生姜土传病原菌五重PCR检测体系的建立OA
Establishment of a quintuple PCR detection method for ginger soil-borne pathogens
[目的]建立一种能够同时快速检测青枯菌(Ralstonia solanacearum)、镰刀菌(Fusarium spp.)、果胶杆菌(Pectobacterium spp.)、肠杆菌(Enterobacter spp.)和腐霉菌(Pythium spp.)的五重PCR体系,为早期准确诊断生姜多种土传病害提供技术支撑.[方法]针对引起生姜青枯病和根茎腐烂病的5类土传病原菌,设计和筛选病原菌的特异性引物组合,探索引物最佳浓度、退火温度和灵敏度,建立最佳的五重PCR检测体系,并应用于田间植株样品的检测,以验证其实用性.[结果]根据青枯菌flic基因、肠杆菌rpoB基因和腐霉菌18S rDNA分别设计特异性引物对Rs1F/Rs1R、En1F/En1R和Py1F/Py1R,并结合已报道镰刀菌特异性引物Fu3/Fu4和果胶杆菌特异性引物23SPecF/23SPecR,建立5种生姜土传病害的多重PCR检测体系(25.00 µL):以上每条引物分别为1.20、0.20、0.60、1.60和0.15 µL,2×PCR Mix 12.50 µL,不同病菌DNA模板各1.00 µL,ddH2O补足至25.00 µL,最适退火温度为55.4℃.建立的五重PCR检测体系可同时扩增出大小分别为516、370、266、207和159 bp的特异性片段,该检测体系对青枯菌、肠杆菌和腐霉菌的最低检测限为10-1 pg/µL,对镰刀菌和果胶杆菌的最低检测限为1 pg/µL,同时检测5类病原菌的灵敏度为103 pg/µL.本研究建立的五重PCR检测体系可对田间病株进行准确检测.[结论]建立的五重PCR检测体系能同时对青枯菌、镰刀菌、果胶杆菌、肠杆菌和腐霉菌进行准确检测,可应用于生姜多种土传病害的早期快速诊断和田间病情监测.
[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.
杨维超;蓝达愉;黄皓;文俊丽;李洪雷;车江旅;周生茂;袁高庆
广西大学农学院,广西 南宁 530004||广东烟草韶关市有限公司乐昌市分公司,广东 韶关 512200广西大学农学院,广西 南宁 530004广西农业科学院蔬菜研究所,广西 南宁 530007广西农业科学院蔬菜研究所,广西 南宁 530007重庆文理学院特色植物研究院,重庆 402160广西农业科学院,广西 南宁 530007广西农业科学院蔬菜研究所,广西 南宁 530007广西大学农学院,广西 南宁 530004
农业科技
生姜土传病原菌五重PCR检测体系病情监测
gingersoil-borne pathogenquintuple PCR detection systemepidemic monitoring
《南方农业学报》 2026 (2)
474-485,12
National Natural Science Foundation of China(32270237)Guangxi Key Research and Development Plan Project(Guike AB21238002)Basic Scientific Research Project of Guangxi Academy of Agricultural Sciences(Guinongke 2024YP082)
评论