摘要
【目的】由丁香假单胞猕猴桃致病变种(Pseudomonas syringae pv.actinidiae,Psa)引起的溃疡病是猕猴桃生产中最具毁灭性的病害,明确病菌的群体遗传特性将对了解病害发生规律和防治策略的制定具有重要理论意义。【方法】采用ERIC-PCR(enterobacterial repetitive inter-genic consensus)和BOX-PCR指纹技术分析了陕西省猕猴桃主要栽培区Psa的遗传特性。【结果】ERIC-PCR聚类结果显示,相似系数为0.626时,72个Psa菌株被分为6个类群,其中供试菌株中的75.7%属于第Ⅱ类群,且菌株无明显的采集地和寄主品种的聚类。BOX-PCR指纹聚类分析可将86个Psa菌株分为8个类群(相似系数为0.668),其中第Ⅰ类群菌株数量最多,占供试菌株数量的69.8%,其地理来源为眉县、周至、杨凌及其全部的意大利供试菌株,其中来源眉县的81.6%的菌株都聚集在该类群;菌株未表现出明显的寄主品种类群。【结论】陕西省猕猴桃溃疡病菌基因组存在丰富的遗传多样性;ERIC-PCR和BOX-PCR多态性分析技术可为我国Psa基因多样性的研究提供一个有效途径。
【Objective】Pseudomonas syringae pv. actinidiae is the widespread pathogenic agent of bacte-rial canker in the green-fleshed kiwifruit（Actinidia deliciosa） and the yellow-fleshed kiwifruit（A. chinensis） that causes severe economic losses in these worldwide cultivated crops. Information about the popula-tion structure of a pathogen during epidemics and preliminary assessment of a microorganism＇s sensitivityto different antimicrobials are fundamental prerequisites for planning effective field strategies to controlthe spread of a bacterium and to reduce the incidence of disease over a long-term period. The relationshipbetween pathogenic bacteria and the host plant（s） involves adaptation to the particular growth environ-ment, the commercial trade routes of individual plant parts, and the subsequent material uses, which leadsto intricate and multifaceted opportunities for global spreading and diversification; thus, commercial agri-cultural plant-pathogen phylogenetic analysis offers a prolific model for assessing the structure of evolu-tionary time and space in host-pathogen relations. Over the last few decades, molecular analysis usingPCR to produce DNA fingerprints has become an ever more refined and more widely accepted techniqueto characterize microorganisms, including P. syringae pathovars. PCR amplification of repetitive bacterialDNA elements（rep- PCR） using ERIC（enterobacterial repetitive intergenic consensus sequences） andBOX primers, has been proved to be a promising genotypic tool for rapid and reliable speciation and typ-ing of bacteria at the strain and pathovar level. The objective of this study was to elucidate the genetic rela-tionships among populations of P. syringae pv. actinidiae isolates present in Shaanxi of China by ERIC-PCR and BOX-PCR.【Methods】A total of 82 strains were obtained from infected vines, branches andtrunks, as well as directly from canker exudates. The selected strains included those from kiwifruit plantsbelonging to different species and cultivars（‘Hayward＇,‘Xuxiang＇and‘Qinmei＇for Actinidia deliciosa and‘Hongyang＇,‘Hort 16A＇,‘Jintao＇and‘Xixuan 2＇for A. chinensis）, grew in Shaanxi＇s main kiwifruitcultivation areas. The molecular characterization and the genetic diversity of P. syringae pv. actinidiae were analyzed using repetitive-sequence PCR performed with BOX and ERIC primer sets. Genomic DNAwas extracted from single colonies using Pure LinkTMGenomic DNA kit. DNA concentrations were estimat-ed by measuring the absorbance at 260 and 280 nm using a Nano Drop 1000 spectrophotometer. Molecularconfirmation of their identity was performed through pathovar-specific amplification with the primers indi-cated by Rees-Georage et al. BOX and ERIC amplification reactions were conducted with two primersets, as described previously by Versalovic et al. Following the amplification step, 10 μL of the productswere separated on 1% agarose gel at 4 V·cm- 1for 4-5 h. The profiles were then visualized and photo-graphed under UV illumination after being stained with ethidium bromide. The images were analyzed andthe amplification profiles were compared by evaluating the presence and size of bands using Phoretix 1Dsoftware. The presence or absence of each band was transformed into binary data, scored 1 for presence,and 0 for absence. A computer assisted analysis was performed using NTSY vision 2.1 software for Win-dows. A genetic similarity matrix was generated for each data set, and for their grouping, using the Dice co-efficient. The dendrograms were generated with unweighted pair-group method analysis（UPGMA） witharithmetic means. The efficiency of UPGMA in estimating the genetic relationships was tested by calculat-ing the cophenetic values and measuring the cophenetic correlation between the similarity matrix and thecophenetic values using Mantel＇s Z-statistics.【Results】The identity of the strain was confirmed by PCRamplification using pathovar-specific primers designed to amplify a specific DNA fragment. All the P. syringae pv. actinidiae strains investigated in this study produced the expected amplification fragments of280 bp with primers Psa F1/F2 in PCR experiments. In the molecular analysis of 82 Pseudomonas strains,all the chosen approaches generated polymorphic profiles and their banding patterns were reproducible inindependently replicated experiments, from DNA extraction onwards. Rep-PCR fingerprinting performedwith ERIC and BOX primer sets clearly showed a relevant diversity among the tested P. syringae pv. actinidiae strains. Differences among banding patterns between strains were clearly visible with each primerset. As regards rep-PCR with three sets of primers, the ERIC primers generated 14 bands, varying from300 to around 1 000 bp. Based on the fingerprint profile of ERIC-PCR, the results showed that 72 P. syringae pv. actinidiae strains were clustered into 6 clusters at the threshold of genetic similar coefficient0.626. Of these clusters, the second cluster was the largest and included 75.7% P. syringae pv. actinidiae strains which were collected from different geographical areas and different kiwifruit varieties. About 49 P. syringae pv. actinidiae strains from Mei county were divided into 6 clusters; and there were the similarcharacteristics of P. syringae pv. actinidiae strains from Yangling and Zhouzhi. Thus, there was no signifi-cant correlation between the genetic relationships and their geographical originals according to the finger-print profile of ERIC-PCR. Furthermore, the BOX A1 R primer produced 9 fragments and clearly showeda relevant diversity among the tested 86 strains. According to BOX-PCR, the dendrogram revealed that86 P. syringae pv. actinidiae strains were divided into 8 clusters（the threshold of genetic similar coeffi-cient 0.668）. The first cluster was the largest and included 69.8% P. syringae pv. actinidiae strains. In thefirst cluster, strains were isolated from Mei county, Yangling, Zhouzhi and Italy. 81.6% of strains isolatedfrom Mei county belonged to the first cluster. Twelve strains from Zhouzhi were clustered into clusterⅠ（6strains）,Ⅱ（1 strain）, Ⅲ（2 strains）, Ⅳ（1 strain） and Ⅴ（2 strains）, respectively. Of these clusters, clusterⅧ just included one strain which isolated from‘Hayward＇in Mei county. Similarly, based on the finger-print profile of BOX-PCR, there was no significant correlation between the genetic diversity and their geo-graphical originals.【Conclusion】The studies suggested that there was the genetic difference in the ge-nome of P. syringae pv. actinidiae from Shaanxi of China. The results also indicated that ERIC-PCR andBOX-PCR can provide an effective tool for the study of genetic diversity of P. syringae pv. actinidiae andoffer a prolific model for assessing the structure of evolutionary time and space in host-pathogen relations.
出处
《果树学报》
CAS
CSCD
北大核心
2016年第3期340-349,共10页
Journal of Fruit Science
基金
高等学校基本科研业务费(QN2013007)
陕西省科技统筹计划工程项目(2012KTJD03-02)
高等学校学科创新引智计划(No.B07049)
