廣藿香論文：廣藿香青枯病病原菌鑒定及致病性測定

1、廣藿香論文：廣藿香青枯病病原菌鑒定及致病性測定【中文摘要】本文主要對廣藿香青枯病菌進行分離培養(yǎng)，并從顯 微形態(tài)、生理生化特征、致病性及其 16s rDNA序列分析等方面進行 了研究,以期了解廣藿香青枯病病原菌的組成及分化情況，為廣藿香 青枯病的綜合防治和抗病育種奠定基礎。本研究主要結果如下：1國內(nèi)外研究評述查閱了與本研究相關的國內(nèi)外文獻，概述了廣藿香的研 究現(xiàn)狀；總結了青枯菌的命名分類、基因組學、致病機理、流行學、 分離鑒定與診斷技術等方面的研究現(xiàn)狀；并介紹了植物病原菌致病性 測定的幾種主要方法。2廣藿香青枯菌的分離培養(yǎng)及生化型測定以感 染了青枯病的廣藿香植株為材料分離青枯菌，從培養(yǎng)特性、形態(tài)

2、學、 生化型等多個方面進行研究，同時采集病區(qū)根際土壤樣品，分析比較 其pH值,探討土壤pH值與病害流行之間的關系。結果表明：在病害 流行期,病株根際土壤pH為6.46與青枯菌室內(nèi)培養(yǎng)最適生長pH6.6 較為接近,表明土壤酸堿度對青枯病發(fā)生及流行有一定的影響。分離 獲得的7個供試菌株（HX1HX7）在 TTC培養(yǎng)基上培養(yǎng)，呈平滑、帶白 色暈圈的紅色菌落；菌體大小不一，大多桿狀,少數(shù)為球形；根據(jù)青枯 菌生化型劃分標準，HX5、HX7屬于生化型I ,HX1、HX6和GIM1.7（番 茄青枯菌）為生化型H ,HX2、HX4劃為生化型皿,HX3屬于生化型V。 這證明田間感染了青枯病的廣藿香植株中，存在多

3、個生理小種的青枯 菌株,它們在培養(yǎng)特性、形態(tài)學、生化型等方面均存在一定的差異。3廣藿香青枯菌致病性測定以廣藿香試管苗及無根苗為材料，分別接種青枯菌菌液及粗毒素，進行致病性測定。結果表明：針刺、傷根和莖 枝浸泡3種不同接種方法的試驗中，從發(fā)病時間、病程發(fā)展速度及操 作簡易性等方面綜合比較，傷根浸泡法較宜用于廣藿香苗期接種致病 性；除HX2外,其余菌株均能引起與田間廣藿香青枯病株相似的青枯、 萎垂等典型癥狀，且大部分菌株致病性均強于參照菌株 GIMI.7,其中 HX4 HX5 HX6和HX7在接種36h時,病級指數(shù)均達3.0以上；在粗 毒素接種外植體的試驗中，除HX1和HX2制備的粗毒素致病性較弱

4、， 病級指數(shù)均在0.7以下,其余菌株制備的粗毒素均先后表現(xiàn)出較強的 致病力。以HX5 HX6的粗毒素致病性最強，在接種第4d平均病級指 數(shù)達3.5以上,經(jīng)處理的無根苗葉色黃褐，后期干枯貼于培養(yǎng)瓶壁。供 試菌株制備的粗毒素對外植體也能引起與病原菌侵染結果相似的癥 狀；且粗毒素的致病性與其對應的菌株致病力強弱相關；粗毒素是廣 藿香青枯菌致病的重要因素。致病性試驗表明，不同青枯菌菌株致病 力有明顯的分化。4廣藿香青枯菌16S rDNA序列分析以致病性較強 的供試菌株為材料，采用不同的提取方法抽提青枯菌基因組 DNA利用 細菌通用引物進行16SrDNA片段PCF擴增，比較不同純度的模板DNA 退火溫度

5、、模板量對PCFT增效果的影響。PCF產(chǎn)物回收測序后的16S rDNA序列在 NCBI (/Blast.cgi)中Blast,應用Mega4.0將相似序列進行多重序列比較后，再用鄰接法構 建系統(tǒng)發(fā)育樹，結果表明：菌落直接PCF雖然經(jīng)濟,但效果不佳；水煮 法和裂解液法操作過程簡易粗放，模板量不易控制，重復性不佳；細菌 基因組DNA提取效果以CTAB/NaC和試劑盒法較為穩(wěn)定；最適退火溫 度為55C； HX4與歐文氏屬（嗜維管束菌）E. stewarti 的16S rDNA 序列同源性為95%,HX7與黃單胞菌屬（黃單胞桿菌）Xanthomo

6、nas的 16S rDNA的序列同源性達99%參照菌株GIM1.7與雷爾氏菌屬（茄青 枯假單胞桿菌）R. solanacearum 16s rDNA 序列的同源性為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.【關鍵詞】廣藿香 青枯菌致病性16S rDNA病原鑒定【英文關鍵詞】P.cablinR.sola nacearumpathoge ni

25、 city16S rDNA pathoge n ide ntificati on【目錄】廣藿香青枯病病原菌鑒定及致病性測定摘要4-6 Abstract 6-9 引言12-131國內(nèi)外研究現(xiàn)狀和評述13-241.1廣藿香的研究概況13-151.1.1廣藿香種質資源與鑒定131.1.2廣藿香栽培和育種13-141.1.3廣藿香青枯病的防治14-151.2青枯菌的研究現(xiàn)狀15-201.2.1青枯菌的命名和分類15-161.2.2青枯菌的基因組學16-171.2.3青枯菌的致病機理17-181.2.4青枯菌的流行與傳播18-191.2.5青枯菌分離鑒定與診斷技術19-201.3植物病原菌致病性測定的研究進展20-241.3.1 田間鑒定211.3.2 室內(nèi)鑒定21-221.3.