Next-generation biocontrol

Impact of female sterility on effectiveness and longevity of biological control.

Both Aspergillus flavus and Fusarium verticillioides are sexual fungi, and sexual reproduction in nature is sufficiently common to affect the population structure of both species. However, female sterility also occurs in both fungi, which is extremely important because it limits a strain to only male functions. Hornok et al postulated that a high frequency of female sterility can eventually drive a sexual population to clonality. Based on this information, we propose three testable hypotheses:

1) Releasing highly fertile biocontrol strains will shift populations towards more sexual reproduction, which causes aflatoxigenicity to be maintained.

2) Releasing highly female-sterile strains will drive populations toward clonality and thus reduce aflatoxigenicity.

3) Longer lasting biocontrol can be achieved by releasing female sterile strains matched to the dominant multilocus haplotypes in the indigenous population.

The project that is currently underway will test these hypotheses. We will determine the frequency of MAT1-1 and MAT1-2 among isolates from soil samples collected from AR, IN, NC, and TX. To ensure that mating type frequencies are not skewed by repeated sampling of the same genotype (clone), multilocus sequence typing (MLST) will be used to genotype the strains for clone correction. This analysis is a prerequisite for testing whether the frequencies of MAT1-1 and MAT1-2 are significantly different, and thus if clonality contributes significantly to a given population. We will determine the predominant haplotypes in the population and categorize as group IB or IC. Strains from ascospores produced in sclerotia sampled from corn ears will be analyzed for putative females by examining mitochondrial (mt) markers and by comparing the sequence of those mt markers to strains isolated from corn and soil. Putative parental strains will be crossed in the lab to confirm patterns of mitochondrial inheritance. For each of the four states, we will select both female-sterile and female-fertile strains belonging to IB and IC. These strains will be applied as biocontrol agents to fields in their respective states. Soil and ears will be sampled and genotyped for 3 years. Aflatoxin and fumoninin analysis will be performed on the ears to assess efficacy of the designed strains.


Impact of biocontrol strain application on population shifts in F. verticillioides and fumonisin contamination.

Similar to A. flavus, female sterility is also associated with clonality in F. verticillioides. F. verticillioides and A. flavus can co-occur in the same kernel,and it is common to have fields contaminated with both aflatoxin and fumonisin. Also, simultaneous inoculation of kernels with both fungi results in less infection by A. flavus and reduced aflatoxin production. A. flavus has been observed to reduce disease severity caused by F. verticillioides on maize,and high density applications of biocontrol strains may further impact this interaction in nature. Other fungi on the corn kernels are also known to reduce infection by both A. flavusand F. verticillioides. Thus, the interaction of A. flavus and F. verticillioides cannot be ignored in the development of an integrated management strategy. We will be examining the effect of biological control on population structure of these two fungi over several years under different environmental conditions. Moreover, it is not known if a shift towards clonality and nonaflatoxigencity in A. flavus induces a corresponding transition in F. verticillioides populations, which are diverse with strains varying greatly in their ability to produce fumonisin. Additionally, information about the structure of indigenous populations of F. verticillioides is needed as a baseline to understand potential interactions between nonaflatoxigenc biocontrol applications and fumonisin contamination of corn.

Research is planned and/or underway to address these questions. Field plots in four states (AR, IN, NC and TX) which are receiving Afla-Guard or AF36 treatment for protection against A. flavus will be used to gather information about F. verticillioides populations. F. verticillioides  will be isolated on dilution plates, population densities will be calculated and fumonisin assays will be performed. F. verticillioides typing will be based on 3 fumonisin genes (FUM1, FUM10, FUM19), translation elongation factor 1 gene (TEF-1α), and the mating-type genes. The impact of the biocontrol treatments will be measured each year for three consecutive years. These data will be subjected to Principal Component Analyses (PCA) to determine changes in population densities of F. verticillioides that occur as the result of A. flavus biocontrol applications.