Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

Abstract

To determine the genetic basis for loss of fumonisin B2 (FB2) biosynthesis in FB2-nonproducing Aspergillus niger and A. awamori strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified from FB2-producing A. niger and A. awamori strains; from FB2-nonproducing strains four amplification patterns arose in which one or more fum gene fragments were absent. Southern hybridization analysis of strains yielding patterns 2 and 3 confirmed that loss of FB2 production in A. awamori is associated with gene deletions within the fumonisin biosynthetic gene cluster. In addition, we observed a fifth multiplex amplification pattern in which all eight fum gene fragments appeared. Reverse transcription-PCR analysis of strains yielding pattern 5 showed that the expression of at least one fum gene was reduced relative to expression in FB2-producing A. niger. This suggests that in these strains loss of FB2 production is a result of structural or regulatory mutations that alter gene expression or function. These results demonstrate a diversity of genotypes within FB2-nonproducing A. niger and A. awamori populations and provide tools useful for identifying certain non-toxigenic strains for industrial or ecological applications.