湘南脐橙柑橘黄龙病的PCR检测及内生细菌的分离

PCR Detection of Citrus Huanglongbing Pathogen and Endogenous Bacteria Isolation for Navel Orange(Citrus sinensis Osb.var.brasliliensis Tanaka)in Hunan

为确保湘南脐橙产业发展安全,严格防控柑橘黄龙病,从湘南地区几个脐橙种植园采集柑橘黄龙病疑似病株样品和健康植株样品,利用文献报道的柑橘黄龙病PCR检测常用引物对样品进行分子检测,并从病株组织中分离内生细菌进行分析鉴定。结果表明:在柑橘黄龙病的常规PCR检测中,湘南脐橙总核酸对P400引物的灵敏度最高,是湘南地区脐橙中柑橘黄龙病害常规PCR检测的最佳引物,其次为P535引物,而OI和16S这2对引物的灵敏度较低;对脐橙中柑橘黄龙病病株组织内生细菌的分离鉴定发现,柑橘中可培养内生细菌主要归类为7个属,分别为短杆菌属(Curtobacterium)、鞘氨醇单胞菌属(Sphingomonas)、异常球菌属(Deinococcus)、假单胞菌属(Pseudomonas)、甲基杆菌属(Methylobacterium)、微杆菌属(Microbacterium)、芽孢杆菌属(Bacillus);其中,LB和1/10LB培养基中的优势菌属为短杆菌属、鞘氨醇单胞菌属和芽孢杆菌属;而脐橙树叶汁培养基中的优势菌属为短杆菌属、鞘氨醇单胞菌属和微杆菌属。

In order to ensure the industry security of navel orange(Citrus sinensis Osb.var.brasliliensis Tanaka)in South Hunan and keep strict prevention or control of citrus Huanglongbing(HLB),the diseased and healthy samples were collected from 4 navel orange ochards of Yizhang County,Chenzhou City,Hunan Province for the PCR detection of citrus HLB pathogen and the isolation of endogenous bacteria.4 kinds of PCR primers were used.In the routine PCR detection of citrus HLB,total nucleic acid of citrus HLB pathogen in the navel orange leaves has the highest sensitivity to primer P400 which is the optimal for PCR detection of citrus HLB for southern Hunan navel orange,followed by primer P535,while OI and 16S primers have lower sensitivity.Curtobacterium,Sphingomonas,Deinococcus,Pseudomonas,Methylobacterium,Microbacterium and Bacillus are seven species of culturable endogenous bacteria isolated from diseased samples.Curtobacterium,Sphingomonas and Bacillus are the dominant genera isolated with LB and 1/10LB,while Curtobacterium,Sphingomonas and Microbacterium are the dominant genera isolated with navel orange leaf juice+agar.