Study On The Isolation Of Antagonistic Bacterium Swu31-2 Against Ralstonia Solanacearum (Tobacco) And Its Biocontrol Effect

Bacterium Ralatonia solanacearum causing tobacco bacterial wilt is a plant disease,which spreads through soil infection.This disease is very serious in tropic,subtropics and some warm areas.In china,this disease generally occurs in Yangzte River drainage area and southern China,and causes tremendous loss. At present,controlling tobacco bacterial wilt is very difficult,and agriculture measure and Chemical control are the main two methods for it.However,environmental affects and cultivation conditions make it unpractical for agriculture control in many cases;Similarly,chemical control has been challenged for its disadvantages such as contamination to environment,harm to human body,inducing resistance of pathogenic bacterium,which halt its application.On the other hand,biological pesticides have merits of no pollution,harmless,low toxicity to mammal and little pressure to environment.Therefore,the development of biological pesticides is valuable and potential.In this work,we tried to develop a biocontrol method for tobacco bacterial wilt.From the tobacco rhizosphere,we obtained an antibiotic-producing bacterium Pseudomonas aeruginos strain swu31-2, which exhibited strong antagonistic effects on Ralatonia solanacearum,the pathogenic bacteria of tobacco wilt.Then,we studied its taxonomy,optimized its fermentation conditions for active substance production, and partially characterized the active substance after purification.We also studied the colonization of the bacterium in tobacco plant body and the control effect in greenhouse.The detailed experimental methods and results are displayed as following:1.Survey on biotypes and virulence of Ralatonia solanacearum in Shilang's tobacco field36 strains of the pathogenic bacteria of tobacco wilt were isolated from collected diseased tobacco plant.Biotypes of 24 strains were used for classification based on Hayward's classification criterion of P. solanacearum.The results showed that 5 strains(about 20.83%)isolated from tobacco could oxidize three kinds of disaccharides,mannitol and sorbitol,but not dulcitol.They were defined as biovarâ…¢-1.And 19 strains isolated from tobacco was hiovarâ…¢,making 79.17%of total amount.The result of virulence detection showed that 8 strains were strong virulence,13 strains were mild,and 15 strains were weak.The strong virulent pathogenic bacteria strains provided advantageous target to screen and control.2.Isolation,screening and identification of antagonistic bacterial swu31-2One antagonistic bacterial swu31-2 was obtained from the soils in the tobacco rhiszosphere,and it had obviously antagonistic effects on 8 strong virulent pathogenic bacteria strains of tobacco wilt and systematically study identification and classifying of the strain.The result showed that active substance of strain swu31-2 was intracellular and antagonistic effect was very steady.Based on the morphological, physiological and biochemical characteristics found swu31-2 was gram-negative aerobic rod bacteria, without spore,with flagellum and mobility.The strain could grew better on PDA solid culture medium with green pigment;It could fluidify glutin and it was catalase positive,oxidase positive and pyocyanine positive,and it couldn't grow at 4℃and could grow at 41℃.Bioinformatics analysis of 16S rDNA gene indicated that 99%identity was shared between swu31-2 strain 16S rDNA gene and many Pseudomonas aeruginos 16S rDNA.In general,according to Bergey's bacteriology identity manual and 16S rDNA gene sequence analysis,swu31-2 strain was identified as Pseudomonas aeruginos,named Pseudomonas aeruginos swu31-2.3.Optimization of fermentation condition of swu31-2 strain for producing active substanceThe cultivation condition was preliminarily optimized through fermentation tests.The appropriate carbon source was glycerol,the appropriate nitrogen source was soybean cake,the metal ion was Mg²⁺, phosphates was K₂HPO₄ and temperature was 30℃,inoculating amount was 3%,primary pH was 8.0, aeration capacity was 30%,incubation time was 36h.By orthogonal design,the appropriate component of fermentation culture medium was obtained.Which was that glycerol was 3%,soybean cake was 5%, MgSO₄·7H₂O was 0.3%,K₂HPO₄ was 0.05%.Producing bacterial amount(6.23×10¹⁰cfu/ml)by this culture medium fermented swu31-2 strain was higher bacterial amount(1.49×10¹⁰cfu/ml)by King's B culture medium,and inhibiting zone was raised from 20.7mm to 28.2mm.4.Primary purification and characterization active substanceBy method of n-butanol precipitation,centrifugalization and silica gel chromatograph,we preliminarily purified the active substance from the culture,and studied its characteristics.The result showed that active substance was stabile to acid,alkali,high temperature and protease K.At the same time,active substance could be solved in methanol,ethyl acetate,acetone,ethanol and water,and solubility was highest in methanol.Chloroform and ethyl acetate extracted active substance from water, respectively.The detection showed that active substance still stayed in aqueous layer.So active substance of swu31-2 strain was preliminarily inferred non-protein molecule with low molecular weight.5.The colonization of swu31-2 strain and its control on tobacco bacterial wiltSpontaneous mutant strain swu31-2^* resisting 300μg/ml streptomycin was obtained by increasing antibiotic concentration in the culture step by step.And the study showed that swu31-2^* still maintained the resistance to Stre and the antagonism activity to Ralatonia solanacearum in petri dish and greenhouse control effects.The mutant strain was used for the colonization analysis.The result indicated swu31-2^* could colonize in surface and inside of tobacco's roots,stems and leaves,swu31-2^* strain could detected in the inside of tobacco roots,stems,leaves one day after watering roots with the suspension.The population peaks of marked swu31-2 strain in tobacco inside roots,stems and leaves were at 8d after inoculation,with numbers 5.57×10⁷,6.79×10⁶ and 3.65×106cfu/g; after colonization population decreased,but the number of bacterium was maintained above 10⁵ cfu/g at 20d after inoculation.From the fluctuation of population observed,the population of strain swu31-2 showed the trend in tobacco inside organs "increased at the beginning and decreased afterward" during 1 to 20d after inoculation,the colonizing number of strain swu31-2 in the root was higher than in the stem, and the population in the stem was slightly higher than in the leaf after watering root treatment. Meantime,the result found that strain swu31-2 was detected in surface of tobacco's roots,stems and leaves at 20d after inoculation.It suggested that they could retain for a long time on the surface of tobacco tissues.In greenhouse experiments,the result showed the bacterial suspension and active substance of strain swu31-2 had controlling effect.And the bacterial suspension and active substance before inoculated R.solanacearum was better among all treatments in controlling the disease with the efficiencies of 60.87%and 60.32%,respectively;and after inoculated R.solanacearum,the control efficiencies of this two treatments reached to 39.50%and 20.90%,respectively.Meantime,the study discovered that the results of active substance by different control treatments were significantly difference,and it suggested that the efficiency of active substance to tobacco bacterial wilt was prevention;and the results of the bacterial suspension by different control treatments weren't significantly difference,so it suggested that the efficiency of the bacterial suspension was both prevention and therapy to tobacco bacterial wilt.