Xanthomonas fragariae is the causal agent of an important bacterial disease in strawberry production regions worldwide and a quarantine plant pathogen in many countries including New Zealand. Xanthomonas fragariae mainly infects the foliage of strawberry plants but can also infect the calyx tissue associated with strawberry fruit. Fresh strawberries are a high‐value internationally traded commodity that has a short shelf‐life. When making biosecurity decisions based on the finding of a quarantine organism such as X. fragariae by PCR, one of the major challenges is the inability to differentiate positive results originating from viable or dead cells. Viability PCR (vPCR) is a technique that selectively inhibits PCR amplification of DNA derived from dead cells through the use of a nucleic acid intercalating dye, for example, PEMAX™. A vPCR protocol has been optimized to enable rapid detection of viable X. fragariae in a tissue sample. PEMAX™ treatment resulted in complete inhibition of PCR amplification of 10⁸–10³ cfu/ml dead X. fragariae cells in strawberry host tissue. The most important parameters for optimization were the dilution of the sample, amplicon length and choice of nucleic acid intercalating dye. This study provides a rapid protocol to discriminate between viable and dead X. fragariae in strawberry in a phytosanitary environment. This test will help timely decisions to be made at the border on imported fresh strawberry consignments that test positive for X. fragariae.

中文翻译：

---

草莓中草莓黄单胞菌活菌与死菌的活力PCR鉴别

草莓黄单胞菌是全球草莓产区重要细菌病害的病原体，也是包括新西兰在内的许多国家的检疫植物病原体。草莓黄单胞菌主要感染草莓植物的叶子，但也可以感染与草莓果实相关的花萼组织。新鲜草莓是一种保质期短的高价值国际贸易商品。在根据通过 PCR 发现草莓角斑病菌等检疫生物做出生物安全决策时，主要挑战之一是无法区分来自活细胞或死细胞的阳性结果。活力 PCR (vPCR) 是一种通过使用核酸嵌入染料（例如 PEMAX™）选择性抑制来自死细胞的 DNA 的 PCR 扩增的技术。vPCR 方案已经过优化，可以快速检测组织样本中的活草莓角斑病菌。PEMAX™ 处理导致草莓宿主组织中 10⁸–10³ cfu/ml 死亡草莓角斑病菌细胞的 PCR 扩增完全受到抑制。最重要的优化参数是样品的稀释度、扩增子长度和核酸嵌入染料的选择。本研究提供了一种快速方案，可用于在植物检疫环境中区分草莓中存活和死亡的草莓角斑病菌。该测试将有助于在边境对草莓角斑病菌检测呈阳性的进口新鲜草莓货物及时做出决定。草莓宿主组织中的草莓细胞。最重要的优化参数是样品的稀释度、扩增子长度和核酸嵌入染料的选择。本研究提供了一种快速方案，可用于在植物检疫环境中区分草莓中存活和死亡的草莓角斑病菌。该测试将有助于在边境对草莓角斑病菌检测呈阳性的进口新鲜草莓货物及时做出决定。草莓宿主组织中的草莓细胞。最重要的优化参数是样品的稀释度、扩增子长度和核酸嵌入染料的选择。本研究提供了一种快速方案，可用于在植物检疫环境中区分草莓中存活和死亡的草莓角斑病菌。该测试将有助于在边境对草莓角斑病菌检测呈阳性的进口新鲜草莓货物及时做出决定。