Abstract Ascochyta blight is the major disease affecting chickpea (Cicer arietinum) around the world. Since the first report of Ascochyta rabiei's isolation in Argentina in 2012, the pathogen has caused severe economic losses in crop production; so, the detection and rapid identification of the pathogen in early stages is key for the management of the disease. In this work, a traditional PCR procedure for detection of A. rabiei directly from plant tissues has been described based on beta-tubulin gene. The TP-6/TP-9 specific primers designed, amplified only a single PCR band of 770 bp from A. rabiei. The specificity of the primers was checked using 12 isolates of A. rabiei and DNA from 10 other different fungi including common pathogens of chickpea as Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum and Phoma medicaginis that cause similar symptoms. The detection sensitivity with primers was 2 x 104 ng.μl-1 genomic DNA. In inoculated plant material, PCR amplification gave a band of the expected size and no amplification was observed when DNA was from healthy and uninoculated plants. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based method developed here can simplify both plant disease diagnosis, and pathogen monitoring in an early phase, as well as aid in effective management practices that avoid the disease advance and minimize losses.

中文翻译：

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传统 PCR 法早期检测鹰嘴豆子叶枯病（Ascochyta rabiei）

摘要 Ascochyta Blight 是影响全球鹰嘴豆（Cicer arietinum）的主要病害。自2012年阿根廷首次报道狂犬病菌分离以来，该病原已给农作物生产造成严重经济损失；因此，在早期阶段检测和快速识别病原体是疾病管理的关键。在这项工作中，基于β-微管蛋白基因描述了一种用于直接从植物组织中检测狂犬病的传统 PCR 程序。设计的 TP-6/TP-9 特异性引物仅从狂犬病菌中扩增出一条 770 bp 的 PCR 带。使用 12 种狂犬病菌的分离物和 10 种其他不同真菌的 DNA 检查引物的特异性，包括鹰嘴豆的常见病原体如链格孢、灰葡萄孢、引起类似症状的核盘菌和药核菌。引物的检测灵敏度为 2 x 104 ng.μl-1 基因组 DNA。在接种的植物材料中，PCR 扩增产生预期大小的条带，当 DNA 来自健康和未接种的植物时，未观察到扩增。结果表明，该测定比标准分离方法更快、更准确地检测到病原体。这里开发的基于 PCR 的方法可以简化早期阶段的植物病害诊断和病原体监测，并有助于有效的管理实践，避免病害的发展并最大限度地减少损失。PCR 扩增产生预期大小的条带，当 DNA 来自健康和未接种的植物时，未观察到扩增。结果表明，该测定比标准分离方法更快、更准确地检测到病原体。这里开发的基于 PCR 的方法可以简化早期阶段的植物病害诊断和病原体监测，并有助于有效的管理实践，避免病害的发展并最大限度地减少损失。PCR 扩增产生预期大小的条带，当 DNA 来自健康和未接种的植物时，未观察到扩增。结果表明，该测定比标准分离方法更快、更准确地检测到病原体。这里开发的基于 PCR 的方法可以简化早期阶段的植物病害诊断和病原体监测，并有助于有效的管理实践，避免病害的发展并最大限度地减少损失。