Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland 1140, New Zealand.
Plant Dis. 2022 Sep;106(9):2348-2354. doi: 10.1094/PDIS-08-21-1819-RE. Epub 2022 Aug 11.
A highly pathogenic fungus characterized as multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase () gene and was shown to detect all tested isolates of MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with MLST1, other species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of MLST2.
一种高致病性真菌，被鉴定为多位点序列型 2（MLST2），是一种新兴的真菌病原体，可导致猕猴桃枯萎病。尽管 MLST2 尚未在智利以外的地区报告，但该病原体有可能通过种质的全球传播而传播到其他国家。目前针对这种真菌的诊断方法依赖于费力且耗时的平板培养鉴定和 DNA 序列分析。在本研究中，我们描述了一种新型实时聚合酶链反应（qPCR）检测方法的开发和验证，用于快速、特异性检测植物组织中的 MLST2。该检测方法针对甘油醛-3-磷酸脱氢酶（）基因，能够检测到所有测试的 MLST2 分离株，检测限约为 2pg 病原体基因组 DNA。与 MLST1、其他种或猕猴桃上发现的非目标真菌种没有交叉反应。该检测方法与植物内参基因复性，用于同时检测病原体和宿主植物的细胞色素氧化酶基因。这种新的特异性和敏感的 qPCR 检测方法是一种快速筛选进口植物材料的有价值的分子诊断工具，也可用于测试田间监测活动中收集的样本，以监测 MLST2 的存在。
