Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR

Authors: Palumbo, Jeffrey - Jeff; O Keeffe, Teresa; FIDELIBUS, MATTHEW - University Of California - Cooperative Extension Service

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2016
Publication Date: 12/1/2016
Publication URL: http://handle.nal.usda.gov/10113/5717468
Citation: Palumbo, J.D., O Keeffe, T.L., Fidelibus, M.W. 2016. Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR. Letters in Applied Microbiology. 63:458–465. doi: 10.1111/lam.12667.

Interpretive Summary: Black-spored Aspergillus fungi are difficult to identify based on visual and microscopic characteristics, and species are usually distinguished based on gene sequencing. To evaluate populations of these coexisting and closely related fungi in soil, growing the fungi in culture and identifying individual strains is time-consuming and inefficient. We developed a culture-free method to detect and measure populations of four black-spored Aspergillus species in soil using a quantitative PCR technique. Using this technique, relative amounts of each fungal species can be estimated based on the relative amount of single gene sequences present from each species in a soil sample. We demonstrated that this method can measure the amount of each species DNA when added as a defined mixture (for example, a mixture of equal parts of DNA from four species) into the PCR reaction. We applied this method to analysis of grape vineyard soil samples, which were collected over two growing seasons from two vineyards. All species tested for were present in different proportions in all soil samples. Together with traditional microbiological methods, this quantitative PCR method provides a way to more quickly define the growth patterns of these fungi in soil communities and other environments.

Technical Abstract: Identification of populations of Aspergillus section Nigri species in environmental samples using traditional methods is laborious and impractical for large numbers of samples. We developed species-specific primers and probes for quantitative droplet digital PCR (ddPCR) to improve sample throughput and simultaneously detect multiple species in each sample. The ddPCR method was used to distinguish A. niger, A. welwitschiae, A. tubingensis and A. carbonarius in mixed samples of total DNA. Relative abundance of each species measured by ddPCR agreed with input ratios of template DNAs. Soil samples were collected at six time points over two growing seasons from two raisin vineyards in Fresno County, California. Aspergillus section Nigri strains were detected in these soils in the range of 10^2 to 10^5 gene copies per gram. Relative abundance of each species varied widely among samples, but in 52 of 60 samples, A. niger was the most abundant species, ranging from 38% to 88% of the total population. In combination with total plate counts, this ddPCR method provides a high-throughput method for describing population dynamics of important potential mycotoxin-producing species in environmental samples.