PDF(Pubmed)
Abstract：
Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.
摘要：
由Didymellasegeticola引起的茶叶斑病是一种重要的疾病，威胁着茶树的健康生长，并导致茶叶的生产力和质量下降。疾病的早期诊断对于控制感染尤为重要。环介导等温扩增(LAMP)分析是一种高效的诊断技术,具有简单的优点,特异性，和敏感性。在这项研究中,我们开发了一种快速的，视觉，以及基于序列表征的扩增区域的D.segeticola检测的高灵敏度LAMP测定法。基于D.segeticola中的特定序列(NCBI登录号:OR987684)设计了两对扩增引物(外部引物F3和B3以及内部引物FIP和BIP)。与茶树中其他属和Didymella属中其他物种的常见病原体相比（Didymellacoffeae-arabicae，Didymellapomorum,和Didymellasinensis)，LAMP方法对检测D.segeticola物种具有特异性。该测定能够在65°C的最佳反应温度下检测到最小浓度为1fg/μL的基因组DNA，持续60分钟。当健康的叶子在实验室接种D.segeticola时，LAMP方法在接种后72小时成功检测到了病茶叶片中的石竹。当从健康叶片提取DNA样品时,LAMP测定为阴性。来自18个茶树种质的具有坏死病变的叶片组织通过LAMP测定对病原体呈阳性。总之,这项研究建立了一个具体的，敏感,和简单的LAMP方法来检测D.segeticola，为估计病害患病率提供了可靠的技术支持，有利于茶叶叶斑病的可持续管理。
