马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

Rapid detection and application of antagonistic bacterium QHZ11 against Rhizoctonia solani in potato by real-time fluorescence quantitative PCR

摘要:

【背景】植物根际促生菌(plant growth-promoting rhizobacteria，PGPR)在根际的定殖是其发挥作用的基础，直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系，并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物，优化反应条件；通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理，T1：对照(无菌水，CK)；T2：QHZ11菌悬液灌土(QHZ11)；T3：将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R；建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好，线性相关系数为0.999 8，检测组内变异系数均在1%以内，扩增效率为0.9，可检测出1×103?1×1010 copies/g-soil的拮抗菌，具有检出限低和扩增效率高的特点。盆栽试验结果发现，T3处理马铃薯根际拮抗菌QHZ11的数量从接种的第10天即高出T2处理一个数量级，并于马铃薯盛花期(接种后第60天)达到峰值，说明二次固体发酵增加了拮抗菌在根际土壤中的存活和繁殖。【结论】建立的实时荧光定量PCR快速检测体系灵敏、高效，可为研究拮抗菌在马铃薯根际原位分布以及与病原菌的互作方面提供便捷有效的方法。

Abstract:

[Background] The colonization of plant growth-promoting rhizobacteria (PGPR) in rhizosphere is the basis of its function. Direct and effective tracking techniques and quantitative methods are important tools to study the distribution of PGPR in rhizosphere. [Objective] To establish a real-time fluorescence quantitative PCR system for rapid detection of antagonistic bacteria QHZ11 against the pathogen, Rhizoctonia solani and to detect the dynamic changes of antagonistic bacterium QHZ11 in potato rhizosphere. [Methods] The specific primers were screened according to the genetic sequence difference between the Paenibacillus and proximal strain gyrB in GenBank. The reaction conditions of fluorescence quantitative PCR were optimized. A soil pot experiment of potato was arranged to detect the number of antagonistic bacteria QHZ11 in potato rhizosphere, with three treatments: T1: CK (sterile water was irrigated); T2: QHZ11 bacterial suspension was irrigated (QHZ11); T3: the biological organic fertilizer, secondary solid fermented with antagonistic bacteria QHZ11 was applied (BOF11). [Results] A pair of special primers gyrB-F/gyrB-R for antagonistic QHZ11 were screened. The established of antagonistic bacteria QHZ11 real-time fluorescence quantitative PCR method could detect the antagonistic bacteria of 1×103?1×1010 copies/g-soil, with such advantages as very good specificity, high sensitivity and better reproducibility, and the linear correlation coefficient was 0.999 8, the coefficient of variation within the detection group was less than 1%, the amplification efficiency was 0.9, characterized by a lower detection limit and higher amplification efficiency. The results of pot experiment showed that the number of antagonistic bacteria QHZ11 in potato rhizosphere of T3 was one order of magnitude higher than that of T2 treatment from the 10th day after inoculation, and reached the peak at the 60th day after inoculation, indicating that the survival rate and reproduction rate of antagonistic bacteria in rhizosphere soil were increased by secondary solid fermentation. [Conclusion] The established real-time fluorescence quantitative PCR system is sensitive and efficient, which provides a convenient and effective method for understanding the distribution of antagonistic bacteria in potato rhizosphere and the interaction between antagonistic bacteria and pathogens.