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(jìn)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(jìn)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(chǔ)。本研究主要結(jié)果如下：1國內(nèi)外研究評述查閱了與本研究相關(guān)的國內(nèi)外文獻(xiàn)，概述了廣藿香的研 究現(xiàn)狀；總結(jié)了青枯菌的命名分類、基因組學(xué)、致病機(jī)理、流行學(xué)、 分離鑒定與診斷技術(shù)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)學(xué)、

2、生化型等多個方面進(jìn)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(guān)系。結(jié)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(biāo)準(zhǔn)，HX5、HX7屬于生化型I ,HX1、HX6和GIM1.7（番茄青枯菌）為生化型n ,HX2、HX4劃為生化型m ,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，存在多個生理小種

3、的青枯菌株,它們在培養(yǎng)特性、形態(tài)學(xué)、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(jìn)行致病性測定。結(jié)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(qiáng)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(dá)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，病級指數(shù)均在0.

4、7以下,其余菌株制備的粗毒素均先后表現(xiàn)出較強(qiáng)的 致病力。以HX5 HX6的粗毒素致病性最強(qiáng)，在接種第4d平均病級指數(shù)達(dá)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(jié)果相似的癥 狀；且粗毒素的致病性與其對應(yīng)的菌株致病力強(qiáng)弱相關(guān)；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(qiáng)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(xì)菌通用引物進(jìn)行16SrDNA片段PCF擴(kuò)增，比較不同純度的模板DNA 退火溫度、模板量對PCRT增效果的

5、影響。PCF產(chǎn)物回收測序后的16SrDNA序列在 NCBI (/Blast.cgi)Blast,應(yīng)用Mega4.0將相似序列進(jìn)行多重序列比較后，再用鄰接法構(gòu)建系統(tǒng)發(fā)育樹，結(jié)果表明：菌落直接PCR雖然經(jīng)濟(jì),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(fù)性不佳；細(xì)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）Xanthomonas的16S rDNA的序列同源

6、性達(dá)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 mainiy describes the characteristicsof p athoge nic bacteria isolated from bacterial-wiltedP ogostem on cabli n( Bla neo)Ben th., in cludi ng their morp hology, biovar type, p athoge ni city and 16S rDNA seque nee. Thi

7、s work aims to explore the in tras pecific differe ntiati on of the bacteria stai ns, which might con tribute to the breedi ng of resista nt cultivars and in tegrated con trol and pr eve nti on over the disease. The con clusi ons of this study are as follows:1Review on releva nt literatures in domes

8、tic and abroadAfter reviewing lots of literatures in China and abroad, the general situatio n of studies of P.cabli n was summarized, in clud ing resource, ide ntificati on ,cultivati on tech niq ue,varity breed and so on. About classificatio n geno mics, p athoge nesis, ep idemiology, isolatio n, i

9、de ntificati on and diag no sis of the pathogenic bacteria was refered. And several major approachesfor the breedi ng of resista ntcultivarsaga inst bacterial wilt disease were in troduced as well.2 Isolati on and biovar determ in atio n of the bacteria strai nsSeve n bacteria stra ins were isolated

10、 from the vascular bun dies of P. cablin which were in fected bacterial wilt in Guangdong P rovi nee, Chi na. Stem segme nts and roots from diseased plants were cultural toisolatepathogenic bacteria. And the cultural characteristics, morp hology, biovar were an alyzed. Rhizos pheric soils were sampi

11、 ed, and their pH were comp aratively an alyzed to in dicate the association between the soil pHand epidemic of the disease.The results in dicated rhizos pheric soil pH of i nfected plants was 6.46 duri ng disease ep idemics. And it was close to the optimal pH(6.6)for R.solanacearum whencultured in

12、laboratory.Rhizos pheric soil pH was associated with p athological state, geogra phical p ositi on and the course of disease. Soil pH is vital for the occurre nee and p revale nee of harmful bacterial wilt. TTC p late colony ies of seve n tested isolates app eared smooth and red, with white rin gs.

13、Most cells were rhabdoid,others 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 n , HX2and HX4belong to biotype 皿,and HX3belongs to biot ype V. Accord ing to th

14、e curre nt data, there were some differe nces betwee n seve n isolates (viz. HX1-HX7) in the cultural characteristics, morp hologya nd biovar typ e.Itin dicated that there is a variety of p hysiological races of bacteria strains from the P.cablin suffering bacterial wilt in the field.3 Pathoge nic t

15、est of the bacteria stra ins fromP.cablinWith the test-tubeplantletsof P.cablin as materials,prick inoculation,root-wounding immersion and stem culture in bacterial soluti on method were empio yed for the suitableino culati onmethod. The results showed root-wo unding immersion was favourable for res

16、ista nceide ntificatio n of P.cabli n seedling in terms of epidemic time, rate of disease progressionand simpie operation.Meanwhile, the pathogenicity of bacteria strains and their crude toxins to the adult plants or non-rooted seedli ngs were evaluated. The results dem on strated that the tested st

17、rains(with the exception of HX2)could actually cause symp toms similar to those in fected by the p athoge nic bacteria in fields, and the virule nee of the crude tox ins were releva nt to the corres ponding p athoge n.Mo reover, most tested stra ins、HX5 HX6 and HX7exhibited strong virule nee over th

18、e refere ncestrainGIM1.7.After 36 h in oculation with HX4 resectively, the disease in dex was over 3.0 .In the explant ino culati on trial, with the exce pti on of HX1 and HX2 of weak virule nt crude tox in (disease in dex was un der 0.7), the crude toxins from the remai ning strai ns man ifested hi

19、gh virule nee.Crude tox ins made from HX5 and HX6 show n the stro ngestp athoge ni city, and the average disease in dex exceeded 3.5 inthe fourth day after ino culati on. In fected non-r ooti ngseedli ngs were yellow brow n or eve n scald-like all through.This in cidated that there was virule nee di

20、ffere ntiati onbetwee n differe nt bacteria stra ins from bacterial-wiltedP .cabli n.4 16S rDNA seque nee an alysis of the bacteriastra in sFour differe nt extracti on methods were in troduced forthe extraction of the genomic DNAof HX4 and HX7. Sequences of16 S rDNA were ampi ified by PCR with the b

21、acterium un iversalp rimers to inv estigate the effects of DNA temp lates ofdiffere ntp urities,ann eali ng temp erature, and temp late volumeon the sp ecific PCR amp lificatio n. The p urified PCR p roducts(16S rDNAsequences) were sequeneed and had an accession totheNCBI (htt p:/blast. ncbi. nim.n

22、/Blast.cgi). And the 16SrDNAphylogenetic tree was constructed by Neighbor-joining(NJ)app roach after mult ip le comp aris on of similar seque nces withMega4.0.The results in dicated that direct colony PCR can notget the satisfactoryoutcome even though it is economical;Thewater-boili ngmethod a

23、nd bacteriolytic soluti on were difficultto con trol the amount of temp lates and atta in goodrep roducibility although sim pie op erati on; CTAB/NaCI and Kit藿香青枯病菌的分離及生化型測定24-322.1材料和方法methods for the bacterial geno mic DNA were fairly stable and recomme nded. The prop erest ann eali ng temp eratur

24、e was 55 C. The tested strai ns HX4 dis played high similarity (homology>=95%) with E.stewarti. And stains HX7was fairly close to Xanthomonas, with 16S rRNA homology 99%. The refere need strain GIM1.7 andR.sola nacearum resulted to the wilti ng of plants from theSola naceae family shared 99% of s

25、eque nee of homology of 16S rRNA.【關(guān)鍵詞】廣藿香 青枯菌致病性16S rDNA病原鑒定【英文關(guān)鍵詞】P.cablin R.sola nacearumP athoge ni city16S rDNAp athoge 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1國內(nèi)外研究現(xiàn)狀和評述4-6 Abstract 6-9 引言 12-1313-241.1廣藿香的研究概況13-151.1.1廣藿香種質(zhì)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(xué)16-171.2.3青枯菌的致病機(jī)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(shù)19-201.3植物病原菌致病性測定的研究進(jìn)