南方农业学报2026，Vol.57Issue(2)：474-485,12.DOI:10.3969/j.issn.2095-1191.2026.02.015
生姜土传病原菌五重PCR检测体系的建立
Establishment of a quintuple PCR detection method for ginger soil-borne pathogens
摘要
Abstract
[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 patho-gens 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 sen-sitivity,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 si-multaneously 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 success-fully 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.关键词
生姜/土传病原菌/五重PCR检测体系/病情监测Key words
ginger/soil-borne pathogen/quintuple PCR detection system/epidemic monitoring分类
农业科技引用本文复制引用
杨维超,蓝达愉,黄皓,文俊丽,李洪雷,车江旅,周生茂,袁高庆..生姜土传病原菌五重PCR检测体系的建立[J].南方农业学报,2026,57(2):474-485,12.基金项目
National Natural Science Foundation of China(32270237) （32270237）
Guangxi Key Research and Development Plan Project(Guike AB21238002) （Guike AB21238002）
Basic Scientific Research Project of Guangxi Academy of Agricultural Sciences(Guinongke 2024YP082) （Guinongke 2024YP082）
