Study On The Relationship Between Endophytic Fungi Community Structure And Its Active Components In

Endophytes are presumably ubiquitous in plants. Fungal endophytes are broadly defined as "which for all or part of their life cycle invade tissues of living plants and cause unapparent and asymptomatic infections entirely within plant tissues, but cause no symptoms of disease". These fungal symbionts can increase host growth, enhance resistance of hosts to pathogens, insects and herbivory in general and enable host plants to counter abiotic stresses. The secondary metabolites play an important role in plant-microbe interactions, particularly in the plants’defenses against pathogenic attacks. Moreover, some fungal endophytes of medicinal plants can not only enhance the host ability to generate secondary metabolites, but also make the same or similar active components as the host plant does. Many studies have revealed the great potential aof endophytes as a major source of biologically active compounds with promising medicinal or agricultural applications. Therefore,"bio-prospecting" of these fungal endophytes of plants has become research ’hot spot’ of great interest to botany, ecology, natural medicine, chemistry and many other related disciplines.Salvia miltiorrhiza Bunge ("danshen" or "tanshen" in Chinese) is a well-known herb and its roots have been widely used in traditional Chinese medicine in the treatment of coronary heart diseases, particularly in angina pectoris and myocardial infarction. Its effective components can be divided into two groups:the lipophilic group of the diterpenoid tanshinones,,and the hydrophilic group of phenolic acids. The main effective components of S. miltiorrhiza has many pharmacological functions, such as antithrombotic, antimicrobiology, antioxidant, antitumor, and antiviral. So every year its synthesis mechanism of the effective components and as the materials with the pharmaceutical raw were iccreased research. At present, the analysis of the effective components of S. miltiorrhiza have been mostly reported, and the relationship between fungal endophytes and effective components accumulation were not reported.The aim of this study was to comprehensively investigate foliar fungal endophyte communities of5. miltiorrhiza and further explore the correlations between fungal endophyte and effective components accumulations. Five plant samples named HUB5(Luotian, Hubei、AH87(Liuan, Anhui、HEN5(Nanyang, Herman)、SD01(Linyi, Shandong) and HEN7(Sanmenxia, Hennan) respectively, were collected from four geological different provinces in China. Foliar fungal endophyte communities were determined by terminal restriction fragment length polymorphism (T-RFLP) of the ITS region. Effective components were analyzed with high performance liquid chromatography (HPLC). After statistical analysis the correlationship between fungal endophytes and effective components, the results showed as follows:1. Based on preliminary results from several restriction endonuclease digestions (Taq Ⅰ, Cfo Ⅰ, Hae Ⅲ and Msp Ⅰ) given the most complex chromatograms, these were chosen for the ITS ribosomal ribonucleic acid (rRNA) based T-RFLP profiling of the five samples. The mean T-RF peak number in T-RFLP profiles of all samples were differented. To determine the structural diversity, the Shannon-Weaver diversity indexes from the T-RFLP community fingerprints of five sample were varences (P<0.05). eg: HUB5>AH87>HEN5>SD01>HEN7. Several T-RFs could be found in all five samples using the same restriction endonuclease digestion. In foliar T-RFLP profiles of Cfol, HaeⅢ, MspⅠ and TaqⅠ in the five samples, there were45,42,38and34same length T-RFs, respectively. Principal component analysis analysis of T-RFLP profiles derived via digestion by four restriction enzymes (Cfo Ⅰ, Hae Ⅲ, Msp Ⅰ, and Taq Ⅰ) showed the fungal endophytes of HEN7and AH87were similar, SD01and HUB5were similar HEN5was varencies with others.2. The lipophilic group of the diterpenoid tanshinones and the hydrophilic group of phenolic acids were analyzed by high performance liquid chromatography (HPLC).The main lipophilic group in foliage of the diterpenoid tanshinones in five samples showed no variences, the contents of tanshinone I and tanshinone IIA were variable. The main hydrophilic group of phenolic acids showed variences in five samples, eg: rosmarinic acid, salvianolic acid B, danshensu, protocatechuic acid and protocatechuic aldehyde, but caffeic acid in HEN7, AH87and HUB5were not variable. Principal component analysis analysis of effective components of foliage showed the HEN5, HEN7and AH87were similar, SD01and HUB5were variable.The main lipophilic group in roots of the diterpenoid tanshinones in five samples showed no variences, eg:dihydrotanshinone I, carnosol, cryptotanshinone, carnosic acid and tanshinone ⅡA were variences, tanshinone I in HUB5and HEN5were not variences. The main hydrophilic group of phenolic acids showed variences in five samples, eg: salvianolic acid B, salvianolic acid A and caffeic acid, but danshensu was not variable. Principal component analysis analysis of effective components of roots showed the SD01and HEN7were similar, HEN5and HUB5were similar, AH87was varienced with others.Principal component analysis analysis of effective components of foliage and roots showed the AH87and HEN5were similar, HUB5and SD01were similar, HEN7was varienced with others.3. In order to explore the possible relationships between these same T-RFs (refer to those appear in five samples that restriction with one endonucleases) and the24effective compounds accumulation, correlations between foliage and root effective compounds contents and those same T-RFs areas were analyzed. The results showed that in profiles of Cfo Ⅰ, Hae Ⅲ, Msp I and Taq I digestion,7,8,5and3T-RFs, respectively, had significant (P<0.05) or very significant correlations (P<0.01) with some foliage or root effective compounds contents.