Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR.
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 Aspergillus niger, Aspergillus welwitschiae, Aspergillus tubingensis and Aspergillus 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 102-105CFU g-1. 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.


CONCLUSIONS
This is the first study to demonstrate the utility of ddPCR as a means to quantify species of Aspergillus section Nigri in soil. This method eliminates the need for isolation and sequence identification of individual fungal isolates, and allows for greater throughput in measuring relative population sizes of important (i.e. mycotoxigenic) Aspergillus species within a population of morphologically indistinguishable species.
Foodborne Toxin Detection and Prevention Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Albany, CA, USA.
J D
JD
Palumbo
Foodborne Toxin Detection and Prevention Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Albany, CA, USA.
T L
TL
O'Keeffe
Kearney Agricultural Research & Extension Center, University of California, Parlier, CA, USA.
M W
MW
Fidelibus
D001230:Aspergillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002140:California; D017931:DNA Primers / Q000235:genetics; D000072480:Farms; D009183:Mycotoxins / Q000235:genetics / Q000378:metabolism; D009793:Ochratoxins / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology; D013045:Species Specificity; D027843:Vitis / Q000254:growth & development* / Q000382:microbiology
D017931:DNA Primers; D009183:Mycotoxins; D009793:Ochratoxins
Aspergillus carbonarius; Aspergillus niger; Aspergillus tubingensis; Aspergillus welwitschiae; droplet digital PCR; fungal ecology; mycotoxins; vineyards