Microbial Resources From Wild Salvia Miltiorrhiza And Their Inducing Effect On Hairy Roots

Salvia miltiorrhiza Bunge(Lamiaceae),generally called Danshen in China,is an important traditional Chinese medicine,which has been widely used in China for the treatment of cardiovascular and cerebrovascular diseases.Enhanceing contents of secondary metabolite is one of the goals in the cultivated S.miltiorrhiza,and microorganisms play an important role in the regulation of secondary metabolite synthesis of plants.In this study,microbes were obtained from the wild S.miltiorrhiza,and their effects on the second metabolites were investigated.Firstly,the microbial resources of wild Danshen in different areas were isolated and the effects of microorganisms isolated from Danshen on the second metabolites were studied.A total of 1022 isolates were obtained from the wild plants of S.miltiorrhiza in 7 regions,and 240 isolates were identified and classified as 63 species of bacteria and 7 fungi.The identified bacteria belong to Pseudomonas,Bacillus,Acinetobacter,arthrobacter,Rhizobium and Sphingobium,while fungi belong to Aspergillus,Purpureocillium,Aspergillus,Mucor,Hypocreaand Trichoderma.About 60 strains were tested the effect on the secondary metabolites in Danshen.Among them,the most promotion of salvianolic acid biosynthesis were performed by bacteria,and the most fungi significantly stimulated tanshinones biosynthesis.Bacteria,including Pantoea dispersa 14DSR23,Pseudomonas chlororaphis subsp.aurantia CaHYR1,Pseudomonas chlororaphis subsp.aurantiaCaHYR26,Pseudomonas chlororaphis subsp.piscium SCR22 and Pseudomonas chlororaphis subsp.aureofaciens LNHR13 exhibited a significant increase of rosmarinic acid(RA,1.21-2.28 times)and of salvianolic acid B(SAB,1.38-2.50 times).Fungi,including Aspergillus japonicus L74,Purpureocillium lilacinum R75,Aspergillus insuetus R77,Mucor irregularis R78,Hypocrea nigricans S79,Aspergillus japonicus S80,Trichoderma atroviride S81,and bacteria Pseudomonas sp.DS3G2,HYR1,Pseudomonas vancouverensis DS6 significantly promoted the contents of total tanshinones,with 1.62-10.14 folds.The best bacteria to stimulate the content of total phenolic acid was Pseudomonas chlororaphis subsp.aureofaciens LNHR13,and the best fungus to stimulate the content of total tanshinones was Aspergillus japonicus L74.Pseudomonas sp.DS3G3,HYR1,and Pseudomonas vancouverensis DS6 promoted the synthesis of both phenolic acid and tanshinones.Secondly,the major components promoting the synthesis of salvianolic acid were studied with Pseudomonas chlororaphis subsp.aureofaciens LNHR13.The fermentation broth of strain LNHR13 was separated and its effect on hairy root of Salvia miltiorrhiza was studied.It was found that the water-soluble portion and the polysaccharides significantly promoted the biomass of hairy roots,and the contents of danshensu,rosmarinic acid,salvianolic acid B,and total salvianolic acids.Some phytohormones,including indole-3-acetic acid(IAA),salicylic acid(SA)and zeatin(tZOG),were identified from the water-soluble portion of Pseudomonas chlororaphis subsp.aureofaciens LNHR13.The bacteria stimulating the contents of salvianolic acids were further identified to have varied phytohormones,with IAA being the most abundant phytohormones in all strains,and salicylic acid(SA)and trans-zeatin-Oglucoside(t-ZOG)being the second abundant phytohormones.The content of phytohormones were 559.182-71543.05 ng/mL for IAA,28.86-379.21 ng/mL for SA,33.619-88.027 ng/m L for t-ZOG.The similar effects of SA were demonstrated on the growth of hairy roots and the biosynthesis of salvianolic acids.The exopolysaccharide was also demonstrated to stimulate the content of salvianolic acid,with the optimal concentration of 20 mg/L and 40 mg/L.Finally,the interaction between the microorganism and the plant was studied.The results showed that bacteria inhibited the growth of hairy root,and generally promoted the biosynthesis of tanshinones.The best concentration of inoculation is 0.01%.In addition,the increasing rate of dihydrotanshinone I and cryptotanshinone is higher than the others.In conclusion,microbes were obtained from the wild plants of Salvia miltiorrhiza.Some bacteria were found to increase the content of salvianolic acid,some fungi improved the content of tanshinone.For the first bacterium LNHR13 to increase the of salvianolic acid,exopolysaccharide and phytohormones were demonstrated to be the active components.These results provide a good basis for further research.