 45% and 43% of the control series not contaminated with Ggt. The decrease of percentage of wheat ears with weight less than 500 mg from 61% in Ggt-series to 25% in Ggt-bacterized-series, and especially the decrease of percentage of wheat ears with weight less than 200 mg from 43% to 14% additionally indicate the partial protection of the winter wheat against Ggt by the rhizobacteria. In the experimental series not contaminated with Ggt the percentage of these wheat ears fractions did not exceed 3% and 0.5%, respectively.

An attempt to protect winter wheat against Fusarium culmorum by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides.

Inoculation of wheat seeds with two strains of Pseudomonas fluorescens (III107 and II21) and two strains of Bacillus mycoides (JC192 and K184) isolated from winter wheat roots, as well as with one strain of P fluorescens (ID13) isolated from oat roots, reduced the negative influence of Fusarium culmorum on winter wheat in a 28 day pot experiment. The bacterial strains (especially III107 and chitinolytic JC 192) markedly increased the plant seedlings emergence and the plant biomass (the shoots weight up to 252%, and the roots weight up to 229%) in comparison to the experimental series with E culmorum alone. Also in a microplot experiment the yield of grain and straw of winter wheat, inoculated with the bacterial strains (especially JC192 and III107) and growing in soil contaminated with F. culmorum, was higher (the grain yield up to 120%, and the straw yield up to 139%) than in a series with F. culmorum alone (100%). In both experiments the highly cyanogenic strain II21 was least effective. A linear correlation (r = 0.926) and a rank Spearman's correlation (rSp = 0.991), both significant at p<0.01, between the weight of plant biomass in the pot experiment and the yield of whole shoots in the microplot experiment were found. It suggests that the same mechanisms worked in both experiments, although with different intensity.