Population Dynamics of Intra- and Inter-Specific Crosses and the Effect of Biocontrol on Natural Populations of Aspergillus Species

My dissertation research evaluates the ramifications of sex in experimental and natural populations of Aspergillus flavus. Experimental crosses were performed in the lab and progeny isolates evaluated for toxin heritability and diversity. I used this knowledge to understand population responses to biocontrol application in a longitudinal sampling design in a field in North Carolina.;In my first project, I examined the genotypes of F1 offspring from several experimental crosses of A. flavus. Linked loci within the aflatoxin gene cluster on chromosome 3 and unlinked loci on different chromosomes were analyzed to detect crossovers and independent assortment. My results indicate that recombination increases the effective population sizes of aflatoxigenic fungi and is driving genetic and functional hyperdiversity in A. flavus. I also observed non-mendelian inheritance of extra-genomic aflatoxin cluster alleles in crosses with at least one parent carrying a partial aflatoxin cluster, which suggests a possible role of cryptic alleles, in addition to sexual recombination, in modulating aflatoxin production.;Aflatoxin production is maintained under balancing selection, however is associated with a cost to production as evidenced by the success of non-aflatoxigenic biocontrol strains used as strategies to reduce aflatoxin contamination in agriculture. These biocontrol strains are applied at high densities to agricultural fields, where they competitively exclude native aflatoxigenic strains from crops and thereby reduce aflatoxin content. These methods are the most effective means of decreasing aflatoxin contamination in the short-term (i.e., several months); however, usage of biocontrol strains is not sustainable and requires repeat applications yearly to remain effective. Furthermore, the effect of these biocontrol strains on the genetic structure of A. flavus populations in these fields is unknown.;In my second project, I sampled A. flavus strains from a cornfield in Rocky Mount, NC to determine the effects of using biocontrol strains on the population diversity of A. flavus. Soil samples were taken before the application of biocontrol and one year afterward. In addition to the soil samples, sclerotia (sexual structures) were harvested from infected corn ears. Eighty A. flavus isolates were collected from the two sets of soil samples and ninety single-ascospore (sexual propogule) isolates were isolated from sclerotia originating from plots treated with biocontrols, for a grand total of 250 isolates. PCR amplification revealed grouping of isolates into three distinct mating-type classes: MAT1-1, MAT 1-2 and MAT 1- 1/MAT1-2. A significant proportion of isolates sampled prior to biocontrol treatments were heterokaryotic for mating type (MAT1-1/MAT1-2), and this same genotype was found in the ascospore isolates. The vertical transmission of MAT1-1/MAT1-2 to progeny ascospore isolates suggests that heterokaryosis can be maintained in subsequent generations, and just like in my first project, is driving genetic diversity in A. flavus. Furthermore, matings were performed to determine functionality of these MAT1-1/MAT1-2 strains and all isolates tested were strictly functional as MAT1-2 , which indicates a non-functional cryptic copy of the mating-type allele. Two biocontrol products are currently approved for commercial application; the population genetic structure before biocontrol application showed the presence of one biocontrol strain and no detection of the other. One year after the application of the two biocontrol strains, the population genetic structure showed a dominance of the biocontrol type found in the field prior to application. These results indicate the degree of relatedness of the biocontrol strain to the predominant indigenous lineage influences the long-term success of a biocontrol strain. These findings will be instrumental in the selection of a strain for use in next-generation biocontrol strategies.