Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Microbiol Spectr. 2024 May 2;12(5):e0010624. doi: 10.1128/spectrum.00106-24. Epub 2024 Mar 27.
Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 ' Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of ' Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.
植物病原细菌会导致宿主植物发生许多疾病，并且可能造成严重的损害。因此，及时准确的诊断技术至关重要。虽然分子技术的进步已经导致能够区分已知植物病原体在种或株水平的诊断系统，但覆盖更大类别的系统大多缺乏。在这项研究中，针对在全球范围内造成重大农业损失的昆虫传播植物病原细菌的一大类——植原体，开发了一种基于 LAMP 的特异性和通用性诊断系统。该系统针对植原体的 23S rRNA 基因，新设计的引物对 CaPU23S-4 在 30 分钟内检测到 31 种“植原体”。该引物对还表现出高度特异性，对其他细菌（包括植原体的近亲）或健康植物没有假阳性结果。该检测方法的灵敏度比用于植原体检测的 PCR 方法高约 10,000 倍。还开发了一种简单、快速的 DNA 提取方法，通过煮沸感染植原体的组织来提取 DNA。当与通用 LAMP 检测方法一起使用时，它可以快速准确地从植物和昆虫中检测到植原体。结果表明，23S rRNA 基因作为细菌属水平通用 LAMP 检测的通用靶标具有潜力，并为探索该基因作为植原体存在检测的分子标记提供了新途径。
重要性：植原体与全球范围内农作物的经济重要疾病有关，包括椰子棕榈的致命黄化病、葡萄的“flavescence dorée”和“bois noir”、核果类的 X 病以及甘蔗的白叶和草状梢。已经报道了许多基于 LAMP 的针对植原体的诊断检测方法，这些方法大多针对 16S rRNA 基因。然而，这些检测方法只能检测到有限数量的“植原体”物种，而该属至少包括 50 种这些物种。在本研究中，开发了一种通用、特异、快速的诊断系统，通过结合针对 23S rRNA 基因的 LAMP 技术和一种简单的 DNA 提取方法，可以在 1 小时内检测到所有暂定分类的植原体。该诊断系统将有助于现场检测植原体，并可能有助于发现新的植原体相关疾病和潜在的昆虫媒介，而不论基础设施和实验资源的可用性如何。
