Screening,Identification And Application Of Brevibacillus Laterosporus Y-3 Strain

Bacterial wilt,is a soil-borne bacterial vascular diseases.It is a soil-borne vascular disease,which is more serious on tomato,pepper and tobacco plants.For prevention and control of the disease mainly adopts the breeding of resistant varieties,as well as the control,biological control bacterial wilt mainly concentrated in the pathogenic bacteria strains,bio-control bacteria.In recent years,research and application of transgenic plants become the focus of the prevention and control of bacterial wilt and hot spots,and has made great progress.Said although the history of bacillus already has more than 100 years,and with the molecular biology research more and more application in the field of bacillus,but that doesn't mean you can ignore looking for distribution in the nature of natural strains.In this paper,soil samples collected from the qinling primeval forest were used as materials to screen out a strain of bacillus that has a strong antagonistic effect against bacterial wilt,and the main results were as follows:1. Through the study of the pretreatment of soil samples,after dilution coating,isolation and save screening to the bacteria under test,then after initial screening,complex screen got has the antagonistic effect on R.solanacearum strain Y-3,the sterile fermentation filtrate of R.solanacearum bacteriostatic diameter of 23.92 mm,after comprehensive morphological identification,determination of physiological and biochemical identification and 16 s r DNA gene sequence analysis,the results show preliminarily identified as brevibacillus laterosporus.2. The broad-spectrum bacteriostatic plate test was used to determine that Y-3 strain had inhibitory effect on a variety of pathogenic bacteria in different degrees and had a broad-spectrum bacteriostatic effect.After that,the enzyme-producing activity of antagonistic strain Y-3 was studied,and it was concluded that the strain Y-3 had the ability to produce cellulase and protease.Through the preliminary study on the stability of antibacterial substance of strain Y-3 fermentation broth,it was found that the antibacterial substance was not sensitive to ultraviolet irradiation and temperature,and still had antibacterial activity after high temperature treatment and ultraviolet irradiation.In order to clarify the action mode of strain Y-3 on bacterial wilt,the morphological changes of bacterial wilt were analyzed by using transmission electron microscopy.Bacterial wilt treated with sterile filtrate of strain Y-3 showed obvious morphological changes,cell depression deformation,bacterial body fracture,and content exosmosis.In this experiment,the leaves treated with Y-3 fermentation superfluid showed pigment deposition after DAB and NBT staining,indicating that the accumulation of reactive oxygen species was generated after the treatment of Y-3strain,which may induce the plant to obtain systemic disease resistance.3. The optimal medium group for fermentation of strain Y-3 by single factor test was divided into:optimal carbon source:glucose;Optimal nitrogen source:peptone;Optimal inorganic salt:Mg SO₄;The optimal fermentation conditions are:optimal p H:7.0;Optimal inoculation dose:1.0%;Optimum temperature:30?;Optimal loading volume:50 m L.After that,the optimal medium formula of Y-3 strain was obtained by the PB test,the steepest climbing test and the response surface analysis method:glucose 13.35 g/L,peptone 20 g/L,Mg SO₄0.7743 g/L;the culture conditions were:The initial p H was 7.30,the loading volume was 50 m L,and the inoculation volume was 1.0%;the temperature of the shaker was 30?,the rotation speed of the shaker was 180 r/min,and the cultivation time was 36 h.4. After the bacterial suspension and fermentation supernatant of strain Y-3 were used to treat pepper seeds,the seed germination rate was significantly improved,indicating that strain Y-3 had a promoting effect on seed germination.After that,the results of the potted control effect test showed that the antagonistic effect was not much different from that of the agent,which could significantly reduce the incidence of bacterial wilt on the plants,and the relative control effect reached 69.35%.