Wang, Wen-Xuan2016-05-042016-05-042016http://hdl.handle.net/2003/3495310.17877/DE290R-17001Endophytic fungi and bacteria are microorganisms harbored in the plant tissues without causing any disease. They are proven to be a rich source of new anticancer and antimicrobial compounds as well as clinically valuable agents identified from plant materials. However, recent research encounters enormous difficulties, which reveals that endophytic systems are complex ecological communities maintained by inter-species and inter-kingdom interactions, including chemical crosstalk. Consequently, investigating the chemical basis of these interactions is essential to interpret the interaction among endophytes and host plant, and exploit the true potential of endophytes. Based on metabolite screening of endophytes from several Chinese traditional medicinal plants with antibacterial related uses, endophytic fungi and bacteria isolated from two plants Narcissus tazetta and Buxus sinica were selected for the investigation in this thesis. From an endophytic fungus, Fusarium solani N06, isolated from a bulb of N. tazetta, nine new hexacyclopeptides (1−9) were discovered as crosstalk molecules between it and another endophytic bacterium, Achromobacter xylosoxidans N12B, from the same tissue. In the matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) experiments, the secretion of these compounds from this F. solani N06 and the accumulation towards A. xylosoxidans N12B was visualized on the border between the fungus and bacterium. From the optimized culture of F. solani N06, sufficient extracts of these hexacyclopeptides were analyzed by liquid chromatography-multiple stage mass spectrometry (LC-MSn) and their sequence was identified to be cyclo((Hyp or Dhp)-Xle-Xle-(Ala or Val)-Thr-Xle) (Dhp: dehydroproline) based on the characteristic a, b, or y ions in MSn spectra. The phenomenon that coexisting endophytes utilize new signal molecules suggests that they have developed communication strategies to survive and function in their distinct ecological niches. Four new compounds, colletotrichones A−D (10−13) and one known compound chermesinone B (14a) were isolated from the fermented rice medium of an endophytic fungus Colletotrichum sp. BS4, which was isolated from the leaves of B sinica. With comprehensive spectroscopic methods including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR as well as single crystal X-ray diffraction and quantum chemistry calculation, their structures were identified to be azaphilones sharing a 3,6a-dimethyl-9-(2-methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione core structure. In the antibacterial bioassay against two environmental strains of Escherichia coli and Bacillus subtilis, as well as two human pathogenic clinical strains Staphylococcus aureus and Pseudomonas aeruginosa, colletotrichone A (10) exhibited extraordinary activity against E. coli and B. subtilis, with a minimum inhibitory concentration lower than positive controls streptomycin and gentamicin. In the cytotoxic assay, interestingly, compounds 10−12, and 14a showed slight cytotoxicity at high concentration (100 µM). Furthermore, the spatial distribution and localization of the compounds produced by the endophyte were visualized by MALDI-imaging-HRMS, which revealed their plausible ecological functions. In the investigation of endophytic fungus Phyllosticta capitalensis harbored in the leaves of B. sinica, two new lactam-fused 4-pyrones (15, 16) were discovered in static PDB fermentation. Their structures were elucidated by 1D and 2D NMR, LC-MSn, DFT 13C NMR calculation and chemical reaction. By 16S rRNA analyses, an endosymbiotic bacterium Herbaspirillum sp. was discovered in the hyphae of P. capitalensis, which was not cultivable in ordinary culturing condition. The PKS/NRPS gene cluster analyses showed these two compounds were biosynthesized by fungal PKS and bacterial NRPS. This cross-species cooperation of secondary metabolites production unveiled another level of interaction between the endophytic bacterium and fungus.enChemical crosstalkEndophytesAntibactrial activityCyclic peptidesAzaphilonesLactam-fused 4-pyrone540570doctoral thesis