广藿香论文：广藿香青枯病病原菌鉴定及致病性测定

1、广藿香论文：广藿香青枯病病原菌鉴定及致病性测定【中文摘要】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

2、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（HX1HX7）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H ,HX2、HX4划为生化型皿,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

3、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 GIMI.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

4、， 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16SrDNA片段PCF扩增，比较不同纯度的模板DNA 退火温度

5、、模板量对PCFT增效果的影响。PCF产物回收测序后的16S rDNA序列在 NCBI (/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PCF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/NaC和试剂盒法较为稳定；最适退火温 度为55C； 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）Xanthomo

6、nas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%【英文摘要】The paper mainly describes the characteristics of pathogenic bacteria isolated from bacterial-wiltedPogostem on cabli n（Bla neo）Ben th., in cludi ng their morphology,biovar type, pathoge ni city and 16S rDNA s

7、eque nee. This work aims to explore the intraspecific differentiation of the bacteria stai ns, which might con tribute to the breedi ng of resista ntcultivarsand in tegratedcon trol and preve nti on overthe disease. The conclusions of this study are as follows:1Review on releva nt literatures in dom

8、estic 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, isolatio n, ide ntificati

9、on and diag no sis of the pathogenic bacteria was refered. And several major approaches for the breedi ng of resista nt cultivarsaga inst bacterial wiltdisease were in troduced as well.2 Isolati on and biovar determ in ati on of the bacteria strai nsSeve n bacteria stra ins were isolated from the va

10、scular bun dies of P. cabli n which were infected bacterial wilt in Guangdong Provinee, China. Stem segme nts and roots from diseased pla nts were cultural to isolate pathogenic bacteria. And the cultural characteristics, morphology, biovar were an alyzed. Rhizospheric soils were sampled, and their

11、pH were comparatively an alyzed to in dicate the association between the soil pHand 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 whencultured in laboratory. Rhizosph

12、eric soil pH was associated with pathological state, geographical position and the course of disease. Soil pH is vital for the occurre nee and prevale nee of harmful bacterial wilt. TTC plate colony ies of seve n tested isolates appeared smooth and red, with white rings. Most cells were rhabdoid, ot

13、hers spherical. According to criteria for the classification of R.solanacearum biotype, HX5and HX7belong to biotyp I ; HX1, HX6 and GIM1.7 (R.sola nacearum from wilted tomato) bel ong to biotype II, HX2and HX4belong to biotype 皿,and HX3belongs to biotype V. Accord ing to the curre nt data, there wer

14、e some differe nces betwee n seve n isolates (viz. HX1-HX7) in the cultural characteristics, morphologya nd 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 f

15、romP.cablinWith the test-tube plantlets of P.cablin as materials, prick inoculation, root-wounding immersion and stem culture in bacterial soluti on method were employed for the suitable ino culati onmethod. The results showed root-wo unding immersio nwas favourable for resistanceidentification of P

16、.cablin seedling in terms of epidemic time, rate of disease progression and simple operation.Meanwhile, the pathogenicity of bacteriastrains and their crude toxins to the adult plants or non-rooted seedli ngs were evaluated. The results dem on strated that the tested strains(with the exception of HX

17、2)could actually cause symptoms similar to those in fected by the pathoge nic bacteria in fields, and the virule nce of the crude tox ins were releva nt to the corresp onding pathoge n.Mo reover, most tested stra ins exhibited strong virule nce over the refere ncestrainGIM1.7.After 36 h inoculation

18、with HX4 、HX5 HX6 and HX7 resectively, the disease in dex was over 3.0 .In the expla nt inoculation trial, with the exception of HX1 and HX2 of weak virule nt crude tox in (disease in dex was un der 0.7), the crude toxins from the remaining strains manifested high virulence.Crude tox ins made from H

19、X5 and HX6 show n the str on gest pathoge ni city, and the average disease in dex exceeded 3.5 in the fourth day after ino culati on. In fected non-rooti ng seedli ngs were yellow brow n or eve n scald-like all through. This in cidated that there was virule nee differe ntiati on between different ba

20、cteria strains from bacterial-wiltedP.cablin.4 16S rDNA sequenee analysis of the bacteria stra in sFour differe nt extracti on methods were in troduced for the extractionof the genomic DNAof HX4 and HX7. Sequences of16 S rDNA were amplified by PCR with the bacterium uni versal primers to inv estigat

21、e the effects of DNA templates ofdifferentpurities,annealing temperature, and template volumeon the specific PCR amplification. The purified PCR products(16S rDNAsequences) were sequeneed and had an accession to theNCBI (http:/blast. ncbi. nlm.n /Blast.cgi). And the 16S rDNAphylogenetic tree w

22、as constructed by Neighbor-joining(NJ)approach after multiple comparis on of similar seque nces with Mega4.0.The results indicated that direct colony PCR can not get the satisfactoryoutcome even though it is economical; Thewater-boili ng method and bacteriolyticsoluti on were difficultto con trol th

23、e amount of templates and atta in good reproducibility although simple operati on; CTAB/NaCI and Kit methods for the bacterial geno mic DNA were fairly stable andrecommended. The properest annealing temperature was 55C. The tested strai ns HX4 displayed high similarity (homology=95%)with E.stewarti.

24、 And stains HX7was fairly close to Xanthomonas, with 16S rRNA homology 99%. The refere need strain GIM1.7 andR.solanacearum resulted to the wilting of plants from theSola naceae family shared 99% of seque nee of homology of 16S rRNA.【关键词】广藿香 青枯菌致病性16S rDNA病原鉴定【英文关键词】P.cablinR.sola nacearumpathoge ni

25、 city16S rDNA pathoge n ide ntificati on【目录】广藿香青枯病病原菌鉴定及致病性测定摘要4-6 Abstract 6-9 引言12-131国内外研究现状和评述13-241.1广藿香的研究概况13-151.1.1广藿香种质资源与鉴定131.1.2广藿香栽培和育种13-141.1.3广藿香青枯病的防治14-151.2青枯菌的研究现状15-201.2.1青枯菌的命名和分类15-161.2.2青枯菌的基因组学16-171.2.3青枯菌的致病机理17-181.2.4青枯菌的流行与传播18-191.2.5青枯菌分离鉴定与诊断技术19-201.3植物病原菌致病性测定的研究进展20-241.3.1 田间鉴定211.3.2 室内鉴定21-221.3.