【作者】 易有金；

【导师】 罗宽； 刘二明；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2007， 博士

【摘要】 烟草青枯病是烟草的一大毁灭性土传病害，至今还没有一种安全有效的药剂能够防治它，因此发掘有效的生防菌进行生物防治，其现实意义十分重大。本研究分离筛选出对烟草青枯病菌有拮抗作用的内生细菌001、009和011，对它们进行分类鉴定，测定它们的生物学特性和抑菌谱；着重对内生细菌001菌株的内生定殖、促生作用及产生的抗菌物质的纯化与其发酵条件等进行了研究，同时开展了对烟草青枯病的田间防治试验，为利用内生细菌防治烟草青枯病奠定了理论和应用基础。从湖南永州、郴州、衡阳和宁乡等的病区健康烟草茎杆中分离到160个内生菌株，其中32个在室内对19株不同青枯菌菌株具有拮抗效果，占到总菌株数的20.0％。在这32个菌株中以001、009、011抑制圈半径较大，分别为3.0mm、3.0 mm、4.5mm，故将内生菌株001、009、011作为重点研究对象。以致病力强的烟草青枯病菌2316菌株，作为测定拮抗菌拮抗性的标准病原菌株。结合001、009、011内生菌株形态学特征、生理生化性状、16S rDNA序列分析和根据MEGA3软件得到系统发育树等，确定001菌株属枯草芽孢杆菌Bacillus subtilis，菌株009和011属短短芽孢杆菌Brevibacillus brevis。菌株001与Bacillus subtilis（DQ415893）处于同一分支，相似性为99.2％；菌株009和011与Brevibacillus brevis（AY591911）处于同一分支，相似性分别为99.5％和99％。001菌株16S rDNA序列全长为1 511bp，009、011菌株16S rDNA序列经测定全长均为1 497bp。001、009、011菌株16S rDNA序列在GenBank数据库中的注册序列号（Accession No）分别为为DQ444283，DQ444284，DQ444285。001，009，011内生菌株的生物学特性研究表明，001、011菌株生长最适pH7.5；适宜在相对较高温环境中生长，最适生长温度为30℃。009菌株生长最适pH6.5；最适生长温度为25℃。009菌株能利用的碳源有蔗糖、葡萄糖、甘露醇等，不能利用α-乳糖、D-木糖、麦芽糖、D-半乳糖、D-果糖；能利用胰蛋白胨、蛋白胨、牛肉浸膏和酵母浸膏这4种有机氮源，不能利用干酪素，氯化铵等无机氮源。菌株001、011菌株都能利用上述所有的碳源；能利用除硝酸钾以外的氮源。说明001，011菌能对碳源和氮源没有严格的要求，适应性很强。在抑菌谱测定中，001、009、011菌株发酵上清液除对烟草青枯病菌有强烈抑制作用外，发酵上清液对多种其他植物病原细菌、真菌，如：柑橘溃疡病菌、马铃薯环腐病菌、白菜软腐病菌、烟草赤星病菌、黄瓜枯萎病菌、花生黑斑病菌等的生长均有抑制作用，表明001、009、011菌株均为广谱拮抗菌。001、009、011菌株的内生定殖测定，以抗Rif300μg／mL为标记，突变菌株接种后从烟株茎秆内回收菌株的培养性状及拮抗性均与原始菌株基本相同。证明了001、009、011均为内生菌株，都能进入烟草植株内定殖。用抗Rif标记001菌株不同菌液浓度接种烟草植株表明，001菌株在浓度为1×10⁸ cfu／mL时就能侵入，菌株的定殖能力随着菌液浓度的升高。采用浸种、浸根、淋根、伤茎和伤叶法将001菌株接种到烟草K326植株上，都能从烟草的根、茎或叶组织回收到001菌株。因此，在大田上利用001菌株来防治烟草青枯病时，可采取浸根、淋根和浸种等方法处理烟草。001菌株接种后在所检测的6个烟草品种NC82、云烟87、红花大金元、翠碧1号、K326、K346烟草植株的根部、茎部、叶部都能回收到001菌株，说明该菌株在烟草植株中生存不受烟草品种的影响。001菌株在烟草K326生长动态表明，在整个生育期中其细菌数量表现出从苗期到旺长期大幅增加，茎部的菌量增长较快，根部次之，叶部最慢：但到了接种150d后即到成熟期，其细菌数量大幅下降，表明在烟草生长前期该菌在烟草植株内的生存能力较强，但到了生长后期减弱。取接种烟草植株的茎部组织超薄切片，用电镜投射观察，发现茎部组织的维管束的导管内及其附近的细胞内有细菌存在。确定001菌株在烟草植株的微管束和细胞内定殖。001菌株对烟草植株的促生试验中，用001菌株悬浮液1×10⁸ cfu／mL浸种不同品种烟草种子，对不同品种烟草苗均有明显的促进生长作用，每株烟草苗平均鲜重增加达43.01％～57.7％。001菌株悬浮液通过不同的接种方法如浸种接种、浸根接种均表现出对烟草苗较明显的促生作用，每株烟草苗平均其鲜重比对照增加56.84％，57.89％。经001菌株悬浮液处理的烟草种子K326，在出苗后1～60d，烟草中IAA、ZR₄、GA₃等与促进植物生长发育有关的内源激素的含量比对照高；但ABA一开始即比对照低，出芽后30d表现最明显，比对照低60.60％。001菌株处理后诱导了寄主植物体内源激素含量的变化，使植物体内与促进生长相关激素的含量增加、抑制生长的激素含量下降，从而促进了植物的生长。B. subtilis 001菌株发酵液抑菌物质粗提液经100℃处理20 min后拮抗活性保持不变，说明该抗菌物质的热稳定性好。粗提液蛋白酶K处理后，拮抗活性保持不变；胰蛋白酶和胃蛋白酶处理后，拮抗活性仍然保持90％。对蛋白酶K不敏感，对胰蛋白酶和胃蛋白酶部分敏感。B. subtilis 001菌株的发酵液经硫酸铵分级沉淀、DEAE-Sepharose FF、FPLC Phenyl FF疏水色谱柱、HPLC C18反相色谱柱后纯化得到电泳纯与质谱纯的单一拮抗物质H16，其分子量为7387.4Da。H16在平板上对烟草青枯病菌有明显的抑菌作用，抑菌圈直径为20mm，而对照没有任何的抑菌迹象。通过正交试验确定菌株001的最佳发酵培养基配方为豆饼粉5％，马铃薯50％，CaCO₃0.5％，Mn₂SO₄0.11‰，KH₂PO₄0.2％。001菌株摇瓶发酵条件的优化表明最佳培养时间为96h，最适温度为30℃，最佳种子菌龄为培养36h的001菌株，最佳接种量7％，最佳转速为200r／min，最佳装液量为100ml，培养基的初始最佳pH值为8.0。001菌株5L发酵罐发酵条件正交试验优化表明，通气量为100L／h，转速为220r／min，温度为30℃，起始pH为8.0，最佳发酵时间为60h。内生拮抗菌B.subtilis001菌株连续4年对烟草青枯病的田间防治效果明显。2003年在宁乡、2004年在桂阳进行小区试验的防治效果分别为82.5％、65.2％；2005年在宁乡与桂阳进行小区试验的防治效果分别为78.14％、40.03％，3年小区试验防效均优于防治对照药剂农用链霉素。2006年在桂阳大面积（1hm²）田间试验平均防治效果为55.8％，防治区处理的病情指数比清水对照（23.8～84.3）减少21.8～53。更多还原

【Abstract】 Tobacco wilt caused by Ralstonia solanacarum is the soil-born disease which isdestructive to tobacco. There are no effective and environmentally harmoniousmethods to control the disease. In the study we screened some bacterial strains tocontrol the disease.The endophytic bacteria strains of 001, 009 and 011 among 160 sampled strainsin this study were found to be able to strongly control the growth of Ralstoniasolanacarum causing tobacco bacterial wilt. The biological characteristics and thespectra of inhibiting plant pathogens of the three strains were identified and tested.In this paper, it was focused on that the strain 001 was used to study the colonization,promoting tobacco seedling growth, purification antagonistic substance, fermentationconditions and bio-efficacy on tobacco R. solanacarum. The goal of this study is toestablish both theoric basis and application technology for tobacco wilt biocontrol.One hundred sixty endophytic bacterial strains were isolated from some healthystems in seriously R. solanacarum-infected fields at Yongzhou, Chenzhou, Hengyangand Ningxiang regions of Hunan. Thirty-two of the strains, accounting for 20% of thetotal samples, showed the inhibition effects on the growth of nineteen R. Solanacarumstrains. The inhibition zone radium of the 001, 009 and 011 strains were 3.0 mm, 3.0mm and 4.5mm, respectively, and were significantly larger than that caused by theother strains. Based on the above result, strains 001, 009 and 011 were mainly studied.The pathogen strain R. solanacarum 2316 causing the most destructive disease to thetobacco plants was chosen to be the standard tester screening the endophytic bacterialstrains biocontrolling effectiveness on the tobacco wilt.Based on the characteristics of morphology, physiology, biochemistry and the16S rDNA phylogenetic tree constructed with MEGA3, strain 001 was identified as astrain of Bacillus subtilis, which had 99.2% sequence similarity with B. subtilis（DQ415893）, stains 009 and 011 were two strains of Brevibacillus brevis, whichhad 99.5% and 99.0% sequence similarity with Brevibacillus brevis（AY591911）,resepectively. It was measured that strains 001, 009 and 011 have the 16S rDNA length of 1 511bp, 1 497bp, and 1 497bp via PCR, respectively. The accessions ofthem in GenBank were DQ444283, DQ444284 and DQ 444285, respectively.The optimal growth pH and temperature were 7.5 and 30℃, 6.5 and 25℃, and7.5 and 30℃for strains 001, 009 and 011, respectively. Strain 009 could grow withcarbonic sources of saccharose, glucose and D-mannitol, and nitrogen sources ofpeptone, tryptone peptone, beef extracts and yeast extracts. The carbonic sources ofD-arabinose, maltose, D-xylose, D-fructose and D-galactose were not suitable for thegrowth of strain 009. Inorganic nitrogen contained in casein, ammonium chloride,potassium nitrate, urea and ammonium oxalate could not be used for the strain 009.However, strains 001 and 011 could use all the mentioned carbonic sources andnitrogen sources except potassium nitrate. Thus, strains 001 and 011 were moreadaptable to the nutrients than the strain 009 was.In their spectra of inhibiting plant pathogens, the fermented liquid of strain 001,009 and 011 effectively inhibited the growth of 2316 strain. They also inhibited thegrowth of other gram-positive bacteria, gram-negative bacteria and pathogen fungi,such as Xanthomonas campestris, Clavibacter michiganenc, Erwinia carotovor,Alternaria sonalia, Fusarium oxysporum and Phaeoisariopsis personata. Therefore,strains 001, 009 and 011 had a wide spectrum of inhibiting plant pathogens.Strains 001, 009 and 011 with resistant to Rif （300ug/mL） were inoculated totobacco plants to test the inner colonization respectively. They were re-isolated fromthe tobacco plant stems. The re-isolated stains kept the same biological characteristicsand inhibition effects to tobacco R. solanacarum.The strain 001 with resistant to Rif （300ug/mL） was inoculated to tobacco plantwith different concentrations. The results showed that it could penetrate the tobaccoplants when its concentration was above 1×10⁸cfu/mL, and the colonization abilityincreased as the inoculating concentration increased. The strain 001 penetratedtobacco cultivar K326 plant tissues using inoculation methods such as: soaking seed,dipping root, watering root, injuring stem and injuring leaf. We also used strain 001tagged with resistant to Rif （300ug/mL） to inoculate other tobacco varieties such as:NC82, Yunyan 87, Honghuadajinyuan, Quanbil, K346. Re-isolation of the strain all recurred on the treated plants. The studies on population dynamics in the total growthstage of tobacco （150 days after inoculation） showed that the number of endophyticbacteria significantly increased from seeding stage t, hrough blossom stage, thensignificantly decreased in mature stage. We observed that strain 001 within vascularand cell after 30 days of inoculation under electron microscope.Thus, the strain 001colonizated within vascular and cell of tobacco plants to control tobacco wilt in thefields. The viability of the bacteria was dependent on the biological activity of thehost plants.The fresh weights of the treated different tobacco seedlings whose seeds treatedwith the strain 001 increased 43.01%～57.7% compared with that of the untreatedtobacco. The fresh weights of the tobacco K326 increased 56.84%, 57.89% comparedwith that of the untreated tobacco with the different treatment. Soaked K326 seeds instrain 001 bacterium suspension 1×10⁸cfu/mL, the hormone concentrations of IAA,ZR₄, GA₃, which promoted plant growth, were higher than that in the untreatedtobacco plants after 60 days of sprouting. The IAA concentrations of the treatedtobacco seedlings was lower than that of the untreated at the emergence stage and30days after spourting it was distinctly less 60% than the CK. The mechanisms ofpromoting tobacco growth were that the bacteria could enhance the tobaccosomatotropic hormone （IAA） and reduce the tobacco ABA hormone.The antagonistic crude extract obtained by ammonium sulfate precipitation of thecell-free culture of the strain 001 was thermo stable （100℃20 min） and resistant toproteinase K, pepsin and trypsin. The fermented liquid of 001 strain was purified withusing ammonium sulfate precipitation, DEAE-Sepharose FF chromatography, FPLCPhenyl FF chromatography and HPLC C18 chromatography. This purificationproduced an antagonistic substance, H16. The peptide showed strong antagonisticactivities to R. solanacearum, its inhibition zone was 20mm. MALDI-TOF-MSanalysis shows the molecular weight of the antagonistic peptide was 7387. 4Da, whichwas the same molecular weight in SDS-PAGE.The flask fermentation study showed that the optimal culture medium for thestrain 001 with orthogonal experiments was soybean powder 5%, potato 50%, Mn₂SO₄ 0.11‰, CaCO₃ 0.5%, and KH₂PO₄ 0.2%. The optimum flask fermentationconditions were: culture temperature 30℃, culture time 96 hours, seed age 36 hours,inoculation volume7%, liquid volume 100ml, pH 8.0, vibration speed 200r/min. Thebatch fermentation in a 5L stirred fermentation container showed that liquid volume3L, stirring rate 220r/min, air flow rate 100L/h, culture temperature 30℃, initial pH8.0 and culture time 60 hours met the optimal condition.Field experiments in Guiyang （2003） and Ningxiang （2004） of Hunan Provinceshowed that the efficacy of the strain 001 on tobacco R.solanacarurn was 82.5% and65.2%, respectively. In 2005, the infestation of the tobacco wilt was reduced by40.03% and 78.14% in the fields in Guiyang and Ningxiang, respectively. Using thestrain 001 to control tobacco wilt was more effective than applying Streptomycini in2003, 2004, 2005. In 2006, its average efficacy on the tobacco wilt was 55.8% atlarger fields （1hm²）in Guiyang. The severity at the treated tobacco was less 21.8～53.0compared with the untreated tobacco（23.8～84.3）.更多还原