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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Aflatoxin Production by Targeting Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

Title: Heritability study of eGFP-transformed Aspergillus flavus strains

Author
item Moore, Geromy

Submitted to: World Mycotoxin Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 2014
Publication Date: N/A

Interpretive Summary: Each of six non-aflatoxigenic Aspergillus flavus isolates, tagged with enhanced green fluorescent protein, was crossed with a toxigenic A. flavus strain. All crosses resulted in viable ascospore production although the heritability of fluorescence was variable. Germinating ascospores resulted in colonies with variation in fluorescence at every level of growth— even within the same conidial chain. Fluorescent offspring showed similar intensity of fluorescence compared to their eGFP-transformed parents. Inheritance of mycotoxins was also detected, with evidence of recombinant offspring exhibiting fluorescence as well as toxin production. A biocontrol strain that maintains fluorescence through subsequent generations is integral to a time-course study that will monitor their movement and persistence in the field. This manuscript continues a research project that aims for easier tracking of biocontrol strains as a preliminary project for future field trials.

Technical Abstract: Pre-harvest prevention of aflatoxin contamination of corn, cottonseed, and peanut through field inoculation with non-aflatoxigenic Aspergillus flavus appears to be the only method for biocontrol currently being used. Until recently, evidence for out-crossing in A. flavus was observed in agar slants. Now there is evidence that out-crossing can occur in a field setting on a similar time scale as in vitro. A previous study tested the ability of non-aflatoxigenic strains, transformed with enhanced green fluorescent protein (eGFP), to maintain fluorescence and to compete against wild type atoxigenic as well as toxigenic strains. The success of long-term field testing of eGFP strains depends on their maintained fluorescence, and to ensure that what will be sampled is indeed the fluorescent atoxigenic strain we must first determine the likelihood of out-crossing between these fluorescent strains and individuals from the native population. In vitro mating experiments paired each of six different eGFP transformed non-aflatoxigenic strains with a toxigenic A. flavus isolate that has proven fecundity in previous mating experiments. Our findings indicate that the eGFP gene is heritable by the F1 progeny. In this study, not all cultured ascospores exhibit fluorescence, and subsequent colony conidiophores show variability in both vegetative and spore fluorescence. Of particular interest is that preliminary toxin assays show some F1 individuals fluoresce while also producing aflatoxin and/or cyclopiazonic acid (CPA). The ultimate objective is to determine if out-crossing between biocontrol strains and native field populations occurs in a natural setting and poses a risk of increasing the future load of aflatoxigenic individuals in treated fields.

Last Modified: 9/20/2014
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