Molecular identification and characterization of Fusarium spp. associated with sorghum seeds.

BACKGROUND: Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp. RESULTS: All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters. CONCLUSION: The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.

Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India.

BACKGROUND: The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In the present study, a multiplex PCR was standardised for the group-specific detection of fumonisin-producing and trichothecene-producing strains of Fusarium species. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions 1 and 2 of rDNA. Primers for group-specific detection were designed from the tri5 and tri6 genes involved in trichothecene biosynthesis and the fum1 and fum13 genes involved in fumonisin biosynthesis. RESULTS: Among the various genera and their strains tested, all the 85 confirmed Fusarium strains were positive for rDNA gene and the rest stayed negative. From among the Fusarium strains, 15 had amplification for trichothecene- and 20 for fumonisin-encoding genes. All PCR positive trichothecene chemotypes of Fusarium species tested were positive for chemical analysis but in the case of fumonisins, of the 20 PCR positive cultures, only 13 showed positive for chemical analysis by HPTLC. CONCLUSION: The assay described here provided a rapid and reliable detection of trichothecene- and fumonisin-producing Fusarium directly from natural food grains and the results were always comparable with a conventional HPTLC detection method. It can, therefore, be used by the food industry to monitor quality and safety.