The Study On The Endophytic Composition And Its Function In Salvia Miltiorrhiza Bge. From Genuine Producing Areas

Salviae Miltiorrhizae Radix et Rhizoma is the dried root and rhizome of Salvia miltiorrhiza Bge.which is belonging to the Labiatae.It mainly contains two categories of Tanshinones and Dan acids. The main place of origin or genuine producing areas of Salvia are Henan, Shandong and Sichuan. The quality of geoherbs not only can be leaded by genetic factors, but also by the suitable environment,which is the necessary conditions for the formation of the good quality and of traditional Chinese medicine. As an important part of the environment within the plant, endophytes co-evoluted with the host plant long-termly, forming a mutually beneficial symbiotic relationship. In this study,we use the combined techniques of pure training-non-pure training to study the community structure of endophytic fungi in Salvia from genuine producing area., and determined the tanshinone compounds content in co-culture system of the Salvia suspension cells with the endophytic fungi,and preliminary verified the endophytic fungi haved an effect on the synthesis of tanshinone metabolites.Firstly, we use the traditional isolation and culture techniques, namely the pure culture method isolated48endophytic fungi in the root of Salvia,which are from FangCheng, Henan Province, Linyi, Shandong province, Zhong Jiang, Sichuan Province. After ITS region of rDNA sequence analysis,the48endophytic fungi were identified four orders, seven families and eight genera and nine species,which are belonging to ascomycetes doors and imperfect fungi subphylum in which the Fusarium subglutinans was the dominantstrains, and27.08%of the total number of strains.Secondly,we adopted the method of DGGE combining with DNA sequencing to study the community structure of endophytic fungi in Salvia from the three genuine producing areas. We put to use the nested-PCR method based on ITS region of Fungal rDNA, analyzed the endogenous fungal community structure of the three genuine producing areas by DGGE, cutted the bands that can be visible in the DGGE profiles and extracted the DNA from the gel and then cloned and sequenced.After BLAST alignment,we got the18kinds of fungi which are identified7orders,7families and8species (among which four were unable to determine the species),in which12kinds of fungi are not included in the pure cure. The Alternaria alternata is the dominant species. The genetic diversity analysis of endophytic fungi in Salvia suggested that the diversity of endophytic fungi in Salvia closely related to the environment,and the more similar environmental conditions are,the more similar is the genetic diversity. We got total of21kinds of endophytic fungi by the methods of Pure culture combining with non-pure culture method.We co-cultured the endophytic fungi that were obtained by pure cultures with the Salvia suspension cells and in which we determined the Tanshinone compounds content by HPLC. The results showed that the endophytic fungi had an impact on the tanshinone synthesis and accumulation of the compounds.Our Studies have shown that endophytic fungi have an impact on the accumulation and synthesis of of tanshinone class of compounds. Different endophytic have a different effect on tanshinone class of compounds,and the same kind of endogenous fungal can also produce different effects on tanshinone class of compounds, we screened a strain which can promote promote the synthesis of tanshinones compounds, and our study laid the foundation for the depth research of endophytic fungi affecting the quality of Salvia.In summary, the pure culture method and non-pure culture method are complementary in the study of endophytic fungi.The community structure of the endophytic fungi in Salvia from the genuine producing areas have similarities but also have their own specificity, and the community diversity of the endophytic fungi change with the the environment. The endophytic fungi in Salvia may impact on the accumulation of its active ingredient, indicating that the impact of the endophytic fungi also one of the causes of the genuineness of Salviae Miltiorrhizae Radix et Rhizoma. We dicussed the the formation mechanism of the Salvia from the point of the micro-ecological,which can supply a new view for the genuineness of Chinese medicine research.