GC-MS based profiling and quantification of bioactive secondary metabolites from Trichoderma harzianum

Introduction

Trichoderma species are widely recognised as potent biocontrol agents in sustainable agriculture because of their ability to antagonise a wide range of phytopathogenic fungi through a variety of mechanisms including mycoparasitism and competition (Harman et al., 2004) [6] . Among these species, Trichoderma harzianum has attracted considerable attention for its robust production of bioactive secondary metabolites that inhibit pathogenic fungi and improve the defense of plants. These secondary metabolites include volatile organic compounds (VOCs), polyketides, terpenes and various fatty acids, many of which directly affect the fungal membrane, inhibit the growth of the fungal spores and interfere with the pathogenic organisms' signalling pathways (Reino et al., 2008; Mukherjee et al., 2013) [7,8] . Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical tool for profiling of metabolites, enables characterization of volatile and semi-volatile compounds with antifungal properties (Siddiquee et al., 2012) [1] . The metabolites identified by GC-MS from T. harzianum have been shown to include compounds such as 6-pentyl-2H-pyran-2-one (6-PP), which is known for its strong antifungal and plant growth-promoting activity (Chaudhary et al., 2022) [11] . Fatty acids like hexadecanoic acid and octadecanoic acid, commonly found in T. harzianum, have been reported to alter membrane fluidity and disrupt cellular integrity of target fungal pathogens (Avis & Bélanger, 2001; Ahluwalia et al., 2015) [5, 4] . Moreover, aromatic compounds and phthalate esters, such as di-n-octyl phthalate, possess documented antifungal activity by affecting ergosterol biosynthesis and inhibiting mitochondrial function in fungi (Zhang et al., 2018) [9] . The biocontrol efficacy of T. harzianum is further enhanced by synergistic interactions among its secondary metabolites, which collectively target multiple fungal pathways, including cell wall biosynthesis, oxidative stress response, and protein synthesis (Vinale et al., 2012) [3] . These compounds not only exhibit direct antifungal activity but also trigger systemic resistance in host plants, activating defense-related gene expression and enhancing tolerance to environmental stressors (Shoresh et al., 2010; Singh et al., 2022) [10,11] . Given the growing concerns about resistance to fungicides and environmental sustainability, the identification and characterization of natural antifungal compounds produced by T. harzianum offers a promising strategy for the development of environmentally friendly crop protection methods. This aim of this study to analyse and quantify the secondary metabolites produced by T. harzianum using GC-MS. By elucidating the chemical profile and concentration of these compounds, this work contributes to a better understanding of the metabolites production potential of T. harzianum, supports its role in sustainable crop protection.

Materials and Methods

Fungal Cultivation

Trichoderma harzianum was grown in potato dextrose broth (PDB) at 25 °C for 14 days with continuous shaking at 140 rpm in a 2 L Erlenmeyer flask containing 1 L of sterile PDB. The culture filtrate was separated from the mycelia using non-absorbent cotton.

Metabolite Extraction

The culture filtrate was subjected to a triple extraction procedure the same volume of ethyl acetate for the purpose of isolating the secondary metabolites. The resulting water fraction was evaporated to dryness by means of a rotary evaporator (Heidolf, Germany) at a reduced pressure of 55 °C. The dry residues of the metabolites were weighed and reconstituted with ethyl acetate at GC-MS grade concentration of 1 g per ml for further analysis.

GC-MS Analysis

The extracted metabolites were analyzed using a Shimadzu GC-MS-QP 2010 system equipped with an Rtx-1 MS column coupled to a triple-axis mass spectrometer. A 1 μL sample was injected, with helium (>99.99% purity) serving as the carrier gas at a head pressure of 10 psi and a flow rate of 0.75 mL/min. The oven temperature was programmed as follows: an initial temperature of 40 °C held for 2 minutes, ramped at 4 °C/min to 120 °C, followed by a 5 °C/min increase to 200 °C (held for 5 minutes), and a final ramp of 10 °C/min to 300 °C, giving a total run time of 51 minutes. Mass spectrometry parameters included an ion source temperature of 250 °C, electron ionization at 60 eV, full scan mode over a mass range of 50-550 AMU, a transfer line temperature of 280 °C, and an electron multiplier voltage of 1222 V. Compounds identification was performed by comparing mass spectra to the National Institute of Standards and Technology (NIST) Mass Spectral Library (Shimadzu Corporation, Kyoto, Japan) with a spectral match factor ≥90%.

Quantification and Data Analysis

Th peak areas of the identified metabolites were determined by Shimadzu LabSolutions and normalized to the internal standard (D4-methanol) in order to exclude instrumental variations. The relative abundance was expressed as a percentage of the total maximum area. The experiment was conducted in three replicates.

Results and Discussion

The GC-MS analysis of Trichoderma harzianum identified metabolites with a high retention time that have antifungal activity and are important contributors to the biocontrol efficacy against phytopathogenic fungi (Table 1.). The gas chromatography-mass spectrometry (GC-MS) identified metabolites with retention time from Trichoderma harzianum which shows antifungal activity against phytopathogenic fungi. The metabolites include 6-pentyl-2H-pyran-2-one (20.44) (Siddiquee et al., 2012) [1] , which inhibits fungal membrane integrity and β-glucan synthase. Hexadecanoic acid was detected at RT (20.654) altering membrane fluidity and inhibiting spore germination (Ahluwalia et al., 2015) [4] . Other fatty acids, tetradecanoic acid and octadecanoic acid at RT (17.76, 22.37) respectively (Lakhdari et al., 2023) [2] , disrupt fungal membranes, causing leakage and growth inhibition (Avis & Bélanger, 2001) [5] . Diethyl benzene-1,2-dicarboxylate (15.85) and 2,4-bis(1,1-dimethylethyl)phenol (13.72) (Lakhdari et al., 2023) [2] disrupt ergosterol biosynthesis and membrane function. Complex metabolites, 2-(2-hydroxypropan-2-yl)-2,3-dihydrofuro[2,3-h]chromen-4-one (16.45) 1monolinoleoylglycerol (23.12) and 2-(7-hydroxyheptyl)-3,5,6-trimethylcyclohex-2-en-1-one (18.63) (Lakhdari et al., 2023) [2] , act synergistically, targeting enzyme inhibition and lipid peroxidation (Vinale et al., 2006) [3] . The antifungal mechanisms involve membrane disruption by fatty acids, which increase permeability (Avis & Bélanger, 2001) [5] , and inhibition of ergosterol and cell wall synthesis by 6-pentyl-2H-pyran-2-one. Volatile alcohols cause oxidative stress, while phenolics like 2,4-bis(1,1-dimethylethyl)phenol are similar to azole antifungal agents. Organic acids induce antagonistic microenvironments and systemic resistance and complex metabolites may target multiple pathways (Vinale et al., 2006) [3] The low peak area of 6-pentyl-2H-pyran-2one suggests high potency despite low abundance (Siddiquee et al., 2012) [1] .

Conclusion

The GC-MS analysis of metabolites produced by Trichoderma harzianum revealed a diverse array of bioactive compounds with significant antifungal properties. Key metabolites such as 6-pentyl-2H-pyran-2-one, hexadecanoic acid, and octadecanoic acid play crucial roles in disrupting fungal cell membranes, inhibiting ergosterol biosynthesis, and impairing spore germination. Additionally, complex compounds like 2-(2-hydroxypropan-2-yl)-2,3dihydrofuro [2,3-h]chromen-4-one and 1monolinoleoylglycerol exhibit synergistic effects targeting lipid peroxidation and enzyme inhibition. The identification of volatile phenolics and organic acids further highlights the multifaceted antifungal mechanisms, including oxidative stress induction and systemic resistance. The phenolic compounds such as 2,4-bis(1,1-dimethylethyl) phenol further supports the multi-targeted antifungal of T. harzianum. These findings highlight the potential of T. harzianum as an effective biocontrol agent against phytopathogenic fungi through synergistic action of its metabolites, paving the way for sustainable and eco-friendly crop protection strategies.

Acknowledgement

AcknowledgmentThe authors gratefully acknowledge ICAR-Indian Agricultural Research Institute (IARI), Pusa, New Delhi for providing the necessary facilities and support to carry out this research work.

References

1. Siddiquee S, Cheong BE, Taslima K, Kausar H, Hasan MM. Separation and identification of volatile compounds from liquid cultures of Trichoderma harzianum by GC-MS using three different capillary columns. Journal of Chromatographic Science. 2012;50:358-367.
2. Lakhdari W, Benyahia I, Bouhenna MM, Bendif H, Khelafi H, Bachir H. Exploration and evaluation of secondary metabolites from Trichoderma harzianum: GC-MS analysis, phytochemical profiling, antifungal and antioxidant activity assessment. Molecules. 2023;28:5025.
3. Vinale F, Marra R, Scala F, Ghisalberti EL, Lorito M, Sivasithamparam K. Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens. Letters in Applied Microbiology. 2006;43:143-148.
4. Ahluwalia V, Kumar J, Rana VS, Sati OP, Walia S. Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity. Natural Product Research. 2015;29:914-920.
5. Avis TJ, Bélanger RR. Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa. Applied and Environmental Microbiology. 2001;67:956-960.
6. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma spp. opportunistic, avirulent plant symbionts. Nature Reviews Microbiology. 2004;2:4356.
7. Reino JL, Guerrero RF, Hernández-Galán R, Collado IG. Secondary metabolites from Trichoderma and their applications in agriculture. Phytochemistry Reviews. 2008;7:89-123.
8. Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley CM. Trichoderma research in the genome era. Annual Review of Phytopathology. 2013;51:105-129.
9. Zhang S, Xu B, Zhang J, Gan Y. Identification of the antifungal activity of Trichoderma longibrachiatum T6 and assessment of bioactive substances in controlling phytopathogens. Pesticide Biochemistry and Physiology. 2018;147:59-66.
10. Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology. 2010;48:2143.
11. Chaudhary P, Singh S, Chaudhary A, Sharma A, Kumar G. Overview of biofertilizers in crop production and stress management for sustainable agriculture. Frontiers in Plant Science. 2022;13:930340.