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: 【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(25.00μL):以上每条引物分别为1.20、0.20、0.60、1.60和0.15μL,2×PCR Mix12.50μL,不同病菌DNA模板各1.00μL,ddH2O补足至25.00μL,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。

     

    Abstract: 【Objective】This study aimed to establish a quintuple PCR method for rapid and simultaneous detection of Ralstonia solanacearumFusarium 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 solanacearumrpoB 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 solanacearumEnterobacter 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 solanacearumFusarium 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.

     

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