Indentification And Pathogenic Test Of Pathogen Of Bacterial Wilt From Pogostemon Cablin (Blanco) Benth.

The paper mainly describes the characteristics of pathogenic bacteria isolated from bacterial-wilted Pogostemon cablin(Blanco) Benth., including their morphology, biovar type, pathogenicity and 16S rDNA sequence. This work aims to explore the intraspecific differentiation of the bacteria stains, which might contribute to the breeding of resistant cultivars and integrated control and prevention over the disease. The conclusions of this study are as follows:1 Review on relevant literatures in domestic and abroadAfter reviewing lots of literatures in China and abroad, the general situation of studies of P.cablin was summarized, including resource, identification,cultivation technique,varity breed and so on. About classification genomics, pathogenesis, epidemiology, isolation, identification and diagnosis of the pathogenic bacteria was refered. And several major approaches for the breeding of resistant cultivars against bacterial wilt disease were introduced as well.2 Isolation and biovar determination of the bacteria strainsSeven bacteria strains were isolated from the vascular bundles of P. cablin which were infected bacterial wilt in Guangdong Province, China. Stem segments and roots from diseased plants were cultural to isolate pathogenic bacteria. And the cultural characteristics, morphology, biovar were analyzed. Rhizospheric soils were sampled, and their pH were comparatively analyzed to indicate the association between the soil pH and epidemic of the disease. The results indicated rhizospheric soil pH of infected plants was 6.46 during disease epidemics. And it was close to the optimal pH(6.6)for R.solanacearum when cultured in laboratory. Rhizospheric soil pH was associated with pathological state, geographical position and the course of disease. Soil pH is vital for the occurrence and prevalence of harmful bacterial wilt. TTC plate colonyies of seven tested isolates appeared smooth and red, with white rings. Most cells were rhabdoid, others spherical. According to criteria for the classification of R.solanacearum biotype, HX5 and HX7 belong to biotypⅠ; HX1, HX6 and GIM1.7 (R.solanacearum from wilted tomato) belong to biotypeⅡ, HX2 and HX4 belong to biotypeⅢ, and HX3 belongs to biotype V. According to the current data, there were some differences between seven isolates (viz. HX1-HX7) in the cultural characteristics, morphologyand biovar type.It indicated that there is a variety of physiological races of bacteria strains from the P.cablin suffering bacterial wilt in the field.3 Pathogenic test of the bacteria strains from P.cablinWith the test-tube plantlets of P.cablin as materials, prick inoculation, root-wounding immersion and stem culture in bacterial solution method were employed for the suitable inoculation method. The results showed root-wounding immersion was favourable for resistanceidentification of P.cablin seedling in terms of epidemic time, rate of disease progression and simple operation. Meanwhile, the pathogenicity of bacteria strains and their crude toxins to the adult plants or non-rooted seedlings were evaluated. The results demonstrated that the tested strains(with the exception of HX2)could actually cause symptoms similar to those infected by the pathogenic bacteria in fields, and the virulence of the crude toxins were relevant to the corresponding pathogen.Moreover, most tested strains exhibited strong virulence over the referencestrain GIM1.7.After 36 h inoculation with HX4,HX5,HX6 and HX7 resectively, the disease index was over 3.0. In the explant inoculation trial, with the exception of HX1 and HX2 of weak virulent crude toxin (disease index was under 0.7), the crude toxins from the remaining strains manifested high virulence. Crude toxins made from HX5 and HX6 shown the strongest pathogenicity, and the average disease index exceeded 3.5 in the fourth day after inoculation. Infected non-rooting seedlings were yellow brown or even scald-like all through. This incidated that there was virulence differentiation between different bacteria strains from bacterial-wilted P.cablin.4 16S rDNA sequence analysis of the bacteria strainsFour different extraction methods were introduced for the extraction of the genomic DNA of HX4 and HX7. Sequences of 16 S rDNA were amplified by PCR with the bacterium universal primers to investigate the effects of DNA templates of different purities, annealing temperature, and template volume on the specific PCR amplification. The purified PCR products (16S rDNA sequences) were sequenced and had an accession to the NCBI (http://blast. ncbi.nlm.nih.gov/Blast.cgi). And the 16S rDNA phylogenetic tree was constructed by Neighbor-joining (NJ) approach after multiple comparison of similar sequences with Mega4.0.The results indicated that direct colony PCR can not get the satisfactory outcome even though it is economical; The water-boiling method and bacteriolytic solution were difficult to control the amount of templates and attain good reproducibility although simple operation; CTAB/NaCI and Kit methods for the bacterial genomic DNA were fairly stable and recommended. The properest annealing temperature was 55℃. The tested strains HX4 displayed high similarity (homology>=95%) with E.stewarti. And stains HX7 was fairly close to Xanthomonas, with 16S rRNA homology 99%. The referenced strain GIM1.7 and R.solanacearum resulted to the wilting of plants from the Solanaceae family shared 99% of sequence of homology of 16S rRNA.