Improvement Of Endophytic Antinomycetes Antimicrobial Activity By Protoplast Fusion

As a new microbial resource, the plant endophytic actinomycetes have wide application prospects. The plant endophytic actinomycetes have a high potential not only as bio-control agents to control plant diseases, but also to find novel pesticides or new medicines produced as secondary metabolites. Improving the biocontrol character of endophytic actinomycetes, the protoplast fusion between endophytic actinomycetes SF4, SG2 and actinomycetes SC1 was studied for getting the recombinants recombined excellent characteristics of both parents.The conditions of protoplast formation and regeneration were optimized by the concentration of Sucrose, concentration of Gly, the growth logarithm period of the parents,the kind and the concentration of the enzyme, the time and the temperature of lysing, the time of thermal and UV light inactivation. Concentration of Sucrose: SF4-30﹪, SG2-20﹪; Concentration of Gly: 0.5﹪; The growth logarithm period: SF4-48-81h, SG2-33-72h; The kind of enzyme: lysozyme; Concentration of lysozyme: SF4-15 mg·mL-1, SG2-10 mg·mL-1; Temperature of lysing: 35℃; Time of lysing: SF4-90min, SG2-60min; Temperature of thermal inactivation: 55℃; Time of thermal inactivation: SF4 -15min, SG2-10min; Time of UV light inactivation: SF4 -4min, SG2-2min;Two parents and three parents were used to fusion, following all conditions optimized, and 25 recombinants were selected according to colony characteristics, and 6 recombinants were selected basied on succession culture of seven generations,antibiotic activity in vitro and growth speed of mycelia.According to the inhibiting mechanism to the target pathogens, the morphological characteristics, the culture properties on different media, biochemical and physiological characteristics and growth speed of mycelium, the recombinants were identified if they were true. The result showed that there were the different as well as the same between the recombinants and strains SF4, SG2 and SC1. Conclusion was gotten that the recombinants inherited characteristics of the parents and produced some new properties. A single band about 1500bp were gained in all strains by amplifying 16S rDNA with universal primers of bacteria, and were digested with endoenzyme respectively, and the different patterns were obtained for each enzyme. In the patterns, some new stripes which were different from the parents'were found.In this study, achieving protoplast fusion between endophytic antinomycetes and biocontrol antinomyectes by the technology of protoplast fusion, which providing a new idea for exploring and utilizing the resources of endophytic actinomycetes, and broadening application area of the technology of protoplast fusion at the same time.