Research On Mutation Breeding Of Streptomyces Sp. LA5

The Actinomycetes LA5 strain was isolated from the capsicum plant in Hainan province, China. 16S rDNA sequence analysis indicated that Actinomycetes LA5 strain shared 98%-99.59% 16S rDNA sequence homology with Stretomyces sp., and belongs genus. The results of former research indicated that the antibiotic produced by Stretomyces sp. LA5 strain is strongly inhibitory in vitro to many plant pathogens such as Phytophthora capsici, Fusarium oxyspoum f.sp.cubense race 4, Colletotrichum musae, C. gloeosporioides and Magnaporthe grisea, etc. when grown on culture plates. The liquid filtrate of LA5 fermentation culture had 90% control effect against capsicum blight in greenhouse tests and 80% control effect in field. The antibiotic includes hydrosoluble and liposoluble active ingredients. The hydrosoluble ingredient when maintained at room temperature for 6 months or at 54℃water bath for 15 days, in a neutral or alkalescence condition, or exposed to sunlight for 8 hours can retain its original potency, thus satisfying the stability requirement of a pesticide. Thus the antibiotic produced by Stretomyces sp.LA5 has great potential of being developed into a plant disease-control pesticide. Optimum culture conditions for LA5 fermentation were pH6.8, temperature 28℃, 200r/min shaking culture 120h. But the antibiotic yield produced by Stretomyces sp. LA5 strain is still low even if under the optimized culture conditions, and can't satisfy the requirement of industrialization. So, It is necessary to carrying out mutant breeding of LA5 strain in order to screen out high-yield mutant strains. The results of study are as follows:The results of analysis on mutation effects of two complex mutant methods to LA5 showed that the best mutation effect was gained from treatment with UV80sec+ NTG80min and the positive mutation rate was 30% in UV+NTG mutation methods; and 26% positive mutate rate was gained from treatment with NTG80min+UV40sec and NTG 60min+UV120sec In NTG+UV complex methods. So, UV80sec + NTG80min, NTG80min + UV40sec and NTG 60min + UV 120sec were selected as the mutation methods.Five antibiotic higher yield mutant strain D-16,F-20,g-14,3h-41,G-14 were screened by first mutant. The inhibitive effects against Phytophthora capsici are increased 7.02%, 10.52%, 12.28%, 16.67%, 17.54% than Streptomyces sp. LA5.D-16,F-20,g-14,3h-41,G-14 were selected as the starting strain for the second mutation by UV80sec+NTG80min. The result of fermentation screening showed that G14-33,F20-43,g14-7,D16-14,3h41-13 are best mutant strains. Their inhibitive effects against Phytophthora capsici are increased 3.51%,4.68%,5.07%,6.24%,9.35% than 3h-41 strain.G14-33,F20-43,g14-7,D16-14,3h41-13 were selected as the starting strain for the third mutation by NTG80min+UV40sec. The result of fermentation screening showed that F20-43-14, g14-7-28, 3h41-13-15, D16-14-36, 3h41-13-32 are best mutant strains. Their inhibitive effects against Phytophthora capsici are increased 1.08%, 1.75%, 2.34%, 4.68%, 7.6% than F20-43 strain.F20-43-14, g14-7-28, 3h41-13-15, D16-14-36, 3h41-13-32 were selected as the starting strain for the fourth mutation by NTG60min+UV120sec. Finally, a positive mutant 3h32-32 was obtain with a 10.18% higher ability of producing antibiotic than D16-14-36 strain, with a 34.5% higher ability of producing antibiotic than Streptomyces sp.LA5.In my study, some biological character of LA5 strain were researched. The results of study are as follows: In different Duojunling concentration at different cultural stage, LA5 growth, producing spores and antibiotic have no change.The amount of spores produced in Streptomyces sp. LA5 fermentating liquid during storage three months have no change.Filling little white mice with LA5 fermentation liquid, do it a week continuously, It turns that these white mice's action and acting is normal. Feeding little white mice with LA5 fermentation liquid, do it three months continuously, It turns that these white mice's action and acting is normal. The new born white mice were normal in activity, and had no difference in appearance, their fur luster.