Study On The Tobacco Endophytic Bacteria And Their Biological Control Of Tobacco Bacterial Wilt

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).