1a.Objectives (from AD-416):
Characterize adaptive differences among aflatoxin-producing fungi in their interactions with host plants and utilize on farm sampling to describe interactions between the life cycle of the causal agent and agronomic practices and in so doing define optimal windows for implementation of biological control and other interventions.
1b.Approach (from AD-416):
Genetic groups identified by ongoing ARS programs will be contrasted in field tests and controlled environment laboratory tests for variation in ability to colonize and decay host tissues using newly developed methods. Host samples from farmer fields in key target regions with severe contamination will be analyzed with novel microbiological assays. Coordination of sampling with farmers will test importance of various agronomic practices on the A. flavus life cycle.
Differences in colonization and competitive ability of aflatoxin-producing fungi were examined in inoculation experiments on sugarcane stems to investigate potential influences of debris from crop rotation on the soil community of aflatoxin-producing fungi and potential roles of fungal adaptation to hosts in these influences. The work in this project is undertaken by a student pursuing a Ph.D. in plant pathology. Work is carried out in the United States Department of Agricultural (USDA) laboratories in Tucson, AZ, on experimental farms, and in commercial sugar cane fields in the Rio Grande Valley. Competitive abilities of the fungi Aspergillus (A.) parasiticus and A. flavus were compared on sugarcane stems because sugarcane fields in the Rio Grande Valley of Texas (RGV) have Aspergillus section Flavi communities that are composed largely of A. parasiticus while those of every other crop, even those neighboring sugarcane fields, are composed exclusively of A. flavus. A. flavus dominates the Aspergillus section Flavi community outside of agricultural in fields in the uncultivated environment. Cotton, maize and sugarcane are common rotation crops in RGV. Stems were wounded and co-inoculated with both A. flavus L strain isolates collected from maize fields and A. parasiticus isolates found in sugarcane field soils or plant tissues collected in the Rio Grande Valley of Texas (RGV). The community of aflatoxin-producing fungi present in the environment where a susceptible crop is grown correlates to the aflatoxin-contamination risk for that crop. In the lab, sugarcane stems were surface sterilized, wounded with a cork borer, and inoculated with spore suspensions of either single isolates or mixtures of A. parasiticus and A. flavus. After 7 days, the wound site was excised from the stem, and shaken for 20 minutes in autoclaved 0.0001% Tween 80 solution. Spores (kind of seeds of the fungus) were separated from the wash solution by centrifugation, and deoxyribonucleic acid (DNA) was extracted from them directly. The washed stem piece was frozen at -80°C and lyophilized, then ground, and DNA was extracted using a cetyl trimethylammonium bromide (CTAB) buffer protocol. In order to quantify the competitive ability of A. parasiticus when co-inoculated with A. flavus on sugarcane, pyrosequencing (method for sequencing DNA) primers were developed based on sequence of the nitrate reductase gene, which includes two single nucleotide polymorphisms (a commonly found change in DNA sequence) (SNP) distinctly conserved for each of the two species. By detecting the ratio of nucleotides at each site, pyrosequencing should be able to determine which isolate is more successful both in the spore wash and in the mycelia infecting the wound site. This study is still underway and pyrosequencing results from the inoculated stems have not yet been obtained. Preliminary results based on fungi grown on culture media support use of this technique in these tests. This work is expected to give insights into how cropping systems select specific aflatoxin-producing fungi and in so doing alter both the structure and the average aflatoxin producing potential of fungal populations. Such information should serve the design of improved aflatoxin management strategies.