Wednesday, November 15, 2006
270-16
Use of Pyrosequencing to Quantify Incidence of a Specific Aspergillus flavus within Complex Fungal Communities Associated with Commercial Cotton Crops.
Modan Das, Kenneth C. Ehrlich, and Peter J. Cotty. USDA-ARS, USDA-ARS U. of Arizona, Plant Sciences Department, AZ
Atoxigenic strains of Aspergillus flavus have been used as aflatoxin management tools on over 100,000 acres of commercial crops since 2000. To assess treatment efficacy, atoxigenic strain incidence is routinely monitored by vegetative compatibility (VC) analyses that require culturing, generation of auxotrophs, and complementation with tester mutants. Two pyrosequencing assays that require no culturing were developed for monitoring atoxigenic strains based on characteristic Single Nucleotide Polymorphisms (SNPs) in the aflR and pksA genes. Cottonseed was collected from commercial gins in South Texas, Arizona, and Southern California where the atoxigenic strain AF36 is used to manage aflatoxin contamination. Cottonseed was washed with 0.005% Tween 80, calcium chloride and diatomaceous earth were added and the wash was centrifuged. DNA was extracted from the pellet. Accuracy and reproducibility of the pyrosequencing assays were contrasted with those for VC analyses. Three samples with high AF36 incidences (87.8% to 98.9% by VC analysis) had 73% to 98.8% AF36 by aflR assay and 89.9% to 96.7% AF36 by pksA assay. Six other samples had AF36 incidence ranging from 23.4% to 42.9% by VC analysis, 22.3% to 46.7% by aflR assay and 22.1% to 78.8% by pksA assay. Correlation coefficients between VC analyses and pyrosequencing assays were high (r = 0.91 for aflR assay and r = 0.80 for pksA assay). Pyrosequencing was highly variable for samples with low quantities of A. flavus DNA and averaging of results from 4 to 5 replicates was required in order to achieve acceptable correlations with VC analyses. Pyrosequencing might allow monitoring of atoxigenic strain incidence without culturing.