Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 110 012, India.
Plant Protection Section, Crop Science Division, Indian Council of Agricultural Research, New Delhi, 110001, India.
World J Microbiol Biotechnol. 2023 Jan 13;39(3):74. doi: 10.1007/s11274-023-03519-1.
Plant diseases cause serious economic losses of agriculture production worldwide. Rapid, accurate and reliable diagnostic methods are required to alleviate the detection of fungal plant pathogens to prevent their spread and achieve effective management. This study was aimed to develop fast, reliable and highly sensitive diagnostics to detect fungal plant pathogens for quarantine processing, safe exchange and conservation of germplasms of pulse crops. Multiplex and real time PCR assays were developed for detection of Rhizoctonia solani, Macrophomina phaseolina, Ascochyta rabiei, Alternaria alternata, A. tenuissima, Fusarium oxysporum f. sp. ciceris, Sclerotium (Athelia) rolfsii, Sclerotinia sclerotiorum, Pseudocercospora cruenta and Cercospora canescens causing various diseases in pulse crops. Twenty-two sets of primers from various genomic regions such as cytochrome oxidase subunit (COX 1), internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF-1α), large subunit (LSU), small subunit (SSU) and β-tubulin as well as two SCAR primers from RAPD profile were designed. The developed markers proved to be species-specific and validated against other fungal plant pathogens associated with pulses for cross-reactivity. The markers proved highly sensitive during conventional and qPCR analysis. Duplex PCR assays for R. solani and M. phaseolina; C. canescens and P. cruenta; A. alternata and A. tenuissima; and a quadruplex PCR assay for A. rabiei, S. sclerotiorum, S. rolfsii and F. oxysporum f. sp. ciceris were developed and validated for simultaneous detection of these pathogens in a single reaction. The assays developed in the present study were able to detect and identify major fungal plant pathogens causing disease in pulse crops.
植物病害在全球范围内对农业生产造成严重的经济损失。为了减轻对真菌植物病原体的检测，防止其传播，并实现有效管理，需要快速、准确和可靠的诊断方法。本研究旨在开发快速、可靠和高度敏感的诊断方法，以检测真菌植物病原体，用于检疫处理、安全交换和豆类种质资源的保存。开发了用于检测 Rhizoctonia solani、Macrophomina phaseolina、Ascochyta rabiei、Alternaria alternata、A. tenuissima、Fusarium oxysporum f. sp. ciceris、Sclerotium (Athelia) rolfsii、Sclerotinia sclerotiorum、Pseudocercospora cruenta 和 Cercospora canescens 的多重和实时 PCR 检测方法，这些病原体可引起豆类作物的各种疾病。从细胞色素氧化酶亚基（COX 1）、内部转录间隔区（ITS）、翻译延伸因子-1α（TEF-1α）、大亚基（LSU）、小亚基（SSU）和β-微管蛋白等各种基因组区域设计了 22 组引物，以及来自 RAPD 图谱的两个 SCAR 引物。开发的标记物被证明是种特异性的，并针对与豆类相关的其他真菌植物病原体进行了交叉反应验证。这些标记物在常规和 qPCR 分析中表现出高度的敏感性。开发了用于检测 R. solani 和 M. phaseolina；C. canescens 和 P. cruenta；A. alternata 和 A. tenuissima 的双重 PCR 检测方法；以及用于同时检测这些病原体的四重 PCR 检测方法 A. rabiei、S. sclerotiorum、S. rolfsii 和 F. oxysporum f. sp. ciceris。本研究开发的方法能够检测和鉴定导致豆类作物发病的主要真菌植物病原体。
