Study On Endophytic Fungi Of Brucea Javanica And Its Active Strain

Abstract:Recently, it is becoming increasingly difficult to obtain active compounds from traditional sources, such as plants, due to gradual exhaustion of medicinal plant resource as a result of adjustment of the industry structure of agriculture and forestry as well as more frequent reclamation for economic fruit trees growing. On the other hand, medicinal plant endophytes can yield bioactive substances which are the same as or similar to those produced by the host, and such endophytes grow fast and their bioactive substances can be produced easily at industrial scale; therefore, it has been increasingly paid attention to with respect to obtaining active chemical compounds from medicinal plant endophytes. In this study, a systematic study was conducted of endophytes in wild Brucea javanica collected around Laohuling Reservoir in Nanning City of Guangxi, mainly related to the isolation, screening and identification of highly active antagonistic strains, the optimization of highly active antagonistic strain fermentation process, the antimicrobial activity of extracellular volatile substances, and the isolation and identification of intracellular antibacterial ingredients. The main findings are as follows:(1) Isolation of endophytes and screening of active strains from Brucea javanicaA total of83endophyte strains were isolated from the root, stem, leaf and fruit of Brucea javanica, including34ones from the stem,32ones from the leaf,18ones from the root, and2ones from the fruit.73strains of endophytic fungi were identified to belong in12genera of6families of5orders, among which14strains showed antagonist activity against at least one kind of plant pathogenic fungi, and the metabolites of9strains exhibited inhibitory action on more than one kind of bacteria. Phomopsis strain YJ-17had relatively strong bacteriostatic activity against several kinds of indicator bacteria.(2) Identification of YJ-17strain.28S rRNA sequence of YJ-17strain was analyzed with BLAST. By homology search, we found that YJ-17strain was most close to other Phomopsis fungi in NCBI database, the similarity being98%or greater. In the phylogenetic tree, YJ-17strain shared the same branch with many Phomopsis fungi strains. YJ-17strain was determined to belong in Phomopsis sp. based on the conventional approaches to observing mycelium, colony, conidiomata and conidiospore morphology, related literature description and28S rRNA sequence analysis. At present, ITS sequence data of Phomopsis in Simaroubaceae bruceae plants are unavailable, neither relevant literature describing Phomopsis of Simaroubaceae bruceae plants are found, so we gave YJ-17strain a tentative name as Phomopsis bruceae Z.N.Liang&K.P.Lai, sp. nov.(3) Optimization of fermentation process of Endophytic fungus YJ-17strain in BruceajavanicaUsing Box-Behnken response surface test, the quadratic regression model of YJ-17strain mycelia’s dry weight vs. various nutritional factors such as corn flour was fitted and optimal level of nutritional factors was calculated, including35.5g·l\L-1for corn flour,16.8g·L-1for glucose,1.61g·L-1for KNO3, and1.58g·L-1for inorganic ions. The maximum predicted value for the response values calculated from the regression model was Y1=1.58g. YJ-17strain mycelia increased by48.45%in optimized culture medium compared to that in original fermentation medium, and the dry weight of mycelia reached1.54g for optimized culture medium and increased by16.29%relative to that for original fermentation medium.(4) Bacteriostatic effect and chemical ingredient identification of volatile substances from Endophytic fungi YJ-17strain in Brucea javanica We explored inhibitory action of YJ-17strain volatile substances against14kinds of plant pathogenic bacteria, finding that these substances possessed high bactericidal activities (above70%) against citrus penicillium bacteria, banana Curvularia lunata, rice sheath blight. We learned that the antibacterial activity of YJ-17strain volatile substances was affected by activated carbon. Some parts of volatile substances of YJ-17strain were absorbed by activated carbon, thus weakening the bacteriostatic activity of volatile substances; but the remaining still showed anti-bacterial function and inhibited hyphal growth of14kinds of pathogenic bacteria and the formation of pigment. The volatile substances produced by YJ-17strain were identified, among which1-Hydroxy-3-cyclohexene had the largest peak area. Among16main volatile substances, β-phellandrene, terpineol and thujone had insecticidal, antimicrobial and anti-inflammatory effects respectively.(5) Separation, purification and identification of bacteriostatic components of Endophytic fungi YJ-17strain in Brucea javanica.We got seven monomeric compounds from YJ-17strain intracellular components using extraction with organic solvents, silica-gel column chromatography and recrystallization. Structural analyses of these6compounds were performed with1H-NMR,13C-NMR, DEPT and EI-MS as well as through document retrieval. They were deduced that (I)3β-hydroxy-5a,8α-ergosterol peroxide-6,22-diene,(Ⅱ)5a,8α-epidioxy-24(S)-methylergosta-trien-3β-ol,(Ⅲ) Ergosta-7,22,dien-3p-ol,(Ⅳ) Ergosterol.