Isolation And Identification Of Antagonistic Bacteria From Tobacco Rhizosphere Soil Of Luoyang

Tobacco is an important economic crop,but tobacco diseases caused by soilborne pathogenic fungi are increasingly heavy.Because of the lack of multi-resistant varities,noneffective agricultural measures,and chemical control easily lead to environmental pollution and fungicide residues in tobacco leaves.Biological control get more and more attention in recent years for which is friendly to ecological environment,and can provide a sustained effect for disease control.Bacteria are one of the most abundant microorganisms in the soil.The study of antagonistic bacteria has been one of the hotspots in the field of biological control.A total of 30 soil samples were collected from different growth stages and different tobacco growth period from four counties(Yiyang,Luoning,Songxian and Ruyang)of Luoyang,dilution plating method was adopted to isolation and count the number of bacterial colonies,plate confrontation test and oxford cup method were adopted to measure inhibitory effect of the pure colony against the pathogenic fungi.The bacterial morphological characteristics,physiological and biochemical characteristics and 16 S r DNA sequence analysis were used to identify antagonistic strains.The research achievements as follows.(1)The average number of bacterial colonies in tobacco rhizosphere soil samples in Luoyang area was determined by dilution plating method,the county of Yiyang is 5.273×107 cfu/g,Songxian is 7.304×107 cfu/g,Luoning county is 7.312×107 cfu/g,Ruyang county is 5.218×107 cfu/g.(2)A total of 180 bacterial colonies were obtained after purification.Screened out 94 strains by the method of plate confrontation test,which had antagonistic action on Phytophthora nicotianae,Pythium aphanidermatum and Fusarium oxysporum.The 10 strains had antagonistic against all this three tobacco pathogens,the 79 strains had antagonistic against Phytophthora nicotianae,the 16 strains had antagonistic against Pythium aphanidermatum,the 69 strains had antagonistic against Fusarium oxysporum.(3)The fermentation broth of 17 strains had certain antagonistic to Phytophthora nicotianae and Fusarium oxysporum by using the oxford cup method,which included G111,G12,G122,G51,G52,J71,J72,L31,O42,S331,S3312,S3341,S61,YJ71,YJ72,YJ73,Y1B2.(4)The 17 pure strains and MS621,MS622,TS1112,TS6114,TS681,TS682,TS683 of laboratory preservation were all gram positive bacteria and can production spores with the method of ccrystal violet staining.(5)The 24 strains of bacteria were tested for their anaerobic and salt tolerance,as well as the use of different carbon sources,citrate and malonate,based on the common bacterial identification manual.The strains of G111,G12,G122,G51,G52,J71,J72,L31,MS621,MS622,O42,S331,S3312,S3341,S61,TS1112,TS6114,YJ71,YJ72,YJ73,Y1B2 were identified as Bacillus spp.;TS681,TS682,TS683 were identified as Paenibacillus spp..(6)The strains of G51,G52,J71,J72,L31,O42,S331,S3312,S3341,S61,TS1112,TS6114,YJ71,YJ72,YJ73,Y1B2 were identified as Bacillus subtilis;strains G111,G12,G122,MS621,MS622 were identified as Bacillus amyloliquefaciens;strains TS681,TS682,TS683 were identified as Paenibacillus polymyxa.By using the 16 S rDNA sequence analysis,combining with the morphological characteristics and physiological and biochemical characteristics.The species of antagonistic bacteria were identified based on the morphological,physiological and biochemical characteristics and molecular biology techniques,and clearly knew their antagonistic ability to tobacco pathogens fungus.And the Paenibacillus polymyxa were first detected in tobacco rhizosphere soil of Luoyang which can provide an important reference for the further study and use of bacteria to prevent and control the soilborne fungal diseases of tobacco.