Isolation And Characterization Of Viola Philippica Producing Luteolin Endophytic Fungus

Endophytic fungi could produce chemical components which was similar or identical to the ones from its host. In this study, endophytic fungi producing luteolin was isolated, classified and purified from Viola philippica. Meanwhile, fungi strain with high antibacterial activity was screened by the antibacterial test to get the antibacterial component within the strain, providing a new source of luteolin. The results were as follows:1. The 51 endophytic fungus strains had been harvested from the roots(n=19), stems(n=15), leaves(n=11) and seeds(n=6) of the plants. Of all the endophytic fungi, G9 showed fluorescent spots with the standard products in similar position(Rf =0.673) using layer chromatography(TLC). Using High Performance Liquid Chromatography(HPLC), G9 had the same peak time with the standard products, with a content of 66.09 μg/g(ingredients/ dry weight). For further study, monomer was isolated from the extracting solution of G9 by TLC. The chemical structure of the monomer was identical to the standard products with Nuclear magnetic resonance spectroscopy(NMR), suggesting luteolin can produced by G9.2. The colony of G9 was grey, with a diameter of 25-30 mm after 10 d. The new hyphae are white, and turned to gray with a neat edge when it was mature. The back of petri dishes was black(Fig. 3-9A). The seprated hyphae with branches of about 2 to 5 microns in diameter could be observed under a light microscope. The conidium was a single cell, shaping as a spindle. According to morphology, G9 was initially classified to be Pyrenochaeta sp.. PCR was conducted based on the ITS sequences for G9, forming a fragment of 600 bp length. Sequences were searched in NCBI with BLAST. G9 strain and Pyrenochaeta sp.(HM208713) were located in the same branch, with a homology of 99%. G9 was identified to be Pyrenochaeta sp. based on morphology and molecular biology.3. Antibacterial test was conducted with the extracting solution of hyphae from 51 endophytic fungus strains, suggesting that G21 had the highest antibacterial activity. Seven components of G21, named as G21-1 to G21-7 were got from the extracting solution of hyphae of G21 to carry out the antibacterial test, indicating G21-6 had the highest antibacterial activity.