Yan Guiping, Smiley Richard W, Okubara Patricia A, Skantar Andrea M, Reardon Catherine 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 99164-6430.
Plant Dis. 2013 Jun;97(6):757-764. doi: 10.1094/PDIS-08-12-0729-RE.
Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.
短体线虫是分布最为广泛且具有重要经济意义的线虫之一，它会侵入植物根系，限制太平洋西北地区的小麦产量。对于需要大规模采样的研究，如轮作作物、小麦品种及其他管理措施的评估，使用显微镜方法对短体线虫进行定量分析具有挑战性。为此开发了一种实时定量聚合酶链反应（qPCR）检测方法，用于从土壤DNA提取物中检测和定量短体线虫。根据核糖体DNA的内部转录间隔区设计的引物，对8个短体线虫分离株的DNA具有高度特异性，表现为单一熔解曲线峰，但不会扩增来自28个其他植物寄生和非植物寄生线虫分离株的DNA。通过扩增添加了线虫的土壤中提取的DNA生成了标准曲线（R = 0.96；P < 0.001）。使用接种了较少数量短体线虫的无菌土壤对土壤标准曲线进行了验证。使用qPCR、怀特黑德托盘法和显微镜方法对15份田间土壤中的线虫数量进行定量分析，结果显示存在显著正相关关系（R = 0.66；P < 0.001），但qPCR通常倾向于给出更高的估计值。实时PCR可能为直接从田间土壤中高效检测和定量短体线虫提供一个有用的平台。
