生姜青枯病病原菌的鉴定与PCR检测方法的建立

Isolation and Identification ofthe Pathogen of Ginger Bacterial Wilt and Establishment of a PCR Detection System

青枯病是一种严重危害生姜种植的毁灭性土传细菌病害,明确引起生姜青枯病的病原菌并对其进行快速检测,对生姜青枯病早期诊断与有效防控具有重要意义.本研究采用组织分离法,对湖北省恩施生姜病样上的病菌进行分离纯化,通过形态学特征、致病性测定和分子生物学的方法对其进行鉴定;筛选并验证了生姜青枯病菌的特异性引物,优化生姜青枯病病原菌的PCR检测方法,并对该方法进行特异性和灵敏度验证,运用该方法对生姜植株和土壤的病原菌进行检测.结果表明,试验分离纯化得到了10株培养性状一致的菌株,从形态学特征、致病性测定和分子生物学方面,将代表菌株ES202023鉴定为青枯雷尔氏菌(Ralstonia solanacearum);引物21F/21R从所获菌株中扩增出长度为125 bp的特异性目的条带;通过优化后的PCR体系(25μL:2×Taq Master PCR Mix 12.5μL, 21F/21R 0.5μL,退火温度61.1℃, 30个循环),使病原菌的检测灵敏度达到10-2 ng/μL,且可以在感病植株及土壤中检测到病原菌.本研究建立的特异性PCR快速检测方法,对生姜青枯病的田间早期预警、姜种带菌检测和绿色防控具有指导作用.

Bacterial wilt is a destructive soil borne disease which is highly harmful to ginger cultivation. Accurate identification and quick detection of the pathogen is of great significance for the early diagnosis and effective control of the disease. In this study, the pathogen of ginger bacterial wilt from Enshi in Hubei Province was isolated and purified with the tissue isolation method, and was identified through morphological observation, pathogenicity determination and molecular biological approaches. First, the specific detection primers of Ralstonia solanacearum were screened and verified, and the PCR amplification system involved was optimized. Then, the specificity and sensitivity of the system were verified. Finally, the optimal PCR system was applied in detection of pathogen on ginger and rhizosphere soil. Ten strains with similar culture characters were isolated and purified. The representative strain ES202023 was identified as R. solanacearum on the basis of morphological characteristics, pathogenicity verification and molecular biology. The primer 21 F/21 R amplified a 125 bp specific band from the obtained strain. The optimized PCR reaction system(25 μL: 2 × Taq master PCR mix 12.5 μL, 21 F/21 R 0.5 μL, annealing temperature 61.1 ℃, 30 cycles) gave a pathogen-detecting sensitivity of 10-2 ng/μL, and is recommended for pathogen detection in diseased ginger plants and the rhizosphere soil.