Palumbo Jeffrey D, O'Keeffe Teresa L, Ho Yvonne S, Fidelibus Matthew W
Foodborne Toxin Detection and Prevention Research Unit, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, USA.
University of California, Kearney Agricultural Research & Extension Center, 9240 South Riverbend Avenue, Parlier, California 93648, USA.
J Food Prot. 2016 Mar;79(3):448-53. doi: 10.4315/0362-028X.JFP-15-437.
Several species of Aspergillus section Nigri, including potential mycotoxin producers, are common residents of grape vineyards, but the relative population size of individual species throughout the growing season is difficult to determine using traditional isolation and identification methods. Using a quantitative droplet digital PCR (ddPCR) method in combination with dilution plating, total Aspergillus section Nigri populations and relative proportions of A. niger, A. welwitschiae, A. carbonarius, and A. tubingensis were measured from vineyard samples without the need for identifying individual fungal isolates. Grapes were sampled from two raisin vineyards (vineyards A and B) at berry set, veraison, harvest, and raisin stages in two consecutive years. Plate counts showed that the total population of Aspergillus section Nigri present on the fruit increased from berry set to raisin and became a larger component of the total recovered fungal population in both vineyards in both years. Results from ddPCR analysis showed that the relative proportion of A. carbonarius among the four species assayed increased later in the season (harvest and raisin) in comparison to earlier in the season (berry set and veraison). Total fungal and Aspergillus section Nigri plate counts were not significantly different between vineyards in either year. However, vineyard A generally showed higher proportions of A. carbonarius in harvest and raisin samples than vineyard B. This coincided with higher incidence and levels of ochratoxin A in vineyard A harvest and raisin fruit than in vineyard B fruit. This work demonstrates that this ddPCR method is a useful tool for culture-independent monitoring of populations of mycotoxigenic Aspergillus species during grape and raisin production.
包括潜在产霉菌毒素菌株在内的几种黑曲霉属真菌是葡萄园中的常见菌种，但使用传统的分离和鉴定方法很难确定整个生长季节中各个菌种的相对种群大小。通过将定量微滴数字PCR（ddPCR）方法与稀释平板法相结合，无需鉴定单个真菌分离株，就能测定葡萄园样品中黑曲霉属真菌的总种群数量以及黑曲霉、魏氏曲霉、炭黑曲霉和管囊曲霉的相对比例。连续两年在坐果期、转色期、收获期和葡萄干期从两个葡萄干葡萄园（葡萄园A和葡萄园B）采集葡萄样本。平板计数显示，果实上黑曲霉属真菌的总种群数量从坐果期到葡萄干期有所增加，并且在这两年中都是两个葡萄园总回收真菌种群中更大的组成部分。ddPCR分析结果表明，与季节早期（坐果期和转色期）相比，在季节后期（收获期和葡萄干期），所检测的四种菌种中炭黑曲霉的相对比例有所增加。在这两年中，两个葡萄园的总真菌和平板计数的黑曲霉属真菌数量均无显著差异。然而，葡萄园A收获期和葡萄干期样本中炭黑曲霉的比例通常高于葡萄园B。这与葡萄园A收获期和葡萄干期果实中赭曲霉毒素A的较高发生率和含量高于葡萄园B果实的情况相吻合。这项工作表明，这种ddPCR方法是在葡萄和葡萄干生产过程中对产霉菌毒素黑曲霉菌种进行非培养监测的有用工具。
