Molecular identification of Neofabraea species associated with bull’s-eye rot on apple using rolling-circle amplification of partial EF-1α sequence

作者：Lin HuiJiao, Xiang Jiang, Jianping Yi, Xinguo Wang, Ranling Zuo, Zide Jiang, Weifang Wang, Erxun Zhou · 发表于：Canadian Journal of Microbiology · 年份：2017 · DOI：10.1139/cjm-2017-0448 · 被引用次数：16 · 研究领域：Plant Pathogens and Fungal Diseases、Plant Pathogenic Bacteria Studies、Plant-Microbe Interactions and Immunity

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.