发生马铃薯立枯病土壤中立枯丝核菌的荧光定量PCR快速检测

Rapid detection of Rhizoctoniain rhizosphere soil of potato using real-time quantitative PCR

连作马铃薯根际土壤中立枯丝核菌的大量累积可能是导致马铃薯连作障碍发生的主要原因之一。为了解根际土壤中土传病害病原菌的积累与连作障碍之间的关系,进一步寻求缓解和克服马铃薯连作障碍土传病害的有效途径,本研究建立优化了荧光定量PCR（real-time PCR）方法,对引起马铃薯立枯病的病原菌立枯丝核菌进行了快速监测和绝对定量,对马铃薯连作1~5年根际土壤立枯丝核菌的动态变化趋势进行了检测分析。结果显示,研究建立优化的荧光定量PCR检测方法,可直接应用土壤DNA进行病原菌的定量检测,能检测到土壤中浓度为1×102个拷贝/g土的马铃薯立枯丝核菌,扩增效率为1.04,具有检出限低、扩增效率高的特点,实现了不通过病土分离培养方法,就可掌握病原菌在根际土壤中的累积状况;在马铃薯立枯病发病严重的连作根际土壤中,立枯丝核菌的累积数量随连作年限的递增呈上升趋势;病原菌的累积随生育进程的推进呈下降趋势;病原菌累积量最大的是连作5年的播前土壤,每g土壤达3.75×107个拷贝数,由此可见,连作导致了土壤微生物种群结构发生改变,土壤致病真菌数量增加,相应地也增加了马铃薯立枯病的初侵染机率。

A leading obstacle to continuous cropping of potatoes is Rhizoctonia solani in the rhizosphere. An understanding of the relationship between potato soil-borne pathogen accumulation and continuous cropping was sought to overcome the obstacles to continuous cropping of soil borne diseases. Synergy Brands （SYBR） Green I Dye （SGI） real time fluorescent quantitative Polymerase Chain Reaction （Rt-PCR） was optimised to perform quick checks and quantification of R. solani in soils with potato damping off disease. The dynamic changes of R. solani in the rhizosphere soil of continuously cropped potato was investigated. Results show that, the methods can be directly applied to soil pathogens with quantitative detection of DNA, which can be detected to a minimum detection level of 1 × 102 copies/g in potato soil concentrations of R. solani. Amplifica- tion efficiency is high （1.04） with a low detection limit. The cumulative status of pathogenic bacteria in the rhizosphere soil can be readily assessed without isolation and culturing. With increasing years of continuous po- tato cropping, the numbers of R. solani in rhizosphere soil had an upward trend with the increasing year of po- tato＇s continuous cropping, by the growth process, the numbers of rhizoctonia solani in rhizosphere soil were downward trend. The largest amount of pathogens accumulated is the pre-seeding period of five years, that is 3.75 × 107 copies/g. This shows that continuous cropping led to a soil microbial population structure that in- creased the amount of change of soil pathogenic fungi. The infection probability of Rhizoctonia solani in early potato blight increased.