香榧树根系病原真菌多重荧光定量PCR检测方法的建立及初步应用

The root rot of Torreya grandis'Merrillii'caused by soil-borne pathogens such as Fusarium oxysporum,Rhizoctonia solani,Sclerotium rolfsii,etc.One of the diseases,the common soil pathogen detection method at present is the plate culture method,but this method has the shortcomings of long detection cycle and inability to quantify the strain.Therefore,this study established a nucleic acid detection system for the rapid detection of these three soil-habiting pathogens and the prevention of Torreya serrata disease.In this study,three sets of primers and TaqMan probes were designed based on the conserved regions of the gene sequences of these three pathogens to establish triple fluorescence quantification.PCR detection method.The sensitivity and reproducibility of standard grains constructed with the gene sequences of the three pathogens were tested,and the genomic DNA of other soil-habiting fungi was used for specific verification.The results showed that the amplification efficiency of triple real-time fluorescence quantitative PCR can reach more than 92.5%,with good specificity and reproducibility.The detection concentration range is 1.5×103～1.5×109 copies/,and the standard curves have good linearity and correlation.The coefficients are all above 0.99.After detecting the pathogenic bacteria in the rhizosphere soil of the inoculated annual Torreya,and the field healthy torreya and the root rot-affected rhizosphere soil of the torreya,the results showed that the multiple real-time fluorescence quantitative PCR can quickly and effectively detect the content of the target pathogen in the soil.