Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay.
Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay.
复制标题
DOI：
10.1038/srep42737
复制
发表时间：
2017-02-20
影响因子：
4.6
通讯作者：
Sharma M
中科院分区：
文献类型：
--
作者：
Ghosh R;Tarafdar A;Sharma M
由真菌bataticola Rhizoctonia （Taub.）引起的干根腐病（DRR）。是鹰嘴豆的一种新兴疾病。该病常与鹰嘴豆枯萎病、颈腐病和黑腐病等其他根腐病相混淆。因此，及时、具体的检测是很重要的。目前的检测方案要么基于真菌学方法，要么基于涉及DNA扩增的聚合酶链反应（PCR）的方案。本文报道了利用环介导等温扩增（LAMP）技术，针对真菌特异性5.8S rDNA序列，对巴塔提科拉进行快速、特异检测。优化反应温度为63°C，反应时间为75 min， DNA最少为10 fg。在LAMP产品中加入SYBR Green I后，发现扩增结果对来自不同地理区域的94株bataticola菌株以及DRR感染植物和病土均具有高度特异性。其他致病真菌感染鹰嘴豆（镰刀菌、茄枯丝核菌、罗尔夫菌核菌和茄枯菌）和鸽子豆（镰刀菌和茄疫霉）均无反应。该方法简便、快速、特异，可用于鹰嘴豆病的目测检测。
Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.