Isolation,identification And Antibacterial Activity Of Antagonistic Bacillus Against Tobacco Brown Spot

As an important economic leaf crop in China, tobacco is quite a remarkable plant inagricultural production. Thus, the pests controlling works in tobacco growth are regarded as astudy emphasis by agronomic researchers. Being one of the main diseases of tobacco, brownspot has brought great loss to tobacco’s production. Nowadays, there are plenty of controllingmethods for this disease, in which the dominating one is chemical pesticide treatment.However, with more concern on pesticides residue and environment pollution, the use ofchemical pesticides should be reduced, and the principle “Prevention-oriented, integratedcontrol” shall be put into application.Bacillus spp., which is a natural bacterial family, has many good characters, such as easynutrition pattern, high rate of propagation, high resistance and rich metabolites. Besides, thisbacterial group is found widespread around the world, aerobian or amphimicrobian, and beingable to yield resistant gemmae. With these traits mentioned above, it thus has a remarkablebiocontrol potential, and could be a great sample for the development of biocontrol inocula. Inthis research, microbe groups in the rhizosphere soil and phyllosphere of tobacco weredirectly screened and isolated, for the purpose of obtaining bacillus with high antagonisticactivity against tobacco brown spot.In this study, tobacco leaves and rhizosphere soil were collected from the productionareas in Shaanxi, Yunnan, Hubei and Henan province, in which408antagonistic bacillusstrains were screened and isolated. Then10bacillus strains with a better antagonistic activitywere found in a further screening by dual cultures. While in these samples, the strain M-07had the best antagonistic effect, which could greatly inhibit the mycelia growth and sporegermination of fungus of brown spot, the width of antifungal stripes reaching14.2mm. Aftersingle spore isolation, culturing and testing by10continuous generations, we can see that alldifferent generations of M-07displayed relatively stability in antagonistic activity.In addition, through the morphological observation to the thallus and colonies, studies on physiological&biochemical characteristics of M-07, as well as16s rDNA sequences analysis,this strain was identified as bacillus amyloliquefaciens. The M-07strain cultured on the LBsolid mediums showed a series of traits, such as unsmoothed colony surfaces,irregular-shaped margins, light yellow color and a certain degree of viscosity. Besides, theelectron microscope observation indicated that its thallus had bacilliform shape, flagellum,ovoid gemmae, and was pram-positive. As for the physiological&biochemical characters,M-07was able to hydrolyze starch, gelatin, caseinate, tyrosine, to produce gas, citrate and outof glucose, and to yield positive results in V-P test, etc.In the microscope observation, we found that the strain M-07could cause amalformation growth in mycelia of tobacco brown spot fungi, in which protoplasts unevenlydistributed, and parts of mycelia’s protoplasm leaked. Moreover, in vitro growth rate methodwas used and the results showed that mycelia growth of this tobacco disease was inhibited, itsEC50being12.82mg/L; likewise, field test outcomes indicated that when the crude extractconcentration of M-07reached80mg/L, the control effect on brown spot was as high as86.0%.Summarily, this paper concluded several results of preliminary studies of the strainM-07’s physiological&biochemical characteristics,16S rDNA sequencing and antagonisticmechanism, to provide a valid theoretical basis for the controlling of tobacco brown spot.