River Basin Research Center, Gifu University, Gifu 501–1193, Japan.
Microbes Environ. 2013;28(2):195-203. doi: 10.1264/jsme2.me12177. Epub 2013 Apr 24.
Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.
腐霉属烟草疫霉和腐霉引起草莓疫霉腐烂病。本研究建立了一种同时检测和定量这两种病原菌的双重实时 PCR 技术。根据 rDNA 的内部转录间隔区（ITS）和 Ras 相关蛋白基因 Ypt1，分别为腐霉属烟草疫霉和腐霉设计了种特异性引物。TaqMan 探针分别用 FAM 标记用于腐霉属烟草疫霉，用 HEX 标记用于腐霉。用 52 种分离物，包括各种土壤传播病原体，验证了特异性。腐霉属烟草疫霉和腐霉 DNA 的灵敏度分别为 10 fg 和 1 pg。该技术应用于自然侵染的土壤和根样本；检测到两种病原菌，并定量了目标 DNA 浓度。发现根和周围土壤中 DNA 数量之间存在显著相关性。估计出导致疾病症状发展的最小土壤 DNA 浓度为 20 pg（g 土壤）（-1）。在所检查的三个草莓温室中，腐霉属烟草疫霉的目标 DNA 浓度范围为 1 至 1,655 pg（g 土壤）（-1），腐霉属的目标 DNA 浓度范围为 13 至 233 pg（g 土壤）（-1）。该方法快速可靠，为监测植物或土壤中的腐霉属烟草疫霉和腐霉提供了有用的工具。
