Identification by PCR of Fusarium culmorum Strains Producing Large and Small Amounts of Deoxynivalenol

Abstract

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.Trichothecenes, including deoxynivalenol, acetyldeoxynivalenol, nivalenol, and fusarenone X, are sesquiterpene toxins produced by Fusarium species, including Fusarium culmorum, which are common fungal contaminants of cereals. Trichothecenes can be found naturally worldwide on cereals (1,9,18,27,38,45,48,54,55,59), and the consumption of these toxins is a potential problem for humans and farm animals (14, 47).It has been established that some Fusarium species, including F. graminearum and F culmorum, are able to produce B trichothecenes, such as deoxynivalenol and acetyldeoxynivalenol, while other species are not (31). These two types of Fusarium strains (producers and nonproducers) can be distinguished on the basis of DNA polymorphism in the ␤-tubulin gene (43) as well as in the large ribosomal subunit or the internal transcribed spacer (21,37,43).According to their trichothecene production, some Fusarium species, such as F graminearum, have been divided into two chemotypes: (i) the nivalenol chemotype, which includes isolates producing nivalenol and fusarenone X, and (ii) the deoxynivalenol chemotype, which includes isolates producing deoxynivalenol and acetyldeoxynivalenol (26,53). Similar observations have been made for F. culmorum strains (28, 39).In addition, it has been demonstrated that, within the same species and in the same culture conditions, toxin production by Fusarium strain may vary sharply; some strains produce large amounts of trichothecenes, whereas others produce small or undetectable amounts of trichothecenes (3,5,19,28,30,35,36,39,53,57). Until now, no method except in vitro culture has been available to distinguish high-producing from low-producing Fusarium strains.Several genes involved in the biosynthesis of trichothecenes have been described, most of them localized in a gene cluster. The tri5 gene encodes the trichodiene synthase, which catalyzes the first step in the biosynthesis of trichothecenes. The nucleotide sequence of the tri5 gene has been characterized in several Fusa...

Assessment ofFusariuminfection in wheat heads using a quantitative polymerase chain reaction (qPCR) assay

“…In a recent work (Terzi et al 2007), a qPCR-based analytical method was developed targeted at the quantification of the biomass of thricothecene-producing F. graminearum and F. culmorum strains using a pair of primers designed on the Tri5-Tri6 intergenic sequence which is involved in DON synthesis. This intergenic sequence has been already used (Bakan et al 2002;Li et al 2005), but the analytical approach developed by Terzi et al (2007) is quantitative and can be used also in samples characterized by a high level of DNA fragmentation, as those derived from food and feed, because of the small size of the amplicon. For this reason, it can be potentially used along the entire production chain.…”

The application of PCR in the detection of mycotoxigenic fungi in foods

“…17 Fusarium culmorum strains studied by Bakan et al (5) produced more than 1 ppm deoxynivalenol (DON) and were considered high-deoxynivalenol-producing strains, whereas 13…”

Detection of toxigenic fungi and quantification of type A trichothecene levels in some food and feed materials from India

“…Positive amplifications were also observed in Fusarium strains isolated from the toxin containing samples. Similar PCR assays have been developed for the detection of trichothecene producing Fusarium (Bakan, Girraud-Delville, Pinson, Richard-Molard, Fournier, & Brygoo, 2002;Niessen, Schmidt, & Vogel, 2004;Niessen & Vogel, 1998;Schnerr, Niessen, & Vogel, 2001). Initial screening of the materials using TLC showed six samples positive for T-2 and DAS, all of which were sorghum, and one poultry feed sample was positive for only T-2 toxin.…”