Department of Entomology and Plant Pathology, Oklahoma State University-Stillwater, Stillwater, OK 74078, USA.
J Microbiol Methods. 2013 Mar;92(3):293-300. doi: 10.1016/j.mimet.2012.12.020. Epub 2012 Dec 30.
Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264 bp PCR product for S. minor, a 218 bp product for S. homoeocarpa, a 171 bp product for S. sclerotiorum, and a 97 bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001 pg/μL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction.
核盘菌（Sclerotinia sclerotiorum (Lib.) de Bary）、小核盘菌（S. minor Jagger）、三叶草核盘菌（S. trifoliorum Eriks）和拟茎点霉（S. homoeocarpa F.T. Benn）是核盘菌属中与植物病原相关的最重要的物种，因为它们有广泛的重要经济宿主，包括番茄、花生、紫花苜蓿和草坪草等。基于形态特征的物种鉴定具有挑战性且耗时，特别是当一种作物宿主多种物种时。本研究旨在设计与多重 PCR 兼容的特异性引物，用于快速、灵敏和准确地检测和区分四种核盘菌。为核盘菌的天冬氨酸蛋白酶基因、三叶草核盘菌的钙调蛋白基因、拟茎点霉的延伸因子-1α基因和小核盘菌的漆酶 2 基因设计了特异性引物。单独和多重 PCR 测试了每个引物对的特异性和灵敏度，并使用感染植物的基因组 DNA 进行了验证。每个引物组都能一致地仅扩增其靶基因的 DNA。不同大小的 DNA 片段被扩增：小核盘菌的 264 bp PCR 产物、拟茎点霉的 218 bp 产物、核盘菌的 171 bp 产物和三叶草核盘菌的 97 bp 产物。这些引物组可单独或多重使用，用于鉴定纯培养或感染植物中的核盘菌属。多重检测比单重检测的灵敏度下限低（每种物种的 0.0001 pg/μL DNA）。开发的多重检测是一种准确快速的工具，可在单个 PCR 反应中区分最相关的植物病原性核盘菌物种。
