Genome-wide nucleotide variation distinguishes Aspergillus flavus from Aspergillus oryzae and helps to reveal origins of atoxigenic A. flavus biocontrol strains

Authors: Chang, Perng Kuang

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2019
Publication Date: 8/20/2019
Citation: Chang, P.-K. 2019. Genome-wide nucleotide variation distinguishes Aspergillus flavus from Aspergillus oryzae and helps to reveal origins of atoxigenic A. flavus biocontrol strains. Journal of Applied Microbiology. 127:1511-1520. https://doi.org/10.1111/jam.14419.
DOI: https://doi.org/10.1111/jam.14419

Interpretive Summary: Populations of Aspergillus flavus are diverse. They are generally divided into L-morphotype and S- morphotype. Isolates of S-type produce, on average, numerous smaller sclerotia and more aflatoxin than isolates of L-type. In this study, a method base on nucleotide variation between genomes was devised to investigate the genetic relatedness of A. flavus and Aspergillus oryzae. Isolates of L-type and S-type were shown to be genetically distant, which reflects their morphological and physiological differences. A subgroup of atoxigenic L-type isolates was found to resemble A. oryzae isolates. A previously misidentified A. oryzae was corrected as Aspergillus parasiticus species. The work also determined genetic origins of atoxigenic A. flavus biocontrol stains currently used in crops to outcompete toxigenic A. flavus isolates. Results of this study have important implications for selecting beneficial atoxigenic A. flavus strains in future biocontrol programs to eliminate aflatoxin contamination.

Technical Abstract: Aim To use total numbers of nucleotide variations between genomes to develop a molecular method for distinguishing isolates of Aspergillus flavus and Aspergillus oryzae. Methods and Results Thirteen A. flavus and eleven A. oryzae genome sequences were obtained from the National Center for Biotechnology Information (NCBI). These sequences were analyzed by MAUVE, a multiple-genome alignment program, to extract genome-wide nucleotide variations (total number of single nucleotide polymorphisms; total SNPs) between isolates from the same species (A. flavus vs A. flavus and A. oryzae vs A. oryzae) or of the two species (A. flavus vs A. oryzae). Results showed that averages of total SNPs of A. flavus isolates belonging to the same sclerotial morphotype (L-type or S-type) (L-type = 178,952 ± 14,033; S-type = 133,188 ± 16,430) and A. oryzae isolates (152,336 ± 49124) were consistently lower than those between A. flavus morphotypes and between the two species. Averages of total SNPs of L-type vs S-type (300,116 ± 1,562) and S-type A. flavus vs A. oryzae (301,797 ± 4,123) were similar but were 36% greater than that of L-type A. flavus vs A. oryzae (226,240 ± 10,779). Based on the devised criterion, ATCC12892, Aspergillus oryzae (Ahlburg) Cohn, which had an averaged total SNPs 10-fold greater than that of other A. oryzae isolates, was determined to be close to Aspergillus parasiticus. In addition, two atoxigenic L-type A. flavus field isolates, WRRL1519 and NRRL35739, were shown to more closely resemble A. oryzae than toxigenic L-type A. flavus. In contrast, two atoxigenic A. flavus biocontrol strains, AF36 and K49, were found to be genetically close to toxigenic L-type. NRRL21882, the active agent of the commercialized biocontrol product Afla-Guard® GR, was unique. It was genetically distant from toxigenic L-type and S-type as well as other atoxigenic L-type A. flavus. Conclusion The close genetic relatedness between A. flavus and A. oryzae was confirmed. ATCC12892, A. oryzae (Ahlburg) Cohn was determined to be not a correctly named species but is closer to A. parasiticus. The evolutionary origins of currently used atoxigenic A. flavus biocontrol strains were revealed. The results demonstrated the utility and efficacy of the devised method. Significance and Impact of the Study The study based on genome-wide SNP analysis provides a greater understanding of genome similarity and dissimilarity between A. flavus and A. oryzae. This method can be reliably used as an auxiliary technique to more accurately identify individual isolates as A. flavus, A. oryzae and A. parasiticus.