Yan Guiping, Smiley Richard W, Okubara Patricia A, Skantar Andrea, Easley Sandra A, Sheedy Jason G, Thompson Alison L
Oregon State University, Columbia Basin Agricultural Research Center, Pendleton 97801.
United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Root Disease and Biological Control Research Unit, Pullman, WA 99164.
Plant Dis. 2008 Nov;92(11):1480-1487. doi: 10.1094/PDIS-92-11-1480.
A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.
开发了一种物种特异性聚合酶链反应（PCR）方法，用于从土壤中检测和鉴定根腐线虫疏忽短体线虫（Pratylenchus neglectus）和索氏短体线虫（P. thornei）。根据短体线虫28S rRNA基因序列的D3扩展域设计了一组引物。用15种线虫的23个分离株以及土壤群落中通常存在的其他植物寄生和非植物寄生线虫，以及与小麦根腐病常见相关的6种真菌物种，确认了引物的特异性。使用市售试剂盒和我们实验室开发的方法获得的DNA产生了可比的扩增效果。优化了PCR条件，通过疏忽短体线虫144 bp和索氏短体线虫288 bp的PCR产物区分这两个物种。通过该检测方法，我们在1克无菌接种土壤中检测到了一条幼虫。对30个田间土壤样本的检测表明，该方法适用于俄勒冈州自然感染这两种病原体的一系列土壤。这种基于PCR的方法快速、高效且可靠，不需要线虫分类学和形态学方面的专业知识，可作为商业和研究应用中疾病预测和管理的快速诊断工具。
