Isolation And Antibacterial Activity Of The Rhizosphere Soil Bacteria And Actinomycetes From Dendrobium Nobile Lindl

Plant rhizosphere soil exist abundant microorganism. Plant can arise secretion from the roots to offered the material energy and nutrition for the rhizosphere microorganisms and affected the rhizosphere microbial community structure and change its metabolic activity. With the new technological development, research and development of microbial metabolism must be a important way to looking for new antibiotics and bioactive compounds.Dendrobium nobile Lindl belongs to Dendrobium species, Orchidaceae. It contains alkaloids, phenols, polysaccharide and other chemicals which have antitumor effects,immunity improvement for cardiovascular and therapeutic effect for digestive and eye diseases. Using different medium, the rhizosphere soil bacteria and actinomycetes which have antibiotic activity were separated, screened out and identified. Moreover,actinomycetes G29 which has broad-spectrum antibacterial activity, was further studied for its optimal fermentation condition and antimicrobial active substance. The detailed rustles were as follows:Using dilution spread plate method, 604 strains of bacteria and 164 strains of actinomycetes were obtained from rhizosphere soil of Dendrobium nobile Lindl. Based on 16 S r RNA gene sequence analysis, rhizosphere bacteria were divided into three groups: Firmicutes, Proteobacteria and Bacteroidetes, including 16 genera, and Bacillus was dominant bacterial community. Rhizosphere soil actinomycetes were divided into Actinobacteria, including Streptomyces and Cellulosimicrobium.To determine the inhibiting activity of fermented product, using the double plate punching method, 32 strains of bacteria and 19 strains of actinomyces were screened and had the inhibiting activity. The bacteria have antibacterial activity mainly belonged to Bacillus. Pseudomonas, Aeromonas, Psychrobacillus and Leclercia., and all the actinomyces which have antibacterial activity were Streptomyces. spp,.The actinomyces G29 which have broad-spectrum antibacterial activity, which can inhibit Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Escherichia carotorora,Candida albicans,Pythium aphanidermatum and Phytophthora infestans. Based on morphology feature, cultural feature, physiological, biochemical features and 16 S rRNA gene sequence analysis, the strain G29 were preliminarily identified as Streptomycescostaricanus.The results indicated that strain G29 is almost homologous with Streptomyces sp. After the fermentation of basal fermentation medium of strain G29 was screened, then, through single factor experiment, Plackett-Burman experiment design method, the steepest climbing experiment design and the response surface method, the optimal fermentation medium and conditions were then detmined as: soluble starch 4.00g/L, malt extract powder 11.27g/L, yeast extract powder 5.83 g/L, CaCO3 3.09 g/L,distilled water 1000 mL, initial pH value 6.5, 5% inoculation quantity under 28°C and120 h, 160 r/min, 100 mL liquid medium in 300 mL fiake,.The activity compositionof the G29 fermentation product were extracted using petroleum ether, ethyl acetate and butanol in turn, and the result showed that the ethyl acetate extraction product had a higher bacterostatic activity. Using thin layer chromatography, column chromatography on silica gel and HPLC methods, the antimicrobial active compounds were further separated and purified, and 3 compounds which have antimicrobial activity were got finally. Among of them, the purity of compound G-1 could reach 99.17%, and its molecular weight was 997 by HPLC-GC mothod.