LAMP快速诊断由拟轮枝镰孢引起的水稻恶苗病

Rapid diagnosis of rice bakanae disease caused by Fusarium verticillioides using a loop-mediated isothermal amplification assay

[目的]水稻恶苗病是为害水稻生长发育的重要病害,在我国的分布非常广泛,经常会造成严重的损失。拟轮枝镰孢是引起水稻恶苗病的重要病原菌,本研究意在建立由拟轮枝镰孢引起的水稻恶苗病的快速诊断技术。[方法]基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以拟轮枝镰孢(Fusarium verticillioides)的3-磷酸甘油酸激酶(3-phosphoglyceric phosphokinase)基因Pgk为靶标,设计筛选LAMP特异性引物。[结果]建立了可直接从水稻恶苗病植株中检测拟轮枝镰孢并诊断由该病原菌引起的水稻恶苗病的LAMP检测技术(FvPgk-LAMP)。该检测技术可以从田间发病的水稻植株中直接对病原菌进行检测,反应条件为62℃、70 min。由于反应前在反应管内加入了羟基萘酚蓝(HNB),反应结束后可对结果进行直接判定,阳性反应呈天蓝色,阴性反应则仍然为紫色。该体系的最低检出限为目标菌纯DNA的100pg·μL^(-1)。应用该技术我们已成功且快速地对来自江苏南京和镇江的水稻恶苗病样本进行了病害诊断。[结论]本研究为拟轮枝镰孢引起的水稻恶苗病的诊断提供了快速且有效的新技术。

[Objectives]Rice bakanae disease is one of the most important diseases,and is appear in all the major rice cultivation areas throughout the world,often causing serious yield losses.Fusarium verticillioides is an important pathogen causing rice bakanae disease,and the purpose of this study is developing the method for the rapid diagnosis of rice bakanae disease caused by F.verticillioides.[Methods]In this study,we used the Pgk（3-phosphoglyceric phosphokinase） gene to design specific primers for F.verticillioides and developed Fv Pgk-LAMP assay.[Results]This assay can be used to detect F.verticillioides in diseased rice tissues from the field directly.The LAMP assay amplified the target gene efficiently at 62 ℃ over 70 min.A positive color（sky blue）was only observed at the presence of F.verticillioides,whereas other species showed no color change with the addition of hydroxynaphthol blue（HNB） dye prior to amplification.The sensitivity of the assay was 100 pg·μL-（-1） of genomic DNA.Suspected diseased rice samples collected from Nanjing and Zhenjiang of Jiangsu Province were diagnosed successfully using the LAMP assay reported here.[Conclusions]This study provides a new and readily available method for the rapid diagnosis of rice bakanae disease caused by F.verticillioides.