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Molecular Identification Strategy for Pathvars of Pseudomonas Syringae and Its Application

Author: WenYi
Tutor: JiangShiJun
School: Henan Agricultural University
Course: Plant Pathology
Keywords: Pseudomonas syringae Disease-causing variants Molecular Identification hrpZ gene 16S rDNA ITS
CLC: S432.4
Type: Master's thesis
Year: 2009
Downloads: 96
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Abstract


Plant pathogen Pseudomonas syringae (Pseudomonas syringae) according to their host plants, as well as due to different symptoms of the disease are divided into many pathogenic variant (pathovar). The identification of the disease-causing variants is an important basic work, usually based on a series of complex physical and chemical characteristics of bacteriology identification. With the rapid development of molecular biology techniques, nucleic acid hybridization, genomic DNA fingerprinting technology, conserved genes, as well as specific gene sequence analysis technology for clove plant pathogenic Pseudomonas the genomic species groups (genomospecies) division and pathogenic Identification of the variant provides state-of-the-art methods. In this study, by analysis of 16S rDNA, the possible role of the 16S-23S ITS, hrp gene in the Pseudomonas syringae pathogenic variants identified, and the results of molecular identification with conventional bacteriology identification results mutual authentication, designed to filter out suitable Pseudomonas syringae rapid identification of disease-causing variants indicator molecules, the establishment of a set of rapid identification of the causative variants of molecular identification technology system. In this study achieved major results are as follows: 1. From the of eight Pseudomonas syringae pathogenic variant P. s. Pv tomato, P. s. Pv. Angulata, P. s pv. Tabaci, P. s . pv. syringae, P. s. pv. garcae, Ps pv. glycinea, P. s. pv. phaseolicola, P. s. pv. maculicola related strains of P. viridiflava, P. gingeri of a total of 16 standard strains genomic DNA as a template, using 16S rDNA universal primers for PCR amplification, a length of about 1560bp specific bands, a recombinant plasmid containing the target fragment obtained by the T / A cloning, get various pathogenic variant of 16S rDNA gene sequencing. The nearly full-length sequences. Sequence Blast homology search and sequence phylogenetic analysis showed: plant Pseudomonas bacteria 16S rDNA sequences are highly conserved, suitable for the genus and species level identification. The more conservative 16S rDNA sequence of Pseudomonas syringae 16S rDNA gene phylogenetic analysis indicated that this highly conserved 16S rDNA gene can not meet the requirements of the Pseudomonas syringae pathogenic variant molecular identification. Using the conserved sequence of the 16S-23S rDNA amplified the ITS full-length sequence degenerate primer is designed, 8 cloves Pseudomonas pathogenic variant of the 16 standard strain genomic DNA as a template for PCR amplification to obtain length for a specific band of 750bp. RFLP analysis showed that the PCR products were cut by limiting enzyme Hinc Ⅱ and Dde Ⅰ digested fragments of the 16S-23S ITS Pseudomonas syringae is also highly conserved, P. syringae pathogenic variant of the ITS amplification product RFLP patterns not enough polymorphism is used to identify disease-causing variants, just a P. gingeri, each P. viridiflava with P. syringae pathogenic variants of the ITS-PCR-RFLP profiles significantly different. Therefore, ITS sequence is not the same Pseudomonas syringae pathogenic variant molecular identification of ideal indicators. 3. HrpZ genes are unique to pathogenic bacteria, their encoded products both pathogenic and allergic induced non-host-related. Using the design of the four pairs of the hrpZ gene primers, to Pseudomonas syringae pathogenic variant of P. s PV Tomato, P. s. PV angulata, P. S PV tabaci, P. S. Pv. Syringae, Ps pv. garcae, P. s. pv. glycinea, P. s. pv. phaseolicola, P. s. pv. maculicola and other tested strains P. viridiflava P. gingeri of genomic DNA for PCR amplification of a template, PCR amplified fragment length polymorphism analysis the hrpZ genes. The results show that the primer pair HrpABF / R effectively distinguish all eight test Pseudomonas syringae pathogenic variant; primer pair HrpZ08F/R1 with HrpZ359/1115 the P. s. Pv. Garcae non-specific amplification, and related strains or polymorphism results. Primer HrpZ09UP/DW tested strains specific or polymorphism amplification results and P. s. Pv syringae different strains between the strip brightness difference. In addition to the primer HrpABF / R primer HrpZ08F / R, HrpZ09UP/DW with HrpZ359/1115 can not of P. s. Pv. Angulala and P. s. Pv. Tabaci effectively distinguish the P. s. Pv . angulata and P. s. pv. the the tabaci both the hrpZ may the same. Compared with 16S rDNA and ITS PCR amplification product hrpZ gene polymorphism in the disease-causing variants is very rich, and pathogenic variants within the conservative and strong, amplified clear and easy defense. hrpZ gene phylogenetic analysis also showed that the gene is Pseudomonas syringae pathogenic variant ideal for rapid identification of the molecular markers. 4. Routine bacteriological identification experiments show that, the bacterial morphological characteristics, the classic LOPAT well GATTa a series of physical and chemical identification results with molecular identification based on hrpZ gene results consistent with fast, accurate, and easy to operate, but hrpZ-based molecular identification results objectively other unique advantages, this study successfully established disease-causing variants based on hrpZ gene the molecular identification technology system provides an effective strategy for the plant pathogen Pseudomonas syringae and its rapid identification of disease-causing variants. In this study, 5. Repeatedly found the wildfire disease-causing variants corner spots pathogenic variant is very similar in morphological characteristics, physical and chemical characteristics, PCR amplification features. In order to differentiate between the two, specific amplification using primers designed tabAF / R to distinguish P. s. Pv. Angulata and P. s. Pv. Tabaci two disease-causing variants. The results show that the wildfire pathogenic variant of P. s. Pv tabaci able to get specific bands of 1263bp fragment length corner spots pathogenic variant of P. s. Pv. Angulata is not the specific bands. Description (ie smoke toxin-encoding gene) amplification strategy to distinguish the tab strains and the strains of tab-specific genes. 6. Established in this study based on the molecular identification hrpZ gene as well tabA gene technology system, the success of western Henan tobacco bacterial leaf spot rapid identification of pathogens, and ultimately determine the bacterial leaf spot pathogen P. s. pv. tabaci tab-strain (ie P. s. pv. angulata) identified the same routine bacteriological identification verification and support.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful
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