Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas

Masroor Qadri1, Sarojini Johri1, Bhahwal Ali Shah2, Anamika Khajuria3, Tabasum Sidiq3, Surrinder K. Lattoo4, M. Z. Abdin5, Rabbani Syed1
1Microbial Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
2Microbial Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, 180001, India
3Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
4Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, 180001, India
5Centre for Transgenic Plant Development, Department of Biotechnology, Jamia Hamdard, New Delhi, 1 10062, India

Tóm tắt

Abstract

This study was conducted to characterize and explore the endophytic fungi of selected plants from the Western Himalayas for their bioactive potential. A total of 72 strains of endophytic fungi were isolated and characterized morphologically as well as on the basis of ITS1-5.8S-ITS2 ribosomal gene sequence acquisition and analyses. The fungi represented 27 genera of which two belonged to Basidiomycota, each representing a single isolate, while the rest of the isolates comprised of Ascomycetous fungi. Among the isolated strains, ten isolates could not be assigned to a genus as they displayed a maximum sequence similarity of 95% or less with taxonomically characterized organisms. Among the host plants, the conifers, Cedrus deodara, Pinus roxburgii and Abies pindrow harbored the most diverse fungi, belonging to 13 different genera, which represented almost half of the total genera isolated. Several extracts prepared from the fermented broth of these fungi demonstrated strong bioactivity against E. coli and S. aureus with the lowest IC50 of 18 μg/ml obtained with the extract of Trichophaea abundans inhabiting Pinus sp. In comparison, extracts from only three endophytes were significantly inhibitory to Candida albicans, an important fungal pathogen. Further, 24 endophytes inhibited three or more phytopathogens by at least 50% in co-culture, among a panel of seven test organisms. Extracts from 17 fungi possessed immuno-modulatory activities with five of them showing significant immune suppression as demonstrated by the in vitro lymphocyte proliferation assay. This study is an important step towards tapping the endophytic fungal diversity from the Western Himalayas and assessing their bioactive potential. Further studies on the selected endophytes may lead to the isolation of novel natural products for use in medicine, industry and agriculture.

Từ khóa


Tài liệu tham khảo

Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25: 3389-3402. 10.1093/nar/25.17.3389

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York City, NY; 1994.

Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H: ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiol 2010, 10: 189-197. 10.1186/1471-2180-10-189

Bergmann S, Schuemann J, Scherlach K, Lange CV, Brakhage AA: Genome driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. Nat Chem Biol 2007, 3: 213-217. 10.1038/nchembio869

Bok JW, Hoffmeister D, Maggio-Hall LA, Renato M, Glasner JD, Keller NP: Genomic mining for Aspergillus natural products. Chem Biol 2006, 13: 31-37. 10.1016/j.chembiol.2005.10.008

Booth C, Spooner BM: Gibberella avenacea, teleomorph of Fusarium avenaceum, from stems of Pteridium aquilinum. T Brit Mycol Soc 1984, 82: 178-180. 10.1016/S0007-1536(84)80231-4

Clardy J, Walsh C: Lessons from natural molecules. Nature 2004, 432: 829-837. 10.1038/nature03194

Cooper JE, Wiseman AC: Novel immunosuppressive agents in kidney transplantation. Clin Nephrol 2010, 73: 333-343.

Demain AL: Pharmaceutically active secondary metabolites of microorganisms. Appl Microbiol Biotechnol 1999, 52: 455-463. 10.1007/s002530051546

Ellof JN: A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998, 64: 711-713. 10.1055/s-2006-957563

Ezra D, Hess WH, Strobel GA: New endophytic isolates of M. albus, a volatile antibiotic-producing fungus. Microbiology 2004, 150: 4023-4031. 10.1099/mic.0.27334-0

Felsenstein J: Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985, 39: 783-791. 10.2307/2408678

Ganley RJ, Newcombe G: Fungal endophytes in seeds and needles of Pinus monticola. Mycol Res 2006, 110: 318-327. 10.1016/j.mycres.2005.10.005

Hanson T, Brooks TM, Da Fonseca GA, Hoffmann M, Lamoreux JF, Machlis G, Mittermeier CG, Mittermeier RA, Pilgrim JD: Warfare in biodiversity Hotspots. Conserv Biol 2009, 23: 578-587. 10.1111/j.1523-1739.2009.01166.x

Hertweck C: Hidden biosynthetic treasures brought to light. Nat Chem Biol 2009, 5: 450-452. 10.1038/nchembio0709-450

Heywood VH (Ed): Global biodiversity assessment. Cambridge University Press, Cambridge, UK; 1995.

Keller NP, Turner G, Bennett JW: Fungal secondary metabolism-from biochemistry to genomics. Nature Rev Microbiol 2005, 3: 937-947. 10.1038/nrmicro1286

Khosla C: Harnessing the biosynthetic potential of modular polyketide synthases. Chem Rev 1997, 97: 2577-2590. 10.1021/cr960027u

Khuroo AA, Rashid I, Reshi Z, Dar GH, Wafai BA: The alien flora of Kashmir Himalaya. Biol Invasions 2007, 9: 269-292. 10.1007/s10530-006-9032-6

Kim JH, Lee HJ, Jeong SJ, Lee MH, Kim SH: Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of Acyl-coenzyme A: cholesterol acyltransferase. Phyther Res 2012. 10.1002/ptr.3734

Knappe TA, Linne U, Zirah S, Rebuffat S, Xie X, Marahiel MA: Isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264. J Am Chem Soc 2008, 13: 11446-11454.

Kusari S, Zühlke S, Spiteller M: An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. J Nat Prod 2009, 72: 2-7. 10.1021/np800455b

McAlpine JB, Bachmann BO, Piraee M, Tremblay S, Alarco AM, Zazopoulos E, Farnet CM: Microbial genomics as a guide to drug discovery, structural elucidation: ECO02301, a novel antifungal agent, as an example. J Nat Prod 2005, 68: 493-496. 10.1021/np0401664

Meca G, Soriano JM, Gaspari A, Ritieni A, Moretti A, Mañes J: Antifungal effects of the bioactive compounds enniatins A, A(1), B, B(1). Toxicon 2010, 56(3):480-485. 10.1016/j.toxicon.2010.04.013

Mohana KP, Zuehlke S, Priti V, Ramesha BT, Shweta S, Ravikanth G, Vasudeva R, Santhoshkumar TR, Spiteller M, Umashaanker R: Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie Van Leeuwenhoek 2012, 101: 323-329. 10.1007/s10482-011-9638-2

Mossmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65: 55-63. 10.1016/0022-1759(83)90303-4

Paul Antony C, Kumaresan D, Hunger S, Drake HL, Murrell J, Shouche YS: Microbiology of Lonar Lake and other soda lakes. ISME J 2012. 10.1038/ismej.2012.137

Porras-Alfaro A, Bayman P: Hidden fungi, emergent properties: endophytes and microbiomes. Annu Rev Phytopathol 2011, 49: 291-315. 10.1146/annurev-phyto-080508-081831

Puri SC, Nazir A, Chawla R, Arora R, Riyaz-Ul-Hassan S, Amna T, Ahmad B, Verma V, Singh S, Sagar R, Sharma A, Kumar R, Sharma RK, Qazi GN: The endophytic fungus Trametes hirsuta as a novel alternative source of podophyllotoxin and related aryl tetralin lignans. J Biotechnol 2006, 122: 494-510. 10.1016/j.jbiotec.2005.10.015

Puri SC, Amna T, Khajuria A, Gupta A, Arora R, Spiteller M, Qazi GN: Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer. Acta Microbiol Immunol Hung 2007, 54: 237-260. 10.1556/AMicr.54.2007.3.2

Qin JC, Zhang YM, Hu L, Ma YT, Gao JM: Cytotoxic metabolites produced by Alternaria no.28, an endophytic fungus isolated from Ginkgo biloba. Nat Prod Commun 2009, 4: 1473-1476.

Rehman S, Shawl AS, Kour A, Sultan P, Ahmad K, Khajuria R, Qazi GN: Comparative studies and identification of camptothecin produced by an endophyte at shake flask and bioreactor. Nat Prod Res 2009, 23: 1050-1057. 10.1080/14786410902750944

Riyaz-Ul-Hassan S, Strobel GA, Booth E, Knighton B, Floerchinger C, Sears J: Modulation of volatile organic compound formation in the Mycodiesel producing endophyte- Hypoxylon sp. C1-4. Microbiology 2012, 158: 464-473.

Saitou N, Nei M: The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Bio Evol 1987, 4: 406-425.

Schroeckh V, Scherlach K, Nützmann HW, Shelest E, Schmidt-Heck W, Schuemann J, Martin K, Hertweck C, Brakhage AA: Intimate bacterial–fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans. Proc Natl Acad Sci USA 2009, 106: 14558-14563. 10.1073/pnas.0901870106

Selvin J, Ninawe AS, Kiran GS, Lipton AP: Sponge-microbial interactions: Ecological implications and bioprospecting avenues. Crit Rev Microbiol 2010, 36: 82-90. 10.3109/10408410903397340

Sharma PR, Shannmugavel M, Saxena AK, Qazi GN: Induction of apoptosis by a synergistic lignan composition from Cedrus deodara in human cancer cells. Phytother Res 2008, 22: 1587-1594. 10.1002/ptr.2511

Sieber SA, Marahiel MA: Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem Rev 2005, 105: 715-738. 10.1021/cr0301191

Singh J, Yadav RR: Dendroclimatic potential of millennium-long ring-width chronology of Pinus gerardiana from Himachal Pradesh, India. Curr Sci 2007, 93: 833-836.

Staley JT, Castenholz RW, Colwell RR, Holt JG, Kane MD, Pace NR, Saylers AA, Tiedje JM: The microbial world: foundation of the biosphere. American Academy of Microbiology, Washington DC; 1997:32.

Staniek A, Woerdenbag HJ, Kayser O: Taxomyces andreanae: A presumed paclitaxel producer demystified? Planta Med 2009, 75: 1561-1566. 10.1055/s-0029-1186181

Stierle A, Strobel GA, Stierle DB: Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 1993, 260: 214-216. 10.1126/science.8097061

Strobel G: Harnessing endophytes for industrial microbiology. Curr Opin Microbiol 2006, 9: 240-244. 10.1016/j.mib.2006.04.001

Strobel GA, Daisy B: Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 2003, 67: 491-502. 10.1128/MMBR.67.4.491-502.2003

Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Bio Evol 2007, 24: 1596-1599. 10.1093/molbev/msm092

Tamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2004, 101: 11030-11035. 10.1073/pnas.0404206101

Twentyman PR: Cyclosporins as drug resistance modifiers. Biochem Pharmacol 1992, 43: 109-117. 10.1016/0006-2952(92)90668-9

Uhlig S, Jestoi M, Parikka P: Fusarium avenaceum- the North European situation. Int J Food Microbiol 2007, 119: 17-24. 10.1016/j.ijfoodmicro.2007.07.021

Vainio EJ, Korhonen K, Hantula J: Genetic variation in Phlebiopsis gigantean as detected with random amplified microsatellite (RAMS) markers. Mycol Res 1998, 2: 187-192.

Wang LW, Zhang YL, Lin FC, Hu YZ, Zhang CL: Natural products with antitumor activity from endophytic fungi. Mini Rev Med Chem 2011, 11: 1056-1074. 10.2174/138955711797247716

Xu L, Wang J, Zhao J, Li P, Shan T, Wang J, Li X, Zhou L: Beauvericin from the endophytic fungus, Fusarium redolens, isolated from Dioscorea zingiberensis and its antibacterial activity. Nat Prod Commun 2010, 5: 811-814.

Yadav RR: Tree ring imprints of long-term changes in climate in Western Himalaya, India. J Biosci 2009, 34: 700-707.

Yuan ZL, Rao LB, Chen YC, Zhang CL, Wu YG: From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis. Fungal Biol 2011, 115: 197-213. 10.1016/j.funbio.2010.11.002

Zhang Y, Han T, Ming Q, Wu L, Rahman K, Qin L: Alkaloids produced by endophytic fungi: a review. Nat Prod Commun 2012, 7(7):963-968.