Trichoderma strains- Silybum marianum hairy root cultures interactions

Document Type: Original paper


1 Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.

2 Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran. Department of Biology, Faculty of Science, Kharazmi University, Karaj, Iran.

3 Department of Microbial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.


Background and objectives: Silymarin is a unique flavonoid complex with documented hepatoprotective properties. Silybum  marianum hairy root culture as a source for producing silymarin has been an important strategy for study the cell signaling pathway. In the present investigation Trichoderma strains- Silybum marianum hairy root cultures interactions have been studied. Methods: The effects of two Trichoderma Strains (KHB and G46-7) (0, 0.5, 1, 2 and 4 mg/ 50 mL culture) in 6 different exposure times (0, 24, 48, 72, 96 and 120 h) have been investigated on flavonolignans production. The flavonolignans were analyzed by High Performance Liquid Chromatography method. Cell signaling pathway was evaluated by determination of H2O2 content, peroxidase and ascorbate peroxidase activities. Results:The elicitation effects of two Trichoderma Strains (KHB and G46-7) were examined on flavonolignans accumulation and the activation of cell defense system in S. marianum hairy root cultures. The results indicated that the highest silymarin accumulation (0.45 and 0.33 mg/g DW) was obtained in media elicited with 0.5 mg/50 mL cultures of T. harzianum Strains (KHB and G46-3, respectively) after 120 h. Feeding time experiments indicated that a significant higher content of silymarin production was achieved after 120 and 72 h in media treated with 0.5 mg/50 mL cultures of KHB and G46-3, respectively. Our results showed that S. marianum treated by KHB strain, increased taxifolin, silychristin, isosilybin and silydianin productions significantly. The H2O2 content in the control hairy root cultures remained lower than the treated cultures. There was significant enhancement in both peroxidase and ascorbate peroxidase activities in treated hairy roots reaching a peak after 72 h. Conclusion: These findings suggested that some Trichoderma strains are positive elicitors for promoting silymarin accumulation in S. marianum hairy root cultures. The results also suggested the presence of H2O2 and oxidative burst induced by T. harzianum as a signaling pathway.


[1] Křen V, Daniela Walterova D. Silybin and silymarin – new effects and applications. Biomed Papers. 2005; 149: 29–41.

[2] Gazák R, Walterová D, Kren V. Silybin and silymarin- new and emerging applications in medicine. Curr Med Chem. 2007; 14: 315-338.

[3] Féher J, Lengyel G. Silymarin in the prevention and treatment of liver diseases and primary liver cancer. Curr Pharm Biotechnol. 2012; 13(1): 210-217.

[4] Deep G, Oberlies NH, Kroll DJ, Agarwal R. Identifying the differential effects of silymarin constituents on cell growth and cell cycle regulatory molecules in human prostate cancer cells. Int J Cancer. 2008; 123: 41-50.

[5] Sanchez-Sampedro MA, Fernandez-Tarago J, Corchete P. Yeast extract and methyl jasmonate induced silymarin production in cell culture of Silybum marianum L. Gaerth. J Biotechnol. 2005; 119: 60-69.

[6] Hasanloo T, Khavari-Nejad RA, Majidi E, Shams-Ardakani MR. Flavonolignan production in cell suspension culture of Silybum marianum (L.) Gaertn. Pharm Biol. 2008; 46: 1-6.

[7]  Rahnama H, Hassanloo T, Shams MR, Sepehrifar R. Silymarin production in hairy root culture of Silybum marianum (L.) Gaetn. Iran J Biotechnology. 2008; 6:113-118.

[8] Tumova L, Gallova K, Rimakova J. Silybum marianum, in vitro. Ceska Slov Farm. 2004;53: 135-140.

[9] Elwekeel A, AbouZid S, Sokkar N, Elfishway A. Studies on flavanolignans from cultured cells of Silybum marianum. Acta Physiol Plant. 2012; 34: 1445- 1449.

[10] Rajendran L, Suvarnalatha G, Ravishankar GA, Venkataraman LV. Enhancement of anthocyanin production in callus cultures of Daucus carota L. under the influence of fungal elicitors. Appl Microbial Biot. 1994;42: 227–231.

[11] Namdeo AG. Plant cell elicitation for production of secondary metabolites: A review. Pharmacog Rev. 2007;1: 69-79.

[12] Rahimi Ashtiani S, Hasanloo T, Bihamta MR. Enhanced production of silymarin by Ag+ elicitor in cell suspension cultures of Silybum marianum. Pharm Biology. 2009; 48: 708-715.

[13] Rahimi Ashtiani S, Hasanloo T, Sepehrifar R, Bihamta MR. Elicitation of silymarin production in cell suspension culture of Silybum marianum (L.) Gaertn. Pharm Sciences. 2012; 4: 253-266.

[14] Bonhomme V, Laurain-Matter D, Lacoux J, Fliniaux MA, Jacquin-Dubreuil A. Tropan alkaloid production by hairy roots of Atropa belladona obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only. J Biotechnol. 2000; 81: 151-158.

[15] Georgiev MI, Agostini E, Ludwig-Müller J, Xu J. Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol. 2012; 30: 528-537.

[16] Dhakulkar S, Bhargava S, Ganapathi TR, Bapat VA. Induction of hairy roots in Gmelina arborea Roxb. using Agrobacterium rhizogenes. BARC Newsletter, Founder’s Day Special Issue. 2005; 100-106.

[17] Ming Q, Su C, Zheng C, Jia M, Zhang Q, Zhang H, Rahman K, Han T, Qin L. Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis. J Exp Bot. 2013;64: 5687-5694.

[18] Savitha BC, Thimmaraju R, Bhagyalakshmi N, Ravishankar GA. Different biotic and abiotic elicitors influence betalain production in hairy root cultures of Beta vulgaris in shake-flask and bioreactor. Process Biochem. 2006; 41: 50–60.

[19] Eskandari Samet A, Piri Kh, Kayhanfar M, Hasanloo T. Influence of jasmonic acids, yeast extract and salicylic acid on growth and accumulation of hyosciamine and scopolamine in hairy root cultures of Atropa Belladonna L. Int J Agric Res Rev. 2012; 2: 403- 409.

[20] Sudha G, Ravishankar GA. Involvement and interaction of various signaling compounds on the plant metabolic events during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects. Plant Cell Tiss Org. 2002; 71: 181-212.

[21] Conrath U, Domard A, Kauss H. Chitosan elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures. Plant Cell Rep. 1989; 8: 152–155.

[22] Tyler RT, Eilert U, Rijnders COM, Roewe IA, Mcnabb CK, Kurz WGW. Studies on benzophenanthrid in alkaloid production in elicited cell cultures of Papaver somniferum L. In: Kurz,W.G.W. ed. Primary and Secondary Metabolism of Plant Cell Cultures. Berlin: Springer-Verlag, 1989.

[23] Khalili M, Hasanloo T, KazemiTabar SK, Rahnama H. Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum. Cell Biol Int. 2009; 33: 988-994.

[24] Khalili M, Hasanloo T, Kazemi Tabar SK. Ag+ enhanced silymarin production in hairy root cultures of Silybum marianum (L.) Greatn. Plant Omics. 2010; 3: 109-114.

[25] Rahimi S, Hasanloo T, Najafi F, Khavari-Nejad RA.Methyl Jasmonate Influences on Silymarin Production and Plant Stress Responses in Silybum marianum Hairy Root Cultures in Bioreactor.Nat Prod Res. 2011; 26: 1662- 1667.

[26] Rahimi S, Hasanloo T, Najafi F, Khavari-Nejad RA. Enhancement of silymarin accumulation using precursor feeding in Silybum marianum hairy root cultures. Plant Omics. 2011; 4: 34-39.

[27] Schumache HM, Gundlach H, Fiedler F, Zenk MH. Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures. Plant Cell Rep. 1987; 6: 410-413.

[28] Wang JW, Kong FX, Tan RX. Improved artemisinin accumulation in hairy root cultures of Artemisia annua by (22S, 23S)––homobrassinolide. Biotechnol Lett. 2002; 24: 1573– 1577.

[29] Polle A, Rennenberg H. Significance of antioxidants in plant adaptation to environmental stress. In: Mansfield T, Fowden L, Stoddard F, ed. Plant Adaptation to Environmental Stress. London: Chapman and Hall, 1993.

[30] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Bioch.  2010; 48: 909- 930.

[31] Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley CM. Trichoderma research in the genome era.  Phytopathology. 2013; 51: 105-129.

[32] Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma species opportunistic, avirulent plant symbionts. Nat Rev Microbiol. 2004; 2: 43-56.

[33] Yedidia I, Shoresh M, Kerem Z, Benhamou N, Kapulnik Y, Chet I. Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Appl Environ Microb. 2003; 69: 7343-7353.

[34] Hanson LE, Howell CR. Elicitors of plant defense responses from biocontrol strains of Trichoderma virens. J Phytopathol. 2004; 94: 171–176.

[35] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15: 473–497.

[36] Kubicek CP, Komon-Zelazowska M, Druzhinina IS. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. J Zhejiang Univ Sc-B. 2008; 9: 753-763.

[37] Chong TM, Abdullah MA, Lai OM, Nor’aini FM, Lajis NH. Effective elicitation factors in Morindaelliptica cell suspension culture. Process Biochem. 2005; 40: 3397-3405.

[38] Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines. Plant Sci. 2000; 151: 59–66.

[39] Chance B, Maehly AC. Assay of catalases and peroxidases, In Methods in enzymology. Academic Press. 1955; 2: 764–775.

[40] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.1976; 248–254.

[41] Nakano Y, Asada K. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 1987; 28: 131–140.

[42] Bhattacharjee S. Reactive oxygen species and oxidative burst: Roles in stress, senescence and signal transduction in plants. Current Science. 2005; 8: 1113- 1121.

[43] Wu J, Ge X. Oxidative burst jasmonic acid biosynthesis and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures. Biotechnol Bioeng. 2004; 85: 714-721.

[44] GoâMez-VaâSquez R, Day R, Buschmann H, Randles S, John R, Beeching J,  Cooper RM. Phenylpropanoids. Phenylalanine Ammonia Lyase and Peroxidases in Elicitor-challenged Cassava (Manihot esculenta) Suspension Cells and Leaves. Ann Bot-London. 2004; 94: 87-97.

[45] Low PS, Merida JR. The oxidative burst in plant defense: function and signal transduction. Physiol Plant. 1996; 96: 533–542.

[46] Karwasara VS, Tomar P, Dixit VK. Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Lin. Pharmacogn Mag. 2011; 7: 307–313.

[47] Radman R, Saez T, Bucke C, Keshavarz T. Elicitation of plants and microbial cell systems. Appl Biochem Biotech.  2003; 37: 91-102.

[48] Mehdy MC.Active oxygen species in plant defense against pathogens. Plant hysiol. 1994; 105: 467-472.

[49] Navazio L, Baldan B, Moscatiello R, Zuppini A, Woo SL, Mariani P, Lorito M. Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biol. 2007; 7: 41-50.

[50] Garcia-Brugger A, Lamotte O, Vandelle E, Bourque S, Lecourieux D, Poinssot B, Wendehn D, Pugin A. Early signaling events induced by elicitors of plant defenses. Mol Plant Microbe In. 2006; 19: 711-724.

[51] Zhu LW, Zhong JJ, Tang YJ. Significance of fungal elicitors on the production of ganoderic acid and Ganoderma polysaccharides by the submerged culture of medicinal mushroom Ganoderma lucidum. Process Biochem. 2008; 43: 1359-1370.

Djonović S, Pozo MJ, Dangott, LJ, Howell CR, Kenerly CM, Sm A. proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance. Mol Plant-Microbe Interact. 2006; 19: 838-853