Rapid Identification Of Aspergillus Spp. Using A Pcr Based Melting Curve Method And Characterization Of A Novel Chitinase In Insect Resistant Maize Lines
Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus.