Abstract
Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.
Similar content being viewed by others
References
Abdel-Fattah, G.M., Shabana, Y.M., Ismail, A.E., and Rashad, Y.M. 2007. Trichoderma harzianum: a biocontrol agent against Bipolaris oryzae. Mycopathologia 164, 81–89.
Alves, E. and Lucas, G.C. 2013. Scanning electron microscopy for fungal sample examination laboratory protocols in fungal biology. In Gupta, V.K., Tuohy, M.G., Ayyachamy, M., Turner, K.M., and O’Donovan, A. (eds.), Laboratory protocol in fungal biology: current methods in fungal biology, pp. 133–150. Springer New York, NY, USA.
Aneja, M., Gianfagna, T.J., and Hebbar, P.K. 2005. Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens. Physiol. Mol. Plant Pathol. 67, 304–307.
Benítez, T., Rincón, A.M., Limón, M.C., and Codón, A.C. 2004. Biocontrol mechanisms of Trichoderma strains. Int. Microbiol. 7, 249–260.
Brader, G., Compant, S., Mitter, B., Trognitz, F., and Sessitsch, A. 2014. Metabolic potential of endophytic bacteria. Curr. Opin. Biotechnol. 27, 30–37.
Dhungana, S. and Crumbliss, A.L. 2005. Coordination chemistry and redox processes in siderophore-mediated iron transport. Geomicrobiol. J. 22, 87–98.
Dias, M.C. 2012. Phytotoxicity: an overview of the physiological responses of plants exposed to fungicides. J. Bot. 2012, 1–4.
Etheridge, D.E. and Craig, H.M. 1973. A bilayer plate technique to detect broad-spectrum antagonism in microorganisms and its application to wood-inhabiting fungi. Can. J. Microbiol. 19, 1455–1458.
Evans, H.C., Holmes, K.A., and Thomas, S.E. 2003. Endophytes and mycoparasites associated with an indigenous forest tree, Theobroma gileri, in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases. Mycol. Prog. 2, 149–160.
Fernando, W.G.D., Ramarathnam, R., Krishnamoorthy, A.S., and Savchuk, S.C. 2005. Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biol. Biochem. 37, 955–964.
Hall, B.G. 2013. Building phylogenetic trees from molecular data with MEGA. Mol. Biol. Evol. 30, 1229–1235.
Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., and Lorito, M. 2004. Trichoderma species—opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43–56.
Hassan, S.B., Gali-Muhtasib, H., Göransson, H., and Larsson, R. 2010. Alpha terpineol: a potential anticancer agent which acts through suppressing NF-kappaB signalling. Anticancer Res. 30, 1911–1919.
Hilgren, J.D. and Salverda, J.A. 2000. Antimicrobial efficacy of a peroxyacetic/octanoic acid mixture in fresh-cut-vegetable process waters. J. Food Sci. 65, 1376–1379.
Ho, C.L., Hua, K.F., Hsu, K.P., Wang, E.I., and Su, Y.C. 2012. Composition and antipathogenic activities of the twig essential oil of Chamaecyparis formosensis from Taiwan. Nat. Prod. Commun. 7, 933–936.
Hosseyni-Moghaddam, M.S. and Soltani, J. 2013. Bioactivity of endophytic Trichoderma fungal species from the plant family Cupressaceae. Ann. Microbiol. 64, 753–761.
Howard, D.H. 1999. Acquisition, transport, and storage of iron by pathogenic fungi. Clin. Microbiol. Rev. 12, 394–404.
Howlett, B.J., Brownlee, A.G., Guest, D.I., Adcock, G.J., and Mc-Fadden, G.I. 1992. The 5S ribosomal RNA gene is linked to large and small subunit ribosomal RNA genes in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea, and Saprolegnia ferax. Curr. Genet. 22, 455–461.
Idris, A.S. 1999. Basal stem rot (BSR) of oil palm (Elaeis guineesis Jacq.) in Malaysia: factors associated with variation in disease severity. Wye College, Wye, UK.
Jagatheesh, K.R., Padarthi, P.K., and Namasivayam, E. 2013. DNA damage protection and haemolytic activity of isolongifolene. Int. Res. J. Pharm. 4, 75–77.
Knudsen, G.R. and Dandurand, L.C. 2014. Ecological complexity and the success of fungal biological control agents. Adv. Agr. 2014, 1–11.
Krishna Reddy, B., Balaji, M., Uma Reddy, P., Sailaja, G., Vaidyanath, K., and Narasimha, G. 2009. Antifeedant and antimicrobial activity of Tylophora indica. African J. Biochem. Res. 3, 393–397.
Lehner, S.M., Atanasova, L., Neumann, N.K., Krska, R., Lemmens, M., Druzhinina, I.S., and Schuhmacher, R. 2013. Isotope-assisted screening for iron-containing metabolites reveals a high degree of diversity among known and unknown siderophores produced by Trichoderma spp. Appl. Environ. Microbiol. 79, 18–31.
Li, W.R., Shi, Q.S., Liang, Q., Huang, X.M., and Chen, Y.B. 2014. Antifungal effect and mechanism of garlic oil on Penicillium funiculosum. Appl. Microbiol. Biotechnol. 98, 8337–8346.
Loper, J.E. and Buyer, J.S. 1991. Siderophore in microbial interaction on plant surface. Mol. Plant Microbe Interact. 4, 5–13.
Macías-Rubalcava, M.L., Hernández-Bautista, B.E., Oropeza, F., Duarte, G., González, M.C., Glenn, A.E., Hanlin, R.T., and Anaya, A.L. 2010. Allelochemical effects of volatile compounds and organic extracts from Muscodor yucatanensis, a tropical endophytic fungus from Bursera simaruba. J. Chem. Ecol. 36, 1122–1131.
Miethke, M. and Marahiel, M.A. 2007. Siderophore-based iron acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71, 413–451.
Milagres, A.M.F., Machuca, A., and Napoleao, D. 1999. Methods detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J. Microbiol. Methods 37, 1–6.
Mo, E.K. and Sung, C.K. 2007. Phenylethyl alcohol (PEA) application slows fungal growth and maintains aroma in strawberry. Postharvest Biol. Technol. 45, 234–239.
Morath, S.U., Hung, R., and Bennett, J.W. 2012. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biol. Rev. 26, 73–83.
Panigrahi, G., Maheshwari, R., Vellaikumar, S., Jayaprakash, S.P., Kumar, S., and Prabakaran, J. 2014. Preparative thin-layer chromatographic separation followed by identification of antifungal compound in Cassia laevigata by RP-HPLC and GC-MS. J. Sci. Food Agric. 94, 308–315.
Paterson, R.R.M. 2007. Ganoderma disease of oil palm–a white rot perspective necessary for integrated control. Crop Prot. 26, 1369–1376.
Pérez-Miranda, S., Cabirol, N., George-Téllez, R., Zamudio-Rivera, L.S., and Fernández, F.J. 2007. O-CAS, a fast and universal method for siderophore detection. J. Microbiol. Methods. 70, 127–131.
Pohl, C.H., Kock, J.L.F., and Thibane, V.S. 2011. Antifungal free fatty acids: a review. In Mendez-Vilas, A. (ed.), Science against microbial pathogens: communicating current research and technological advances, pp. 61–71. Formatex, Badajoz, Spain.
Rees, R.W., Flood, J., Hasan, Y., Potter, U., and Cooper, R.M. 2009. Basal stem rot of oil palm (Elaeis guineensis); mode of root infection and lower stem invasion by Ganoderma boninense. Plant Pathol. 58, 982–989.
Reino, J.L., Guerrero, R.F., Hernández-Galán, R., and Collado, I.G. 2007. Secondary metabolites from species of the biocontrol agent Trichoderma. Phytochem. Rev. 7, 89–123.
Schoeman, M.W., Webber, J.F., and Dickinson, D.J. 1996. The effect of diffusible metabolites of Trichoderma harzianum on in vitro interactions between basidiomycete isolates at two different temperature regimes. Mycol. Res. 100, 1454–1458.
Shakeri, J. and Foster, H.A. 2007. Proteolytic activity and antibiotic production by Trichoderma harzianum in relation to pathogenicity to insects. Enzyme Microb. Technol. 40, 961–968.
Shi, M., Chen, L., Wang, X.W., Zhang, T., Zhao, P.B., Song, X.Y., Sun, C.Y., Chen, X.L., Zhou, B.C., and Zhang, Y.Z. 2012. Antimicrobial peptaibols from Trichoderma pseudokoningii induce programmed cell death in plant fungal pathogens. Microbiology 158, 166–175.
Siddiquee, S., Cheong, B.E., Taslima, K., Kausar, H., and Hasan, M.M. 2012. Separation and identification of volatile compounds from liquid cultures of Trichoderma harzianum by GC-MS using three different capillary columns. J. Chromatogr. Sci. 50, 358–367.
Singh, D., Kumar, T.R., Gupta, V.K., and Chaturvedi, P. 2012. Antimicrobial activity of some promising plant oils, molecules and formulations. Indian J. Exp. Biol. 50, 714–717.
Stević, T., Berić, T., Šavikin, K., Soković, M., Gođevac, D., Dimkić, I., and Stanković, S. 2014. Antifungal activity of selected essential oils against fungi isolated from medicinal plant. Ind. Crops Prod. 55, 116–122.
Stoppacher, N., Kluger, B., Zeilinger, S., Krska, R., and Schuhmacher, R. 2010. Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HSSPME-GC-MS. J. Microbiol. Methods 81, 187–193.
Sundram, S. 2005. Msc thesis. Performance of Trichoderma harzianum Rifai as a biological control agent for basal stem rot of oil palm (Elaeis guineensis Jacq.) caused by Ganoderma boninense Pat. Universiti Putra Malaysia, Serdang, Malaysia.
Sundram, S. 2013. First report: isolation of endophytic Trichoderma from oil palm (Elaeis guineensis Jacq.) and their in vitro antagonistic assessment on Ganoderma boninense. J. Oil Palm Res. 25, 368–372.
Sundram, S., Abdullah, F., Ahmad, Z.A.M., and Yusuf, U.K. 2008. Efficacy of single and mixed treatments of Trichoderma harzianum as biocontrol agents of Ganoderma basal stem rot in oil palm. J. Oil Palm Res. 20, 470–483.
Sundram, S., Angel, L.P.L., Tay, B.Y.P., Roslan, N.D., Mohamed Azni, I.N.A., and Idris, A.S. 2016. Trichoderma virens, an effective biocontrol agent against Ganoderma boninense. MPOB TT No. 587.
Sundram, S., Meon, S., Idris, A.S., and Othman, R. 2011. Symbiotic interaction of endophytic bacteria with arbuscular mycorrhizal fungi and its antagonistic effect on Ganoderma boninense. J. Microbiol. 49, 551–557.
Symeonidis, A. and Marangos M. 2012. Iron and microbial growth. In Roy, P.K. (ed.), Insight and control of infectious disease in global scenario, pp. 289–332. InTech, Rijeka, Croatia.
Tan, C.J., How, K.C., Loh-Mia, P.P., Getha, K., Seki, T., and Vikineswary, S. 2002. Bioactivity of selected actinomycetes against Ganoderma boninense. Asia Pac. J. Mol. Biol. Biotechnol. 10, 119–125.
van der Helm, D. and Winkelmann, G. 1994. Hydroxamates and polycarboxylates as iron transport agents (siderophore) in fungi. In Winkelmann, G. and Winge, D.R. (eds.), Metal ions in fungi, 2nd ed, pp. 39–98. CRC Press, NY, USA.
Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Ruocco, M., Woo, S., and Lorito, M. 2012. Trichoderma secondary metabolites that affect plant metabolism. Nat. Prod. Commun. 7, 1545–1550.
Wilhite, S.E., Lumsden, R.D., and Straney, D.C. 2001. Peptide synthetase gene in Trichoderma virens. Appl. Environ. Microbiol. 67, 5055–5062.
Yanai, H. 2015. Green synthetic approaches for biologically relevant heterocycles. In Brahmachari, G. (ed.), pp. 257–289. Green synthetic approaches for biologically relevant heterocycles. Elsevier, Waltham, MA, USA.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplemental material for this article may be found at http://www.springerlink.com/content/120956.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Angel, L.P.L., Yusof, M.T., Ismail, I.S. et al. An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense . J Microbiol. 54, 732–744 (2016). https://doi.org/10.1007/s12275-016-6304-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-016-6304-4