Antifungal metabolite profiling and inhibition assessment of Trichoderma-derived compounds during initial spore germination stage against postharvest pathogens

According to the United Nations Food and Agriculture organization (FAO), postharvest losses of agricultural commodities account for approximately 14% of total production^[1]. Pathogenic fungi are a major cause of these losses, affecting the quality, nutritional value, and marketability of stored produce. Aspergillus species, notably A. flavus and A. niger, are significant due to their production of aflatoxins, classified as Group 1 human carcinogens by the International Agency For Research on Cancer (IARC)^[2]. Penicillium molds infect fruits, vegetables, grains, and stored products such as apples and citrus fruits, reducing marketability and posing health risks through mycotoxins like ochratoxin A^[3]. Botrytis cinerea is a versatile pathogen causing grey mold across multiple crops, including grapes and strawberries^[4]. Certain Fusarium species produce harmful mycotoxins, such as deoxynivalenol and fumonisins affecting cereals like wheat and maize^[5]. Alternaria species, like A. alternata, induce spoilage and may contaminate crops such as tomatoes and carrots with alternariol^[6]. Cladosporium species contribute to crop decay and mycotoxin contamination, leading to further storage losses in cereals and fruits^[7]. The use of chemical fungicides, such as Thiophanate-methyl, Prochloraz, Propiconazole, and others, is one of the most effective strategies for controlling postharvest plant pathogenic fungi in storage^[8]. However, many of these fungicides raise health concerns. For example, Thiophanate-methyl is classified by the United States Environmental Protection Agency (US EPA) as a probable human carcinogen (Group C), while Prochloraz and Propiconazole are considered suspected endocrine disruptors and have shown carcinogenic potential in animal studies^[9]. Prolonged exposure can cause skin, eye, and respiratory irritation, as well as affect the liver, blood, thyroid, endocrine function, reproductive health, and development. Overuse has also led to resistance in fungal pathogens^[10].

Biological control offers an alternative, minimizing risks to human health and the environment^[11,12]. Trichoderma spp. are well-known biocontrol agents in plant pathology^[13,14], engaging in competitive interactions with pathogenic fungi for resources, thus inhibiting their growth^[15]. These fungi can directly attack pathogens by entwining around their hyphae and producing lytic enzymes, such as chitinases, glucanases, and proteases, as well as antifungal metabolites^[16]. Key antifungal compounds produced by Trichoderma include harzianic acid, trichodermin, gliotoxin, and others, which have demonstrated significant antifungal activity against various postharvest pathogens^[17,18]. All studies have focused on competition and secondary metabolite production during the mature stages of Trichoderma growth. However, no research has explored the interactions during the early stages. Focusing on the spore germination stage may reveal unique metabolite profiles that are absent or undetectable during later stages of growth. This study aimed to identify secondary metabolites produced by T. harzianum, T. virens, and T. atroviride during their spore germination stage using Gas Chromatography-Mass Spectrometry (GC-MS), and to examine their effects on the germination of 18 postharvest pathogenic fungi, including Alternaria spp., Aspergillus spp., B. cinerea, Cladosporium spp., Fusarium spp., and Penicillium spp. The spore germination stage is a critical phase in fungal life cycles, representing a window for rapid establishment and early competition, which is essential for effective biocontrol.

To prepare standardized spore suspensions and characterize germination behavior, three species of the genus Trichoderma (T. atroviride, T. harzianum, T. virens) and 18 post-harvest plant pathogenic fungi were obtained from Gorgan University of Agricultural Sciences and Natural Resources, Iran (Table 1). The propagation of fungi was carried out on autoclaved potato dextrose agar (PDA) medium (Merck 110130). A mycelial disc (5 mm) from the stock culture was aseptically transferred onto PDA plates (pH 7.0). The plates were then incubated (Memmert IN110plus) at 25 ± 2 °C for three days, with a relative humidity of 60%. All culture and germination experiments were performed in triplicate to ensure reproducibility. The spore suspensions were prepared by scraping spores from actively growing cultures with a sterile loop into sterile distilled water. The suspensions were filtered through sterile 200-mesh gauze to remove mycelial fragments. The suspension was transferred into sterile 15 mL tubes and subjected to centrifugation at 5,000 rpm for 20 min. Following centrifugation, the supernatant was carefully removed using a sampler, and the resultant pellet, which contained the spores, was resuspended in sterile potato dextrose broth (PDB) medium (Himedia GM403) by transferring it to new tubes. The final spore concentration was adjusted to 1 × 10⁵ spore/mL, using a hemocytometer. For each fungal species, 100 µL of spore suspension was placed on a sterile glass slide within a petri dish, covered with a sterile coverslip, and incubated at 25 ± 2 °C in continuous darkness. Spore germination tests were conducted in triplicate, and appropriate negative controls (slides without spores and without medium) were included to monitor background effects. Observations of spore germination and size (average of 100 spores) were made at 40× magnification using an Olympus CH20i light microscope, with spore size and germination recorded every 30 min.

Extraction of secondary metabolites

According to Siddiquee et al.^[19], with slight modifications, each Trichoderma sp. was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile PDB medium using 10 mL of spore suspension (10⁵ spores/mL). Three biological replicates (flasks) were prepared per species. The flasks were incubated at 26 ± 2 °C on an orbital shaking incubator (Benchmark B00BUA672E) with an agitation speed of 4 m/s² for 12 h, a duration selected to capture the early spore germination stage, before activating mycelial growth. PDB medium was then filtered through a 400-mesh filter to separate the mycelial and conidial biomass from the culture broth. An equal volume of analytical-grade ethyl acetate (Merck 109623) was added to the filtered PDB medium, and the mixture was incubated for 12 h at 25 ± 2 °C to ensure the inactivation of any remaining fungal components. The ethyl acetate was separated from the medium using a Buchner vacuum filtration funnel. The extracts were then evaporated at 60 °C using a rotary evaporator (Chemglass CG-1334-X64). The resulting extract was immediately diluted in 100 mL of HPLC analytical-grade n-hexane (Merck 104391) and either subjected to GC-MS analysis or stored at −20 °C in a laboratory-grade freezer (Thermo Fisher Scientific) until analysis.

Identification of secondary metabolites

The GC-MS analysis was conducted using an Agilent 7890A gas chromatograph interfaced with an Agilent 5975C mass spectrometer. The GC-MS system employed electron ionization at 70 eV. A non-polar capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, DB-5MS or its equivalent) was utilized for chromatographic separation. A new and preconditioned DB-5MS column was used to ensure optimal performance and accuracy. The oven temperature was initially set to 60 °C for 1 min, followed by an increase of 10 °C/min until reaching 300 °C, where it was held for 5 min. The injector operated at 300 °C in splitless mode, and the detector temperature was maintained at 320 °C. Helium (99.99% purity, supplied by Linde Gas) served as the carrier gas, flowing at 1.0 mL/min. The mass spectrometer was set with an ion source temperature of 200 °C, scanning in the range of 35–450 m/z at a rate of 0.50 scans per second. A 3-min solvent delay was applied to avoid solvent-related interference.

The system was calibrated using a standard tuning mixture (PFTBA, perfluorotributylamine, Sigma-Aldrich), following the manufacturer's calibration guidelines. Metabolites were identified by comparing mass spectra against the National Institute of Standards and Technology (NIST) library version 23, with a match probability of ≥ 85%. Retention indices (RI) were calculated using a C8–C40 n-alkane series (Sigma-Aldrich) run under the same conditions, and values were compared with literature data for further confirmation. Only compounds with ≥ 85% library match and consistent retention indices were considered positively identified. The identification was qualitative; no internal standard was used for absolute quantification. However, relative abundance was inferred from peak areas in the total ion chromatogram (TIC). Each sample, including both positive (caffeine) and negative (sterile media) controls, underwent triplicate injections.

Evaluation of the spore inhibition activity of secondary metabolites

The extract of each Trichoderma sp. was diluted in sterile PDB medium to achieve a concentration of 200 μg/mL. A sterile glass slide was placed in a sterile petri dish, followed by the addition of 1 mL of prepared PDB medium onto the slide. Subsequently, 100 μL of a post-harvest fungal pathogen spore suspension (10⁵ spores/mL) was added onto the slide. The slide was covered with a sterile coverslip and incubated at 26 ± 2 °C with a relative humidity of 60% under dark conditions for 12 h. Spore germination was defined as the emergence of a visible germ tube that was at least half the diameter of the spore. After incubation, germination was assessed under a light microscope, and the percentage of germinated spores was calculated based on a count of 100 spores per replicate. The percentage of spore germination in PDB medium without secondary metabolites served as the control treatment (Table 1). Each experiment was conducted with five replicates, and the inhibition rate was calculated using the following formula:

where, Gc is the spore germination percentage in the control treatment, and Gt is the spore germination percentage in the presence of secondary metabolites extracted from Trichoderma species during the spore germination phase.

Statistical analysis

A completely randomized design was employed to investigate the impact of each Trichoderma sp. extract on the spore germination rate of postharvest plant pathogenic fungi. The collected data were analyzed using one-way analysis of variance (ANOVA). The Shapiro-Wilk test (p < 0.05) was used to assess the normality of data, and Levene's test (p < 0.05) was conducted to confirm the homogeneity of variances. ANOVA was performed using SPSS statistical software (Version 21.0, SPSS Inc., Chicago, IL, USA). Mean values of the treatments were compared using Duncan's multiple range test at α = 0.05. Graphs were created using Microsoft Excel (Version 2211).

Microscopic evaluations revealed that the spore size of the fungi differed in terms of length, width, and shape, as detailed in Table 1. The highest and lowest spore length was recorded for A. alternata (49.795 µm) and T. virens (2.626 µm), respectively. In general, based on Duncan's multiple range test, Trichoderma spp., Penicillium spp., Aspergillus spp., Cladosporium limoniforme, and C. tenuissimum were grouped as fungi with spore lengths smaller than 5.2 µm, and no significant difference was observed among them. Approximately 42.85% of the other fungi exhibited a spore length greater than 7.9 µm. Furthermore, A. alternata had the largest spore width (8.58 µm), while C. tenuissimum had the smallest spore width (2.09 µm).

In terms of the end germination time, defined as the time when the length of the germination tube reaches one and a half times the length of the spore, species-specific differences were observed among the fungi in Table 1. T. atroviride and T. harzianum completed their germination period faster than the other species, taking approximately 8.7 and 8.8 h, respectively. C. cladosporioides exhibited the longest time (22 h) to complete spore germination. Approximately 61.90% of the studied fungi could complete the spore germination period within a time range exceeding 14 h. Only T. virens, P. glabrum and A. alternata completed the spore germination period within a time range of 11 to 12 h, and Duncan's multiple range test indicated a non-significant difference in germination time between these species. Additionally, A. niger and A. flavus exhibited a similar spore germination period, ranging from 10 to 11 h. By dividing the final length of the germination tube by the elapsed time, it was determined that F. proliferatum, C. cladosporioides, and F. solani showed the highest germination speeds, measuring 1.9, 1.5, and 1.3 μm/h, respectively. The lowest germination speed was observed in P. digitatum, with a rate of 0.287 μm/h. Among the Trichoderma species, T. atroviride exhibited the highest speed (0.681 μm/h), followed by T. harzianum (0.536 μm/h), and T. virens (0.334 μm/h).

Inhibitory effects of secondary metabolites on post-harvest pathogenic fungi

The inhibitory abilities of the secondary metabolites produced by each Trichoderma species varied among the post-harvest plant pathogenic fungi (Fig. 1). This ability displayed a notable distinction even in terms of inhibiting the spore germination of species within the same genus. According to Duncan's multiple range test, the secondary metabolites of T. atroviride displayed the highest inhibitory effect on the spore germination of A. alternata and A. tenuissima, with 39.93% and 37.97%, respectively. No significant difference was observed between them (Fig. 1a). However, according to Duncan's multiple range test, the inhibitory ability of T. atroviride against the spore germination of A. niger, A. flavus, and F. proliferatum was grouped as the least effective treatments, and no significant difference observed between them (Fig. 1a). The secondary metabolites of T. harzianum exhibited the highest inhibitory ability against the spore germination of F. oxysporum, resulting in a reduction of 40.84% (Fig. 1b). On the other hand, the inhibitory capacity of T. harzianum secondary metabolites against the spore germination of A. flavus, C. cladosporioides, and C. ramotenellum reached a minimum level, and Duncan's multiple range test did not indicate a significant difference among them (Fig. 1b). Furthermore, the secondary metabolites of T. virens exhibited the highest inhibitory ability against the spore germination of F. oxysporum, A. alternata, A. tenuissima, and A. arborescens, resulting in reductions of 37.02%, 35.05%, 37.05%, and 35.06%, respectively. No significant difference was observed among these inhibitory effects (Fig. 1c). However, according to Duncan's multiple range test, the inhibitory effect of T. virens secondary metabolites against the spore germination of C. cladosporioides was classified as the least effective when compared to the other post-harvest plant pathogenic fungi (Fig. 1c).

Figure 1. 

Mean comparisons of the inhibition percentage of spore germination of post-harvest fungi as affected by secondary metabolites from initial spore germination stage of each Trichoderma sp. after 12 h. Values are means of five replicates. (a) The effect of T. atroviride on the inhibition of spore germination (Sum of squares: 7,659.592; df: 17; Mean square: 450.564; F-value: 169.764; p-value: 0.0001). (b) The effect of T. harzianum on the inhibition of spore germination (Sum of squares: 8316.157; df: 17; Mean square: 489.186; F-value: 142.419; p-value: 0.0001). (c) The effect of T. virens on the inhibition of spore germination (Sum of squares: 5410.919; df: 17; Mean square: 318.289; F-value: 86.543; p-value: 0.0001). Bars (standard error) with different letters indicate significant differences (Duncan range test subset for α = 0.05).

In terms of grouping the inhibitory ability of the secondary metabolites of Trichoderma spp. against spore germination of a post-harvest pathogenic fungus (Fig. 2; Table 2), Duncan's multiple range test did not reveal any significant difference (p > 0.05) in inhibiting A. flavus and A. tenuissima. Secondary metabolites of T. atroviride showed the highest inhibitory ability against spore germination in 50% of the post-harvest plant pathogenic fungi, in comparison to T. virens and T. harzianum. However, T. harzianum revealed highest inhibitory ability against spore germination of P. expansum, P. digitatum, A. niger, F. oxysporum, F. solani, in comparison to T. virens and T. atroviride. Furthermore, secondary metabolites of T. virens only demonstrated the highest inhibitory ability against spore germination of F. proliferatum and A. arborescens, in comparison to T. atroviride and T. harzianum.

Figure 2. 

Mean comparisons of the inhibition percentage of spore germination of each post-harvest fungus as affected by secondary metabolites from initial spore germination stage of Trichoderma spp. after 12 h. Values are means of five replicates. Bars (standard error) with different letters indicate significant differences. Sections (A)−(R) are independent experiments that grouped separately by Duncan's range test (subset for α = 0.05), which the results of their analysis of variance are presented in Table 2.

GC-MS profiling of secondary metabolites in T. atroviride

The results of GC-MS analysis revealed the presence of 23 secondary metabolites in the 12-h extract of PDB medium inoculated with T. atroviride spores, as detailed in Table 3 and illustrated in Fig. 3. The molecular weight of these secondary metabolites ranged from 86.134 g/mol (Pentanal) to 390.62 g/mol [1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester]. Secondary metabolites with a weight range between 200 and 300 g/mol exhibited the highest abundance, accounting for 43.47% of the identified metabolites from T. atroviride. Metabolites with a weight range between 100 and 200 g/mol followed, representing a frequency of 34.78%. Only four metabolites, accounting for 17.39% abundance, were detected in the molecular weight range between 300 and 400 g/mol in the 12-h extract of T. atroviride. Metabolites with a molecular weight range below 100 g/mol exhibited the lowest abundance, representing 4.34%. Furthermore, all the metabolites identified in the 12-h extract of T. atroviride was recorded for four chemical classes: simple hydrocarbons, organo-oxygens, organo-sulfurs, and organo-nitrogens. Among the identified metabolites, organo-oxygens and simple hydrocarbons were the most prevalent, accounting for 60.86 and 30.43%, respectively. Organic sulfurs and organo-nitrogens, on the other hand, exhibited the lowest frequency, each accounting for 4.34%. Among the metabolites, 1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester and 2-Hexanone, 5-methyl had the highest (47.78%) and lowest (0.11%) area percentages on the chromatogram graph, respectively.

Figure 3. 

Clustered column charts illustrating the distribution percentages of various metabolites across Trichoderma spp. extracts based on GC-MS analysis. (a) and (b) represent the distribution percentages categorized by molecular weight. (c) and (d) display the distribution percentages classified by molecular structure.

Metabolite characterization of T. virens through GC-MS analysis

The quantity of secondary metabolites detected in the 12-h extract derived from PDB medium inoculated with T. virens spores was 9.52% lower compared to those found in T. atroviride, as detailed in Table 4 and illustrated in Fig. 3. GC-MS analysis revealed a total of 21 distinct secondary metabolites present in the extract of germinated T. virens spores. Among these compounds, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, demonstrated the highest chromatographic area percentage at 71.94%, whereas 2,4-di-tert-butylphenol exhibited the minimal relative abundance, registering only 0.02%. Notably, none of the metabolites identified within the 12-h T. virens extract possessed molecular weights below 100 g/mol. The most prevalent class of secondary metabolites fell within the molecular weight range of 100 to 200 g/mol, constituting 47.61% of the total detected compounds. This was followed by metabolites with molecular weights ranging from 200 to 300 g/mol, which represented the second most abundant category. Conversely, compounds with molecular masses between 300 to 400 g/mol and 400 to 500 g/mol were comparatively scarce, comprising 9.52% and 4.78%, respectively. Among the metabolites characterized, 2-methyloctacosane exhibited the greatest molecular weight at 408.8 g/mol, while Propene, 1,1'-oxybis was the lightest, at 100.16 g/mol. Interestingly, no organo-sulfur compounds were detected in the extract. The chemical class of organo-oxygen compounds dominated the profile, accounting for 66.66% of the metabolites identified, followed by simple hydrocarbons which constituted 28.57%. Only a single metabolite belonging to the organo-nitrogen class was present within the 12-h T. virens extract.

Characterization of secondary metabolites from T. harzianum extract

According to the data presented in Table 5 and Fig. 3, the quantity of secondary metabolites detected during the initial spore germination phase of T. harzianum was 15% and 5% lower than that observed in T. atroviride and T. virens, respectively. Nonetheless, the molecular weight distribution of the metabolites identified in T. harzianum exhibited greater heterogeneity compared to the other two species. Overall, GC-MS analysis revealed the presence of 20 secondary metabolites in the 12-h extract of PDB medium inoculated with T. harzianum spores. Among these, only Cyclopentanol was characterized by a molecular weight below 100 g/mol. The most prevalent metabolites fell within the 200 to 300 g/mol and 100 to 200 g/mol weight intervals, constituting approximately 45% and 30% of the total compounds identified, respectively. Metabolites with molecular weights ranging from 300 to 400 g/mol and 400 to 500 g/mol were equally represented, each comprising 10% of the total profile. The heaviest compound identified was 2-methyloctacosane, with a molecular mass of 408.8 g/mol. On the chromatographic profile, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl) ester accounted for the greatest relative abundance (49.48%), whereas Cyclopentanol was the least abundant, registering only 0.11%. Chemically, organo-oxygen compounds dominated the metabolite spectrum at 50%, followed closely by simple hydrocarbons at 40%. Notably, organo-sulfur compounds were absent from the metabolite profile of T. harzianum at this stage, while organo-nitrogen compounds were detected at a comparatively lower frequency of 10%.

The results demonstrate that the secondary metabolites derived from the initial spore germination stage of Trichoderma spp. actively inhibit the spore germination of all post-harvest plant pathogenic fungi. On average, T. atroviride, T. harzianum, and T. virens exhibited inhibitory activities of 27%, 24%, and 25%, respectively, against spore germination of 18 post-harvest fungal pathogens. According to the literature, secondary metabolites derived from the mycelial growth stage of Trichoderma spp. have shown efficacy against Penicillium spp.^[18,20], Aspergillus spp.^[14,15,20], B. cinerea^[20−22], Cladosporium spp.^[20,23], Fusarium spp.^{[15,20,24−26]}, and Alternaria spp.^[27,28]. Although these studies, in line with our findings, highlight the significant bioactivity of Trichoderma spp. metabolites, none have investigated the compounds produced specifically during the spore germination stage.

This study provides the first evidence that spore germination metabolites of Trichoderma spp. serve as key competitive factors in the environment, enabling rapid niche colonization and suppression of competing spores before hyphal establishment, thereby positioning Trichoderma spp. as early ecological dominants in microhabitats. GC-MS analysis identified 39 secondary metabolites produced by T. harzianum, T. virens, and T. atroviride during this early developmental stage. Of these, 30.76% were unique to T. atroviride, while 12.82% and 15.38% were specifically produced by T. virens and T. harzianum, respectively. Additionally, 23.07% of the compounds were shared across all three species. Another 12.82% were common to T. virens and T. harzianum, whereas only 5.12% were shared between T. atroviride and T. virens. These early-stage Trichoderma metabolites show potential for biocontrol, particularly in postharvest settings where fungal spores are present but mycelial growth hasn't begun.

Diversity of initial spore germination stage metabolites of Trichoderma spp.

Several metabolites identified in this study have previously demonstrated antimicrobial activity during the mycelial growth stage of Trichoderma spp. For example, we exclusively detected lauric acid in T. atroviride, a compound that has also been reported during the mycelial stage of T. atroviride, T. harzianum, and T. asperellum^[29−31]. Studies have confirmed that monolaurin, a derivative of lauric acid, inhibits various fungal species, including Candida albicans^[32] and Trichophyton rubrum^[33]. Researchers attribute this antifungal activity to monolaurin's ability to disrupt fungal lipid membranes, leading to cell lysis and death^[34]. We also identified palmitic acid, methyl ester exclusively from T. atroviride, which belongs to the class of fatty acid methyl esters (FAMEs). Studies have shown that FAMEs derived from vegetable oils exhibit antifungal activity against pathogens such as Paracoccidioides spp., C. glabrata, C. krusei, and C. parapsilosis^[35]. Additionally, FAMEs extracted from Sesuvium portulacastrum leaves have demonstrated antimicrobial effects against both bacterial and fungal human pathogens^[36]. The production of palmitic acid and its methyl ester has also been observed during the mycelial stage of T. longibrachiatum and T. reesei^[37,38]. Palmitic acid was also detected in all Trichoderma species examined in this study. In vitro experiments have revealed that palmitic acid effectively inhibits C. tropicalis and its biofilm formation, a critical factor in fungal pathogenicity^[39]. Previous research has reported the biosynthesis of palmitic acid during the mycelial phase of T. virens and T. harzianum^[16].

We identified undecanoic acid exclusively during the spore germination stage of T. virens. However, prior studies have reported this metabolite during the mycelial stage of T. harzianum, T. koningii, T. koningiopsis, and T. asperellum^[31,40−42]. Rossi et al.^[43] highlighted undecanoic acid's potential as a therapeutic antifungal agent. Similarly, the study of Lee et al.^[44] demonstrated its ability to interfere with fungal communication and exert antibiofilm and anti-virulence effects against C. albicans. Moreover, Avrahami & Shai^[45] explained that conjugating undecanoic acid with amino acid-containing antimicrobial peptides enhanced their antifungal activity. Although earlier studies have identified decanoic acid during the mycelial stage of T. viride, T. koningii, T. koningiopsis, T. reesei, and T. virens^[40,46−48], we detected it only during the spore germination stage of T. virens.

The antifungal mechanism of decanoic acid is believed to involve membrane disruption, causing cellular leakage and fungal cell death^[49]. Itaconic acid was uniquely detected in T. harzianum in this research, whereas previous studies have reported its presence during the mycelial stage of T. reesei^[50]. Itaconic acid has been found to exert anti-inflammatory and antibacterial effects^[51]. Multiple studies have explored itaconic acid's antimicrobial roles in macrophages and its function as an endogenous antimicrobial metabolite^[52]. A study reported the antibacterial activities of hexadecanoic acid methyl ester against multidrug-resistant bacteria^[53]. We also identified hexadecanoic acid, methyl ester similar from T. virens and T. harzianum, which is commonly referred to as methyl palmitate and has been isolated from the mycelial stage of T. pseudokoningii^[17,20]. Differences in metabolite profiles between germination and mycelial stages suggest that Trichoderma probably regulates secondary metabolism in a phase-specific manner, likely influenced by environmental and developmental signals, an area worth further study.

Metabolites without an antifungal background

Several metabolites identified during the initial spore germination stage of Trichoderma spp. in this study, as well as those reported during the mycelial phase in previous investigations, have not been shown to possess direct antifungal activity. For example, we exclusively detected tridecane in T. atroviride during spore germination, although Siddiquee et al.^[19] previously reported it during the mycelial stage of the same species. Similarly, we identified dodecane during the spore germination stage of all Trichoderma spp., while earlier studies have documented its presence only during the mycelial phase of T. harzianum^[29]. Phenol, 2,4-bis(1,1-dimethylethyl), was detected exclusively in T. atroviride in our current analysis, whereas Mulatu et al.^[54] found this compound in the mycelial stage of T. asperellum and T. longibrachiatum. Although we identified hexadecane in T. atroviride, T. harzianum, and T. asperellum, previous literature has confirmed its occurrence only in the mycelial stage of T. harzianum^[55]. In alignment with our findings, the literature also confirms that octadecane appears commonly in both spore germination and mycelial stages of T. atroviride, T. harzianum, and T. virens^[56,57].

We exclusively identified eicosane during the spore germination stage of T. atroviride, while earlier studies reported this compound only in the mycelial phase of T. harzianum^[58]. Furthermore, literature findings indicate that tetradecane occurs during the mycelial stage of T. biocontroller, T. harzianum, T. viride, T. asperellum, and T. virens^[59−61]. However, we detected it during the spore germination stage of T. virens and T. harzianum. Both phthalic acid, di(2-propylphenyl) ester and 2,4-Di-tert-butylphenol were identified in the spore germination stage of T. virens and T. harzianum in this study. In contrast, previous reports have described phthalic acid, di(2-propylphenyl) ester only during the mycelial stage of T. pinnatum^[62], and 2,4-Di-tert-butylphenol from T. asperellum^[63]. Consistent with our results, the literature also confirms the presence of 2,4-diamino-6-hydroxypyrimidine in both the spore germination and mycelial phases of T. harzianum^[64]. Importantly, 56.41% of the metabolites identified in this study have not been previously reported in any Trichoderma species. However, the study is limited to extracellular compounds and spore germination assays. Future research should explore metabolite synergy, molecular regulation, broader pathogen targets, and real-world efficacy.

These findings elucidate the crucial role of these metabolites in mediating the initial competitive interactions of Trichoderma spp. with other fungi, thereby enhancing their dominance in the environment. By unraveling the ecological significance of these metabolites in Trichoderma interactions with other organisms and the surrounding environment, our study contributes to a deeper understanding of microbial ecology and secondary metabolite biology. Importantly, these insights also have practical implications for postharvest disease management. The early production of antifungal metabolites suggests that Trichoderma spp. can be strategically employed to suppress pathogenic fungi during vulnerable postharvest stages. Understanding the timing and profile of metabolite release may inform the development of more effective biocontrol formulations, enabling targeted application to reduce spoilage and losses in storage and supply chains.