Ristaino J B, Madritch M, Trout C L, Parra G
Department of Plant Pathology, North Carolina State University, Raleigh 27695, USA.
Appl Environ Microbiol. 1998 Mar;64(3):948-54. doi: 10.1128/AEM.64.3.948-954.1998.
We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.
我们开发了一种聚合酶链式反应（PCR）程序，用于扩增DNA，以便快速鉴定植物病原菌疫霉属六个分类组中每个组的重要经济物种。该程序包括使用ITS引物ITS 5和ITS 4扩增5.8S核糖体DNA基因和内部转录间隔区（ITS）。用限制性内切酶RsaI、MspI和HaeIII对扩增的DNA产物进行限制性酶切。用RsaI酶切后，以下物种的限制性片段模式相似：辣椒疫霉和柑桔褐腐疫霉；致病疫霉、恶疫霉和奇异疫霉；草莓疫霉、樟疫霉和桃上的大豆疫霉；棕榈疫霉、柑桔褐腐疫霉、番茄红腐疫霉和隐地疫霉；以及树莓上的大豆疫霉和大豆疫霉。用MspI酶切可将辣椒疫霉与柑桔褐腐疫霉分开，并将恶疫霉与致病疫霉和奇异疫霉分开。用HaeIII酶切可将柑桔褐腐疫霉与隐地疫霉分开，樟疫霉与草莓疫霉和桃上的大豆疫霉分开，棕榈疫霉与柑桔褐腐疫霉分开，树莓上的大豆疫霉与大豆疫霉分开。致病疫霉和奇异疫霉的酶切结果相同，隐地疫霉和番茄红腐疫霉的酶切结果与所有测试的限制性内切酶相同。利用辣椒疫霉ITS区域I的独特DNA序列开发了一种名为PCAP的引物。PCAP引物与ITS 1一起用于PCR，仅扩增辣椒疫霉、柑桔褐腐疫霉和柑桔褐腐疫霉的分离株，而不扩增该属的其他13个物种。用MspI酶切可将辣椒疫霉与其他两个物种分开。在田间样本中，PCR在检测感染甜椒组织中的辣椒疫霉方面优于传统分离方法。所描述的技术将为疫霉属主要物种的鉴定提供一个强大的工具。
