Development of TaqMan assays for the quantitative detection of Fusarium avenaceum/Fusarium tricinctum and Fusarium poae esyn1 genotypes from cereal grain

Abstract

Fungi of the genus Fusarium are important plant pathogens and contaminants of cereal grains producing different types of mycotoxins. Enniatins are a group of mycotoxins with ionophoric properties frequently detected in North European grains. Within the Fusarium complex responsible for grain infection, Fusarium avenaceum, Fusarium poae and Fusarium tricinctum are the most potential enniatins producers. This study presents the development of two quantitative TaqMan MGB (Minor Groove Binder) assays for the specific quantification of F. avenaceum/F. tricinctum and F. poae esyn1 genotypes, respectively. Two sets of genotype-specific primers/probes were designed on the basis of esyn1 gene homologues encoding multifunctional enzyme enniatin synthetase. The specificity of the assays developed has been tested successfully on 111 Fusarium isolates from different geographical origins. The detection limits for F. avenaceum/F. tricinctum esyn1 genotype and F. poae genotype were 19 and 0.3 pg, respectively. The application of the assays developed on asymptomatic wheat grain samples revealed significant positive correlations between the enniatins levels and the amount of F. avenaceum/F. tricinctum esyn1 genotype (R=0.61) and F. poae esyn1 genotype (R=0.42).

Use of QuantitativePCRDetection Methods to Study Biocontrol Agents and Phytopathogenic Fungi and Oomycetes in Environmental Samples

“…), while Fusarium avenaceum , F. tricinctum and F. poae esyn1 genotypes were detected on asymptomatic wheat grain samples and revealed a significant positive correlation between the amount of this genotype and enniatin levels (Kulik et al. ). A multiplex qPCR method to quantify aflatoxin, ochratoxin A, patulin and trichothecene producing moulds in foods was recently developed using specific genes involved in the biosynthesis of the three toxins (Rodríguez et al.…”

“…Several studies have reported a correlation between the biomass of producing fungi and specific mycotoxin contaminations (Gil-Serna et al 2009;Boutigny et al 2012. However, as the toxigenic ability of each strain needs to be considered, various mycotoxin genotyping assays have been developed to directly quantify genes responsible for mycotoxin synthesis, from both fungal culture and plant material (Kulik et al 2011). The availability of toxin-specific qPCR assays can be used to study the pathogen population structure, competition between toxin-producing and non-producing subpopulations and the effects of disease management strategies on reducing toxin contaminations (Luo et al 2009;Sanzani et al 2009).…”

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants: Quantitative PCR and ELISA Accurately Determine Fusarium Biomass

“…Furthermore, both methods observe the same effect, as visual disease symptoms are mainly caused by mycotoxin intoxication and not by the growth of mycelium. Studies have shown that the amount of fungal mycelia formed during infection not always correlates well with these parameters (Waalwijk et al, 2004;Hill et al, 2006;Brunner et al, 2009;Kulik et al 2011). Asymptomatic kernels may contain significant amounts of mycotoxins while symptomatic kernels within the same sample may not.…”

“…For some of these studies species specific assays were used to assess the biomass of various Fusarium species (Schnerr et al, 2000;Waalwijk et al, 2004;Brandfass & Karlovsky, 2006;Yli-Mattila et al 2008) or group specific assays were used to identify of quantify certain toxin producers (Schnerr et al 2001(Schnerr et al , 2002Bluhm et al 2004;Waalwijk et al, 2008;Kulik et al, 2011).…”

Transcriptional regulation of enniatins production by Fusarium avenaceum

“…Recent studies showed high intraspecific variability of this species with a lack of host specialization and a not clear association with a particular geographical area (Kulik et al . ,b). A wide range of variation in pathogenicity and mycotoxin production between isolates has been reported (Yli‐Mattila et al .…”