Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2005
Publication Date: 8/15/2005
Citation: Chang, P.-K., Horn, B.W., Dorner, J.W. 2005. Sequence Breakpoints in the Aflatoxin Biosynthesis Gene Cluster and Flanking Regions in Nonaflatoxigenic Aspergillus flavus Isolates. Fungal Genetics and Biology. 42(11):914-923. Interpretive Summary: Aspergillus flavus populations are extremely diverse genetically, and isolates vary considerably in their capacity to produce aflatoxins and cyclopiazonic acid (CPA). Surveys of various geographic locations have shown differences in the proportions of isolates that do not produce or produce low, intermediate, or high amounts of aflatoxins. Estimates are that 20 to 30 percent of A. flavus field isolates do not produce aflatoxins. In this study, we investigated the defect(s) in the aflatoxin gene cluster of 16 AFB1-negative/CPA-positive and 24 AFB1-negative/CPA-negative A. flavus isolates collected from southern United States. The latter group includes A. flavus NRRL 21882, the active ingredient of the biopesticide Afla-Guard®. We found diverse deletion patterns of the aflatoxin gene cluster in the A. flavus isolates. The chromosomal breakpoints responsible for some of the deletions were determined. NRRL 21882 and 17 isolates all have the entire aflatoxin gene cluster deleted. Since some of these A. flavus isolates produce abundant conidia and others copious over-winter sclerotia, they could serve as candidate biocontrol strains if future biopesticide formulation with more than one strain is needed. This work verifies the biosafety of certain field isolates with regard to aflatoxin production potential. It would expedite the progress of field trials using nonaflatoxigenic biocompetitive A. flavus isolates to control aflatoxin contamination of crops.
Technical Abstract: Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxin (AF) and cyclopiazonic acid (CPA) are found in the field. A 66-kb gene cluster is responsible for the synthesis of AF. However, factors associated with nontoxigenicity of A. flavus isolates are not understood. To investigate if defects are present in the AF gene cluster of AF-negative A. flavus isolates collected from southern United States, we performed PCR assays on 40 isolates using AF-gene specific primers. The 16 CPA-positive and 24 CPA-negative isolates were grouped into four (A to D) and five (E to I) deletion patterns, respectively. Chromosomal breakpoints of the patterns C, F, and I were determined. One breakpoint of the 3 isolates of pattern C is in the 3’ non-translated region of the cypA gene, 217 nt downstream of the TGA stop codon. Another breakpoint is in the coding region of the verA gene, at the first nt encoding the amino acid residue #378 of VerA. The size of the deleted region is 40 kb. Pattern F, two isolates of different vegetative compatibility groups (VCGs), has a breakpoint at the #300 nt upstream of the start codon of the ver1 gene. The breakpoints of the two isolates at the other end are identical, although the position in the chromosome is not known. Pattern I consists of 18 isolates belonging to different VCGs and includes the active ingredient of the biopesticide Afla-Guard®, A. flavus NRRL 21882. For pattern I in which the entire AF gene cluster is deleted, the breakpoint beyond the hypA end of the cluster is in the hexA gene of the downstream sugar utilization gene cluster, at the first nt encoding the amino acid residue #62 of HexA. The breakpoint beyond the norB gene extends to an end of the chromosome as suggested by the presence of the 12 repeats of the telomeric sequence, TCAACATTAGGG reported for Aspergillus oryzae. Isolates in the same VCG have identical deletion patterns, but isolates belonging to different VCGs also have identical deletion patterns. Deletions in the AF gene cluster among A. flavus isolates thus are not rare and the patterns appear to be diverse.