Molecular Identification Strategy For Pathvars Of Pseudomonas Syringae And Its Application

Pseudomonas syringae group includes a number of pathovars that vary in their host range and/or disease symptoms. The traditional bacteriological identification is a basic but important work, including a series of physiological and morphyological characteristics tests. With the development of molecular biological technology, DNA-DNA hybridization, genomic DNA fingerprint analysis and DNA sequence analysis of conservative genes and special genes have been used to distinguish the strains or pathovars of P. syringae and to classify P. syringae into several groups-genomospecies.In order to screen some useful molecular identification marker and build up a new available strategy,8 pathovars and their 16 identified strains were used. Their 16S rDNA genes were sequenced, the 16S-23S rDNA internal transcriptional spacer sequences were analysed by PCR-RFLP, and one important hypersensitive-response and pathogenesis related gene, hrpZ gene was cloned and analysied. At the same time, the traditional bacteriological identification were performed for the validity of the established molecular identification strategy.The main results of this study were followed:1. Sixteen standard strains belonging to eight Pseudomonas syringae pathvars including P. s. pv. tomato, P. s. pv. angulata, P. s. pv. tabaci, P. s. pv. syringae, P. s. pv. garcae, P. s. pv. glycinea, P. s. pv. phaseolicola, P. s. pv. maculicola and two strains from P. viridiflava and P. gingeri were used in the experiments. The full sequences of 16S rDNA gene were cloned with the universal 16S rDNA primers. The unique, band of about 1560bp in size were recovered, cloned and sequenced. These sequences were blasted in Genebank database for researching their homologies. According to the results of phylogenetic analysis,16S rDNA sequences were so highly conservative that they only were an available molecular marker for genus and species identification of Pseudomonas syringae. However, the 16S rDNA sequence was not a useful molecular marker for pathvar determination.2. The full-length ITS of the tested strains were amplified by PCR with the degenerate primers. Unique product of about 750bp in size were recovered and degested with HincⅡand DdeⅠ, respectively, in order to analyse the PCR-RFLP. The results showed that there just were some differences at inter-species level, while revealing highly consistent at inter-pathovar and intra-pathovar level. Therefore, the ITS sequence was neither an ideal molecular marker.3. The hrp genes in plant pathogenic bacteria play important roles in causing disease on host plants and eliciting hypersensitive response on non-host plants. The sequences of hrpZ gene of the tested strains were amplified by PCR with the degenerate primers. The sequence polymorphism of hrpZ gene were analyzed and the NJ-tree was constructed. The target fragments coule be amplified with HrpABF/HrpABR, HrpZ09UP/HrpZ09DW primers, and also distinguished by HrpZ08F/HrpZ08Rl and HrpZ359/HrpZl 115 but for P. s. pv. garcae and related strain P. viridiflava and P. gingeri. We also found that there were some differences between the tested strains of P. s. pv. syringae. Comparing with the 16S rDNA and ITS molecular marker, as a new molecular identification marker hrpZ gene was very conservative in different strains of a certain Pseudomonas syringae pathvar and highly homologous in different pathvars. The differences between the tested pathvars could be detected clearly. In conclusion, hrpZ gene was a perfect molecular marker for the rapid identification of Pseudomonas syringae pathvars.4. Consistent with the trantional identification results, which were based on characteristics of physical and chemical, hrpZ-based molecular identification with clear and objective result was much more rapid, accurate and easy to carry out. It revealed us that as the special gene hrpZ was a new perfect molecular identification marker and the strategy based on this gene was viable.5. During the study, usually, P. s. pv. angulata and P. s. pv. tabaci were not only familiar with most of the characteristics of physical and chemical, but also with the same band amplified by PCR. To differentiate P. s. pv. angulata from P. s. pv. tabaci, tabAF/tabAR primers were designed for amplification by PCR. P. s. pv. angulata lacked this special band as a important feature and was distinguished from P. s. pv. tabaci. Thus the tab A gene was another useful molecular marker, which was used to distinguish the two pathvars. 6. Using the molecular identification technology strategy, it was successful to identify the pathogen causing bacterial leaf spot on tobacco in the west of Henan province. The pathogen was tab- strain of P. s. pv. tabaci. At the same time the identified result was validated by the characteristics of physical and chemical.