Establishment of a quintuple PCR detection method for ginger soil-borne pathogens

【Objective】This study aimed to establish a quintuple PCR method for rapid and simultaneous detection of Ralstonia solanacearum， Fusarium spp.， Pectobacterium spp.， Enterobacter spp.， and Pythium spp.， which provided technical support for early diagnosis of various soil-borne diseases on ginger.【Method】For five types of soil-borne pathogens causing ginger bacterial wilt and rhizome rot， specific primer combinations were designed and screened， the optimal quintuple reaction system was established by exploring optimal primer concentrations， annealing temperature， and sensitivity， and was applied to detect field plant samples to verify its utility.【Result】Specific primers pairs Rs1F/Rs1R， En1F/En1R， and Py1F/Py1R were designed according to flic gene of Ralstonia solanacearum， rpoB gene of Enterobacter spp.， and 18S rDNA of Pythium spp.， and combined with reported Fusarium spp. specific primers Fu3/Fu4 and specific primers 23SPecF/23SPecR of Pectobacterium spp.， a quintuple PCR reaction system for ginger soil-borne pathogens has been established （25.00 μL）： above primer dosage was 1.20 ， 0.20， 0.60， 1.60， and 0.15 μL respectively； 2×PCR Mix 12.50 μL； DNA templates of different pathogens were 1.00 μL each； added ddH₂O to 25.00 μL. Annealing temperature was optimized to 55.4 ℃. The specific fragments with sizes of 516， 370， 266， 207， and 159 bp could be amplified simultaneously in the established quintuple PCR system， and the detection limit of this system for Ralstonia solanacearum， Enterobacter spp. and Pythium spp. reached 10^-1 pg/μL， for Fusarium spp. and Pectobacterium spp. was 1 pg/μL， and for detecting five pathogens simultaneously was 10³ pg/μL. The multiplex PCR system established in this study could successfully detect the diseased plant samples from the field.【Conclusion】The quintuple PCR system established is able to rapid-ly and accurately detect Ralstonia solanacearum， Fusarium spp.， Pectobacterium spp.， Enterobacter spp.， and Pythium spp.， which provides a useful tool for timely diagnosis and epidemic monitoring of various soil-borne diseases of ginger.