1a.Objectives (from AD-416):
Characterize influences of agrichemicals, including glyphosate and copper, on communities of aflatoxin-producing fungi and evaluate the potential for improved aflatoxin management through altered use of agrichemicals and improved biocontrol with chemical resistant atoxigenic strains. Improve understanding of the genetic relationships among aflatoxin-producing and closely related fungi with varying sensitivity to agrichemicals.
1b.Approach (from AD-416):
Collections of Aspergillus flavus from agricultural environments in Arizona and Texas will be screened for sensitivity to agrichemicals. Relationships between chemical sensitivity and chemical exposure will be assessed. Genetic relationships among sensitive and resistant fungi will be assessed and barriers to gene flow among resistant and sensitive isolates evaluated. Competitiveness of resistant and sensitive fungi will be compared and attempts will be made to select atoxigenic strains with improved resistance to agrichemicals as elite biocontrol strains.
Research activities are carried out in close proximity to Agricultural Research Service (ARS) laboratories in Tucson, Arizona. Initial screening of Aspergillus (A.) flavus (a producer of the potent carcinogen, aflatoxin) fungal isolates from Texas and Arizona for sensitivity to a common herbicide failed to detect significant differences in sensitivity. Isolates with varying sensitivity to boron (chemical name)-containing compounds continued to be detected. To assess molecular details of differential sensitivity to agrichemicals, a deoxyribonucleic acid (DNA)-based transformation (method for transferring genes) system is needed. In our previous progress report, we noted difficulties in transforming A. flavus isolate AF36 to zeocin resistance. Further because there was variability in sensitivity to zeocin (an antibiotic) among A. flavus isolates, several different selectable marker systems were tested this year as well as different transformation protocols. We developed an improved system using a specific protoplast (cell without cell-wall) transformation which gave much less variability and background non-transformed strains than using the gene niaD with a nitrate reductase mutant. To demonstrate the utility of the transformation system we are characterizing the role in A. flavus of a transcription regulator thought to play a role in morphological switching in fungi. We have demonstrated by polymerase chain reaction (PCR) (an instrument that multiplies DNA pieces) that the gene replacement strains lack the wild type gene copy. Phenotypic characterization of these strains is underway, and the affect on colonization of host tissue will be assessed. Phenotypic variation of transformed strains has been noted, both in terms of sporulation (spore formation) and sclerotial (over wintering bodies) formation but it has not been associated with the gene knockout yet.