Coniochaeta ligniaria: antifungal activity of the cryptic endophytic fungus associated with autotrophic tissue cultures of the medicinal plant Smallanthus sonchifolius (Asteraceae)

Abstract

Few studies have addressed the presence and bioactivity of endophytic fungi living in plantlets growing under in vitro conditions. After unfruitful attempts to grow axenic shoot cultures of the medicinal plant Smallanthus sonchifolius (yacon) were made, healthy shoots grew on half strength Murashigue and Skoog media supplemented with 2.2 μM benzylaminopurine without sucrose. We isolated a fungus UM109 from these autotrophic tissue cultures and it was identified as Coniochaeta ligniaria using molecular, physiological and morphological methods. Dichloromethane extracts from C. ligniaria and its host S. sonchifolius exhibited antifungal activity against phytopathogenic fungi Colletotrichum acutatum, C. fragariae and C. gloeosporioides. Both extracts of C. ligniaria and S. sonchifolius were subjected to antifungal bioassay-directed fractionation using NMR spectroscopy and GC-FID analysis. Twelve antifungal fatty acids were identified and 8 out of the total were produced by the fungus and the plant including caproic, caprylic, myristic, palmitic, heptadecanoic, stearic, oleic and linoleic acids. Additionally, caproic, caprylic and palmitic acids were produced at high concentrations by the endophytic fungus and its host. The detection of these antifungal fatty acids produced by both C. ligniaria and S. sonchifolius suggests that these bioactive compounds may be partially responsible for the high resistance of S. sonchifolius to phytopathogenic fungal attacks. This finding also indicates the existence of an interesting chemical symbiosis between an endophytic fungus and its host. Furthermore, the isolation of C. ligniaria from tissue culture of S. sonchifolius demonstrates that plantlets growing in vitro as autotrophic cultures can shelter specific endophytic fungal communities. The use of autotrophic tissue cultures may become an important tool for studies on the taxonomy, ecology, evolution and biotechnological application of endophytes.

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References

  1. Agoramoorthy G, Chandrasekaran M, Venkatesalu V, Hsu MJH (2007) Antibacterial and antifungal activities of fatty acids methyl esters of the blind-your-eye mangrove from India. Braz J Microbiol 38:739–742

    Article  Google Scholar 

  2. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLASTand PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    PubMed  Article  CAS  Google Scholar 

  3. Aráoz MVC, Mercado MI, Grau A, Catalán CAN (2010) Ent-kaurane derivatives from the root cortex of yacon and other three Smallanthus species (Heliantheae, Asteraceae). Biochem Syst Ecol 38:1042–1048

    Article  Google Scholar 

  4. Ayer WA, Kawahara N (1995) Lecythophorin, a potent inhibitor of blue-stain fungi, from the hyphomycetous Fungus Lecythophora hoffmannii. Tetrahed Lett 36:7953–7956

    Article  CAS  Google Scholar 

  5. Carballeira NM (2008) New advances in fatty acids as antimalarial, antimycobacterial and antifungal agents. Prog Lip Res 47:50–61

    Article  CAS  Google Scholar 

  6. Casieri L, Hofstetter V, Viret O, Gindro K (2009) Fungal communities living in the wood of different cultivars of young Vitis vinifera plants. Phytopathol Mediterr 48:73–83

    Google Scholar 

  7. Chakravarty P, Hiratsuka Y (1994) Evaluation of Lecythophora hoffmannii as a potential biological control agent against a blue stain fungus on Populus tremuloides. J Plant Dis Prot 101:74–79

    CAS  Google Scholar 

  8. Chen X, Qi Y, Wei J, Zhang Z, Wang D, Feng J, Gan B (2011) Molecular identification of endophytic fungi from medicinal plant Huperzia serrata based on rDNA ITS analysis. World J Microbiol Biotechnol 27:495–503

    Article  Google Scholar 

  9. Clay K, Holah J (1999) Fungal endophyte symbiosis and plant diversity in successional fields. Science 285:1742–1744

    PubMed  Article  CAS  Google Scholar 

  10. Clay K, Schardl CL (2002) Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. Am Nat 160:S99–S127

    PubMed  Article  Google Scholar 

  11. Damm U, Fourie PH, Crous W (2010) Coniochaeta (Lecythophora), Collophora gen. nov. and Phaeomoniella species associated with wood necroses of Prunus trees. Persoonia 24:60–80

    PubMed  Article  CAS  Google Scholar 

  12. de Hoog GS, Guarro J, Genó J, Figueras MJ (2000) Atlas of clinical fungi. CBS Baarn and Delft and Universitat Rovira i Virgili

  13. Drees M, Wickes BL, Gupta M, Hadley S (2007) Lecythophora mutabilis prosthetic valve endocarditis in a diabetic patient. Med Mycol 45:463–467

    PubMed  Article  Google Scholar 

  14. Dugan FM, Lupien SL, Grove GG (2002) Incidence, aggressiveness, and in planta interactions of Botrytis cinerea and other filamentous fungi quiescent in grape berries and dormant buds in central Washington state. J Phytopathol 150:375–381

    Article  Google Scholar 

  15. Ellis R, Kock JLF, Van Wyk PWJ, Botes PJ, Pohl CH (2009) Arachidonic acid increases antifungal susceptibility of Candida albicans and Candida dubliniensis. J Antimicrobl Chemoth 63:124–128

    Article  Google Scholar 

  16. Errasti A, Carmarán CC, Novas MV (2010) Diversity and significance of fungal endophytes from living stems of naturalized trees from Argentina. Fungal Div 41:29–40

    Article  Google Scholar 

  17. Gazis R, Rehner S, Chaverri P (2011) Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences. Mol Ecol 20:3001–3013

    PubMed  Article  Google Scholar 

  18. Hawksworth D, Crous PW, Redhead SA et al (2011) The Amsterdam declaration on fungal nomenclature. IMA Fungus 2:105–112

    PubMed  Article  Google Scholar 

  19. Inoue A, Tamogami S, Kato H, Nakazato Y, Akiyama M, Kodama O, Akatsuka T, Hashidoko Y (1995) Antifungal melampolides from leaf extracts of Smallanthus sonchifolius. Phytochemistry 39:845–848

    Article  CAS  Google Scholar 

  20. Kabara JJ, Swieczkowski DM, Conley AJ, Tuant JP (1972) Fatty acids and derivatives as antimicrobial agents. Antimicrob Agents Chemother 2:23–28

    PubMed  Article  CAS  Google Scholar 

  21. Kakuta H, Seki T, Hashidoko Y, Mizutani J (1992) Entkaurenic acid and its related-compounds from glandular trichome exudate and leaf extracts of Polymnia sonchifolia. Biosci Biotechnol Biochem 56:1562–1564

    Article  CAS  Google Scholar 

  22. Ko TWK, Stephenson SL, Bahkali AH, Hyde KD (2011) From morphology to molecular biology: can we use sequence data to identify fungal endophytes? Fungal Div 50:113–120

    Article  Google Scholar 

  23. Kokaew J, Manoch L, Worapong J, Chamswarng C, Singburaudom N, Visarathanonth N, Piasai O, Strobel G (2011) Coniochaeta ligniaria an endophytic fungus from Baeckea frutescens and its antagonistic effects against plant pathogenic fungi. Thai J Agric Sci 44:123–131

    Google Scholar 

  24. Lin F, Morifumi H, Kodama O (2003) Purification and identification of antimicrobial sesquiterpene lactones from Yacon (Smallanthus sonchifolius) leaves. Biosci Biotechnol Biochem 67:2154–2159

    PubMed  Article  CAS  Google Scholar 

  25. Liu S, Weibin R, Jing L, Hua X, Jingan W, Yubao G, Jingguo W (2008) Biological control of phytopathogenic fungi by fatty acids. Mycopathologia 166:93–102

    PubMed  Article  CAS  Google Scholar 

  26. Lucero M, Barrow JR, Osuna P, Reyes I (2008) A cryptic microbial community persists within micropropagated Bouteloua eriopoda (Torr.) Torr. Cultures Plant Sci 174:570–575

    Article  CAS  Google Scholar 

  27. Moerman DE (1991) The medicinal flora of native North America- an analysis. J Ethnopharmacol 31:1–42

    PubMed  Article  CAS  Google Scholar 

  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Google Scholar 

  29. Pirttila AM, Pospiech H, Laukkanen H, Myllyla R, Hohtola A (2003) Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.). Microb Ecol 45:53–62

    PubMed  Article  CAS  Google Scholar 

  30. Pohl CH, Kock JLF, Thibane VS (2011) Antifungal free fatty acids: a review, in: Science against microbial pathogens: communicating current research and technological advances. Microbiology Book Series - Number 3 A. Méndez-Vilas, pp. 61–71

  31. Rivera-Orduña NF, Suarez-Sanchez RA, Flores-Bustamante ZR, Gracida-Rodriguez JN, Flores-Cotera LB (2011) Diversity of endophytic fungi of Taxus globosa (Mexican yew). Fungal Div 47:65–74

    Article  Google Scholar 

  32. Rosa LH, Gonçalves VN, Caligiorne RB, Alves TMA, Rabello A, Sales PA, Romanha AJ, Sobral MEG, Rosa CA, Zani CL (2010) Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil. Braz J Microbiol 41:114–122

    Article  Google Scholar 

  33. Rosa LH, Vieira MLA, Cota BB, Johann S, Alves TMA, Zani CL, Rosa CA (2011) Endophytic fungi of tropical forests: a promising source of bioactive prototype molecules for the treatment of neglected diseases. In: Ekinci D (ed) Drug development - a case study based insight into modern strategies. Intech, Croatia

    Google Scholar 

  34. Rosa LH, Tabanca N, Techen N, Wedge DE, Pan Z, Moraes RM (2012) Biological activity of endophytic fungal community associated with Smallanthus maintained in vitro as autotrophic cultures and as pot plants in the greenhouse. Can J Microbiol 58:1202–1211

    PubMed  Article  CAS  Google Scholar 

  35. Sakaeyama SI, Sano A, Murata Y, Kamei K, Nishimura KH (2007) Lecythophora hoffmannii isolated from a case of canine osteomyelitis in Japan. Med Mycol 45:267–272

    PubMed  Article  Google Scholar 

  36. Segeth MP, Bonnefoy A, Brönstrup M, Knauf M, Schummer D, Toti L, Vértesy L, Wetzel-Raynal MC, Wink J, Seibert G (2003) Coniosetin, a novel tetramic acid antibiotic from Coniochaeta ellipsoidea DSM 13856. J Antibiot 56:114–122

    PubMed  Article  CAS  Google Scholar 

  37. Stone JK, Polishook JD, White JF (2004) Endophytic fungi. In: Mueller GM, Bills GF, Foster MS (eds) Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press, San Diego

    Google Scholar 

  38. Strobel G, Daisy B (2003) Bioprospecting for Microbial Endophytes and their natural products. Microbiol Mol Biol Rev 67:491–502

    PubMed  Article  CAS  Google Scholar 

  39. Sugijanto NE, Diesel A, Ebel R, Indrajanto G, Zaini NC (2009) Chemical constituents of the endophytic fungus Lecythophora sp. isolated from Alyxia reinwardtii. Nat Prod Commun 4:1485–1488

    PubMed  CAS  Google Scholar 

  40. Sugijanto NE, Diesel A, Rateb M, Pretsch A, Gogalic S, Zaini NC, Ebel R, Indrayanto G (2011) Lecythomycin, a new macrolactone glycoside from the endophytic fungus Lecythophora sp. Nat Prod Commun 6:677–678

    PubMed  CAS  Google Scholar 

  41. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    PubMed  Article  CAS  Google Scholar 

  42. Taylor JW (2011) One fungus = one name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2:113–120

    PubMed  Article  Google Scholar 

  43. Thibane VS, Ells R, Hugo A, Albertyn J, van Rensburg WJ, Van Wyk PW, Kock JL, Pohl CH (2010) Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms. Biochim Biophys Acta 1820:1463–1468

    Article  Google Scholar 

  44. Wedge DE, Kuhajek JM (1998) A microbioassay for fungicide discovery. SAAS Bull Biochem Biotechnol 11:1–7

    CAS  Google Scholar 

  45. Wedge DE, Klun JA, Tabanca N, Demirci B, Ozek T, Baser KHC, Liu Z, Zhang S, Cantrell CL, Zhang J (2009) Bioactivity-guided fractionation and GC-MS fingerprinting of Angelica sinensis and A. archangelica root components for antifungal and mosquito deterrent activity. J Agric Food Chem 57:464–470

    PubMed  Article  CAS  Google Scholar 

  46. White TJ, Bruns TD, Lee SB (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis NA, Gelfand J, Sninsky J (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego

    Google Scholar 

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Acknowledgements

This work received support of the Conselho Nacional of the Desenvolvimento Científico and Tecnológico (processo 200774/2011-5), Fundação of Amparo the Pesquisa of Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PGCI 036/13). The authors thank Ms. J.L. Robertson, Mrs. R. Pace, Mrs. Amber Reichley, Mr. Solomon Green III and Mrs. Marilyn Ruscoe for technical support. We also thank Dr. N. Tabanca and Dr. A. L. Cerdeira for valuable comments on the manuscript. The National Center for Natural Products Research, University of Mississippi is partially supported through a cooperative scientific agreement 58-6408-2-0009 from the USDA, Agricultural Research Service.

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Correspondence to Luiz H. Rosa or David E. Wedge.

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Rosa, L.H., Queiroz, S.C.N., Moraes, R.M. et al. Coniochaeta ligniaria: antifungal activity of the cryptic endophytic fungus associated with autotrophic tissue cultures of the medicinal plant Smallanthus sonchifolius (Asteraceae). Symbiosis 60, 133–142 (2013). https://doi.org/10.1007/s13199-013-0249-8

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Keywords

  • Antifungal
  • Fatty acids
  • Medicinal plant
  • Plant tissue culture
  • Symbiosis