连作马铃薯根际干腐病优势病原菌荧光定量PCR快速检测及在根际的动态变化

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,本研究建立了以马铃薯根际土壤为研究对象的干腐病病原菌荧光定量PCR快速检测体系。结果显示,研究建立优化的荧光定量PCR检测方法,对引起马铃薯干腐病的优势病原菌茄病镰孢菌和接骨木镰孢菌进行快速检测和绝对定量,主要优化参数为:上下游引物各0.4 μL(10 mmol/L),DNA模板3 μL,退火温度60℃。连作马铃薯根际土壤茄病镰孢菌和接骨木镰孢菌随连作年限的动态变化趋势均表现为随连作年限的递增呈现上升趋势,其中CP₅的累积量最大,为1.45×10⁴拷贝/g,比CK增加了27.8倍。CP₄、CP₃、CP₂、CP₁根际病原菌累积量分别比对照增加了10.5,16.31,8.32,3.51倍。由此可见,茄病镰孢菌和接骨木镰孢菌的数量均随连作年限的递增呈上升趋势。根际镰刀菌随生育进程的动态变化因病原菌种类而异,茄病镰孢菌以收获期累积量最大,平均为1.2×10⁴拷贝/g,随生育进程的推进呈上升趋势。而接骨木镰刀菌是播前累积量最大,平均为1.55×10⁴ 拷贝/g,随生育进程的推进呈下降趋势。因此,研究区马铃薯根际土壤中镰刀菌的大量积累可能是导致马铃薯连作障碍发生的主要原因之一。

Changes in the dominant pathogens causing Fusarium dry rot of potato in rhizospheric soil under continuous potato cropping systems based on real-time quantitative PCR

The relationship between continuous cropping obstacles and accumulation of pathogens in rhizospheric soil was evaluated, and the mechanisms of continuous potato cropping obstacles explored, using an optimized SYBR Green I dye real time fluorescent quantitative PCR approach. The methods were used to perform quick checks and quantification of Fusarium sambucim and F. solani in soils infested with potato Fusarium dry rot disease and to investigate the dynamic changing trends of F. sambucim and F. solani in rhizospheric soil of continuously cropped potato. The concentration of primers was 0.4 μL (10 mmol/L), of DNA template was 3 μL, and the annealing temperature was 60℃. With an increase in years of continuous cropping potato, the numbers of F. solani and F. sambucim in rhizospheric soil showed an upward trend. The largest amount (1.45×10⁴ copies/g) of F. solani accumulated during the squaring period of five years and it increased 27.8 times compared with the control group (CK). F. solani cumulants CP4, CP3, CP2, CP1 were more than those of RP and increased by 11.4, 35.5, 6.9 and 2.8 times respectively. The largest amount of F. sambucim cumulants (2.21×10⁴ copies/g) was also during the squaring period of five years and compared to the CK they increased 25.0 times. The F. sambucim cumulants CP4, CP3, CP2, CP1 were more than those of the CK and increased by 10.5, 16.31, 8.32 and 3.51 times respectively. Accumulation of rhizospheric Fusarium may be one of the main reasons for the occurrence of obstacles to continuous potato cropping.