Rep-PCR analysis of genotypic diversity of Pseudomonas syringae pv. actinidiae strains from Shaanxi province of China

Abstract:【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.