Improvement Of Biocontrol Actinomycetes By Protoplast Fusion

The protoplast fusion between hyperparasitic Streptomyces F46 and actinomycetes SC1 and SC11 was studied for getting the fusants recombined excellent characteristics of both parents.The key factors affecting the protoplasts formation and fusant regeneration were determined between hyperparasitic Streptomyces F46 and actinomycetes SC1 and/or SC11. The results shown that the optimized concentration of Gly and Sucrose for strain F46, SC1 and SC11 were respectively 0.5% and 30%. The high rate of protoplasts formation and fusants regeneration could been obtained for strain F46 lysing 75min with 10 mg/mL snailase and 10 mg/mL lysozyme (1:1) in 35℃water bath, and for strains SC1 and SC11 lysing 60 min and 100min with 10 mg/mL lysozyme and 15 mg/mL lysozyme respectively in 35℃water bath. The period of thermal inactivation in 55℃was 45min for strain F46, and was 15min for strain SC1 and SC11. The optimized inactivation period with UV light was respectively 16 min, 2 min and 3min for strain F46, SC1 and SC11.Two parents and three parents were used to fusion, following all conditions optimized, and 35 fusants were selected according to colony characteristics, and 7 fusants were selected in succession culture of six generations. Three fusants were selected basied on antibiotic activity in vitro and growth speed of mycelia. The fusants F1-1 derive from the strain F46 inactivated by UV and the strain SC1 inactivated by heating. The fusant F11-2 was derived from the strain F46 inactivated by heating and the strain SC11 inactivated by UV-treatment. The fusant A-1 was derived from the strain F46 inactivated by heating, the strain SC1 inactivated by UV-treatment and the strain SC11 inactivated by UV-treatment.The fusants were identified according to the cultural properties on different media, biochemical and physiological characteristics and spore production of the fusants. A single band about 1500bp were observed in all strains by amplifying 16S rDNAs with universal primers of bacteria, and were digested with HaeⅢ, EcoRⅠand EcoRⅤrespectively. The two new bands were observed in fusant F1-1 different from the parents; the pattern of the fusant F11-2 were similar to SC11'pattern; the fusant A-1 had the same bands as F46 and SC11, but not had the same bands as the SC1'bands, and the fusant A-1'pattern was similar to parents SC11'pattern.