Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots

  • Ramesh Kumar Kushwaha
  • Sucheta Singh
  • Shiv Shanker Pandey
  • Alok Kalra
  • C. S. Vivek BabuEmail author
Original Paper


Endophytes have been reported from all plant species from different parts of tissue including root, stem and leaves. Here we report, three fungal endophytes, Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) from Withania somnifera, which could enhance withanolides content in leaf and root. Upon treatment with the above endophytes to 4 weeks old plants in field conditions, W. somnifera elicited withanolide A content (97 to 100%) in leaves without considerable changes in withaferin A content. Furthermore, withanolide A content in roots of 5aWF and 10aWF endophyte treated W. somnifera plants increased up to 52% and 65% respectively. Incidentally, expression profile of withanolide and sterol biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were significantly upregulated in 2aWF, 5aWF and 10aWF fungal endophyte treated plants. Besides, modulation of withanolide biosynthetic pathway genes, fungal endophytes also induce a host resistant related gene, NPR1 resulting in 2, 4 and 16 fold expression levels in 2aWF, 10aWF and 5aWF endophyte treatments respectively, compared to control plants. Overall, our results illustrate that application of native-fungal endophytes 2aWF (96.60%), 5aWF (95%) and 10aWF (147%) enhances plant biomass in addition to withanolide content.


Endophytes Withania somnifera Aspergillus terreus Penicillium oxalicum Sarocladium kiliense Withaferin A and Withanolide A 



This work was supported by NWP BSC0117 (XII Five Year Plan Network Project) from the Council of Scientific and Industrial Research (CSIR), India. Authors express sincere thanks to the Director, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India for his support and encouragement. RKK acknowledges Indian Council of Medical Research (ICMR), India for financial assistance in the form of fellowship and contingency grant for research activity. CSV and RKK greatly acknowledges Dr. Dinesh A Nagegowda for providing withanolides standards & primers and Dr. D.K. Venkata Rao for sharing his lab facilities.

Compliance with ethical standards

Conflict of interest

Authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (DOCX 4396 KB)


  1. Ait Barka E, Nowak J, Clement C (2006) Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN. Appl Environ Microbiol 72:7246–7252. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Babiychuk E, Bouvier-Nave P, Compagnon V, Suzuki M, Muranaka T, Van Montagu M, Kushnir S, Schaller H (2008) Albinism and cell viability in cycloartenol synthase deficient Arabidopsis. Plant Signal Behav 3:978–980CrossRefGoogle Scholar
  3. Carroll G (1988) Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 69:2–8. CrossRefGoogle Scholar
  4. Closa M, Vranova E, Bortolotti C, Bigler L, Arro M, Ferrer A, Gruissem W (2010) The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis, and complete loss of FPP synthase activity causes early developmental arrest. Plant J 63:512–525. CrossRefPubMedGoogle Scholar
  5. Dhar N, Razdan S, Rana S, Bhat WW, Vishwakarma R, Lattoo SK (2015) A decade of molecular understanding of Withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: prospects and perspectives for pathway engineering. Front Plant Sci 27:1031. CrossRefGoogle Scholar
  6. Dutt M, Barthe G, Irey M, Grosser J (2015) Transgenic citrus expressing an arabidopsis NPR1 gene exhibit enhanced resistance against Huanglongbing (HLB; Citrus Greening). PLoS ONE 11:e0137134. CrossRefGoogle Scholar
  7. El-Fattah Adnan Dababat A, Alexander Sikora R (2007) Induced resistance by the mutualistic endophyte, Fusarium oxysporum strain 162, toward Meloidogyne incognita on tomato. J Biol Sci Tech 17:969–975CrossRefGoogle Scholar
  8. Fouda AH, Hassan SE, Eid AM, Ewais EE (2015) Biotechnological applications of fungal endophytes associated with medicinal plant Asclepias sinaica (Bioss.). Ann Agric Sci 60:95–104Google Scholar
  9. Franken P (2012) The plant strengthening root endophyte Piriformospora indica: potential application and the biology behind. Appl Microbiol Biotechnol 96:1455–1464. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gao FK, Dai CC, Liu XZ (2010) Mechanisms of fungal endophytes in plant protection against pathogens. Afr J Microbiol Res 4:1346–1351Google Scholar
  11. Gouda S, Das G, Sen SK, Shin HS, Patra JK (2016) Endophytes: a treasure house of bioactive compounds of medicinal importance. Front Microbiol 7:1538. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Grover A, Samuel G, Bisaria VS, Sundar D (2013) Enhanced withanolide production by overexpression of squalene synthase in Withania somnifera. J Biosci Bioeng 115:680–685. CrossRefPubMedGoogle Scholar
  13. Han JY, In JG, Kwon YS, Choi YE (2009) Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng. Phytochemistry 71:36–46. CrossRefPubMedGoogle Scholar
  14. Hankin L, Zucker M, Sands DC (1971) Improved solid medium for the detection and enumeration of pectolytic bacteria. Appl Microbiol 22:205–209PubMedPubMedCentralGoogle Scholar
  15. Hardoim PR, van Overbeek LS, Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hiruma K, Gerlach N, Sacristan S, Nakano RT, Hacquard S, Kracher B, Neumann U, Ramirez D, Bucher M, O’Connell RJ, Schulze-Lefert P (2016) Root endophyte Colletotrichum tofieldiae confers plant fitness benefits that are phosphate status dependent. Cell 165:464–474. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hoffman MT, Gunatilaka MK, Wijeratne K, Gunatilaka L, Arnold AE (2013) Endohyphal bacterium enhances production of indole-3-acetic acid by a foliar fungal endophyte. PLoS ONE 8:e73132. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Holmberg N, Harker M, Wallace AD, Clayton JC, Gibbard CL, Safford R (2003) Co-expression of N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase and C24-sterol methyltransferase type 1 in transgenic tobacco enhances carbon flux towards end-product sterols. Plant J 36:12–20CrossRefGoogle Scholar
  19. Hosny MH, Farouk HH (2012) Protective effect of Withania somnifera against radiation-induced hepatotoxicity in rats. Ecotoxicol Environ Saf 80:14–19. CrossRefGoogle Scholar
  20. Jadaun JS, Sangwan NS, Narnoliya LK, Singh N, Bansal S, Mishra S, Sangwan RS (2016) Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis. Physiol Plantarum 59:381–400. CrossRefGoogle Scholar
  21. Khan R, Shahzad S, Choudhary MI, Khan SA, Ahmad A (2010) Communities of endophytic fungi in medicinal plant Withania somnifera. Pak J Bot 42:1281–1287Google Scholar
  22. Khan AL, Hamayun M, Kang SM, Kim YH, Jung HY, Lee JH, Lee IJ (2012) Endophytic fungal association via gibberellins and indole acetic acid can improve plant growth under abiotic stress: an example of Paecilomyces formosus LHL10. BMC Microbiol 12:3. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Khan AR, Ullah I, Waqas M, Shahzad R, Hong SJ, Park GS, Jung BK, Lee IJ, Shin JH (2015) Plant growth-promoting potential of endophytic fungi isolated from Solanum nigrum leaves. World J Microbiol Biotechnol 31:1461–1466. CrossRefPubMedGoogle Scholar
  24. Kumar A, Patil D, Rajamohanan PR, Ahmad A (2012) Isolation, purification and characterization of vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus. PLoS ONE 8:e71805. CrossRefGoogle Scholar
  25. Kumar S, Kaushik N, Proksch P (2013) Identification of antifungal principle in the solvent extract of an endophytic fungus Chaetomium globosum from Withania somnifera. Springerplus 2:37. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kusari S, Verma VC, Lamshoeft M, Spiteller M (2012) An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin. World J Microbiol Biotechnol 28:1287–1294. CrossRefPubMedGoogle Scholar
  27. Lata R, Chowdhury S, Gond SK, White JF Jr (2018) Induction of abiotic stress tolerance in plants by endophytic microbes. Lett Appl Microbiol 66:268–276. CrossRefGoogle Scholar
  28. Leon-Reyes A, Spoel SH, De Lange ES, Abe H, Kobayashi M, Tsuda S, Millenaar FF, Welschen RA, Ritsema T, Pieterse CM (2009) Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling. Plant Physiol 149:1797–1809. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lichtenthaler HK, Wellburn AR (1971) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11:591–592. CrossRefGoogle Scholar
  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆Ct method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  31. Maggini V, De LM, Mengoni A, Gallo ER, Miceli E, Reidel RVB, Biffi S, Pistelli L, Fani R, Firenzuoli F, Bogani P (2017) Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model. Sci Rep 7:16924. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J (2009) Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 14:2373–2393. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mishra S, Bansal S, Mishra B, Sangwan RS, Asha, Jadaun JS, Sangwan NS (2016) RNAi and homologous over-expression based functional approaches reveal triterpenoid synthase gene-cycloartenol synthase is involved in downstream withanolide biosynthesis in Withania somnifera. PLoS ONE 11: e0149691. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mishra A, Singh SP, Mahfooz S, Singh SP, Bhattacharya A, Mishra N, Nautiyal CS (2018a) Endophyte-mediated modulation of defense-responsive genes and systemic resistance in Withania somnifera (L.) Dunal under Alternaria alternata stress. Appl Environ Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Mishra A, Singh SP, Mahfooz S, Bhattacharya A, Mishra N, Shirke PA, Nautiyal CS (2018b) Bacterial endophytes modulates the withanolide biosynthetic pathway and physiological performance in Withania somnifera under biotic stress. Microbiol Res 212–213:17–28CrossRefGoogle Scholar
  36. Molitor A, Kogel KH (2009) Induced resistance triggered by Piriformospora indica. Plant Signal Behav 4:215–216CrossRefGoogle Scholar
  37. Möller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20:6115–6116CrossRefGoogle Scholar
  38. Ngamau CN, Matiru VN, Tani A, Muthuri CW (2014) Potential use of endophytic bacteria as biofertilizer for sustainable banana (Musa spp.) production. Afr J Hort Sci 8:1–11Google Scholar
  39. Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, Dowling DN (2015) Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front Microbiol 6:745. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Pal S, Yadav AK, Singh AK, Rastogi S, Gupta MM, Verma RK, Nagegowda DA, Pal A, Shasany AK (2016) Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal. Protoplasma 254:389–399 CrossRefPubMedGoogle Scholar
  41. Pandey V, Niranjan A, Atri N, Chandrashekhar K, Mishra MK, Trivedi PK, Misra P (2014) WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco. Planta 239:1217–1231. CrossRefPubMedGoogle Scholar
  42. Pandey SS, Singh S, Babu CS, Shanker K, Srivastava NK, Kalra A (2016a) Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids. Planta 243:1097–1114. CrossRefPubMedGoogle Scholar
  43. Pandey SS, Singh S, Babu CS, Shanker K, Srivastava NK, Shukla AK, Kalra A (2016b) Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis. Sci Rep 6:26583. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Patel N, Patel P, Kendurkar SV, Thulasiram HV, Khan BM (2015) Overexpression of squalene synthase in Withania somnifera leads to enhanced withanolide biosynthesis. Plant Cell Tissue Organ Cult 122:409–420CrossRefGoogle Scholar
  45. Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiologiya 17:362–370Google Scholar
  46. Qadri M, Johri S, Shah BA, Khajuria A, Sidiq T, Lattoo SK, Abdin MZ, Riyaz-Ul-Hassan S (2013) Identification and bioactive potential of endophytic fungi isolated from selected plants of the Western Himalayas. Springerplus 2:8. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Raghavan A, Shah ZA (2015) Withania somnifera: a pre-clinical study on neuroregenerative therapy for stroke. Neural Regen Res 10:183–185. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rai M, Acharya D, Singh A, Varma A (2001) Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 11:123–128. CrossRefPubMedGoogle Scholar
  49. Rana S, Bhat WW, Dhar N, Pandith SA, Razdan S, Vishwakarma R, Lattoo SK (2014) Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations. BMC Biotechnol 14:89. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rodriguez RJ, Henson J, Van VE, Hoy M, Wright L, Beckwith F, Kim YO, Redman RS (2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416. CrossRefPubMedGoogle Scholar
  51. Saema S, ur Rahman L, Niranjan A, Ahmad IZ, Misra P (2015) RNAi-mediated gene silencing of WsSGTL1 in W. somnifera affects growth and glycosylation pattern. Plant Signal Behav 10:e1078064. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Salini TS, Dibu D, Shabanamol S, Sharrel R, Jisha MS (2014) Antimicrobial and immunomodulatory potential of endophytic fungus fusarium solani isolated from Withania somnifera. WJPR 10:879–890Google Scholar
  53. Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686CrossRefGoogle Scholar
  54. Seo JW, Jeong JH, Shin CG, Lo SC, Han SS, Yu KW, Harada E, Han JY, Choi YE (2005) Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 66:869–877. CrossRefPubMedGoogle Scholar
  55. Singh S, Pal S, Shanker K, Chanotiya CS, Gupta MM, Dwivedi UN, Shasany AK (2014) Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis. Physiol Plant 152:617–633. CrossRefPubMedGoogle Scholar
  56. Singh AK, Dwivedi V, Rai A, Pal S, Reddy SG, Rao DK, Shasany AK, Nagegowda DA (2015) Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance. Plant Biotechnol J 13:1287–1299. CrossRefPubMedGoogle Scholar
  57. Singh G, Tiwari M, Singh SP, Singh S, Trivedi PK, Misra P (2016) Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera. Sci Rep 6:25562. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sivanandhan G, Dev GK, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114:121–129CrossRefGoogle Scholar
  59. Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M (2014) Improved production of withanolides in shoot suspension cultureof Withania somnifera (L.) Dunal by seaweed extracts. Plant Cell Tissue Organ Cult 119:221–225CrossRefGoogle Scholar
  60. Teather RM, Wood PJ (1982) Use of Congo redpolysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780PubMedPubMedCentralGoogle Scholar
  61. Tenguria RK, Khan NF (2015) Biodiversity of endophytic fungi in Withania Somnifera leaves of panchmarhi biosphere reserve, Madhya Pradesh. JIPBS 2:222–228Google Scholar
  62. Toward Sustainable Agricultural Systems in the 21st Century (2010) The National Academies Press, 500 Fifth Street, NW, Washington, DC, pp 624–6242Google Scholar
  63. Trivedi MK, Panda P, Sethi KK, Jana S (2016) Metabolite profiling of Withania somnifera roots hydroalcoholic extract using LC-MS, GC-MS and NMR spectroscopy. Chem Biodivers. CrossRefGoogle Scholar
  64. Van Deenen N, Bachmann AL, Schmidt T, Schaller H, Sand J, Prufer D, Schulze GC (2011) Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Mol Biol Rep 39:4337–4349. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Ramesh Kumar Kushwaha
    • 1
    • 3
  • Sucheta Singh
    • 2
    • 3
  • Shiv Shanker Pandey
    • 3
  • Alok Kalra
    • 2
    • 3
  • C. S. Vivek Babu
    • 1
    • 3
    Email author
  1. 1.Microbial Technology LaboratoryCSIR-Central Institute of Medicinal and Aromatic Plants, Research CentreBangaloreIndia
  2. 2.Microbial Technology DivisionCSIR-Central Institute of Medicinal and Aromatic PlantsLucknowIndia
  3. 3.Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Institute of Medicinal and Aromatic PlantsLucknowIndia

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