Chapter 9. Summary & conclusion

Transcription

1 Chapter 9 Summary & conclusion

2 Endophytes are the microbes that inhabit the internal tissues of plant without causing substantial harm to the host plant and forms association usually mutualistic. These are relatively unstudied and potential sources of chemically novel and biologically important natural products for exploitation in medicine, agriculture and industry Plants growing in unique environmental settings with ethnobotanical value and longevity or endemic location are likely to be a source of rarely occurring novel microbial endophytes of commercial significance. Hanging roots of ethnobotanically important, medicinal tree Ficus bengalensis (Banyan tree) were selected for the isolation of endophytic bacteria. Seven gram positive spore forming rod shaped bacilli species exhibiting the antifungal activity were isolated from the aerial roots of Banyan tree. All bacilli exhibited broad spectrum activity against various fungal strains, which include Aspergillus niger, Alternaria brunsii (causative agent of blight in cumin), Fusarium oxysporum (causative agent of yellow disease in Ginger and wilt in Tomato), Aspergillus parasiticus, Aspergillus flavus, Cladosporium herbarum 1112, Lasiodiplodia theobromae, Candida albicans, Trichosporon 1110 and chrysosporium indicum. The presence of rod shaped bacteria in cross sections of Banyan hanging roots could be demonstrated by light microscopy as well as Transmission Electron Microscopy. The isolates designated as K1, A2, A4, A12 were identified as Bacillus subtilis whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using BioLog s Microbial Identification System. All cultures exhibited differentiable carbon source utilization pattern on GP2 (Gram positive plates, Biolog Inc., USA) plates, which consist of 95 different carbon sources, thus indicating that none of these isolates shared 100% identity to any other isolate. The extracellular metabolites from Banyan endophytes were analyzed using Intact Cell Mass spectrometry (ICMS). ICMS was performed using Matrix Assisted Laser Desorption Ionization Time of Flight Mass spectrometer (MALDI-TOF) (Ultraflex TOF/TOF, Bruker, Daltonics). These isolates were able to produce compounds in the mass range of m/z The metabolites in a mass range from m/z 1000 to 1500 belonged to cyclic lipopeptides which could be identified as surfactin, iturin and fengycin (Vater et al. 2002). On the basis of the mass spectral pattern also, endophytes could be 231 Summary & Conclusion

3 differentiated from each other. Among all endophytic isolates, Bacillus subtilis K1 exhibiting maximum antifungal activity was selected for further characterization. Antifungal methanolic extract of Bacillus subtilis K1 exhibited broad spectrum antifungal activity against plant and animal pathogens. The antifungal activity of crude extract was found to be stable over wide range of ph (2-10) and could withstand autoclaving (121 C, 15 lbs pressure) temperature for 30 min. without loss of activity. The LC-ESI-MS enabled the separation of compounds with m/z ranging from from crude antifungal extract produced by B. subtilis K1. The three groups of lipopeptides m/z 994 to1195 and , the former mass group metabolites were identified as isoforms of surfactin and iturins while the later molecular mass ions with m/z were found to be isoforms of fengycins. The protonated (m/z , , )and alkali metal ion adducts (m/z , , , 1073) of surfactin isoforms were characterized with a limitation of inability to differentiate between Leu/Ile, a major source of heterogeneity in surfactin family. The sequence deduced from the tandem mass spectrometry of protonated and alkali metal ion adducts of surfactins was ß hydroxy fatty acid-glu- Ile/Leu-Leu- Val- Asp- Leu- Leu/ Ile. These isoforms exhibited variation in the length of the ß-OH FA chain from carbon units. The extracellular crude extract of B. subtilis K1 obtained after 72 hours of incubation, was subjected to purification using HPLC. The purified fractions were then analyzed by MALDI-TOF MS for identification of metabolites based on their mass numbers. The mass spectrometric analyses of pure fractions showed presence of metabolites with two sets of peaks with m/z values from 1000 to 1109 and 1421 to The m/z values ranging from 1000 to 1109 could be attributed to the isoforms of surfactin and iturin groups of cyclic lipopeptides. The structural investigation of these cyclic lipopeptides was carried out using Tandem mass spectrometry. The purified fractions were subjected to Electro Spray Ionization MS 2 (ESI-MS n ) (Esquire 3000 plus, Bruker Daltonics) and MALDI TOF MS-MS(Bruker Daltonics TOF/TOF) for sequence determination of cyclic lipopeptides. The compounds with m/z values , , and could 232 Summary & Conclusion

4 be identified as isoforms of iturin A. The structure of iturin A was derived from series of b n and y n from Post Source Decay MALDI TOF MS spectra. The sequence of iturin A i derived was P-N-S-ßAA-Fattyacid-N-Y-N-Q-CO +. Under Collision Induced Dissociation (CID) condition, ring opening reaction in iturin A was observed at the glutamine- proline peptide bond which resulted into linear acylium ions. The variation in isoforms of iturin A was observed at the fatty acid chain portion. The fatty acid chains of iturin A isoforms with corresponding m/z values , , and were composed of 13, 14, 15 and 16 CH 2 residues, respectively. The second set of compounds with m/z values ranging from 1421 to 1542 could be identified as isoforms of fengycins. Mass spectrometric analysis of fengycin isoforms gave two types of marker ions patterns for fengycin A (966, 1080 m/z) and fengycin B (994, 1108 m/z). During our investigation of fengycin molecules using MALDI-MS/MS, we observed additional two different types of marker ions patterns in CID spectra. The new marker ions observed in CID spectra of fengycin molecules were m/z and m/z. To determine the complete sequences, saponified fengycin molecules were subjected to MALDI MS/MS analysis. The CID spectra of saponified linear fengycins showed four different series of b n and y n ions and using these series of fragment ions, four sequences of fengycins were derived. The complete sequence of fengycin homologues were summarized as: fatty acid-e-ornithine-y-t-e-x 1 -P-E-Y-X 2, where X 1 =Ala/Val and X 2 =Val/Ile. In new sequences of fengycins, variations were observed at 6 th and 10 th amino acid starting from glutamate residue in the cyclic peptide. The new fengycins were classified as fengycin A2 (Alanine 6th & Valine 10th ) and fengycin B2 (Valine 6th & Valine 10th ). Iturin A isoforms produced by B. subtilis K1 inhibited the growth of Aspergillus niger 40211, A. flavus, A. parasiticus, F. oxysporum 1072, L.theobromae ABFK1, Alternaria. brunsii(2), Trichosporon spp.1110, Chrysosporium indicum and Candida albicans while Fengycin isoforms inhibited the growth of A. flavus, F. oxysporum 1072, L. theobromae ABFK1, A. brunsii(2), Trichosporon spp. 1110, Chrysosporium indicum, Candida albicans, Helmethosporium graminum 1126, Cladosporium herbarum1112. Although, 233 Summary & Conclusion

5 iturins and fengycins both exhibited broad spectrum antifungal activity but iturins were found to be more potent in comparison to fengycins. The optimization studies for production of antifungal cyclic lipopeptides by B. subtilis K1 were undertaken for improving yield. The optimum carbon and nitrogen source for maximum antifungal activity were glucose (50 g/l) and KNO 3 (5 g/l). The culture produced maximum antifungal activity in medium with initial ph 9.0 at 30 C under shaking conditions (150 rpm) upon 90 h of incubation. Under the optimized conditions, highest antifungal activity observed was 36.4 AAU/mL which was 12.3-fold higher in comparison to that obtained using Luria broth (3.0AAU/ml) at ph 7.0 and 30 C. The culture supernatant of B. subtilis K1 was able to reduce surface tension and emulsify water immiscible substrates. The surface active property and emulsifying activity were investigated using culture supernatant of B. subtilis K1. The surface active property of the culture supernatant was highly stable and resisted extreme change in ph (2-10), temperature (100 C for 1hr) and NaCl concentration upto 15% (w/v). The emulsion formed remained stable for more than 15days without any shaking under ambient conditions. To demonstrate the application of biosurfactant produced by B. subtilis K1 in microbially enhanced oil recovery, the sand column experiment was performed. It was found that culture supernatant upon 48 h of incubation exhibited efficient emulsification enhanced recovery of oil from the sand column resulting in recovery 43.3±2.2% oil. The applications of lipopeptides viz. surfactins, iturins and fengycins produced by B. subtilis K1 were also been investigated as biocontrol agents against rice blast disease caused by Magnaporthe oryzae. The mixed lipopeptide preparation (LP) containing surfactins, iturins and fengycins was found to inhibit germination of M. oryzae conidiospore. Upon prolong incubation with lipopeptides (0.1mg/mL) disruption of spores was observed. The protective effect on lipopeptide on paddy leaves against rice blast disease was also dose dependant. Microscopy studies of fungal infected paddy leaves revealed extensive colonization of infection structures of M. oryzae (appressorium) in the lower epidermis. The lower epidermal, mesophyll as well as vascular bundles of paddy leaf were extensively colonized and damaged due to penetration and progression of infection hypha from one cell to another cell. In 234 Summary & Conclusion

6 lipopeptide treated cells, the penetration as well as colonization of fungal hypha was significantly controlled. The light microscopy studies of thin section of LP treated leaves (without challenged with fungal spores) showed the thickening of epidermal cell walls, probably due to lignin deposition suggesting the role of lipopeptides in induction of defense system of rice leaf. Thus on the basis of detached leaf assay the biocontrol potential of lipopeptides produced by B. subtilis K1 was demonstrated, however further pot and field trials are needed to further comment in this aspect. Thus, novel Bacilli with ability to co-produce three families of lipopeptides with lot of microheterogeneity were isolated from Banyan hanging roots. It would be interesting to further investigate the biological role of these non-ribosomally synthesized lipopeptides for the producing organism. Nevertheless, Bacillus subtilis K1 holds commercial prospect as a biocontrol agent as well as a source of stable detergent with various applications ranging from use in detergents to enhanced oil recovery process. 235 Summary & Conclusion