Table_3_Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.xlsx

Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.ml−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

苛养木杆菌（Xylella fastidiosa，Xf）是一种虫传细菌，仅定殖于植物的木质部导管内。该植物病原菌寄主范围广泛，据统计可侵染560余种植物。该病原菌已被报道存在5个亚种，各亚种的寄主范围既有差异又存在重叠，但目前学界广泛认可的仅有3个亚种，分别为fastidiosa亚种、multiplex亚种以及pauca亚种。该菌最初仅分布于美洲地区，自2013年起在欧洲陆续检出Xf。鉴于欧洲地区的苛养木杆菌疫情防控依赖于亚种鉴定，因此尽早准确确定染病植株中的亚种类型具有重要意义。因此，本研究开发了多种四重与三重定量聚合酶链式反应（quantitative PCR，qPCR）检测体系，可在单管反应中同时完成苛养木杆菌的检测及其植株体内亚种的鉴定。本研究基于SkIf——一款基于k-mer的生物信息学工具——设计引物与探针，从代表苛养木杆菌多样性的58个基因组序列数据集中提取该菌物种及亚种的特异性特征序列用于检测。本研究利用39株靶标菌株与30株非靶标菌株、13种分别接种了不同苛养木杆菌亚种的植物样本，以及多种环境来源和接种寄主植物的样本，对上述qPCR检测体系进行了性能验证。单重检测体系对纯化DNA的检测灵敏度与参考方法相当，甚至略优于参考方法。四重qPCR检测体系的灵敏度与参考方法一致，可在所有接种样本中检出低至10³个·毫升⁻¹的苛养木杆菌。此外，该四重qPCR检测体系可在同一样本中同时检出2至3个亚种的混合侵染。在环境植物样本中，当基于多位点序列分型（multilocus sequence typing, MLST）的现有方法无法完成鉴定时，四重qPCR检测体系仍可实现亚种鉴定。本研究开发的qPCR检测体系稳定性强、模块化程度高，可直接在植物样本中高效区分苛养木杆菌的不同亚种。