应用TaqMan探针实时荧光定量PCR技术早期检测稻瘟病

Early detection of rice blast by TaqMan real-time flourescence quantitative polymerase chain reaction

稻瘟病是一种严重危害水稻生产的真菌病害,早期监测和防治是关键。温室接种大田主栽品种合系39和感病水稻蒙古稻,定时观察发病症状。提取水稻病样总DNA,根据稻瘟菌28S rDNA基因序列,设计特异引物和TaqMan探针,进行荧光定量PCR检测。结果表明稻瘟菌在蒙古稻和大田主栽品种合系39上症状最初出现时间为接种后72 h,蒙古稻典型症状出现时间为接种后168 h,在合系39上出现典型症状的时间为接种后190 h以后。利用TaqMan探针荧光定量PCR技术,在接种12 h的蒙古稻和合系39上均能检测到稻瘟菌DNA,接种48 h,菌量拷贝数达到最高峰,接种72 h,菌量拷贝数开始下降。不同品种中病原菌拷贝数存在差别,在接种12 h的蒙古稻中稻瘟菌含量为7.2×103个拷贝数,在合系39中为4.9×103个拷贝数。本研究结果表明,应用实时荧光定量PCR技术可以在接种后12 h症状未显现之前检测到稻瘟菌,为稻瘟病流行监测和早期防治提供了科学依据。

Rice blast caused by Magnaporthe grisea is an important rice disease.It causes serious damage to both yield and grain quality.Early detection and monitoring of the pathogen is crucial for disease control.After inoculating the pathogen on the susceptible cultivar Menggu and the widely planted cultivar Hexi 39,the symptom was observed and the total DNA of the diseased rice tissues was purified and detected by real time PCR.It was showed that the little necrotic spot occurred at 72 hours after inoculation on Mengu and Hexi 39.The typical symptom was observed at 168 hours after inoculation on Menggu rice,but at 190 hours after inoculation on Hexi 39.The amount of the pathogen in both rice cultivars increased and reached the highest level at 48 hours after inoculation and decreased after 72 hours.There was some difference between susceptible and widely planted cultivar in pathogen DNA amount.Pathogen DNA in both rice cultivars could be detected firstly at 12 hours after inoculation,with 7.2×103 copies in Menggu and 4.9×103 copies in Hexi 39.The results proved that the real time PCR technique could be used to detect M.grisea at early stage of infection.