Targeting Fumonisin Biosynthetic Genes
Methods in molecular biology, January 2017
Robert H. Proctor, Martha M. Vaughan
The fungus Fusarium is an agricultural problem because it can cause disease on most crop plants and can contaminate crops with mycotoxins. There is considerable variation in the presence/absence and genomic location of gene clusters responsible for synthesis of mycotoxins and other secondary metabolites among species of Fusarium. Here, we describe a quantitative real-time PCR (qPCR) method for distinguishing between and estimating the biomass of two closely related species, F. proliferatum and F. verticillioides, that are pathogens of maize. The qPCR assay is based on differences in the two species with respect to the genomic location of the gene cluster responsible for synthesis of fumonisins, a family of carcinogenic mycotoxins. Species-specific qPCR primers were designed from unique sequences that flank one end of the cluster in each species. The primers were used in qPCR to estimate the biomass of each Fusarium species using DNA isolated from pure cultures and from maize seedlings resulting from seeds inoculated with F. proliferatum alone, F. verticillioides alone, or a 1:1 mixture of the two species. Biomass estimations from seedlings were expressed as the amount of DNA of each Fusarium species per amount of maize DNA, as determined using maize-specific qPCR primers designed from the ribosomal gene L17. Analyses of qPCR experiments using the primers indicated that the assay could distinguish between and quantify the biomass of the two Fusarium species. This finding indicates that genetic diversity resulting from variation in the presence/absence and genomic location of SM biosynthetic gene clusters can be a valuable resource for development of qPCR assays for distinguishing between and quantifying fungi in plants.
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