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.
This serves as the final report for project 6435-42000-022-04S. 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-containing compounds continued to be detected. To assess molecular details of differential sensitivity to agrichemicals, a deoxyribonucleic acid (DNA)-based transformation system is needed. In our previous progress report, difficulties in transforming A. flavus isolate AF36 to zeocin resistance were noted. Further because there was variability in sensitivity to zeocin among A. flavus isolates, several different selectable marker systems were tested this year as well as different transformation protocols. An improved system was developed using protoplast transformation. This protocol gave much less variability and background in non-transformed strains than using niaD (name of a gene) gene with a nitrate reductase (enzyme) mutant. To demonstrate the utility of the specific transformation system the role in A. flavus of a transcription regulator thought to play a role in morphological switching in fungi was tested. Gene replacement mutants have been developed as well as ectopic (occuring in abnormal place) transformed strains of the AFL2G_05757 gene, known as RYP1 in the organism Histoplasma capsulatum. The method, polymerase chain reaction (PCR), demonstrated that the gene replacement strains lacked the wild type gene copy. Phenotypic (outside appearance) characterization of these strains is underway, and the effect on colonization of host tissue will be assessed. Phenotypic variation of transformed strains has been noted, both in terms of sporulation (ability to produce spores for reproduction) and sclerotial (overwintering bodies) formation but it has not been associated with the gene knockout yet. Continuation of this project will be dependent on identification of additional funding.