Actinomycetes In The Rhizosphere Bacteria And Halophilic Microbial Metabolites Preliminary Study,

According to the principle of rhizosphere talk, a screening model against seed germination and root growth of Arabidopsis thaliana in vitro was set up to study and discover plant growth regulator or herbicide secreted by rhizosphere actinomycetes.To testify the validation of screening model and to study the inhibitive activity of Toyocamycin, which was purified by our lab with the similar structure as 6-BA, a known plant hormone, against the root growth of Arabidopsis thaliana, a series of experiments were carried out using 6-BA as the positive control, while distilled water as the negative control. The results showed that the screening model was successed and 6-BA had stronger inhibitive activities on the root growth of Arabidopsis thaliana than Toyokamycin had. The concentration of Toyocamycin required for 50% of root growth inhibition in Arabidopsis thaliana is 0.200 mg/L compared with 0.096 mg/L of 6-BA.Based on research above, the fermentation broths of 628 rhizosphere actinomycetes collected from rhizospheric soil of Chinese medicinal plants in Yunnan Province as screening samples were evaluated. The results showed the fermentation broth of 25 strains were positive. Further more, the ethyl acetate (EA) extracts of two strains coded as I06A-02110 and I06A-02381, separativly, showed the highest inhibitive activities by optimized assay model.The research on the isolation and purification of the EA extract of strain I06A-02381 guided by assay model showed the most active part was the minor component, but the weak active part was the major component coded as compound 2381.Based on silica gel column chromatography, Thin-layer chromatography (TLC), Semi-Prepartive High performance Liquid chromatography (HPLC), compound 2381 was purified. HPLC analysis showed the purity of compound 2381 was 100% under UV detector at 254 nm. On the basis of spectral analysis including UV, NMR and HR-MS, and the comparison with literature, compound 2381 was determined as a formylation of Nanaomycin A, a known antibiotic.Using 6-BA as the positive control, meanwhile, distilled water and DMSO as the negative controls, the concentration of compound 2381 required for 50% of root growth inhibition is more than 1 mg/L , the theoretical value of which is 7.285 mg/L, while 6-BA is 0.139 mg/L.The research work established a good foundation for further studies on metabolites of rhizosphere actinomycetes with root growth inhibition of Arabidopsis thaliana. Based on the hypothesis: "New species may produce new compounds", a moderately halophilic actinomycete I06A-01708 collected from the soil of Aiding Lake Basin of Xinjiang Province was studied to find new compouds from metabolites, since the evidence of systematic taxonomy showed that I06A-01708 was a novel specie of genus Actinopolyspora.The metabolites of I06A-01708 can inhibit the growth of Staphylococcus aureus 209P and Escherichia coli, especially Bacillus subtilis. On the basis of inhibitive activity against Bacillus subtilis, time course of the strain I06A-01708 was determined.Bioactive-guided fractionation by different methods such as silica gel column chromatography, Sephadex LH-20 column chromatography, Thin-layer chromatography(TLC), High performance Liquid chromatography(HPLC) were carried out to follow and separate active components produced by the strain, after extracted by ethyl acetate(EA).One active component (MW=1110) against Bacillus subtilis was purified and the other highly active component against Bacillus subtilis was proved to be compounds with no UV absorption, further studies on the EA extract was still on the way.