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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity ab
a
a
Vivek Ahluwalia , Jitendra Kumar , Virendra S. Rana , Om P. Sati & S. Walia
b
a
a
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi110 012, India
Click for updates
b
Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand246 174, India Published online: 24 Sep 2014.
To cite this article: Vivek Ahluwalia, Jitendra Kumar, Virendra S. Rana, Om P. Sati & S. Walia (2015) Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity, Natural Product Research: Formerly Natural Product Letters, 29:10, 914-920, DOI: 10.1080/14786419.2014.958739 To link to this article: http://dx.doi.org/10.1080/14786419.2014.958739
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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Natural Product Research, 2015 Vol. 29, No. 10, 914–920, http://dx.doi.org/10.1080/14786419.2014.958739
Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity Vivek Ahluwaliaab*, Jitendra Kumara, Virendra S. Ranaa, Om P. Satib and S. Waliaa a
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, India; Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand 246 174, India
b
Downloaded by [New York University] at 14:53 22 April 2015
(Received 26 June 2014; final version received 21 August 2014) This investigation was undertaken to identify the major secondary metabolite, produced by two Trichoderma harzianum strains (T-4 and T-5) with their antifungal activity against phytopathogenic fungi using poison food technique. The ethyl acetate extract was subjected to column chromatography using n-hexane, ethyl acetate and methanol gradually. Chromatographic separation of ethyl acetate extract of T. harzianum (T-4) resulted in the isolation and identification of palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)-furanone (4), stigmasterol (5) and b-sitosterol (6), while T. harzianum (T-5) gave palmitic acid (1), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone (8), 6-pentyl-2Hpyran-2-one (3), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) as major metabolites. Among compounds screened for antifungal activity, compound 10 was found to be most active (EC50 35.9– 50.2 mg mL21). In conclusion, the present investigation provided significant information about antifungal activity and compounds isolated from two different strains of T. harzianum obtained from two different Himalayan locations. Keywords: Trichoderma; secondary metabolite; antifungal activity
1. Introduction Trichoderma species are considered as free living opportunistic plant symbionts and widely used as biofertilisers and biopesticides. Some of the Trichoderma species used as biocontrol agents develop the ability to interact simultaneously with plants and fungal pathogens, thus are used as model microorganisms to study complex and multiple-player plant –microbe interactions (Marra et al. 2006). These species are useful to agriculture due to their ability to parasitise on different pathogenic fungi namely. Botritis spp., Fusarium spp., Pythium spp., Rhizoctonia spp., Verticillium spp. and Sclerotinia spp. Trichoderma species are also widely used for their biological control (Jelen´ et al. 2013). It has been found that the production of secondary metabolite in Trichoderma species is one major reason that contributes to its biological activities during interactions (Sivasithamparam & Ghisalberti 1998; Reino et al. 2008; Vinale et al. 2008). The metabolite production varies according to the species or strain used and their biosynthesis and biotransformation rates (Vinale et al. 2013). More than 200 secondary metabolites have been reported from different species and strains of Trichoderma, some of which have shown promising biological activities (Reino et al. 2008; Vinale et al. 2013). The work on the identification of different secondary metabolites from different species and strains of Trichoderma has continued, but to the best of our knowledge no work has been found
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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on the comparative evaluation of secondary metabolite production from Trichoderma harzianum collected from the Himalayan region of India. This work describes the isolation, characterisation and comparative study of major secondary metabolites produced by two strains (T-4 and T-5) of T. harzianum obtained from two different locations with varied environmental conditions. 2. Results and discussion The structures of the pure compounds (1–11) isolated from the ethyl acetate extract of Trichoderma strains (T-4 and T-5) were determined by using 1H NMR, 13C NMR, DEPT, HMQC, HMBC and ESI-MS data and were identified as palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2) (Liu et al. 2009), 6-pentyl-2H-pyran-2-one (3) (Claydon et al. 1987), 2(5H)-furanone (4), stigmasterol (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7) (Liu et al. 2009), d-decanolactone (8) (Hill et al. 1995), ergosterol (9), harzianopyridone (10) (Dickinson et al. 1989) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) (Slater et al. 1967) (Figure 1).
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OH
O
OH
O OH
13
O
O O 2.
1.
3.
H H
H
H
O H O
H
4.
5. O
H
H HO
HO
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OH H
O
H
H
HO
O 7. MeO
O
H N
O
OH
O
8.
9. OH
O
OH
MeO OH O 10
11
Figure 1. Chemical structures of secondary metabolites isolated from T. harzianum strains T-4 and T-5. Palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)furanone (4), stigmasterol (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone (8), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11).
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Table 1. Antifungal activity# (EC50 mg mL21). Compounds
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1 2 3 4 5 6 7 8 9 10 11 Bavistin
R. solani
S. rolfsii
M. phaseolina
F. oxysporum
498.9 1515.4 64.6 124.3 683 886.8 655.7 70.6 434.2 35.9 529.7 10.7
563.9 1126.5 38.8 237.4 521.8 779.4 542.8 122.2 464.5 42.2 468.8 14.8
623.1 1093.1 90.6 193.8 695.8 490.4 363.6 141.4 449.6 60.4 314.5 13.5
578.8 1619.8 74.3 154.8 635.8 589.0 727.6 124.1 531.7 50.2 356.1 15.8
Pure compounds were further screened for their antifungal activity against four phytopathogenic fungi, namely Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum and the EC50 values of the compounds determined are given in Table 1. Results showed that harzianopyridone (10) was most effective and inhibited more than 90% growth of R. solani, F. oxysporum and S. rolfsii (EC50 35.9– 50.2 mg mL21) but was less active than Bavistin, a standard fungicide (Table 1). Cutler and Jacyno (1991) reported that harzianopyridone was only moderately active against bacteria and relatively inactive against fungi, although Dickinson et al. (1989) have reported significant activity against Botrytis cinerea and R. solani. Vinale et al. (2006) showed that harzianopyridone inhibited the growth of Gaeumannomyces graminis var. tritici, R. solani and Pythium ultimum. Although harzianopyridone has been evaluated for its biological activities, but to our knowledge its activity against phytopathogenic fungi M. phaseolina, S. rolfsii and F. oxysporum has not been reported. Among anthraquinones, 1-hydroxy-3-methylanthraquinone (7) was more active than 1,8-dihydroxy-3-methylanthraquinone (2). Its activity was found highest against M. phaseolina (363.6 mg mL21) and least against F. oxysporum (727.6 mg mL21). Introduction of one hydroxyl function in 1-hydroxy-3-methylanthraquinone (7) led to a decrease in antifungal activity of 1,8dihydroxy-3-methylanthraquinone (2). Our results differ strikingly from an earlier report in which compound 2 was found to be having more fungal growth inhibition against R. solani and B. cinerea and less antibacterial activity against Staphylococcus aureus than compound 7 (Liu et al. 2009). According to Agarwal et al. (2000), 1,8-dihydroxy-3-methylanthraquinone (2) showed encouraging antifungal activity (MIC 25– 250 mg mL21) against Candida albicans, Cryptococcus neoformans, Trichophyton mentagrophytes and Aspergillus fumigatus, while Vinale et al. (2006) reported very weak antifungal activity of both compounds against G. graminis var. tritici, R. solani and P. ultimum. Earlier reports suggested that the same compounds which differ in number and position of hydroxyl groups can have varying antimicrobial properties. These discrepancies could arise due to difference in assays being used, sizes of fungal inoculums, volume of broth or agar, type of broth or agar, size of wells, size of paper discs, strains of a particular bacterial species used and incubation period (Cushnie & Lamb 2005). 6-Methyl-1,3,8-trihydroxyanthraquinone (11) isolated from T-5 strain also showed antifungal activity with EC50 value ranging between 314.5 and 529.7 mg mL21 against tested fungi (Figure 2). It is known to exhibit antimicrobial, antineoplasic and cathartic action (Chukwujekwu et al. 2006; Wu et al. 2007). It also showed monoamine oxidase (Fujimoto et al. 1998) and tyrosine kinase inhibiting activity (Kumar et al. 1998). The compounds with low oxidation state have potential to change to compounds with high oxidation state by the host
EC50 (µg mL–1)
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2000 1800 1600 1400 1200 1000 800 600 400 200 0
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Compounds
Figure 2. Antifungal activity (EC50 mg mL21) of isolated compounds. Palmitic acid# (1), 1,8-dihydroxy-3methylanthraquinone (2), 6-pentyl-2H-pyran-2-one# (3), 2(5H)-furanone (4), stigmasterol# (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone# (8), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11).
reactive oxygen species that are released in response to attack by a microbial pathogen. These high oxidation state compounds have high antimicrobial activity and may increase the effective efficiency of Trichoderma against host resistance to other pathogens when this symbiotically inhabits a plant’s roots system (Liu et al. 2009). 2(5H)-Furanone (4) isolated from strain T-4 caused 52.14 –62.17% fungal growth inhibition at 250 mg mL21 (Figure 3). 6-Pentyl-2H-pyran2-one (3) and d-decanolactone (8) isolated earlier from different species of Trichoderma when evaluated for their potential as antifungal compound had shown good activity (Parker et al. 1997). Our group had previously reported the antifungal activity of palmitic acid (1), 6-pentyl-apyranone (3), stigmasterol (5) and d-decanolactone (8) from Trichoderma koningii (Ahluwalia et al. 2014#) and their activity has also been compared with newly isolated compounds (Figures 2 and 3 and Table 1). 6-Pentyl-2H-pyran-2-one has been reported active against R. solani and F. oxysporum f. sp. lycopersici (Scarselletti & Faull 1994). It was also found to reduce rotting of stored kiwi fruits caused by B. cinerea (Poole et al. 1998).
Figure 3. Fungal growth percentage inhibition of compounds at 250 mg mL21 concentration.
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2.1 Conclusion Eleven compounds were isolated and characterised from the two different T. harzianum strains collected from two separate Himalayan locations, of which harzianopyridone (10) was found to be the most effective, while 1,8-dihydroxy-3-methylanthraquinone (2) was least effective. Harzianopyridone and 6-pentyl-2H-pyran-2-one need to be further evaluated against the more phytopathogenic fungi of agricultural importance to explore their further use as antifungal agents.
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3. Experimental 3.1 General Chemicals and organic solvents used were of chromatographic and analytical grades (Merck India Ltd.). Column chromatography was performed using silica gel (60 –120 mesh) (Merck). NMR spectra of the pure compounds were recorded on Bruker (400 MHz) instrument using tetramethyl silane as internal standard in CDCl3 or DMSO-d6 and ESI-MS data on LC – MS Spectrometer (Thermo-Finnigan LCQ, USA).
3.2 Fungal cultures Two fungal strains of T. harzianum (T-4 and T-5) were obtained from CSK Himachal Pradesh Krishi Vishavidalaya, Palampur, Himachal Pradesh (India). All the fungal strains were maintained on potato dextrose agar (PDA) slants at room temperature and sub-cultured bimonthly. Four phyto-pathogenic fungi, M. phaseolina, R. solani, S. rolfsii and F. oxysporum were procured from Indian Type Culture Collection Centre, Division of Mycology and Plant Pathology, IARI, New Delhi, India.
3.3 Liquid culture and metabolite production Two 7 mm diameter plugs of each T. harzianum strain were taken from actively growing margins of PDA cultures and were inoculated into 5 L conical flasks containing 1 L of sterile liquid broth containing KH2PO4 (7 g L21), K2HPO4 (2 g L21), MgSO4·7H2O (0.1 g L21), (NH2) SO4 (1 g L21), yeast extract (0.6 g L21) and glucose (10 g L21). The stationary cultures were incubated for 28 days at 25 ^ 18C. The mycelium of the fungal strain was removed by filtering through a muslin cloth.
3.4 Isolation and purification of secondary metabolites The culture filtrate (20 L) of strain T-4 was extracted with EtOAc three times at room temperature. The solvent was evaporated in vacuo at 408C to a get red-brown residue (0.28 g L21). The crude extract was subjected to column chromatography using a glass column packed with silica gel (60 –120 mesh) and eluted with n-hexane, and mixture of n-hexane – ethyl acetate and ethyl acetate– methanol with increasing polarity, and 10 fractions were obtained. The elution of the column with different solvents was monitored by TLC and the fractions having similar spots were mixed and dried. Fraction 2 was further subjected to preparative TLC using hexane –ethyl acetate (90:10) as developing solvent to yield 1 (6 mg). Fraction 4 was also subjected to silica gel column chromatography using hexane and ethyl acetate mixture from which compound 2 was obtained (8 mg). Compounds 3 (14 mg) and 4 (3 mg) were isolated from fractions 5 and 6 by repeated column chromatography followed by preparative TLC. Similarly, fraction 7 was column
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chromatographed using petroleum ether –acetone mixture (7:3) followed by preparative TLC from which white crystalline compounds 5 (6 mg) and 6 (3 mg) were obtained. Similarly the culture filtrate of strain T-5 was also extracted with ethyl acetate. The solvent was removed under reduced pressure and ethyl acetate extract (0.32 g L21) was obtained. Column chromatography of the ethyl acetate extract over silica gel (60 –120 mesh) was carried out. The column was packed with n-hexane and eluted with mixture of n-hexane – ethyl acetate and ethyl acetate –methanol with increasing polarity from which 15 fractions were collected. Fraction 3 showed a major spot in TLC and was subjected to preparative-TLC and a pure compound 1 (15 mg) was separated. Fraction 5 was found to contain three spots on the TLC plate which were again subjected to repeated column chromatography over silica gel using n-hexane – ethyl acetate mixture and two pure compounds 7 (11 mg) and 8 (9 mg) were crystallised. Fraction 9 was also subjected to column chromatography using petroleum ether – acetone (6:4) mixture followed by preparative-TLC from which two compounds were isolated. The less polar compound was separated as a colourless oily compound 9 (4 mg) and the more polar compound gave pale yellow compound 3 (4 mg). Fractions 12 and 14 were further purified by preparativeTLC to afford compounds 10 (7 mg) and 11 (12 mg). 3.5 Antifungal activity The antifungal activity of pure secondary metabolites was carried out by poison food technique as described earlier (Sati et al. 2013). Briefly, compound(s) were dissolved in minimum quantity of acetone/DMSO and were added to molten PDA medium (65 mL) to get the concentration of 250, 125, 62.5 and 31.25 mg mL21. Pathogen disc (5 mm) from a 7-day-old culture was inoculated at the centre of each PDA plate. PDA plates treated with acetone/DMSO without compound(s) were used as negative control and those with Bavistinw as a positive control. Each treatment consisted of three replicates and the experiment was repeated twice. EC50 (effective concentration required for 50% inhibition of mycelial growth) of each compound was calculated statistically by Probit analysis. The data were analysed with the help of SAS Package (statistical analysis system package) software. Supplementary material Spectral data of pure compounds can be obtained from the corresponding author. Acknowledgements The authors are thankful to Head, Division of Agricultural Chemicals, IARI, New Delhi, India and Head, Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand, India for providing necessary facilities. Financial assistance from NAIP is duly acknowledged.
References Agarwal SK, Singh SS, Verma S, Kumar S. 2000. Antifungal activity of anthraquinone derivatives from Rheum emodi. J Ethnopharmacol. 72:43–46. Ahluwalia V, Walia S, Sati OP, Kumar J, Kundu A, Shankar J, Paul YS. 2014. Isolation, characterization of major secondary metabolites of Trichoderma koningii and their antifungal activity. Arch Phytopathol Plant Prot. 47:1063–1071. Chukwujekwu JC, Coombes PH, Mulholland DA, Staden J. 2006. Emodin, an antibacterial anthraquinone from the roots of Cassia occidentalis. South Afr J Bot. 72:295–297. Claydon N, Allan M, Hanso JR, Avent AG. 1987. Antifungal alkyl pyrones of Trichoderma harzianum. Trans Br Mycol Soc. 88:503–513. Cushnie TPT, Lamb AJ. 2005. Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 26:343–356.
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Cutler HG, Jacyno JM. 1991. Biological activity of (2) – harzianopyridone isolated from Trichoderma harzianum. Agric Biol Chem. 55:2629– 2631. Dickinson JM, Hanson JR, Hitchcock PB, Claydon N. 1989. Structure and biosynthesis of harzianopyridone, an antifungal metabolite of Trichoderma harzianum. J Chem Soc. 1:1885–1887. Fujimoto H, Satoh Y, Yamaguchi K, Yamazaki M. 1998. Monoamine oxidase inhibitory constituents from Anixiella micropertusa. Chem Pharm Bull. 46:1506–1510. Hill RA, Cutler HG, Parker SR. 1995. Trichoderma and metabolites as control agents for microbial plant diseases. PCT Int Appl 9520879. Jelen´ H, Błaszczyk L, Chełkowski J, Rogowicz K, Strakowska J. 2013. Formation of 6-n-pentyl-2H-pyran-2-one (6-PAP) and other volatiles by different Trichoderma species. Mycol Prog. :1 –12. doi:10.1007/s11557-013-0942-2. Kumar A, Dhawan S, Aggarwal BB. 1998. Emodin (3-methyl-1,6,8-trihydroxy-anthraquinone) inhibits TNF-induced NF-kB activation, IkB degradation, and expression of cell surface adhesion proteins in human vascular endothelial cells. Oncogene. 17:913–918. Liu SY, Lo CT, Shibu MA, Leu YL, Jen BY, Peng KC. 2009. Study on the anthraquinones separated from the cultivation of Trichoderma harzianum strain Th-R16 and their biological activity. J Agric Food Chem. 57:7288–7292. Marra R, Ambrosino P, Carbone V, Vinale F, Woo SL, Ruocco M, Ciliento R, Lanzuise S, Ferraioli S, Soriente I, et al. 2006. Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Curr Genet. 50:307–321. Parker SR, Cutler HG, Jacyno JM, Hill RA. 1997. Biological activity of 6-pentyl-2H-pyran-2-one and its analogs. J Agric Food Chem. 45:2774–2776. Poole PR, Ward BG, Whitaker G. 1998. The effects of topical treatments with 6-pentyl-2-pyrone and structural analogs on stem end postharvest rots in kiwi fruit due to Botrytis cinerea. J Agric Food Chem. 77:81–86. Reino JL, Guerriero RF, Hernandez-Galan R, Collado IG. 2008. Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem Rev. 7:89–123. Sati SC, Sati N, Ahluwalia V, Walia S, Sati OP. 2013. Chemical composition and antifungal activity of Artemisia nilagirica essential oil growing in northern hilly areas of India. Nat Prod Res. 27:45– 48. Scarselletti R, Faull JL. 1994. In vitro activity of 6-pentyl-a-pyrone, a metabolite of Trichoderma harzianum, in the inhibition of Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici. Mycol Res. 98:1207–1209. Sivasithamparam K, Ghisalberti EL. 1998. In: Harman GE, Kubicek CP, editors. Trichoderma and Gliocladium. Vol. 1. London: Taylor and Francis; p. 139–191. Slater GP, Haskins RH, Hogge LR, Nesbitt LR. 1967. Metabolic products from a Trichoderma viride Pers. ex Fries. Can J Chem. 45:92–96. Vinale F, Marra R, Scala F, Ghisalberti EL, Lorito M, Sivasithamparam K. 2006. Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens. Lett Appl Microbiol. 43:143–148. Vinale F, Nigro M, Sivasithamparam K, Flematti G, Ghisalberti EL, Ruocco M, Varlese R, Marra R, Lanzuise S, Eid A, et al. 2013. Harzianic acid: a novel siderophore from Trichoderma harzianum. FEMS Microbiol Lett. 347:123–129. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Barbetti MJ, Li H, Woo SL, Lorito M. 2008. A novel role for Trichoderma secondary metabolites in the interactions with plants. Physiol Mol Plant Pathol. 72:80–86. Wu Y, Tu X, Lin G, Xia H, Huang H, Wan J, Cheng Z, Liu M, Chen G, Zhang H, Fu J, Liu Q, Liu D-X. 2007. Emodinmediated protection from acute myocardial infarction via inhibition of inflammation and apoptosis in local ischemic myocardium. Life Sci. 81:1332–1338.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity ab
a
a
Vivek Ahluwalia , Jitendra Kumar , Virendra S. Rana , Om P. Sati & S. Walia
b
a
a
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi110 012, India
Click for updates
b
Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand246 174, India Published online: 24 Sep 2014.
To cite this article: Vivek Ahluwalia, Jitendra Kumar, Virendra S. Rana, Om P. Sati & S. Walia (2015) Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity, Natural Product Research: Formerly Natural Product Letters, 29:10, 914-920, DOI: 10.1080/14786419.2014.958739 To link to this article: http://dx.doi.org/10.1080/14786419.2014.958739
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
Downloaded by [New York University] at 14:53 22 April 2015
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 10, 914–920, http://dx.doi.org/10.1080/14786419.2014.958739
Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity Vivek Ahluwaliaab*, Jitendra Kumara, Virendra S. Ranaa, Om P. Satib and S. Waliaa a
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, India; Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand 246 174, India
b
Downloaded by [New York University] at 14:53 22 April 2015
(Received 26 June 2014; final version received 21 August 2014) This investigation was undertaken to identify the major secondary metabolite, produced by two Trichoderma harzianum strains (T-4 and T-5) with their antifungal activity against phytopathogenic fungi using poison food technique. The ethyl acetate extract was subjected to column chromatography using n-hexane, ethyl acetate and methanol gradually. Chromatographic separation of ethyl acetate extract of T. harzianum (T-4) resulted in the isolation and identification of palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)-furanone (4), stigmasterol (5) and b-sitosterol (6), while T. harzianum (T-5) gave palmitic acid (1), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone (8), 6-pentyl-2Hpyran-2-one (3), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) as major metabolites. Among compounds screened for antifungal activity, compound 10 was found to be most active (EC50 35.9– 50.2 mg mL21). In conclusion, the present investigation provided significant information about antifungal activity and compounds isolated from two different strains of T. harzianum obtained from two different Himalayan locations. Keywords: Trichoderma; secondary metabolite; antifungal activity
1. Introduction Trichoderma species are considered as free living opportunistic plant symbionts and widely used as biofertilisers and biopesticides. Some of the Trichoderma species used as biocontrol agents develop the ability to interact simultaneously with plants and fungal pathogens, thus are used as model microorganisms to study complex and multiple-player plant –microbe interactions (Marra et al. 2006). These species are useful to agriculture due to their ability to parasitise on different pathogenic fungi namely. Botritis spp., Fusarium spp., Pythium spp., Rhizoctonia spp., Verticillium spp. and Sclerotinia spp. Trichoderma species are also widely used for their biological control (Jelen´ et al. 2013). It has been found that the production of secondary metabolite in Trichoderma species is one major reason that contributes to its biological activities during interactions (Sivasithamparam & Ghisalberti 1998; Reino et al. 2008; Vinale et al. 2008). The metabolite production varies according to the species or strain used and their biosynthesis and biotransformation rates (Vinale et al. 2013). More than 200 secondary metabolites have been reported from different species and strains of Trichoderma, some of which have shown promising biological activities (Reino et al. 2008; Vinale et al. 2013). The work on the identification of different secondary metabolites from different species and strains of Trichoderma has continued, but to the best of our knowledge no work has been found
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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on the comparative evaluation of secondary metabolite production from Trichoderma harzianum collected from the Himalayan region of India. This work describes the isolation, characterisation and comparative study of major secondary metabolites produced by two strains (T-4 and T-5) of T. harzianum obtained from two different locations with varied environmental conditions. 2. Results and discussion The structures of the pure compounds (1–11) isolated from the ethyl acetate extract of Trichoderma strains (T-4 and T-5) were determined by using 1H NMR, 13C NMR, DEPT, HMQC, HMBC and ESI-MS data and were identified as palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2) (Liu et al. 2009), 6-pentyl-2H-pyran-2-one (3) (Claydon et al. 1987), 2(5H)-furanone (4), stigmasterol (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7) (Liu et al. 2009), d-decanolactone (8) (Hill et al. 1995), ergosterol (9), harzianopyridone (10) (Dickinson et al. 1989) and 6-methyl-1,3,8-trihydroxyanthraquinone (11) (Slater et al. 1967) (Figure 1).
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OH
O
OH
O OH
13
O
O O 2.
1.
3.
H H
H
H
O H O
H
4.
5. O
H
H HO
HO
6.
OH H
O
H
H
HO
O 7. MeO
O
H N
O
OH
O
8.
9. OH
O
OH
MeO OH O 10
11
Figure 1. Chemical structures of secondary metabolites isolated from T. harzianum strains T-4 and T-5. Palmitic acid (1), 1,8-dihydroxy-3-methylanthraquinone (2), 6-pentyl-2H-pyran-2-one (3), 2(5H)furanone (4), stigmasterol (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone (8), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11).
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Table 1. Antifungal activity# (EC50 mg mL21). Compounds
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1 2 3 4 5 6 7 8 9 10 11 Bavistin
R. solani
S. rolfsii
M. phaseolina
F. oxysporum
498.9 1515.4 64.6 124.3 683 886.8 655.7 70.6 434.2 35.9 529.7 10.7
563.9 1126.5 38.8 237.4 521.8 779.4 542.8 122.2 464.5 42.2 468.8 14.8
623.1 1093.1 90.6 193.8 695.8 490.4 363.6 141.4 449.6 60.4 314.5 13.5
578.8 1619.8 74.3 154.8 635.8 589.0 727.6 124.1 531.7 50.2 356.1 15.8
Pure compounds were further screened for their antifungal activity against four phytopathogenic fungi, namely Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum and the EC50 values of the compounds determined are given in Table 1. Results showed that harzianopyridone (10) was most effective and inhibited more than 90% growth of R. solani, F. oxysporum and S. rolfsii (EC50 35.9– 50.2 mg mL21) but was less active than Bavistin, a standard fungicide (Table 1). Cutler and Jacyno (1991) reported that harzianopyridone was only moderately active against bacteria and relatively inactive against fungi, although Dickinson et al. (1989) have reported significant activity against Botrytis cinerea and R. solani. Vinale et al. (2006) showed that harzianopyridone inhibited the growth of Gaeumannomyces graminis var. tritici, R. solani and Pythium ultimum. Although harzianopyridone has been evaluated for its biological activities, but to our knowledge its activity against phytopathogenic fungi M. phaseolina, S. rolfsii and F. oxysporum has not been reported. Among anthraquinones, 1-hydroxy-3-methylanthraquinone (7) was more active than 1,8-dihydroxy-3-methylanthraquinone (2). Its activity was found highest against M. phaseolina (363.6 mg mL21) and least against F. oxysporum (727.6 mg mL21). Introduction of one hydroxyl function in 1-hydroxy-3-methylanthraquinone (7) led to a decrease in antifungal activity of 1,8dihydroxy-3-methylanthraquinone (2). Our results differ strikingly from an earlier report in which compound 2 was found to be having more fungal growth inhibition against R. solani and B. cinerea and less antibacterial activity against Staphylococcus aureus than compound 7 (Liu et al. 2009). According to Agarwal et al. (2000), 1,8-dihydroxy-3-methylanthraquinone (2) showed encouraging antifungal activity (MIC 25– 250 mg mL21) against Candida albicans, Cryptococcus neoformans, Trichophyton mentagrophytes and Aspergillus fumigatus, while Vinale et al. (2006) reported very weak antifungal activity of both compounds against G. graminis var. tritici, R. solani and P. ultimum. Earlier reports suggested that the same compounds which differ in number and position of hydroxyl groups can have varying antimicrobial properties. These discrepancies could arise due to difference in assays being used, sizes of fungal inoculums, volume of broth or agar, type of broth or agar, size of wells, size of paper discs, strains of a particular bacterial species used and incubation period (Cushnie & Lamb 2005). 6-Methyl-1,3,8-trihydroxyanthraquinone (11) isolated from T-5 strain also showed antifungal activity with EC50 value ranging between 314.5 and 529.7 mg mL21 against tested fungi (Figure 2). It is known to exhibit antimicrobial, antineoplasic and cathartic action (Chukwujekwu et al. 2006; Wu et al. 2007). It also showed monoamine oxidase (Fujimoto et al. 1998) and tyrosine kinase inhibiting activity (Kumar et al. 1998). The compounds with low oxidation state have potential to change to compounds with high oxidation state by the host
EC50 (µg mL–1)
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2000 1800 1600 1400 1200 1000 800 600 400 200 0
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Compounds
Figure 2. Antifungal activity (EC50 mg mL21) of isolated compounds. Palmitic acid# (1), 1,8-dihydroxy-3methylanthraquinone (2), 6-pentyl-2H-pyran-2-one# (3), 2(5H)-furanone (4), stigmasterol# (5), b-sitosterol (6), 1-hydroxy-3-methylanthraquinone (7), d-decanolactone# (8), ergosterol (9), harzianopyridone (10) and 6-methyl-1,3,8-trihydroxyanthraquinone (11).
reactive oxygen species that are released in response to attack by a microbial pathogen. These high oxidation state compounds have high antimicrobial activity and may increase the effective efficiency of Trichoderma against host resistance to other pathogens when this symbiotically inhabits a plant’s roots system (Liu et al. 2009). 2(5H)-Furanone (4) isolated from strain T-4 caused 52.14 –62.17% fungal growth inhibition at 250 mg mL21 (Figure 3). 6-Pentyl-2H-pyran2-one (3) and d-decanolactone (8) isolated earlier from different species of Trichoderma when evaluated for their potential as antifungal compound had shown good activity (Parker et al. 1997). Our group had previously reported the antifungal activity of palmitic acid (1), 6-pentyl-apyranone (3), stigmasterol (5) and d-decanolactone (8) from Trichoderma koningii (Ahluwalia et al. 2014#) and their activity has also been compared with newly isolated compounds (Figures 2 and 3 and Table 1). 6-Pentyl-2H-pyran-2-one has been reported active against R. solani and F. oxysporum f. sp. lycopersici (Scarselletti & Faull 1994). It was also found to reduce rotting of stored kiwi fruits caused by B. cinerea (Poole et al. 1998).
Figure 3. Fungal growth percentage inhibition of compounds at 250 mg mL21 concentration.
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2.1 Conclusion Eleven compounds were isolated and characterised from the two different T. harzianum strains collected from two separate Himalayan locations, of which harzianopyridone (10) was found to be the most effective, while 1,8-dihydroxy-3-methylanthraquinone (2) was least effective. Harzianopyridone and 6-pentyl-2H-pyran-2-one need to be further evaluated against the more phytopathogenic fungi of agricultural importance to explore their further use as antifungal agents.
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3. Experimental 3.1 General Chemicals and organic solvents used were of chromatographic and analytical grades (Merck India Ltd.). Column chromatography was performed using silica gel (60 –120 mesh) (Merck). NMR spectra of the pure compounds were recorded on Bruker (400 MHz) instrument using tetramethyl silane as internal standard in CDCl3 or DMSO-d6 and ESI-MS data on LC – MS Spectrometer (Thermo-Finnigan LCQ, USA).
3.2 Fungal cultures Two fungal strains of T. harzianum (T-4 and T-5) were obtained from CSK Himachal Pradesh Krishi Vishavidalaya, Palampur, Himachal Pradesh (India). All the fungal strains were maintained on potato dextrose agar (PDA) slants at room temperature and sub-cultured bimonthly. Four phyto-pathogenic fungi, M. phaseolina, R. solani, S. rolfsii and F. oxysporum were procured from Indian Type Culture Collection Centre, Division of Mycology and Plant Pathology, IARI, New Delhi, India.
3.3 Liquid culture and metabolite production Two 7 mm diameter plugs of each T. harzianum strain were taken from actively growing margins of PDA cultures and were inoculated into 5 L conical flasks containing 1 L of sterile liquid broth containing KH2PO4 (7 g L21), K2HPO4 (2 g L21), MgSO4·7H2O (0.1 g L21), (NH2) SO4 (1 g L21), yeast extract (0.6 g L21) and glucose (10 g L21). The stationary cultures were incubated for 28 days at 25 ^ 18C. The mycelium of the fungal strain was removed by filtering through a muslin cloth.
3.4 Isolation and purification of secondary metabolites The culture filtrate (20 L) of strain T-4 was extracted with EtOAc three times at room temperature. The solvent was evaporated in vacuo at 408C to a get red-brown residue (0.28 g L21). The crude extract was subjected to column chromatography using a glass column packed with silica gel (60 –120 mesh) and eluted with n-hexane, and mixture of n-hexane – ethyl acetate and ethyl acetate– methanol with increasing polarity, and 10 fractions were obtained. The elution of the column with different solvents was monitored by TLC and the fractions having similar spots were mixed and dried. Fraction 2 was further subjected to preparative TLC using hexane –ethyl acetate (90:10) as developing solvent to yield 1 (6 mg). Fraction 4 was also subjected to silica gel column chromatography using hexane and ethyl acetate mixture from which compound 2 was obtained (8 mg). Compounds 3 (14 mg) and 4 (3 mg) were isolated from fractions 5 and 6 by repeated column chromatography followed by preparative TLC. Similarly, fraction 7 was column
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chromatographed using petroleum ether –acetone mixture (7:3) followed by preparative TLC from which white crystalline compounds 5 (6 mg) and 6 (3 mg) were obtained. Similarly the culture filtrate of strain T-5 was also extracted with ethyl acetate. The solvent was removed under reduced pressure and ethyl acetate extract (0.32 g L21) was obtained. Column chromatography of the ethyl acetate extract over silica gel (60 –120 mesh) was carried out. The column was packed with n-hexane and eluted with mixture of n-hexane – ethyl acetate and ethyl acetate –methanol with increasing polarity from which 15 fractions were collected. Fraction 3 showed a major spot in TLC and was subjected to preparative-TLC and a pure compound 1 (15 mg) was separated. Fraction 5 was found to contain three spots on the TLC plate which were again subjected to repeated column chromatography over silica gel using n-hexane – ethyl acetate mixture and two pure compounds 7 (11 mg) and 8 (9 mg) were crystallised. Fraction 9 was also subjected to column chromatography using petroleum ether – acetone (6:4) mixture followed by preparative-TLC from which two compounds were isolated. The less polar compound was separated as a colourless oily compound 9 (4 mg) and the more polar compound gave pale yellow compound 3 (4 mg). Fractions 12 and 14 were further purified by preparativeTLC to afford compounds 10 (7 mg) and 11 (12 mg). 3.5 Antifungal activity The antifungal activity of pure secondary metabolites was carried out by poison food technique as described earlier (Sati et al. 2013). Briefly, compound(s) were dissolved in minimum quantity of acetone/DMSO and were added to molten PDA medium (65 mL) to get the concentration of 250, 125, 62.5 and 31.25 mg mL21. Pathogen disc (5 mm) from a 7-day-old culture was inoculated at the centre of each PDA plate. PDA plates treated with acetone/DMSO without compound(s) were used as negative control and those with Bavistinw as a positive control. Each treatment consisted of three replicates and the experiment was repeated twice. EC50 (effective concentration required for 50% inhibition of mycelial growth) of each compound was calculated statistically by Probit analysis. The data were analysed with the help of SAS Package (statistical analysis system package) software. Supplementary material Spectral data of pure compounds can be obtained from the corresponding author. Acknowledgements The authors are thankful to Head, Division of Agricultural Chemicals, IARI, New Delhi, India and Head, Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand, India for providing necessary facilities. Financial assistance from NAIP is duly acknowledged.
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