Identification Of Endophytic Bacteria HT-5 And Its Biological Control Effect On Alternaria Solani

Entophytic bacteria is a class of microorganisms with antibacterial activity that colonization in healthy plants, which is a potential bacteria resourse in agricultural biocontrol. It has played an irreplaceable role in replacing pesticides, improving agricultural ecosystems, keeping the diversity of microoganisms and maintaining agriculture sustainable development, it has been a hot topic in recent years. Endophytic bacteria HT5 was isolated from healthy wulnuts stem which had certain antimicrobial activity on a variety of pathogens. In this paper, the strain was indentified, the antibacterial activity, biocontrol and promoting effect, optimization of fermentation, antibacterial mechanism and stability of endophytic bacteria HT-5 metabolites were studied, aim at the futher application of it.By the methods of mycelium growth rates, antimicrobial activity of strain HT-5 was measured, the results showed that aseptic filtrate of endophytes HT-5 had certain inhibitory effect on 8 kinds of fungus, and the effect on Alternaria solani was the best, reached 72.58%. Strain HT-5 was identified as Bacillus amyloliquefaciens on the basis of the morphology and DNA sequence analysis of 16SrDNA molecular biology identification.Growth-promoting research showed that:The fermentation broth of 50 times dilution,80 times dilution had significant effect on the germination of tomato seeds, the emergence of tomato seedling and the growth of tomato seedling; 50-fold dilution can remarkable promote the growth of germ, promoting rate was 27.6%,80-fold dilution can remarkable promote the growth of radicle, promoting rate was 43.3%; 80-fold dilution had significant effect on the emergence of tomato seedling, growth rate of wet weight and dry weight were 162.4% and 130.3%; 80-fold dilution had significant effect on the growth of tomato seedling, Growth rate of stem length was 72.4%, growth rate of wet weight and dry weight were 78.4% and 56.9%.The tomato fruit in vitro was treated with different dilution time of fermentation filtrate,20-fold dilution and 50-fold dilution can remarkable inhibited the growth of bacteria, inhibition rate were 55.5% and 52.2%; Pot experiment showed that the effect of treatment againest spraying by 20-fold dilution and 50-fold dilution were better, the control effect could reach 59.2% and 52.7%.Alternaria solani as indicator pathogen, by single factor experiment and uniform experiment, the medium composition was optimized, the result showed that the optimal carbon source, nitrogen source and salt were Glucose, Yeast extract and MgSO4·7H2O respectively. The composition of best medium was 22.3 (g/L) sucrose,29.9 (g/L)yeast extract,5(g/L)MgSO47H2O. The optimal fermentation conditions were optimized by orthogonal experiment, the optimal fermentation conditions were the combination of temperature 28℃,4% inoculums, initial pH 6.5, speed 150 r.min-1, liquid volume 50 mL/250 mL, incubation time 4 d.A preliminary study showed antimicrobial agent can be determined as protein. Crude extracts of antifungal protein was found to be thermostable,121 ℃ treatment 30min still had 66.5% antibacterial activity, resistant to Ultraviolet irradiation, and sensitive to acid and alkali; Crude protein can inhibit the gemination of conidia and the growth of mycelial, make the spores germiate abnormally, cause hypha wizened and the end of hypha enlarged.