Development and application of multiple polymerase chain reaction assays to detect ochratoxin-producing Aspergillus and Penicillium, and trichothecene-producing Fusarium in stored and processed grain.

摘要

Cereal crop plants and stored grains are colonized by many mycotoxigenic fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecenes. Polymerase chain reaction based assays were developed and used to detect and quantify these mycotoxin-producing fungi in stored and processed barley. The multiplex real-time PCR method developed in the present study can simultaneously detect ochratoxin-producing A. ochraceus and P. verrucosum and trichothecene-producing F. graminearum. It can also quantify genomic DNA over five orders of magnitude (3 pg to 30 ng). We also observed that the biomass and volatile content of these fungi increased in barley stored at various moisture contents (13%, 18%, 20%, and 25%) over time. The volatiles, 3-methyl-1-butanol, 1-octen-3-ol and 3-octanone, significantly (P 0.05) increased, reaching as high as 5.7 microg/g, 14.0 microg/g and 12.3 microg/g in naturally infected barley on day 22 of storage at 25% moisture content. We observed that the mycotoxin-producing gene (Tri5) expression occurred during the third day of the germination stage of barley malting. We found, by random polymorphic DNA analysis that electron beam irradiation may have caused mutations in F. graminearum and that the mycotoxicity of the fungus was reduced by irradiation.