【作者】 刘丹；

【导师】 贺红；

【作者基本信息】 广州中医药大学 ， 生药学， 2011， 硕士

【摘要】 本文主要对广藿香青枯病菌进行分离培养,并从显微形态、生理生化特征、致病性及其16s rDNA序列分析等方面进行了研究,以期了解广藿香青枯病病原菌的组成及分化情况,为广藿香青枯病的综合防治和抗病育种奠定基础。本研究主要结果如下：1国内外研究评述查阅了与本研究相关的国内外文献,概述了广藿香的研究现状；总结了青枯菌的命名分类、基因组学、致病机理、流行学、分离鉴定与诊断技术等方面的研究现状;并介绍了植物病原菌致病性测定的几种主要方法。2广藿香青枯菌的分离培养及生化型测定以感染了青枯病的广藿香植株为材料分离青枯菌,从培养特性、形态学、生化型等多个方面进行研究,同时采集病区根际土壤样品,分析比较其pH值,探讨土壤pH值与病害流行之间的关系。结果表明：在病害流行期,病株根际土壤pH为6.46与青枯菌室内培养最适生长pH6.6较为接近,表明土壤酸碱度对青枯病发生及流行有一定的影响。分离获得的7个供试菌株(HX1～HX7)在TTC培养基上培养,呈平滑、带白色晕圈的红色菌落；菌体大小不一,大多杆状,少数为球形；根据青枯菌生化型划分标准,HX5、HX7属于生化型Ⅰ,HX1、HX6和GIM1.7(番茄青枯菌)为生化型Ⅱ,HX2、HX4划为生化型Ⅲ,HX3属于生化型V。这证明田间感染了青枯病的广藿香植株中,存在多个生理小种的青枯菌株,它们在培养特性、形态学、生化型等方面均存在一定的差异。3广藿香青枯菌致病性测定以广藿香试管苗及无根苗为材料,分别接种青枯菌菌液及粗毒素,进行致病性测定。结果表明：针刺、伤根和茎枝浸泡3种不同接种方法的试验中,从发病时间、病程发展速度及操作简易性等方面综合比较,伤根浸泡法较宜用于广藿香苗期接种致病性；除HX2外,其余菌株均能引起与田间广藿香青枯病株相似的青枯、萎垂等典型症状,且大部分菌株致病性均强于参照菌株GIMl.7,其中HX4、HX5、HX6和HX7在接种36h时,病级指数均达3.0以上；在粗毒素接种外植体的试验中,除HX1和HX2制备的粗毒素致病性较弱,病级指数均在0.7以下,其余菌株制备的粗毒素均先后表现出较强的致病力。以HX5、HX6的粗毒素致病性最强,在接种第4d平均病级指数达3.5以上,经处理的无根苗叶色黄褐,后期干枯贴于培养瓶壁。供试菌株制备的粗毒素对外植体也能引起与病原菌侵染结果相似的症状；且粗毒素的致病性与其对应的菌株致病力强弱相关；粗毒素是广藿香青枯菌致病的重要因素。致病性试验表明,不同青枯菌菌株致病力有明显的分化。4广藿香青枯菌16S rDNA序列分析以致病性较强的供试菌株为材料,采用不同的提取方法抽提青枯菌基因组DNA,利用细菌通用引物进行16S rDNA片段PCR扩增,比较不同纯度的模板DNA、退火温度、模板量对PCR扩增效果的影响。PCR产物回收测序后的16S rDNA序列在NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi)中Blast,应用Mega4.0将相似序列进行多重序列比较后,再用邻接法构建系统发育树,结果表明：菌落直接PCR虽然经济,但效果不佳；水煮法和裂解液法操作过程简易粗放,模板量不易控制,重复性不佳；细菌基因组DNA提取效果以CTAB/NaCl和试剂盒法较为稳定；最适退火温度为55℃；HX4与欧文氏属(嗜维管束菌)E. stewarti的16S rDNA序列同源性为95%,HX7与黄单胞菌属(黄单胞杆菌)Xanthomonas的16S rDNA的序列同源性达99%,参照菌株GIM1.7与雷尔氏菌属(茄青枯假单胞杆菌)R. solanacearum 16s rDNA序列的同源性为99%。更多还原

【Abstract】 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.更多还原