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首页> 外文期刊>Plant Pathology >Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems
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Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

机译：基于PCR的“分子工具箱”的开发和应用，用于鉴定破坏森林和自然生态系统的疫霉菌

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A PCR-based 'molecular tool box',based on a region of the ras-related protein gene Ypt1,was developed for the identification of 15 Phytophthora species that damage forests and trees:P.cactorum,P.cambivora,P.cinnamomi,P.citricola,P.europaea,P.inundata,P.lateralis,P.megasperma,P.nemorosa,P.kernoviae,P.pseudosyringae,P.psychrophila,P.quercina,P.ramorum and P.ilicis.Most primers proved highly specific in BLAST analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium.Exceptions were primers designed for P.cactorum and P.ilicis,which cross-reacted with P.idaei and P.nemorosa,respectively.Amplification with Phytophthora-genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only:detection limits ranged between 100 and 10 pg target DNA pL~(-1) in the latter,compared with 100 fg muL~(-1) in nested PCR.Using existing methods for rapid extraction and purification of DNA,single-round amplification was appropriate for detection of target Phytophthora species in leaves,but nested PCR was required for soil and water samples.The quarantine pathogens P.ramorum and P.kernoviae were detected in a number of naturally infected leaves collected in England and Wales,whereas P.citricola was commonest in water and soil samples from natural Scottish ecosystems.
机译：基于ras相关蛋白基因Ypt1的一个区域，开发了一种基于PCR的“分子工具箱”，用于鉴定15种破坏森林和树木的疫霉菌：仙人掌，斑节对虾，肉桂，柠檬酸假单胞菌，欧洲不透明假单胞菌，unundata假单胞菌，侧生假单胞菌，巨大精子，nemorosa假单胞菌，kernoviae假单胞菌，拟南芥假单胞菌，quercina假单胞菌，ramorum和Piliculis在BLAST分析和从35种疫霉菌和9种典型的腐霉菌中分离出的DNA的DNA的测试中证明具有高度特异性。分别使用疫霉属特异性引物进行扩增，然后再使用各种物种特异性引物进行扩增（嵌套PCR），与仅使用物种特异性引物进行扩增相比，检出灵敏度更高：检测限范围为100至10 pg目标DNA后者的〜（-1）与巢式PCR中的100 fg muL〜（-1）相比。使用现有的快速提取和纯化DNA的方法，单轮扩增适合检测叶片中的目标疫霉菌种，但需要对土壤和水样品进行巢式PCR。在英格兰和威尔士收集了许多自然感染的叶子，而柠檬酸假单胞菌在苏格兰自然生态系统的水和土壤样品中最常见。

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