Advertisement

Microbial Ecology

, Volume 78, Issue 4, pp 914–926 | Cite as

Functional Fungal Endophytes in Coleus forskohlii Regulate Labdane Diterpene Biosynthesis for Elevated Forskolin Accumulation in Roots

  • Anthati Mastan
  • RKB Bharadwaj
  • Ramesh Kumar Kushwaha
  • Chikkarasanahalli Shivegowda Vivek BabuEmail author
Plant Microbe Interactions

Abstract

Coleus forskohlii is a perennial medicinal shrub cultivated mainly for its forskolin content. The plant has been used since ancient times in ayurvedic traditional medicines for the treatment of hypertension, glaucoma, asthma, congestive heart failures, obesity, and cancer. Use of endophytic microorganisms presents a special interest for the development of value-added bioactive compounds through agriculture. Limited investigations have been undertaken on in planta enhancement of forskolin content using endophytic fungus in sustainable agriculture. Here we report specific roles of three fungal endophytes, Fusarium redolens (RF1), Phialemoniopsis cornearis (SF1), and Macrophomina pseudophaseolina (SF2), functionally acting as plant probiotic fungus, regulating secondary metabolite (forskolin) biosynthesis in C. forskohlii. The root endophyte, RF1, and shoot endophytes, SF1 and SF2, were found to enhance forskolin content by 52 to 88% in pot and 60 to 84% in field experiments as compared to uninoculated control plants. The three endophytes also enhanced total biomass owing to plant growth promoting properties. The expression of diterpene synthases (CfTPSs) like CfTPS1, CfTPS2, CfTPS3, and CfTPS4 were significantly upregulated in endophyte-treated C. forskohlii plants. Elevated expression of key diterpene synthases (CfTPS2) in the forskolin biosynthesis pathway, exclusively present in the root cork of C. forskohlii, was observed following SF2 endophyte treatment. Furthermore, endophyte treatments conferred a variety of antagonistic activity against nematode galls (80%) and plant pathogens like Fusarium oxysporum, Colletotricum gloeosporioides, and Sclerotium rolfsii. RF1 and SF1 fungal endophytes showed positive for IAA production; however, SF1 also indicated phosphate solubilization activity. Overall, the qualitative and quantitative improvement of in planta forskolin enhancement represents an area of high commercial interest, and hence, our work focused on novel insights for the application of three fungal endophytes for in planta enhancement of forskolin content for C. forskohlii cultivation by a sustainable approach.

Keywords

Fungal endophyte Forskolin Coleus forskohlii Agriculture Diterpene synthases 

Notes

Acknowledgments

The authors wish to thank the Director, CSIR- Central Institute of Medicinal and Aromatic Plants, Lucknow, India, for providing necessary facilities and encouragement during the course of investigation.

Funding Information

This work was supported by, Council of Scientific and Industrial Research (CSIR), India funded projects BSC 117 and BSC-203. Department of Science and Technology (DST –INSPIRE), Council of Scientific and Industrial Research (CSIR), and Indian Council of Medical Research (ICMR), New Delhi, India, provided financial support in the form of fellowships to A.M., R.K.B.B., and R.K.K., respectively.

Supplementary material

248_2019_1376_MOESM1_ESM.docx (2 mb)
ESM 1. Effect of endophytes RF1, SF1 and SF2 on C. forskohlii plants grown for 180 daysin pot condition and comparison with control plant. (DOCX 2084 kb)
248_2019_1376_MOESM2_ESM.docx (3.2 mb)
ESM 2. Effect of selected endophytes (RF1, SF1 and SF2) on plant growth response of C. forskohlii in field condition. The control plants bed (a), RF1 (b), SF1 (c), and SF2 (d). The photographic images (Beds) represent the field view of endophytes treated C. forskohlii plants (n = 16) at the time of harvesting. (DOCX 3290 kb)
248_2019_1376_MOESM3_ESM.docx (3.2 mb)
ESM 3. The three fungal endophytes isolated from Coleus forskohlii which could regulates biosynthesis of forskolin in roots. (DOCX 3318 kb)
248_2019_1376_MOESM4_ESM.docx (336 kb)
ESM 4. Analysis of forskolin content in fresh roots of endophyte treated and control plants under pot condition. Std: forskolin standard, M: mixture of forskolin types (If: isoforskolin, 7d: 7 deacetyl forskolin, FK: forskolin and 1,9 DF: 1,9 dideoxy forskolin), C : control, 1: RB1, 2: LB1, 3: RF1, 4: RF2, 5: RF3, 6: RF4, 7: SF1, 8: SF2, 9: SF3, 10: SF5 and 11: LF1. (DOCX 335 kb)
248_2019_1376_MOESM5_ESM.docx (637 kb)
ESM 5. Quantification of forskolin by HPLC method. (DOCX 636 kb)
248_2019_1376_MOESM6_ESM.docx (4.3 mb)
ESM 6. Antagonistic activity of endophytes [E1-Fusarium redolens (RF1), E2- Phialemoniopsis cornearis (SF1) and E3-Macrophomina pseudophaseolina (SF2)] against potent plant pathogens (P1- F. oxysporum, P2- S. rolfsiiand P3-C. gloeosporioides). (DOCX 4413 kb)
248_2019_1376_MOESM7_ESM.docx (15 kb)
ESM 7. List of primers used in this study. (DOCX 14 kb)
248_2019_1376_MOESM8_ESM.docx (14 kb)
ESM 8. Colonization of endophytes in C. forskohlii plants (DOCX 14 kb)
248_2019_1376_MOESM9_ESM.docx (14 kb)
ESM 9. NCIM accession numbers of fungal endophytes and their NCBI GenBank accession number(s) for ITS nucleotide sequence(s). (DOCX 14 kb)

References

  1. 1.
    Rho H, Hsieh M, Kandel SL, Cantillo J, Doty SL, Kim SH (2017) Do endophytes promote growth of host plants under stress? A meta-analysis on plant stress mitigation by endophytes. MicrobEcol 75:407–418Google Scholar
  2. 2.
    Chowdhary K, Kaushik N (2015) Fungal endophyte diversity and bioactivity in the indian medicinal plant Ocimum sanctum Linn. PLoS One 10:e0141444PubMedPubMedCentralGoogle Scholar
  3. 3.
    Turner TR, James EK, Poole PS (2013) The plant microbiome. Genome Biol 14:209PubMedPubMedCentralGoogle Scholar
  4. 4.
    Wielkopolan B, Obrępalska-Stęplowska A (2016) Three-way interaction among plants, bacteria, and coleopteran insects. Planta 244:313–332PubMedPubMedCentralGoogle Scholar
  5. 5.
    Pandey SS, Singh S, Babu CS, Shanker K, Srivastava NK, Kalra A (2016) Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids. Planta 243:1097–1114PubMedGoogle Scholar
  6. 6.
    Loftus HL, Astell KJ, Mathai ML, Su XQ (2015) Coleus forskohlii extract supplementation in conjunction with a hypocaloric diet reduces the risk factors of metabolic syndrome in overweight and obese subjects: a randomized controlled trial. Nutrients 7:9508–9522PubMedPubMedCentralGoogle Scholar
  7. 7.
    Schneyer CR, Piiieyro MA, Gregerman RI (1983) Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes. Life Sci 33:275–279PubMedGoogle Scholar
  8. 8.
    Ahmad S, Rizwan M, Parveen R, Mujeeb M, Aquil M (2008) A validated stability-indicating TLC method for determination of forskolin in crude drug and pharmaceutical dosage form. Chroma 67:441–447Google Scholar
  9. 9.
    Singh R, Soni SK, Kalra A (2013) Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions. Mycorrhiza1:35–44Google Scholar
  10. 10.
    Laurberg P (1984) Forskolin stimulation of thyroid secretion of T4 and T3. FEBS Lett 170:273–276PubMedGoogle Scholar
  11. 11.
    Henderson S, Magu B, Rasmussen C, Lancaster S, Kerksick C, Smith P, Melton C, Cowan P, Greenwood M, Earnest C, Almada A, Milnor P, Magrans T, Bowden R, Ounpraseuth S, Thomas A, Kreider RB (2005) Effects of Coleus forskohlii supplementation on body composition and hematological profiles in mildly overweight women. J Int Soc Sports Nutr 2:54–62PubMedPubMedCentralGoogle Scholar
  12. 12.
    Mariya P, Radha A, Suresh Kumar D (2013) On the high value medicinal plant, Coleus forskohlii Briq. Hygeia J D Med 5:64–73Google Scholar
  13. 13.
    Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089PubMedGoogle Scholar
  14. 14.
    Bhat SV, Dohadwalla AN, Bajwa BS, Dadkar NK, Dornauer H, de Souza NJ (1983) The antihypertensive and positive inotropic diterpene forskolin: effects of structural modifications on its activity. J Med Chem (4):486–492PubMedGoogle Scholar
  15. 15.
    Nishizawa Y, Seamon KB, Daly JW, Aronstam RS (1990) Effects of forskolin and analogues on nicotinic receptor-mediated sodium flux, voltage-dependent calcium flux, and voltage-dependent rubidium efflux in pheochromocytoma PC12 cells. Cell MolNeurobiol 3:351–368Google Scholar
  16. 16.
    De Souza NJ, Dohadwalla AN, Reden J (1983) Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory and adenylate cyclase activating properties. Med Res Rev 2:201–219Google Scholar
  17. 17.
    Singh R, Kalra A, Ravish BS, Divya S, Parameswaran TN, Srinivas KVNS, Bagyaraj DJ (2012) Effect of potential bioinoculants and organic manures on root-rot and wilt, growth, yield and quality of organically grown Coleus forskohlii in a semiarid tropical region of Bangalore (India). Plant Pathol 61:700–708Google Scholar
  18. 18.
    Bhattacharya A (2013) Adaptive expression of host cell wall degrading enzymes in fungal disease: an example from Fusarium root rot of medicinal Coleus. Pak J BiolSci 16:2036–2040Google Scholar
  19. 19.
    Santamaria O, Lledó S, Rodrigo S, Poblaciones MJ (2017) Effect of fungal endophytes on biomass yield, nutritive value and accumulation of minerals in Ornithopus compressus. MicrobEcol 74:841–852Google Scholar
  20. 20.
    Ming Q, Su C, Zheng C, Jia M, Zhang Q, Zhang H, Rahman K, Han T, Qin L (2013) Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis. J Exp Bot 64:5687–5694PubMedGoogle Scholar
  21. 21.
    Gangopadhyay M, Gantait S, Palchoudhury S, Ali MN, Mondal C, Pal AK (2016) UVC-priming mediated modulation of forskolin biosynthesis key genes against Macrophomina root rot of Coleus forskohlii - a tissue culture based sustainable approach. Phytochem Lett 17:36–44Google Scholar
  22. 22.
    Sessitsch A, Hardoim P, Döring J, Weilharter A, Krause A, Woyke T, Mitter B, Hauberg-Lotte L, Friedrich F, Rahalkar M, Hurek T, Sarkar A, Bodrossy L, van Overbeek L, Brar D, van Elsas JD, Reinhold-Hurek B (2012) Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol Plant Microbe In1:28–36PubMedGoogle Scholar
  23. 23.
    Pateraki I, Andersen-Ranberg J, Hamberger B, Heskes AM, Martens HJ, Zerbe P, Bach SS, Møller BL, Bohlmann J, Hamberger B (2014) Manoyl Oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii. Plant Physiol 164:1222–1236PubMedPubMedCentralGoogle Scholar
  24. 24.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108Google Scholar
  25. 25.
    Krause K, Henke C, Asiimwe T, Ulbricht A, Klemmer S, Schachtschabel D, Boland W, Kothe E (2015) Biosynthesis and secretion of indole-3-acetic acid and its morphological effects on Tricholoma vaccinum-spruce ectomycorrhiza. Appl Environ Microbiol 81:7003–7011PubMedPubMedCentralGoogle Scholar
  26. 26.
    Meng X, Yan D, Long X, Wang C, Liu Z, Rengel Z (2014) Colonization by endophytic OchrobactrumanthropiMn1 promotes growth of Jerusalem artichoke. Microb Biotechnol (6):601–610PubMedPubMedCentralGoogle Scholar
  27. 27.
    Goswami D, Dhandhukia P, Patel P, Thakker JN (2014) Screening of PGPR from saline desert of Kutch: growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiol Res 169:66–75PubMedGoogle Scholar
  28. 28.
    Elias F, Woyessa D, Muleta D (2016) Phosphate solubilization potential of rhizosphere fungi isolated from plants in jimma zone, southwest ethiopia. Int J Microbiol 2016:5472601.  https://doi.org/10.1155/2016/5472601 Google Scholar
  29. 29.
    Naik PR, Raman G, Narayanan KB, Sakthivel N (2008) Assessment of genetic and functional diversity of phosphate solubilizing fluorescent Pseudomonads isolated from rhizospheric soil. BMC Microbiol 8:230PubMedPubMedCentralGoogle Scholar
  30. 30.
    Prapagdee B, Kuekulvong C, Mongkolsuk S (2018) Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi. Int J BiolSci 4:330–337Google Scholar
  31. 31.
    Rahman MA, Begum MF, Alam MF (2009) Screening of Trichoderma isolates as a biological control agent against Ceratocystisparadoxa causing pineapple disease of sugarcane. Mycobiology 37:277–285PubMedPubMedCentralGoogle Scholar
  32. 32.
    Lahlali R, Hijri M (2010) Screening, identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants. FEMS Microbiol Lett 311:152–159PubMedGoogle Scholar
  33. 33.
    Kanne H, Burte NP, Prasanna V, Gujjula R (2015) Extraction and elemental analysis of Coleus forskohlii extract. Pharm Res 7:237–241Google Scholar
  34. 34.
    Nidiry ESJ, Ganeshan G, Lokesha AN (2015) Antifungal activity of the extractives of Coleus forskohlii roots and forskolin. Pharm Chem J 49:624–626Google Scholar
  35. 35.
    Robert-Seilaniantz A, Grant M, Jones JD (2011) Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev Phytopathol 49:317–343PubMedGoogle Scholar
  36. 36.
    Pandey SS, Singh S, Babu CS, Shanker K, Srivastava NK, Shukla AK, Kalra A (2016) Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis. Sci Rep 6:26583PubMedPubMedCentralGoogle Scholar
  37. 37.
    Das A, Kamal S, Shakil NA, Sherameti I, Oelmüller R, Dua M, Tuteja N, Johri AK, Varma A (2012) The root endophyte fungus Piriformosporaindica leads to early flowering, higher biomass and altered secondary metabolites of the medicinal plant, Coleus forskohlii. Plant Signal Behav 7:103–112PubMedPubMedCentralGoogle Scholar
  38. 38.
    Das A, Tripathi S, Varma A (2014) In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica. World J Microbiol Biotechnol 30:1075–1084PubMedGoogle Scholar
  39. 39.
    Garyali S, Kumar A, Reddy MS (2013) Taxol production by an endophytic fungus, Fusarium redolens, isolated from Himalayan yew. J Microbiol Biotechnol 23:1372–1380PubMedGoogle Scholar
  40. 40.
    Venugopalan A, Potunuru UR, Dixit M, Srivastava S (2016) Effect of fermentation parameters, elicitors and precursors on camptothecin production from the endophyte Fusarium solani. Bioresour Technol 206:104–111PubMedGoogle Scholar
  41. 41.
    Clay K (1987) Effects of fungal endophytes on the seed and seedling biology of Loliumperenne and Festucaarundinacea. Oecologia 73:358–362PubMedGoogle Scholar
  42. 42.
    Hiruma K, Gerlach N, Sacristán S, Nakano RT, Hacquard S, Kracher B, Neumann U, Ramírez D, Bucher M, O’Connell RJ, Schulze-Lefert P (2016) Root endophyte Colletotrichum tofieldiae confers plant fitness benefits that are phosphate status dependent. Cell 165:464–474PubMedPubMedCentralGoogle Scholar
  43. 43.
    Gupta R, Singh A, Srivastava M, Singh V, Gupta MM, Pandey R (2017) Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress. Sci Rep 7:41867Google Scholar
  44. 44.
    Zamioudis C, Pieterse CM (2012) Modulation of host immunity by beneficial microbes. Mol Plant-Microbe Interact 25:139–150PubMedGoogle Scholar
  45. 45.
    Zhang CL, Zheng BQ, Lao JP, Mao LJ, Chen SY, Kubicek CP, Lin FC (2008) Clavatol and patulin formation as the antagonistic principle of Aspergillus clavatonanicus, an endophytic fungus of Taxus mairei. Appl Microbiol Biotechnol 78:833–840PubMedGoogle Scholar
  46. 46.
    Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, Tremblay J, Engelbrektson A, Kunin V, Del Rio TG, Edgar RC, Eickhorst T, Ley RE, Hugenholtz P, Tringe SG, Dangl JL (2012) Defining the core Arabidopsis thaliana root microbiome. Nature 488:86–90PubMedPubMedCentralGoogle Scholar
  47. 47.
    Finkel OM, Castrillo G, Herrera Paredes S, Salas González I, Dangl JL (2017) Understanding and exploiting plant beneficial microbes. Curr Opin Plant Biol 38:155–163PubMedPubMedCentralGoogle Scholar
  48. 48.
    Rabha AJ, Naglot A, Sharma GD, Gogoi HK, Veer V (2014) In vitro evaluation of antagonism of endophytic Colletotrichum gloeosporioides against potent fungal pathogens of Camellia sinensis. Indian J Microbiol 54:302–309PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Anthati Mastan
    • 1
    • 2
  • RKB Bharadwaj
    • 1
    • 2
  • Ramesh Kumar Kushwaha
    • 1
    • 2
  • Chikkarasanahalli Shivegowda Vivek Babu
    • 1
    • 2
    Email author
  1. 1.Microbial Technology LaboratoryCSIR- Central Institute of Medicinal and Aromatic Plants, Research CenterBangaloreIndia
  2. 2.Academy of Scientific and Innovative Research(AcSIR)GhaziabadIndia

Personalised recommendations