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United States Department of Agriculture

Agricultural Research Service

Research Project: Environmental and Ecological Approaches to Eliminate Fungal Contamination and Mycotoxin Production in Plant Products

Location: Foodborne Toxin Detection and Prevention

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

Authors
item Palumbo, Jeffrey
item O Keeffe, Teresa
item Gorski, Lisa

Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 6, 2012
Publication Date: March 1, 2013
Repository URL: http://www.mycologia.org/content/early/2012/09/06/11-418.full.pdf+html.
Citation: Palumbo, J.D., O Keeffe, T.L., Gorski, L.A. 2013. Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains. Mycologia. 105:277-284.

Interpretive Summary: In a previous study, 65 strains of Aspergillus niger that do not make the mycotoxin fumonisin B2 (FB2) were isolated from California raisin vineyard samples. To discover why these strains do not make FB2, we designed PCR primers to amplify and detect eight genes in the fumonisin biosynthesis gene cluster. Six groups of FB2 non-producing strains were derived from PCR results, each having a different pattern of gene amplification, as a result of presence or absence of these genes. One or more genes were missing from 45 strains; 20 strains contained all of the genes examined. We examined expression of fumonisin biosynthesis genes in these 20 strains, using reverse transcription-PCR to amplify the gene fragments from RNA. Under conditions permissive for FB2 production, all of the genes were expressed in FB2-producing strains. On the other hand, each of the 20 non-producing strains showed that at least one gene was not expressed, suggesting multiple causes for FB2 non-production. This work shows that loss of FB2 production in A. niger may be the result of a large number of genetic variations, so using any single fumonisin biosynthesis gene to predict whether a strain of A. niger is capable of making FB2 may be inaccurate.

Technical Abstract: In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger 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 in FB2-producing A. niger strains; in FB2 non-producing strains, five amplification patterns arose in which one or more fum gene fragments were absent. In these strains, it is likely that loss of FB2 production is the result of gene deletions within the fumonisin biosynthetic gene cluster. In addition, we observed a sixth multiplex amplification pattern in which all eight fum gene fragments appeared. Analysis of these strains by reverse transcription-PCR 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 the result of structural or regulatory mutations that alter gene expression or function. These results demonstrate the diversity of genotypes within FB2 non-producing A. niger populations, and provide tools useful for identifying non-toxigenic strains for industrial or ecological applications.

Last Modified: 11/21/2014
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