Table_2_Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.docx

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）是一种寄生于植物木质部导管的虫传细菌。该植物病原菌宿主范围极广，据估计可侵染560余种植物。目前已报道该病原菌存在5个亚种，各亚种宿主范围各异但存在重叠，但仅3个亚种被广泛认可，即fastidiosa亚种、multiplex亚种与pauca亚种。该病原菌最初仅分布于美洲，自2013年起在欧洲被检出。由于欧洲地区针对苛养木杆菌暴发疫情的防控工作依赖于亚种鉴定，因此尽早准确确定染病植株的亚种具有重要意义。为此，本研究开发了多种四重与三重定量PCR（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在植株体内的亚种鉴定。研究团队基于SkIf——一款基于k-聚体的生物信息学工具——设计引物与探针，从代表苛养木杆菌遗传多样性的58个基因组序列数据集中，筛选得到该物种及各亚种的特异性分子标记。我们对39株靶标菌株与30株非靶标菌株开展了qPCR检测验证，同时利用13种不同植物样本分别接种不同亚种的苛养木杆菌，并对各类环境寄主植物及人工接种寄主植物的样本进行了测试。单重qPCR检测的灵敏度与基于纯化DNA的参考方案相当，或略优于后者；四重qPCR检测的灵敏度与参考方案一致，可在所有人工侵染样本中检出最低至10³细胞·毫升⁻¹的苛养木杆菌。此外，四重qPCR检测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的现有方法无法完成鉴定时，四重qPCR检测仍可实现亚种识别。本研究开发的qPCR检测体系为稳健且模块化的工具，可直接在植物样本中高效区分苛养木杆菌的亚种。