Svircev Antonet M, Kim Won-Sik, Lehman Susan M, Castle Alan J
Agriculture and Agri-Food Canada, Vineland Station, ON, Canada.
Methods Mol Biol. 2009;508:115-29. doi: 10.1007/978-1-59745-062-1_10.
Erwinia amylovora is the causative agent of fire blight, a very destructive disease of numerous members of the rosaceae. The primary route of infection for host species, including commercially grown apple and pear, is the newly opened blossom. Susceptibility of flowers to infection for only a few days creates narrow window for infection. Not surprisingly, the risk of disease is related to E. amylovora population size. As a result, methods that supply quick, accurate and sensitive quantification of the pathogen population are important tools for determining the need for and the efficacy of disease control intervention. Plating samples and assessing colony-forming units constitutes an accurate and sensitive but slow method. Endpoint PCR is quick and sensitive but is not particularly amenable to quantification. We describe a real-time PCR procedure that provides all requirements. This method is based on chromosomal genes rather than on the pEa29 plasmid and so can be used to measure isolates that have been cured of the plasmid. The method has been used very successfully in directly quantify whole E. amylovora cells, in a variety of tissues from the orchard environment.
梨火疫病菌是火疫病的致病因子，火疫病是蔷薇科众多植物的一种极具破坏性的病害。包括商业种植的苹果和梨在内的寄主物种的主要感染途径是新开的花朵。花朵仅在几天内易受感染，这为感染创造了狭窄的窗口期。毫不奇怪，病害风险与梨火疫病菌的种群数量有关。因此，能够快速、准确且灵敏地定量病原菌种群的方法是确定病害控制干预措施的必要性和有效性的重要工具。对样品进行平板接种并评估菌落形成单位是一种准确、灵敏但耗时的方法。终点PCR快速且灵敏，但不太适合定量分析。我们描述了一种满足所有要求的实时PCR方法。该方法基于染色体基因而非pEa29质粒，因此可用于检测已消除该质粒的分离株。该方法已非常成功地用于直接定量果园环境中各种组织中的完整梨火疫病菌细胞。
