Peterson G L, Bonde M R, Phillips J G
USDA-ARS Foreign Disease-Weed Science Research Unit, Fort Detrick, MD 21702.
USDA-ARS, North Atlantic Area, Wyndmoor, PA 19038.
Plant Dis. 2000 Sep;84(9):999-1007. doi: 10.1094/PDIS.2000.84.9.999.
A method was developed to isolate teliospores of Tilletia indica from infested grain. The technique was evaluated to determine its sensitivity for detection and quantification of teliospores, the time required to conduct an individual test, and its utility for the detection and identification of the pathogen for phytosanitary regulation and seed certification. A seed wash of a 50-g grain sample was washed through 53-μm and 20-μm pore size nylon screens to remove unwanted debris and to concentrate and isolate teliospores. The material retained in the 20-μm screen was suspended for direct microscopic examination or plated on water agar for teliospore germination and identification by polymerase chain reaction (PCR) utilizing two pairs of T. indica-specific primers. The reliability of detection for both light microscopy and PCR are 100% at an infestation of five teliospores per 50-g sample. The proportion of teliospores recovered from grain samples artificially infested with T. indica was 0, 82, 88, 81, and 82%, respectively, at infestation levels of 0, 1, 2, 5, and 10 teliospores per 50-g wheat sample. Extraction efficiency was comparable to the centrifuge seed-wash method currently used by most seed health laboratories. Sample analysis using size-selective sieving was more than 83% faster than the standard centrifuge seed wash.
已开发出一种从受侵染谷物中分离印度腥黑粉菌冬孢子的方法。对该技术进行了评估，以确定其对冬孢子检测和定量的灵敏度、进行单个测试所需的时间，以及其在植物检疫监管和种子认证中用于病原体检测和鉴定的效用。将50克谷物样品的种子洗液通过孔径为53微米和20微米的尼龙筛冲洗，以去除不需要的杂质，并浓缩和分离冬孢子。保留在20微米筛上的物质悬浮起来用于直接显微镜检查，或接种在水琼脂上，用于冬孢子萌发以及利用两对印度腥黑粉菌特异性引物通过聚合酶链反应（PCR）进行鉴定。在每50克样品中有五个冬孢子的侵染水平下，光学显微镜和PCR检测的可靠性均为100%。在每50克小麦样品中分别有0、1、2、5和10个冬孢子的侵染水平下，从人工接种印度腥黑粉菌的谷物样品中回收的冬孢子比例分别为0%、82%、88%、81%和82%。提取效率与目前大多数种子健康实验室使用的离心种子洗涤法相当。使用尺寸选择筛分法进行样品分析比标准离心种子洗涤法快83%以上。
