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Insights into in vitro phenotypic plasticity, growth and secondary metabolites of the mycobiont isolated from the lichen Platygramme caesiopruinosa

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Abstract

The choice of inoculum for successful isolation and establishment of axenic lichen mycobiont culture is a key step towards eliminating endolichenic and lichenicolous fungi and other microbial contamination. The nutritional requirements of each lichen species are unique. This work reports on the isolation, phenotypic plasticity, growth and secondary metabolites from mycobiont culture of the pantropical lichen Platygramme caesiopruinosa. Media composition [Malt yeast extract (MY), Modified Murashige and Skoog (MMS) and Lilly and Barnett (LB) media] was optimized to determine nutritional requirements for optimal growth of this species as assessed by dry biomass and the occurrence of secondary metabolite. Furthermore, the role of different carbon sources in affecting growth, growth stages, phenotypic plasticity, biomass and spectrum of secondary metabolites produced of this mycobiont was examined. The molecular identity of the mycobiont culture was confirmed by amplifying mitochondrial small subunit (mtSSU) sequences. Cultures showed optimum biomass production in MY medium with 10% sucrose. The secondary metabolite profiles for each culture treatment were characterized using High-performance Thin-Layer Chromatography (HPTLC) and Gas Chromatography with Mass Spectrometric (GC–MS) analysis. The HPTLC spectral comparison, phenolic and iodine confirmatory analysis revealed the absence of phenolic metabolites and the presence of non-phenolic metabolites in mycobiont extracts, while GC–MS analysis revealed the biosynthesis of side chain fatty acids, hydrocarbons and sugar alcohol in mycobiont cultures treated with 10% supplemented sucrose as a carbon source.

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Abbreviations

HPTLC:

High performance thin layer chromatography

GC–MS:

Gas chromatography with mass spectrometry

MYC:

Mycobiont culture

MY :

Malt yeast extract medium

MYF:

Malt yeast extract medium with fructose

MYG:

Malt yeast extract medium with glucose

MYM:

Malt yeast extract medium with mannitol

MYR:

Malt yeast extract medium with ribitol

MYS:

Malt yeast extract medium with sucrose

MYSR:

Malt yeast extract medium with sorbitol

References

  • Andrews JH (1992) Fungal life history strategies. In: Carroll GC, Wicklow DT (eds) The fungal community, 2nd edn. Dekker, New York

    Google Scholar 

  • Arup U, Ekman S, Lindblom L, Mattsson JE (1993) High performance thin layer chromatography (HPTLC), an improved technique for screening lichen substances. Lichenol 25:61–71

    Article  Google Scholar 

  • Awasthi DD (1991) A key to the microlichens of India, Nepal and Sri Lanka. Biblioth Lichenol 40:1436–1698

    Google Scholar 

  • Bago B, Cano C, Azcon-Aguilar C, Samson J, Coughlan AP, Piche´ Y, (2004) Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoexenically on spatially heterogenous culture media. Mycologia 96:452–462

    Article  Google Scholar 

  • Behera BC, Adawadkar B, Makhija U (2006) Tyrosinase-inhibitory activity in some species of the lichen family Graphidaceae. J Herb Pharmacother 6(1):55–69

    Article  Google Scholar 

  • Bemmann W (1981) Dimorphism of fungi—review of the literature. Zentralbl Bakteriol Naturwiss 136:369–416

    CAS  PubMed  Google Scholar 

  • Brunauer G, Hager A, Grube M, Türk R, Stocker Wörgötter E (2007) Alteration in secondary metabolism of aposymbiotically grown mycobionts of Xanthoria elegans and culture resynthesis stages. Plant Physiol Biochem 45:146

    Article  CAS  Google Scholar 

  • Černajová I, Škaloud P (2020) Lessons from culturing lichen soredia. Symbiosis 82:109–122. https://doi.org/10.1007/s13199-020-00718-4

    Article  CAS  Google Scholar 

  • Cordeiro LMC, Iacomini M, Stocker-Wörgötter E (2004) Culture studies and secondary compounds of six Ramalina species. Mycol Res 108:489–497

    Article  CAS  Google Scholar 

  • Crittenden PD, David JC, Hawksworth DL, Campbell FS (1995) Attempted isolation and success in the culturing of a broad spectrum fungi. New Phytol 130:267–297

    Article  Google Scholar 

  • Culberson CF, Armaleo D (1992) Induction of a complete secondary-product pathway in a cultured lichen fungus. Fungal Genet Biol 16:52

    CAS  Google Scholar 

  • Díaz EM, Zamora JC, Ruibal C et al (2020) Axenic culture and biosynthesis of secondary compounds in lichen symbiotic fungi, the Parmeliaceae. Symbiosis 82:79–93. https://doi.org/10.1007/s13199-020-00719-3

    Article  CAS  Google Scholar 

  • Fazio AT, Bertoni MD, Adler MT et al (2009) Culture studies on the mycobiont isolated from Parmotrema reticulatum (Taylor) Choisy: metabolite production under different conditions. Mycol pro 8:359. https://doi.org/10.1007/s11557-009-0609-1

    Article  Google Scholar 

  • Fazio AT, Adler M, Maier M (2014) Usnic acid and triacylglycerides production by the cultured lichen mycobiont of Ramalina celastri. Nat Prod Com 9:213–214

    CAS  Google Scholar 

  • Fazio AT, Adler MT, Parnmen S et al (2018) Production of the bioactive pigment elsinochrome A by a cultured mycobiont strain of the lichen Graphis elongata. Mycol pro 17:479–487. https://doi.org/10.1007/s11557-017-1374-1

    Article  Google Scholar 

  • Gargas A, Taylor JW (1992) Polymerase chain reaction (PCR) primers for amplifying and sequencing nuclear 18S rDNA from lichenized fungi. Mycologia 84:589–592. https://doi.org/10.1080/00275514.1992.12026182

    Article  CAS  Google Scholar 

  • Hamada N (1996) Induction of the production of lichen substances by non-metabolites. Bryologist 99:68–70

    Article  CAS  Google Scholar 

  • Jennings DH (1993) Understanding tolerance to stress: laboratory culture versus environmental actuality. In: Jennings DH (ed) Stress tolerance of fungi. Marcel Dekker, New York

    Google Scholar 

  • Karthik S (2020) Species diversity, in vitro biosynthesis of secondary metabolites of selected lichen species from habitats in India, and lichen inhabiting-fungal communities of selected lichens from Germany. PhD Thesis, University of Madras, Chennai

  • Kinoshita Y (1993) The production of lichen substances for pharmaceutical use by lichen tissue culture. Nippon Paint Publications, Osaka

    Google Scholar 

  • Kukwa M, Schiefelbein U, Flakus A (2013) A contribution to the lichen family Graphidaceae (Ostropales, Ascomycota) of Bolivia. 2. Herzogia 26:231–252. https://doi.org/10.2478/pbj-2014-0017

    Article  Google Scholar 

  • Le DH, Takenaka Y, Hamada N, Mizushina Y, Tanahashi T (2014) Polyketides from the cultured lichen mycobiont of a Vietnamese Pyrenula sp. J Nat Prod 77:1404–1412

    Article  CAS  Google Scholar 

  • Lilly VG, Barnett HL (1951) Physiology of the fungi. McGraw-Hill, New York

    Google Scholar 

  • Lücking R, Archer AW, Aptroot A (2009) A world-wide key to the genus Graphis (Ostropales: Graphidaceae). Lichenol 41:363–452. https://doi.org/10.1017/S0024282909008305

    Article  Google Scholar 

  • Lücking R, Hodkinson BP, Leavitt SD (2017) The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota—Approaching one thousand genera. Bryologist 119(4):361–416. https://doi.org/10.1639/0007-2745-119.4.361

    Article  Google Scholar 

  • Lumbsch HT, Leavitt SD (2011) Goodbye morphology? A paradigm shift in the delimitation of species in lichenized fungi. Fungal Divers 50:59. https://doi.org/10.1007/s13225-011-0123-z

    Article  Google Scholar 

  • McDonald TR, Gaya E, Lutzoni F (2013) Twenty-five cultures of lichenizing fungi available for experimental studies on symbiotic systems. Symbiosis 59:165–171

    Article  Google Scholar 

  • Miranda-González R, McCune B (2020) The weight of the crust: Biomass of crustose lichens in tropical dry forest represents more than half of foliar biomass. Biotropica 52:1298–1308

    Article  Google Scholar 

  • Molina M, Crespo A, Vicente C, Elix J (2004) Differences in the composition of phenolics and fatty acids of cultured mycobiont and thallus of Physconia distorta. Plant Physio Biochem. https://doi.org/10.1016/S0981-9428(02)00017-7

    Article  Google Scholar 

  • Molina MC, Divakar PK, González N (2015) Success in the isolation and axenic culture of Anaptychia ciliaris (Physciaceae, Lecanoromycetes) mycobiont. Mycoscience 56:351–358

    Article  CAS  Google Scholar 

  • Muggia L, Pérez-Ortega S, Fryday A et al (2014) Global assessment of genetic variation and phenotypic plasticity in the lichen-forming species Tephromela atra. Fungal Divers 64:233–251. https://doi.org/10.1007/s13225-013-0271-4

    Article  Google Scholar 

  • Muthukumar S, Karthik S, Hariharan GN (2016) Mycobiont and whole thallus cultures of Roccella montagnei Bél. for the biosynthesis of secondary compounds. Cryptogam Biodivers Assess. https://doi.org/10.21756/cba.v0i.11014

    Article  Google Scholar 

  • Orange A, James PW, White FJ (2001) Microchemical methods for the identification of lichens. British Lichen Society, London

    Google Scholar 

  • Osyczka P, Rola K (2013) Phenotypic plasticity of primary thallus in selected Cladonia species (lichenized Ascomycota: Cladoniaceae). Biologia 68:365–372. https://doi.org/10.2478/s11756-013-0169-3

    Article  CAS  Google Scholar 

  • Pérez-Ortega S, Fernández-Mendoza F, Raggio J, Vivas M, Ascaso C, Sancho L, Los Ríos A (2012) Extreme phenotypic variation in Cetraria aculeata (lichenized Ascomycota), adaptation or incidental modification? Ann Bot 109:1133–1148

    Article  Google Scholar 

  • Pichler G, Candotto CF, Muggia L, Holzinger A, Tretiach M, Kranner I (2021) Enhanced culturing techniques for the mycobiont isolated from the lichen Xanthoria parietina. Mycol Prog 20:797–808. https://doi.org/10.1007/s11557-021-01707-7

    Article  PubMed  PubMed Central  Google Scholar 

  • Pittayakhajonwut P, Sri-indrasutdhi V, Dramae A, Lapanun S, Suvannakad R, Tantichareon M (2009) Graphisins A and B from the lichen Graphis tetralocularis. Aust J Chem 62:389–391

    Article  CAS  Google Scholar 

  • Promislow D (2005) A regulatory network analysis of phenotypic plasticity in yeast. Am Nat 165:515–523

    Article  Google Scholar 

  • Reis RA, Iacomini M, Gorin PAJ, Souza LM, Grube M, Cordeiro LMC, Sassaki GL (2005) Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts. FEMS Microbiol Lett 247:1–6. https://doi.org/10.1016/j.femsle.2005.04.023

    Article  CAS  PubMed  Google Scholar 

  • Romano AH (1966) Dimorphism. In: Ainsworth GC, Sussman AS (eds) The fungi: An advanced treatise 2. Academic Press, New York, pp 181–209

    Google Scholar 

  • Sangvichien E, Hawksworth DI, Whalley AJS (2011) Ascospore discharge, germination and culture of fungal partners of tropical lichens, including the use of a novel culture technique. IMA Fungus 2:143–153

    Article  Google Scholar 

  • Shanmugam K, Srinivasan M, Hariharan GN (2016) Developmental stages and secondary compound biosynthesis of mycobiont and whole thallus cultures of Buellia subsororioides. Mycol Prog 15:41

    Article  Google Scholar 

  • Singh P, Singh KP (2014) Two new species of Graphis (Ascomycota: Ostropales: Graphidaceae), from Indo-Burma biodiversity hotspot. Mycosphere 5:504–509. https://doi.org/10.5943/mycosphere/5/4/2

    Article  Google Scholar 

  • Singh KP, Sinha GP (2010) Indian lichens: an annotated checklist. Botanical Survey of India, Kolkata

    Google Scholar 

  • Slepecky RA, Starmer WT (2009) Phenotypic plasticity in fungi: a review with observations on Aureobasidium pullulans. Mycologia 101(6):823–832. https://doi.org/10.3852/08-197

    Article  Google Scholar 

  • Srinivasan M, Shanmugam K, Kedike B, Narayanan S, Shanmugam S, Gopalasamudram Neelakantan H (2020) Trypethelone and phenalenone derivatives isolated from the mycobiont culture of Trypethelium eluteriae Spreng and their anti-mycobacterial properties. Nat Prod Res 34(23):3320–3327. https://doi.org/10.1080/14786419.2019.1566823

    Article  CAS  PubMed  Google Scholar 

  • Stocker-Wörgötter E (2007) Polyketides and PKS genes in lichen-forming fungi. The impact of algal transfer metabolites (polyols and glucose) on the production of “lichen substances”. (SEB Main Meeting, Symbiosis, Glasgow: 31.3.-4.4.2007). Comp Biochem Physiol (Part A) 146(2007):215–223

    Google Scholar 

  • Stocker-Wörgötter E (2008) Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimate metabolite production, and PKS genes. Nat Prod Rep 25(1):188–200. https://doi.org/10.1039/b606983p

    Article  CAS  PubMed  Google Scholar 

  • Stocker-Wörgötter E, Elix JA (2006) Morphogenetic strategies and induction of secondary metabolite biosynthesis in cultured lichen forming ascomycota, as exemplified by Cladia retipora (Labill.) Nyl. and Dactylina arctica (Richards) Nyl Symbiosis 41:9–20

  • Stomp M, van Dijk MA, van Overzee HMJ, Wortel MT, Sigon CAM, Egas M, Hoogvel H, Grons HJ, Huisman J (2008) The time scale of phenotypic plasticity and its impact on competition in fluctuating environments. Am Nat 172:169–185

    Article  Google Scholar 

  • Takenaka Y, Hamada N, Tanahashi T (2008) Biosynthetic origin of graphenone in cultured lichen mycobionts of Graphis handelii. Zeitschrift für Naturforschung 63C:565–568

    Article  Google Scholar 

  • Takenaka Y, Morimoto N, Hamada N, Tanahashi T (2011) Phenolic compounds from the cultured mycobionts of Graphis proserpens. Phytochemistry 72:1431–1435. https://doi.org/10.1016/j.phytochem.2011.04.017

    Article  CAS  PubMed  Google Scholar 

  • Takenaka Y, Naito Y, Le DH, Hamada N, Tanahashi T (2013) Napthoquinones and phenaleone derivatives from the cultured mycobiont of Trypethelium sp. Heterocycles 87:1897–1902

    Article  CAS  Google Scholar 

  • Tanahashi T, Kuroishi M, Kuwahara A, Nagakura N, Hamada N (1997) Four phenolics from the cultured lichen mycobionts of Graphis scripta var. pulverulenta. Chem Pharm Bull 45:1183–1185

    Article  CAS  Google Scholar 

  • Tanahashi T, Takenaka Y, Nagakura N, Hamada N, Miyawaki H (2000) Two isocoumarins from the cultured lichen mycobiont of Graphis sp. Heterocycles 53:723–728

    Article  CAS  Google Scholar 

  • Tanahashi T, Takenaka Y, Nagakura N, Hamada N (2003) 6H Dibenzo[b,d]pyran-6-one derivatives from the cultured lichen mycobionts of Graphis spp. and their biosynthetic origin. Phytochemistry 62(1):71–75. https://doi.org/10.1016/S0031-9422(02)00402-8

    Article  CAS  PubMed  Google Scholar 

  • Tretiach M, Brown DH (1995) Morphological and physiological differences between epilithic and epiphytic populations of the lichen Parmelia pastillifera. Ann Bot 75:627–632

    Article  Google Scholar 

  • Valarmathi R, Hariharan GN (2007) Soredial culture of Dirinaria applanata (Fée) Awasthi: observations on developmental stages and compound production. Symbiosis 43:137–142

    Google Scholar 

  • Valarmathi R, Hariharan GN, Gayatri V, Ajay P (2009) Characterization of a non-reducing polyketide synthase gene from lichen Dirinaria applanata. Phytochemistry 70:721–729. https://doi.org/10.1016/j.phytochem.2009.04.007

    Article  CAS  PubMed  Google Scholar 

  • Valarmathi R (2009) Bioprospecting secondary compounds from selected lichen species in vitro production, characterization and molecular basis of compound production. PhD thesis, University of Madras, Chennai

  • Vallardes F, Gianoli E, Gómez JM (2007) Ecological limits to plant phenotypic plasticity. New Phytol 167:749–776

    Google Scholar 

  • Vo V, Le DH, Thanh NT, Nguyen NH, Vo TP, Sichaem J, Nguyen VK, Duong TH (2020) A new eremophilane-sesquiterpene from the cultured lichen mycobiont of Graphis sp. Nat Prod Res. https://doi.org/10.1080/14786419.2020.1779717

    Article  PubMed  Google Scholar 

  • Williams L, Colesie C, Ullmann A, Westberg M, Wedin M, Büdel B (2017) Lichen acclimation to changing environments: Photobiont switching vs. climate—specific uniqueness in Psora decipiens. Ecol Evol 7:2560–2574

    Article  Google Scholar 

  • Yamamoto Y, Mizuguchi R, Takayama S, Yamada Y (1987) Effects of culture conditions on the growth of Usneaceae lichen tissue cultures. Plant Cell Physiol 28:1421–1426

    CAS  Google Scholar 

  • Zoller S, Scheidegger C, Sperisen C et al (1999) PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenol 31(5):511–515

    Article  Google Scholar 

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Acknowledgements

Prof. M.S. Swaminathan, Founder Chairman of M.S. Swaminathan Research Foundation (MSSRF) and the Executive Director (MSSRF) M.S. Swaminathan Research Foundation, are thanked for their unwavering encouragement and support. Dr. Gayatri Venkataraman, MSSRF and Suni Sebastian, for correcting the language; Mr. G.K. Dayanandham and S. Kannapan assisted the authors in revising figures, formatting and uploading the final version of the manuscript. The authors are grateful for the financial support provided by the Department of Biotechnology, Government of India.

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  1. Lichen Ecology and Bioprospecting Laboratory, Biotechnology Programme, M.S. Swaminathan Research Foundation, 3rd Cross Street, Institutional Area, Taramani, Chennai, 600 113, India

    Karthik Shanmugam, Muthukumar Srinivasan & Hariharan Gopalasamudram Neelakantan

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  1. Karthik Shanmugam
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Contributions

KS and MS collected the lichen samples, conceptualized and conducted the in vitro experiments, secondary metabolites profiling and data analysis and wrote the original draft. GNH supervised the experiments, corrected and approved the manuscript for publishing.

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Correspondence to Hariharan Gopalasamudram Neelakantan.

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Shanmugam, K., Srinivasan, M. & Neelakantan, H.G. Insights into in vitro phenotypic plasticity, growth and secondary metabolites of the mycobiont isolated from the lichen Platygramme caesiopruinosa. Arch Microbiol 204, 90 (2022). https://doi.org/10.1007/s00203-021-02685-w

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