Detection and discrimination of four aspergillus section nigri species by pcr

Authors: Palumbo, Jeffrey - Jeff; O Keeffe, Teresa

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2014
Publication Date: 2/1/2015
Publication URL: http://handle.nal.usda.gov/10113/60887
Citation: Palumbo, J.D., O Keeffe, T.L. 2015. Detection and discrimination of four aspergillus section nigri species by pcr. Letters in Applied Microbiology. 60(2):188-195. doi: 10.1111/LAM.12358.

Interpretive Summary: Species identification of black-spored Aspergillus is difficult using traditional microbiological methods. Current techniques for species identification rely on comparisons of common gene sequences to known species. To make species identification faster and simpler, se designed a set of PCR primers to amplify specific gene fragments from individual Aspergillus species. We chose A. niger, A. awamori, A. carbonarius, and A. tubingensis as representative of the major species found on grapes. Primers designed to amplify DNA from A. niger will not amplify the same gene fragment from A. awamori, A. carbonarius or A. tubingensis, and so on. When DNA from multiple species was mixed, the primers successfully amplified DNA in a species-specific manner. Because these fungi naturally exist in soil, we inoculated soil with combinations of these species, extracted DNA from the soil, and were able to use the designed PCR primers to amplify DNA fragments from each inoculated species. These results suggest that the PCR primers could be used for detection and differentiation of Aspergillus species in soil and other environmental samples.

Technical Abstract: Species of Aspergillus section Nigri are not easily distinguished by traditional morphological techniques, and typically are identified by DNA sequencing methods. We developed four PCR primers to distinguish between A. niger, A. awamori, A. carbonarius and A. tubingensis, based on species-conserved differences in the calmodulin gene sequence. PCR amplification from total DNA using these primers was species-specific; no amplification occurred from non-target species DNA for each primer pair. Species-specific PCR could distinguish between species in mixed DNA templates, indicating a utility in determining culture uniformity of isolated Aspergillus strains. In addition, with these primer sets, each species could be detected in soil following mixed-species inoculation with Aspergillus spores. This indicates that PCR with these species-specific primers may be useful in determining the distribution of Aspergillus species in environmental samples without the need for species identification from isolated strains, as well as detecting species that may be infrequently isolated by culture-based methods.