Testen Anna Louise, Puri Purnima, Shaw R Scott, Domsic Evan Christopher, Griffin-LaHue Deirdre, Murphy Kevin Matthew, Mattupalli Chakradhar
USDA-ARS Application Technology Research Unit, Wooster, OH.
Department of Plant Pathology, Washington State University, Northwestern Washington Research and Extension Center, Mount Vernon, WA.
Plant Dis. 2024 Sep;108(9):2887-2893. doi: 10.1094/PDIS-11-23-2308-RE. Epub 2024 Sep 3.
Quinoa downy mildew, caused by , is the most devastating disease of quinoa globally. Rapid, sensitive diagnostic methods are needed to detect and quantify this pathogen in seeds and plant tissue. A hydrolysis probe-based quantitative real-time PCR (qPCR) assay including a competitive internal control was developed for detection. This assay could detect as low as 20 ag of DNA or approximately 25 internal transcribed spacer (ITS) copies per reaction with efficiencies ranging from 93.9 to 98.2%. No nontarget amplification was observed when tested against DNA from other downy mildew pathogens and related oomycetes. . strains from multiple countries were detected using this assay. The assay was successfully applied to quantify the pathogen in quinoa seeds from a field trial conducted in the state of Washington. Downy mildew disease was recorded on all 14 genotypes, with the genotypes 104.88 and 106.49 recording the highest area under the disease progress curve values (mean ± SE; 3,236 ± 303 and 2,851 ± 198, respectively) and J6 and Dutchess recording the lowest (441 ± 107 and 409 ± 129, respectively). Seed washes obtained from field samples were subjected to the qPCR assay, and the pathogen was detected in all samples. The highest pathogen ITS copy number was recorded with 106.49 (194,934 ± 38,171), and the lowest was observed in Pasto (5,971 ± 1,435) and Riobamba (9,954 ± 4,243). This qPCR assay could lead to improved detection and quantification of as well as increased understanding of quinoa- interactions and epidemiology.
藜麦霜霉病由[病原体名称缺失]引起，是全球藜麦最具毁灭性的病害。需要快速、灵敏的诊断方法来检测和定量种子及植物组织中的这种病原体。为此开发了一种基于水解探针的定量实时PCR（qPCR）检测方法，其中包括竞争性内对照用于[病原体名称缺失]的检测。该检测方法每个反应能检测低至20 ag的DNA或约25个内转录间隔区（ITS）拷贝，效率范围为93.9%至98.2%。用来自其他霜霉病病原体和相关卵菌的DNA进行测试时，未观察到非靶标扩增。使用该检测方法检测了来自多个国家的[病原体名称缺失]菌株。该检测方法已成功应用于量化华盛顿州进行的田间试验中藜麦种子中的病原体。在所有14个基因型上均记录到了霜霉病，基因型104.88和106.49的病害进展曲线下面积值最高（平均值±标准误；分别为3236±303和2851±198），而J6和达奇斯的最低（分别为441±107和409±129）。对田间样本获得的种子洗涤液进行qPCR检测，在所有样本中均检测到了病原体。病原体ITS拷贝数最高的是106.49（194934±38171），最低的是帕斯托（5971±1435）和里奥班巴（9954±4243）。这种qPCR检测方法可提高对[病原体名称缺失]的检测和定量能力，以及增进对藜麦与[病原体名称缺失]相互作用及流行病学的了解。
