Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum.

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2⁻106.0%, R²-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Sinapic Acid Affects Phenolic and Trichothecene Profiles of F. culmorum and F. graminearum Sensu Stricto.

Plant-derived compounds for reducing the mycotoxin load in food and feed have become a rapidly developing research field of importance for plant breeding efforts and in the search for natural fungicides. In this study, toxigenic strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to sinapic acid on solid YES media at levels close to those reported in wheat bran. Fusaria produced phenolic acids, whose accumulation was decreased by exogenous sinapic acid. Strains exposed to the lowest doses of sinapic acid showed more efficient reduction of phenolic acid production than fungi kept at higher concentrations of this compound. Fungi reduced exogenous sinapic acid, leading to the formation of syringic aldehyde. Treatment with sinapic acid led to a dramatic accumulation of its parent compound ferulic acid, presumably due to inhibition of the further conversion of this phenolic compound. Exogenous sinapic acid decreased the production of trichothecenes by fungi. Higher doses of sinapic acid resulted in more efficient reduction of mycotoxin accumulation in the media. Gene expression studies of Tri genes responsible for trichothecene biosynthesis (Tri4, Tri5 and Tri10) proved that the inhibition of mycotoxin production by sinapic acid occurred at the transcriptional level. Fusaria respond to sinapic acid by stimulation of ergosterol biosynthesis.

trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto.

Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time.

ToxGen: an improved reference database for the identification of type B-trichothecene genotypes in Fusarium.

Type B trichothecenes, which pose a serious hazard to consumer health, occur worldwide in grains. These mycotoxins are produced mainly by three different trichothecene genotypes/chemotypes: 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) and NIV (nivalenol), named after these three major mycotoxin compounds. Correct identification of these genotypes is elementary for all studies relating to population surveys, fungal ecology and mycotoxicology. Trichothecene producers exhibit enormous strain-dependent chemical diversity, which may result in variation in levels of the genotype's determining toxin and in the production of low to high amounts of atypical compounds. New high-throughput DNA-sequencing technologies promise to boost the diagnostics of mycotoxin genotypes. However, this requires a reference database containing a satisfactory taxonomic sampling of sequences showing high correlation to actually produced chemotypes. We believe that one of the most pressing current challenges of such a database is the linking of molecular identification with chemical diversity of the strains, as well as other metadata. In this study, we use the Tri12 gene involved in mycotoxin biosynthesis for identification of Tri genotypes through sequence comparison. Tri12 sequences from a range of geographically diverse fungal strains comprising 22 Fusarium species were stored in the ToxGen database, which covers descriptive and up-to-date annotations such as indication on Tri genotype and chemotype of the strains, chemical diversity, information on trichothecene-inducing host, substrate or media, geographical locality, and most recent taxonomic affiliations. The present initiative bridges the gap between the demands of comprehensive studies on trichothecene producers and the existing nucleotide sequence databases, which lack toxicological and other auxiliary data. We invite researchers working in the fields of fungal taxonomy, epidemiology and mycotoxicology to join the freely available annotation effort.

Depicting the Discrepancy between Tri Genotype and Chemotype on the Basis of Strain CBS 139514 from a Field Population of F. graminearum Sensu Stricto from Argentina.

Recent studies on a field population of F. graminearum sensu stricto from Argentina revealed an atypical panel of strains identified through PCR genotyping as 15ADON genotypes, but producing high levels of 3ADON. Based on representative strain CBS 139514, we asked if the discrepancy between the trichothecene genotype and chemotype might result from an inter-chemotype recombination of the chemotype-determining genes. To answer this, we sequenced the complete core Tri gene cluster (around 30,200 bp) from this strain and compared its sequence to sequence data of typical type B trichothecene genotypes/chemotypes. Sequence alignment showed that CBS 139514 has an identical sequence within the entire core Tri cluster to the 15ADON genotype. The revealed discrepancy underlines the need for using both molecular and chemical methods for reliable characterization of toxigenic strains of Fusarium.