Fungal Molecular Biology Laboratory, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India.
Can J Microbiol. 2011 Nov;57(11):934-42. doi: 10.1139/w11-089. Epub 2011 Oct 21.
Spot blotch of wheat caused by Bipolaris sorokiniana is an important disease of wheat, especially in slightly warm (25 ± 1 °C) and humid weather conditions. A quick and reliable PCR-based diagnostic assay has been developed to detect B. sorokiniana using a pathogen-specific marker derived from genomic DNA. A PCR-amplified band of 650 bp obtained in B. sorokiniana isolates using universal rice primer (URP 1F) was cloned in pGEMT easy vector and sequenced. Based on sequences, six primers were designed, out of which a primer pair RABSF1 (GGTCCGAGACAACCAACAA) and RABSR2 (AAAGAAAGCGGTCGACGTAA) amplified a sequence of 600 bp in B. sorokiniana isolates. The specificity of the marker when tested against 40 isolates of B. sorokiniana, seven isolates of other species of Bipolaris, and 27 isolates of other pathogens infecting wheat and other crops showed a specific band of 600 bp only in B. sorokiniana. The detection limit was 50 pg of genomic DNA. The marker could detect the pathogen in soil and wheat leaves at presymptomatic stage. This sequence characterized amplified region (SCAR) marker designated as SCRABS(600) could clearly distinguish B. sorokiniana from other fungal plant pathogens, including Bipolaris spp. The utilization of this diagnostic PCR assay in analysis of field soil and wheat leaves will play a key role in effective management of the disease.
小麦条斑叶枯病由旋孢腔菌引起，是一种重要的小麦病害，尤其在温暖（25±1°C）和潮湿的天气条件下更为严重。为了快速、可靠地检测病原菌，本研究基于从旋孢腔菌基因组 DNA 中提取的病原菌特异性标记，开发了一种基于 PCR 的检测方法。使用通用水稻引物（URP1F）对旋孢腔菌分离株进行 PCR 扩增，得到一条 650bp 的扩增带，将其克隆到 pGEMT easy 载体中并测序。根据序列设计了 6 对引物，其中一对引物 RABSF1（GGTCCGAGACAACCAACAA）和 RABSR2（AAAGAAAGCGGTCGACGTAA）可在旋孢腔菌分离株中扩增出 600bp 的序列。该标记对 40 个旋孢腔菌分离株、7 个其他种属的弯孢菌和 27 个感染小麦和其他作物的其他病原菌进行了特异性检测，仅在旋孢腔菌中检测到 600bp 的特异性条带。检测限为 50pg 基因组 DNA。该标记可在土壤和小麦叶片的无症状期检测到病原菌。本研究将此序列特征扩增区域（SCAR）标记命名为 SCRABS（600），它可以明确地区分旋孢腔菌与其他真菌植物病原菌，包括弯孢菌属。该诊断 PCR 检测方法在分析田间土壤和小麦叶片中的应用将在病害的有效管理中发挥关键作用。
