Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89124 Reggio Calabria, Italy.
J Microbiol Methods. 2012 Mar;88(3):356-68. doi: 10.1016/j.mimet.2011.12.012. Epub 2011 Dec 23.
Current molecular detection methods for the genus Phytophthora are specific to a few key species rather than the whole genus and this is a recognized weakness of protocols for ecological studies and international plant health legislation. In the present study a molecular approach was developed to detect Phytophthora species in soil and water samples using novel sets of genus-specific primers designed against the internal transcribed spacer (ITS) regions. Two different rDNA primer sets were tested: one assay amplified a long product including the ITS1, 5.8S and ITS2 regions (LP) and the other a shorter product including the ITS1 only (SP). Both assays specifically amplified products from Phytophthora species without cross-reaction with the related Pythium s. lato, however the SP assay proved the more sensitive and reliable. The method was validated using woodland soil and stream water from Invergowrie, Scotland. On-site use of a knapsack sprayer and in-line water filters proved more rapid and effective than centrifugation at sampling Phytophthora propagules. A total of 15 different Phytophthora phylotypes were identified which clustered within the reported ITS-clades 1, 2, 3, 6, 7 and 8. The range and type of the sequences detected varied from sample to sample and up to three and five different Phytophthora phylotypes were detected within a single sample of soil or water, respectively. The most frequently detected sequences were related to members of ITS-clade 6 (i.e. P. gonapodyides-like). The new method proved very effective at discriminating multiple species in a given sample and can also detect as yet unknown species. The reported primers and methods will prove valuable for ecological studies, biosecurity and commercial plant, soil or water (e.g. irrigation water) testing as well as the wider metagenomic sampling of this fascinating component of microbial pathogen diversity.
目前，针对疫霉属的分子检测方法主要针对少数几个关键物种，而不是整个属，这是生态研究和国际植物卫生法规协议中公认的一个弱点。在本研究中，开发了一种使用针对内部转录间隔区（ITS）区域设计的新型属特异性引物来检测土壤和水样中疫霉属物种的分子方法。测试了两种不同的 rDNA 引物组：一种测定法扩增了包括 ITS1、5.8S 和 ITS2 区的长产物（LP），另一种测定法扩增了仅包括 ITS1 的短产物（SP）。这两种测定法都特异性地扩增了来自疫霉属物种的产物，与相关的节旋体属没有交叉反应，但 SP 测定法被证明更敏感和可靠。该方法使用苏格兰因弗戈里的林地土壤和溪流水进行了验证。现场使用背包喷雾器和在线水过滤器比在采样时离心更快速和有效地提取疫霉菌繁殖体。共鉴定出 15 种不同的疫霉属菌系，它们聚类在报道的 ITS 群 1、2、3、6、7 和 8 中。检测到的序列的范围和类型因样本而异，在单个土壤或水样中，分别检测到多达三个和五个不同的疫霉属菌系。最常检测到的序列与 ITS 群 6（即类似于 P. gonapodyides 的成员）的成员有关。新方法在区分给定样本中的多个物种方面非常有效，并且还可以检测到未知的物种。所报道的引物和方法将对生态研究、生物安全以及商业植物、土壤或水（例如灌溉水）测试以及更广泛的微生物病原体多样性这一迷人组成部分的宏基因组采样具有重要价值。
