Phenolic Compounds Synthesized by Trichoderma longibrachiatum Native to Semi-Arid Areas Show Antifungal Activity against Phytopathogenic Fungi of Horticultural Interest

Abstract

Fungal diseases are a major threat to the horticultural industry and cause substantial postharvest losses. While secondary metabolites from Trichoderma sp. have been explored for their antifungal properties, limited information exists on the phenolic compounds produced by less studied species like Trichoderma longibrachiatum. In this study, phenolic compounds were extracted from a liquid culture of T. longibrachiatum using various solvents and methods (conventional and ultrasonic-assisted). Phenolic compounds were quantified by spectrophotometry and identified by high-performance liquid chromatography with diode array detection (HPLC-DAD). The antifungal activity against Alternaria alternata and Fusarium oxysporum was determined by mycelial growth inhibition assays, maximum growth rate (µ_max) by the Gompertz equation, and spore germination tests. Although no significant differences (p ≥ 0.05) were found between the extraction methods, the type of solvent significantly influenced the phenolic content (p ≤ 0.05). Extraction with 70% ethanol showed the highest content of phenolic compounds and flavonoids. More than eight phenolic compounds were detected. Further, this is the first report of the phenolics ferulic, chlorogenic and p-coumaric acids identification in T. longibrachiatum, along with flavonoids such as epicatechin and quercetin, among others. The 70% ethanolic extracts notably inhibited the mycelial growth of A. alternata and F. oxysporum, reducing their maximum growth rate by 1.5 and 1.4 mm/h, respectively. Furthermore, p-coumaric and ferulic acids significantly inhibited spore germination of both pathogens, with a minimum inhibitory concentration (MIC) of 1.5 mg/mL and a minimum fungicidal concentration (MFC) of 2 mg/mL. These findings demonstrate the potential of T. longibrachiatum and its phenolic compounds as viable alternatives for biological control in horticulture and postharvest disease management.

1. Introduction

One of the main problems in the horticultural industry is the loss of fruits and vegetables postharvest due to infections with phytopathogenic fungi, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Monilinia sp., Penicillium sp., and Rhizopus sp. [1,2]. Fungi infections are controlled with chemical compounds that are not one hundred percent effective. In addition, due to the prolonged and excessive use of synthetic fungicides, there is a growing concern in the population because of the potential negative impact on human health and the environment [3]. Furthermore, the intensive use of synthetic fungicides induces the development of resistant strains of microorganisms [4,5]. For this reason, it is important to carry out studies that lead to the development of safe alternatives to control diseases in horticultural products. One of the alternatives is the use of biocontrol agents among which the genus Trichoderma predominates [1].

Trichoderma is a widely studied fungus that lives in the soil, and it is characterized by its saprophytic or parasitic behavior. It presents mechanisms of action that give it advantages over phytopathogenic fungi, such as mycoparasitism, competition for nutrients and space, induction of defense systems in plants, and antibiosis through secondary metabolites (SMs) [6]. SMs have various biological functions, such as anticancer [7], herbicidal [8], nematicidal [9], and antimicrobial activity [10]. Based on the work published by Mukherjee et al. [11], some Trichoderma species have the ability to synthesize metabolites with potent antimicrobial activity that can have a considerable impact on the control of plant diseases. Among the bioactive metabolites produced by Trichoderma are polyketides, terpenes, terpenoids, pyrogens, phenols, alkaloids derived from indolic compounds, and non-ribosomal peptides [12,13]. Indeed, the antifungal effects of SM produced by various species of Trichoderma, such as T. asperellum, T. atroviride, T. viride, T. harzianum, and T. koningiopsis have been reported for the control of diseases in different fruits, such as tomato [14], melon [15], and chili pepper [16]. However, it has been reported that different species of the same family and different isolates of the same species can often produce different SMs [17], which suggests the need to study every Trichoderma isolate.

Although various SMs participating in the antifungal activity of Trichoderma have been identified, there is little information on the antifungal activity of phenolic compounds against phytopathogenic fungi. Gajera [18] and Al-Askar [19] found a relationship between the production of phenols by Trichoderma species and antifungal activity against various fungal species. Tchameni, et al. [20] identified more than 20 compounds in extracts of four species of Trichoderma, showing a significant correlation between the production of phenolic compounds and flavonoids and the antifungal activity against Pythium myriotylum. The minimum inhibitory concentrations were 80, 40, 20, and 10 μg/μL for extracts obtained from T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11), respectively. These results demonstrate the antifungal effect of phenolic compounds produced by Trichoderma species. However, the type of phenolic compounds that have antifungal activity has not been reported. Therefore, there is a need to identify specific phenolic compounds with the capacity to reduce or inhibit the growth of phytopathogens.

There are several methods for extracting phenolic compounds. Different methods have been reported, such as solid-liquid extraction [21], Soxhlet extraction [22], liquid-liquid extraction [23], pressurized liquid extraction [24], microwave-assisted extraction, and ultrasound-assisted extraction (UAE) [25]. Moreover, not only the methodology but also the solvent system plays a crucial role in the extraction process, since different phenolic compounds have variable chemical miscibilities, polarities, and characteristics. During the isolation process, organic solvents, such as acetone, benzene, chloroform, methanol, n-hexane, and petroleum ether, are often required. However, the use of these solvents has several disadvantages, including being flammable, explosive, poorly biodegradable, and toxic to the final product [26,27]. The most commonly used solvents for the extraction of phenolic compounds are water, ethanol, methanol, acetone, and their mixtures with water [28]. Currently, there is no standard method for the extraction of phenolic compounds, so it is important to establish a procedure, mainly for the extraction of phenols from fungi, such as Trichoderma.

Trichoderma harzianum is one of the most studied and characterized species [29]. However, there are other species that have been less studied as producers of SMs, such as T. longibrachiatum. This Trichoderma species is a fungus that has been isolated in different geographic regions, such as forests, arid areas, and aquatic environments [30]. Due to its ability to adapt to arid conditions, T. longibrachiatum has been isolated from desert areas, such as the Sahara Desert [31] and the Sonoran Desert in North America [32]. This ability to adapt to arid conditions is due to the fact that they can resist and, in turn, produce secondary metabolites in response to abiotic factors such as soil pH, temperature and salinity, among others, typical of desert areas [33]. The authors reported that T. longibrachiatum inhibited the growth of Fusarium solani and induced the activation of the phenylpropanoid pathway and oxidative stress enzymes in transgenic cotton plants [32]. Further, Zhang, et al. [34] observed that non-volatile SM from T. longibrachiatum isolated from forest soil presented antifungal activity against various phytopathogenic fungi such as Valsa mali, the causal agent of apple canker. Although it has been reported that these species of Trichoderma produce certain SMs with antifungal activity, there are few studies reporting the production of phenolic compounds with antifungal activity. Therefore, this study aimed to analyze the capacity of T. longibrachiatum to produce phenolic compounds with antifungal activity against fungi that deteriorate horticultural products.

2. Materials and Methods

2.1. Microorganisms Used and Culture Conditions

Strains of T. longibrachiatum previously isolated from olive tree cultivation soil (Latitude N: 30°50′02.7″, Longitude: 112°54′02.7″) located in the Altar desert, northwest Mexico, were used. The T. longibrachiatum strain was characterized taxonomically and molecularly [35]. The sequence was deposited in GenBank (PP538035, PP538036). The strains of phytopathogenic fungi used belong to isolates from infected fruits, which were identified according to their morphological and phenotypic characteristics as A. alternata and F. oxysporum [36,37,38]. The strains preserved at 4 °C in the Plant Biotechnology and Postharvest laboratory of CIAD, AC, Sonora, Mexico, were reactivated in PDA at a temperature of 28 °C for 15 days. In the case of T. longibrachiatum, the reactivation time was 4 days.

2.2. Obtaining the Crude Extract of T. longibrachiatum in Submerged Culture

A spore suspension of T. longibrachiatum was inoculated at a concentration of 10⁸ spores/mL in 250 mL of PDB medium and incubated in an orbital shaker (Shaker Environ 3527) for 14 days at 28 ± 1 °C and 150 rpm/min in darkness. Subsequently, the culture medium was filtered with Whatman No. 40 filter paper, followed by vacuum filtration through a cellulose acetate membrane, 0.45 µm (Corning, Glendale, AZ, USA). The resulting filtrate was called the crude extract. The crude extract was frozen at −80 ± 1 °C and lyophilized to dryness. The dried crude extract was stored at −80 ± 1 °C for later use.

2.3. Extraction of Phenolic Compounds from Extract of T. longibrachiatum

Two extraction methods were tested: conventional (CE, based on the use of different extraction solvents) and ultrasound-assisted (UAE). For each method, six extraction solvents were evaluated: 100% ethanol, 70% ethanol, 100% methanol, 80% methanol, water, and 100% acetone. The dry crude extract was resuspended in a 1:20 (w/v) ratio with each of the different solvents. In CE, the mixture was kept under constant stirring in a water bath at 40 ± 1 °C for 30 min. UAE was carried out in an ultrasonic bath (Branson 2510) with the power and working temperature set to 250 W and 40 ± 1 °C, respectively, for 30 min [25]. The extraction process was carried out in duplicate. Finally, the content of total phenolics and flavonoids was determined for each of the extracts obtained.

2.4. Quantification of Total Phenolics and Flavonoids

The total phenolic content was determined with three replicates per treatment using the Folin-Ciocalteau method according to Lopez, et al. [39], based on a calibration curve modeled by the regression equation: y = 1.8157x − 0.0105, with R² = 0.9929. For the quantification of total phenolics, the results were expressed as microgram equivalents of gallic acid per milliliter (µg EAG/mL). The flavonoid content was determined with three replicates per treatment according to the Aluminum Chloride method according to Kamali, et al. [40] from the regression equation: y = 8.2821x − 0.0182, with R² = 0.9925. The results were expressed as micrograms of quercetin equivalent per milliliter (µg QE/mL).

2.5. Determination of the Profile of Phenolic Compounds by HPLC

From the phenolic extract with a major content of phenols and flavonoids, the type of phenolic compounds present in the extract was determined by HPLC-DAD. The chromatographic test of the phenolic extract was carried out according to the technique described by Mradu, et al. [41] with some modifications. Chromatographic separation was carried out in a reversed-phase liquid chromatograph (RP-HPLC) (Agilent Technologies, Glendale, AZ, USA) equipped with a diode array detector (DAD) (Agilent Technology, 1290 Infinity). The injection volume was 20 µL from the extracts previously filtered (0.22 µm nylon membrane) (Millipore Co., Bedford, MA, USA). For separation of analytes, a Luna C₁₈ reverse phase analytical column (5 µ, 250 × 4.6 mm) with a binary gradient (A: 100% acetonitrile and B: 0.1% v/v orthophosphoric acid in MilliQ water) was used at a flow rate of 1 mL/min. The elution gradient for the separation of phenolic acids began with 8% of A and 92% of B for 5 min, then 40 min with 22% of A and 78% of B, and finally 1 min with 8% of A and 92% B. The analytes were detected at a wavelength of 280 nm. Phenolic acids were identified by comparing their retention times with those of commercial standards: gallic, shikimic, protocateic, hydroxybenzoic, vanillic, caffeic, syringic, chlorogenic, ferulic, isoferulic, o-coumaric, and p-coumaric acids (Sigma-Aldrich, St. Louis, MO, USA).

For the analysis of flavonoids, the same binary gradient used for phenolic acids (A: 100% acetonitrile and B: 0.1% v/v orthophosphoric acid in MilliQ water) was used at a flow rate of 1 mL/min. An elution gradient was used that consisted of 15% A and 85% B from 0 to 5 min, 30% A and 70% B at 10 min, 32% A and 68% B at 15 min, 33% A and 67% B at 22 min, 50% A at 27 min, 95% A and 5% B at 39–46 min, and 15% A and 95% B at 50 min. The analytes were detected at a wavelength of 270 nm. The resulting flavonoids were identified by comparing their retention times with those of commercial standards of catechin, esculetin, epicatechin, naringenin, scopoletin, morin, kampferol, luteolin, quercetin, rutin, kamferol-3-O-glucoside, kamferol-3-O-rutinoside, naringenin chalcone, and naringenin-7-O-glucoside (Sigma-Aldrich, USA).

2.6. Antifungal Capacity of the Ethanolic Extract on Mycelial Growth

In 50 mm diameter Petri dishes containing PDA culture medium, two 5 mm diameter wells were made with a separation of 1.5 cm between them. In the central well, 100 µL of spore suspension of 10⁴ spores/mL of the phytopathogen to be evaluated (A. alternata and F. oxysporum) was added, and 100 µL of ethanolic extract of T. longibrachiatum was added to the other well (Figure 1). The control was carried out under the same treatment conditions, and 70% ethanol was added. The plates were incubated at 28 °C for 5 days (120 h). The diameter of the colony was measured every 24 h using a vernier caliper. Two biological replicates were carried out, with five replicates per treatment.

The percentage of inhibition of mycelial growth (IMG) was calculated using the following equation:

% IMG = (1 − Dp/CD) × 100)

(1)

where CD is the diameter of the control colony, and Dp is the diameter of the phytopathogen colony.

In addition, the effect of the extract on mycelial growth was determined using the Gompertz model. The maximum growth rate was estimated with the DMFit Excel tool using the following equation:

$$y=A\mathrm{e}\mathrm{x}\mathrm{p}(-\exp \left({\displaystyle \frac{\mathrm{\mu }\mathrm{m}\mathrm{a}\mathrm{x}}{A}}\left(\mathsf{\lambda }-\mathrm{t}\right)+1\right))$$

(2)

where y is the diameter of the colony at a given time, A is the maximum growth of the fungus in the stationary phase, t is time (h), λ: latency phase (h), µ_max maximum growth rate (mm/h). The maximum growth rate indicates the maximum rate at which the population grows during the exponential phase.

2.7. Antifungal Capacity of the Ethanolic Extract on Spore Germination

For spore germination analysis, 50 µL of ethanolic extract and 10⁴ spores/mL (v:v) suspension were placed in an Eppendorf tube. The mixture was incubated for 24 h at 28 °C. An aliquot of 20 µL of the mixture was taken, which was placed on a slide and observed under a microscope (Binocular Primo Star Carl Zeiss, Jena, Germany) at 40× magnification at different incubation times (0, 6, 12, 24, and 48 h). Spore counting was performed by counting the number of germinated spores with respect to the total number of spores in the visual field. Spores were considered germinated when the germ tube reached half the size of the spore. The results were reported as the percentage of spore germination. After treatment, the fungicidal activity of the ethanolic extract was evaluated by sub-culturing 20 µL of Eppendorf tube content on PDA plates. The results were recorded after incubating the plates for 24 h at 28 °C.

2.8. Antifungal Activity of Phenolic Acids Identified on Spore Germination

The antifungal activity of the phenolic acids identified in T. longibrachiatum was tested using commercial reagents. Based on the phenolic acids detected in T. longibrachiatum extract, chlorogenic, ferulic, and p-coumaric acids (Sigma-Aldrich, USA) were used in this experiment. The preparations were made in 1.7 mL conical Eppendorf tubes, to which different concentrations of each phenolic acid were added (0.5, 1.0, 1.5, 2.0, 4.0, and 6.0 mg/mL in 10% DMSO). A suspension of 10⁴ spores/mL in the PDB medium of the phytopathogen to be evaluated (A. alternata and F. oxysporum) was prepared from plates after 10 days of incubation. The final volume was 100 µL. To the control, only a 10% DMSO solution was added. Observation and quantification of germinated spores were performed at different incubation times (0, 6, 12, 24, and 48 h), as described previously. The results were reported as the percentage of spore germination. In addition, the minimum inhibitory concentration (MIC) of phenolic acids on phytopathogenic fungi was determined according to the proposal of the National Committee for Clinical Laboratory Standards (NCCLS) with some modifications [42]. The same procedure mentioned above was followed using the same concentrations of phenolic acids. After 24 h of spore exposure to phenolic acids, the minimum fungicidal concentration (MFC) values for phenolic acids were evaluated by sub-culturing 20 µL showing no turbidity on PDA plates. Results were recorded after incubating the plates for 24 h at 28 °C. This experiment was carried out twice with three replicates.

2.9. Statistical Analysis

A completely randomized design with a factorial arrangement was used to analyze the effect of extraction methods (two levels: conventional and ultrasound-assisted) and solvents (six levels: 100% ethanol, 70% ethanol, 100% methanol, 80% methanol, water, and 100% acetone) on total phenolics and flavonoids content. For the antifungal activity of T. longibrachiatum extract, means, and kinetic parameters were reported as mean ± standard deviation and compared using Student’s t-test at a 95% confidence level. Furthermore, a completely randomized design with a factorial arrangement was employed to investigate the antifungal activity of different concentrations of phenolic acids (seven levels: 0, 0.5, 1.0, 1.5, 2.0, 4.0, and 6.0 mg/mL) as a function of time (five levels: 0, 6, 12, 24, and 48 h) on the percentage of germinated spores. Significant differences in means were determined using the Tukey-Kramer multiple comparison method with a confidence level of 95% (p ≤ 0.05). All treatments were performed in triplicate. Analysis of variance (ANOVA) was performed using the NCSS 2007/GESS 2006 statistical package, and graphs were prepared using GraphPad Prism 9^®.

3. Results

3.1. Effect of Extraction Method on the Concentration of Phenolic Compounds

The type of solvent, extraction method, and sample are crucial factors for obtaining phenolic compounds. In this study, two extraction methods (CE and UAE) were evaluated using different solvents from the lyophilized crude extract of T. longibrachiatum. Analysis of the results showed that there were no significant differences between the extraction methods (p ≥ 0.05). However, significant differences were observed between the solvents analyzed (p ≤ 0.05). Figure 2 shows that methanol, 80% methanol, water, and 70% ethanol were equally effective for the extraction of total phenols (p ≥ 0.05). However, with 70% ethanol, a concentration of 144 µg GAE/mL was obtained, which is the highest amount recorded. In contrast, extraction with acetone turned out to be the least effective for obtaining phenolic compounds (Figure 2A). In the case of flavonoid extraction (Figure 2B), 70% ethanol allowed a greater extraction (126 µg QE/mL) with respect to the other solvents (p ≤ 0.05), but it was not statistically different with respect to ethanol. In this study, 70% ethanol was selected because it was the most effective solvent for extracting both phenolics and flavonoids.

3.2. Phenolic Profile of T. longibrachiatum Extract

According to the results obtained in the previous section, in this study, the ethanolic extract obtained from T. longibrachiatum after 14 days of incubation was analyzed by HPLC-DAD. Chromatographic analysis revealed the presence of nineteen well-defined and separated peaks corresponding to various phenolic compounds (Figure 3). Based on the retention times of the commercial standards of phenolic acids, it was possible to identify only seven compounds. These compounds were identified as gallic, protocateic (derived from hydroxybenzoic acid), chlorogenic, p-coumaric, ferulic, isoferulic, and o-coumaric acids (derived from hydroxycinnamic acid) (Figure 3A). Likewise, based on the retention times of commercial flavonoid standards, the presence of catechin, scopoletin, kaempferol, naringenin, phloridzin, morin, and quercetin was determined (Figure 3B). In addition, ferulic acid and morin were the phenolic compounds that presented peaks of greater height than the other compounds.

The chromatograms revealed the presence of other well-defined peaks that could not be identified, suggesting the presence of additional phenolic compounds or derivatives not covered by the standards used. The application of HPLC-DAD proved highly effective for the separation and identification of these compounds, which suggests the usefulness of this technology in the comprehensive analysis of fungal metabolites. Future studies should aim to identify unknown peaks, potentially through mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy, to fully elucidate the phenolic profile of T. longibrachiatum. The identified compounds could have antifungal and antioxidant properties and several of these phenolic acids and flavonoids have been documented to exhibit such activities. These findings not only expand our understanding of the metabolic capabilities of T. longibrachiatum but also suggest potential applications in biological control and plant protection.

3.3. Effect of the Ethanolic Extract of T. longibrachiatum on Mycelial Growth of A. alternata and F. oxysporum

The ethanolic extract of T. longibrachiatum significantly reduced (p ≤ 0.05) the mycelial growth of A. alternata and F. oxysporum under in vitro conditions. In Figure 4A,B, the inhibition of mycelial growth occurs from the first 24 h after treatment, reaching a maximum in the percentage of inhibition of 33.9% in A. alternata and 24.8% in F. oxysporum, 120 h after treatment. According to the Gompertz model, the ethanolic extract of T. longibrachiatum significantly decreased the µ_max of both phytopathogens (

Table 1. Kinetic parameters of the effect of the ethanolic extract of T. longibrachiatum on mycelial growth of A. alternata and F. oxysporum for 120 h.

Phytopathogen	Treatment	µmax (mm/h) *	R2	p Value
A. alternata	Control	0.39 ± 0.02 a	0.9912	0.0001
	Extract	0.24 ± 0.00 b	0.9913
F. oxysporum	Control	0.41 ± 0.00 a	0.9898	0.0001
	Extract	0.28 ± 0.00 b	0.9985

* µ_max: maximum growth rate. Values represent the mean ± standard deviation (n = 5). Different literals per phytopathogen indicate significant differences between treatments (p ≤ 0.05).

). In A. alternata exposed to the ethanolic extract, the mycelial µ_max was reduced by 62.8%, while the ethanolic extract was reduced by 70% in F. oxysporum (Table 1). These results suggest that F. oxysporum is more susceptible to the components of the ethanolic extract of T. longibrachiatum. In addition, a reduction in mycelial growth was observed compared to the control (Figure 4), where a smaller diameter of the colony was observed in the treatment.

3.4. Effect of the Ethanolic Extract of T. longibrachiatum on Spore Germination of A. alternata and F. oxysporum

Figure 5 shows the effects of T. longibrachiatum extract on spore germination of A. alternata and F. oxysporum. After 24 h of incubation, it was observed that there was no germination in any of the phytopathogens. To determine the fungicidal capacity of the ethanolic extract, spores exposed to the extract were transferred to a PDA Petri plate. After 24 h of incubation, no fungal growth was observed. It is important to mention that, in the controls, the germination process began at 3 h for A. alternata and for F. oxysporum after six hours in PDB. Based on the results obtained, the ethanolic extract of T. longibrachiatum had a fungicidal effect on spore germination in both phytopathogens.

3.5. Antifungal Activity of Phenolic Acids on Spore Germination

Figure 6 shows the effects of ferulic, chlorogenic, and p-coumaric acids previously identified in the extract of T. longibrachiatum on the germination of A. alternata and F. oxysporum spores. As can be seen, the phenolic acids analyzed decreased the percentage of spore germination in both fungi evaluated. Low concentrations (1.0 mg/mL) of ferulic acid did not decrease the germination percentage of both phytopathogens. In contrast, a concentration of 1.5 mg/mL of this acid delayed the start of germination by 3 h in A. alternata and 6 h in F. oxysporum. No spore germination was observed at concentrations of 2.0, 4.0, and 6.0 mg/mL in both fungi. A similar behavior was observed when testing 1.0 mg/mL of p-coumaric acid, which did not reduce spore germination. In comparison, 1.5 mg/mL delayed the start of germination by 12 h in A. alternata and 6 h in F. oxysporum. Furthermore, 90–100% germination was observed 24 h post-treatment in both fungi.

Regarding chlorogenic acid, 1.0 mg/mL concentration reduced the germination process during the first 6 h in A. alternata, reaching 100% germination at 12 h post-treatment. While the application of 1.5 mg/mL delayed the start of germination by 3 h, achieving 50% germination at 12 h and 100% at 48 h post-treatment. No spore germination of A. alternata was observed when applying 4.0 and 6.0 mg/mL of p-coumaric acid. The germination of F. oxysporum spores was similar when applying the three phenolic acids. Based on these results, the minimum inhibitory concentration (MIC) for ferulic and p-coumaric acids was 1.5 mg/mL, except for chlorogenic acid against A. alternata, where the MIC was 2.0 mg/mL. These results suggest that, although phenolic acids did not completely prevent spore germination at these concentrations (0.5, 1.0 mg/mL), they did affect their development.

This inhibitory effect on the development of germ tubes could be indicative of the fungistatic action of phenolic acids, which interfere with cell elongation and cell wall formation in germinated spores. In our study, spores exposed to phenolic acids for 48 h were inoculated onto PDA agar. After 24 h, it was observed that exposure to higher concentrations of phenolic acids lowered the growth on the plates. This suggests that phenolic acids presented fungistatic activity at concentrations 1.5 mg/mL; while at higher concentrations, from 2.0 mg/mL, the effect was fungicidal for p-coumaric acid where no fungal growth was observed. In the case of ferulic and chlorogenic acids, the fungicidal activity was from 4 mg/mL (Figure 7).

4. Discussion

The native strain of T. longibrachiatum from the semi-arid zone of northwest Mexico has demonstrated the ability to synthesize phenolic compounds in submerged liquid culture. The literature reports several extraction methods for phenolic compounds using different organic solvents, with the efficiency of the extraction method depending on the type of solvent and sample [43]. In this study, 70% ethanol was the most efficient solvent for the extraction of phenolic compounds from the T. longibrachiatum culture. In contrast, Hameed, et al. [44] found greater extraction efficiency using acetone followed by ethanol and methanol in Mucor circinelloides. This is most likely due to the nature of the phenolic compounds present in the extract, which have high and intermediate polarities. Phenolic compounds present in mycelia may require stronger solvents for extraction [45]. This indicates that the combination of water and ethanol is the best condition for the extraction of phenolic compounds from the culture extract of T. longibrachiatum.

The analysis of the phenolic profile of T. longibrachiatum by HPLC-DAD allowed us to identify, for the first time, twelve phenolic compounds in this species. Among these, the phenolic acids are gallic, protocateic, chlorogenic, p-coumaric, ferulic, isoferulic, and o-coumaric acids, and the flavonoids are catechin, epicatechin, phloridzin, morin, and quercetin. So far, certain polyphenols (6-ethoxy-3(40-hydroxyphenyl)-4-methylcoumarin and 2-hydroxycinnamic acid), as well as several flavonoids (dihydromyricetin, isorhamnetin, 40-hydroxy5, 7-dimethoxyflavanone, and afzelequin) have been reported in 24 h cultures of T. asperellum [13]. In a similar study, the presence of four-methoxymethylphenol was detected in T. longibrachiatum isolated from the rhizosphere of P. incarnata [46]. Based on the results obtained, the detection of these compounds in the present study expands our knowledge of the diversity of metabolites produced by the Trichoderma genus. It is important to mention that some compounds detected in this study could not be identified. Therefore, further analysis is needed to identify these compounds.

The study of phenolic compounds is of great interest since antifungal and antibacterial properties have been reported [47]. These compounds have been shown to be effective in inhibiting the growth of several phytopathogens [48], including fungi and bacteria [49]. According to the results of the present study, T. longibrachiatum produces phenolic compounds that can help protect plants and fruits from infection by phytopathogenic fungi. Furthermore, phenolic acids such as caffeic, chlorogenic, and vanillic acid are compounds that have been detected in tomatoes infected with phytopathogens, such as A. alternata [50]. Also, in the study carried out by Ruelas, et al. [51], it was observed that caffeic and chlorogenic acids reduced the germination of A. alternata spores by up to 76.96 and 79.27%. This suggests a natural way in which these compounds could contribute to antifungal activity. Therefore, it is crucial to explore the potential of these compounds as antifungal agents for horticultural applications.

With the Gompertz model, the parameter µmax can be obtained to evaluate how it affects the proliferation capacity of phytopathogens when exposed to the extract [52]. The decrease in µmax of the phytopathogenic fungus shows the ability of the T. longibrachiatum extract to reduce the growth and development of the phytopathogenic fungus. Furthermore, the inhibition of spore germination suggests the fungicidal nature of this extract, which supports its application as an antifungal agent for the control of plant diseases. This finding is consistent with the antifungal activity previously reported for phenolic compounds present in Trichoderma extracts. Imran, et al. [53] evaluated culture filtrates of three Trichoderma isolates, including T. longibrachiatum, to mitigate tomato early blight caused by A. solani and found that T. longibrachiatum decreased by 48.7% the growth of this phytopathogen. In addition, an alteration in mycelial growth was observed, which coincides with the results observed in the present study. In a similar study, Abdelmoteleb et al. [31] reported that cell-free culture filtrate of T. longibrachiatum isolated from rhizospheric soil of common bean (Phaseolus vulgaris L.) plants significantly inhibited mycelial growth of F. solani, with growth reduction ranging from 28.8 to 97.3% and inhibited spore germination up to 96%. Recently, Rauf, et al. [54] evaluated the effect of different Trichoderma spp. extracts on the percentage decrease in the growth of F. oxysporum f.sp. pisi. Among the different Trichoderma species evaluated (T. longibrachiatum, T. hamatum, T. harzianum, T. koningii, and T. viridie), T. harzianum at concentrations extract of 40–60%, showed the maximum reduction of fungal biomass (58–87%); while T. longibrachiatum reduced 64–83% of fungal growth. These results are slightly higher than those obtained in our study, which may be because they used the crude extract obtained from the culture on malt extract broth medium. Although the chemical composition of Trichoderma extract was not reported in that study, other studies have reported that this type of extract contains several secondary metabolites (alkaloids, terpenes, polyphenols, saponins, etc.), as well as hydrolytic enzymes that act directly on phytopathogens [55].

These findings are consistent with those of previous studies that have demonstrated the antifungal activity of phenolic acids against a variety of plant pathogens. Zabka and Pavela [56] reported the efficacy of individual phenols against a group of six toxigenic and phytopathogenic fungi of the genera Fusarium, Penicillium, and Aspergillus. The authors obtained 100% growth inhibition of these phytopathogens using phenolic compounds, such as thymol, carvacrol, eugenol, isoeugenol, and p-coumaric acid. On the other hand, p-coumaric acid also inhibits anthracnose caused by C. gloeosporioides [57]. Ferulic acid has been reported as an antifungal agent against Fusarium graminearum at concentrations less than 100 µg/mL [58]. While chlorogenic acid has shown inhibitory activity against important phytopathogens such as Sclerotinia sclerotiorum, Fusarium solani, Verticillium dahliae, Botrytis cinerea, and Cercospora sojina [59] at a concentration of 15 mg/mL. In contrast, in our work, a lower concentration (2.0 mg/mL) of phenolic acids was required to observe antifungal activity against the phytopathogens analyzed in this study. It is noteworthy that the extract had a greater effect on inhibiting spore germination as compared to each of the phenolic acids evaluated. Therefore, the inhibition of spore germination was higher in the extract of T. longibrachiatum with respect to each of the phenolic acids evaluated. This suggests that a possible synergistic effect of phenolic acids occurs in the extract. Nevertheless, further studies are required to confirm or deny this hypothesis.

The differences in the antifungal activity of the phenolic acids analyzed can be attributed to the different affinities of the cellular components of the fungi and/or the ability of phytopathogenic fungi to resist the effects of antifungals. Examples of resistance mechanisms to antifungals are the presence of transporters of the Major Facilitating Superfamily (MFS) and the ATP Binding Cassette (ABC) that have been observed in A. alternata [60] and in Fusarium sp. [61], which allows it to resist antifungals. Furthermore, the same phenolic compound, depending on the species or even the strain, may act in different ways. For example, p-coumaric acid in Colletotrichum gloeosporioides induces membrane damage and inhibits the secretion of organic acids [57]. In contrast, against Botrytis cinerea it acts as a mitochondrial uncoupler, interrupting the production of ATP [62]. The differences in the antifungal activity of phenolic compounds are influenced by the structure and cellular composition of the different fungi and their molecular response mechanisms, as well as the type of action mechanism that the phenolic compound presents for that specific fungus [63].

The structure-activity relationship has an influence on the biological activity of phenolic compounds. Soto, et al. [64] performed a study of the effect of ring substitution and side chain hydration on the activity of geranylated phenols against B. cinerea. They found a relationship between the position of the functional group (OH) and the antifungal activity of these compounds, where the –OH position provided greater binding affinity for the succinate dehydrogenase enzyme. Likewise, Shirai, et al. [65] reported that lipophilicity depends on the methoxy substituents of the ring; therefore, the greater the lipophilicity, the greater the ability to internalize the cell membrane and accumulate in the phospholipid bilayer and, thus, alter the permeability of the membrane. With respect to chlorogenic acid, it has the lowest lipophilicity compared to ferulic and p-coumaric acids, therefore, it has lower antifungal activity. These findings highlight the importance of considering the structure and affinity of phenolic compounds when designing strategies for plant disease control. The ability of phenolic acids to inhibit spore germination of A. alternata and F. oxysporum at relatively low concentrations is promising and suggests their potential application as fungicidal agents in the control of plant diseases. However, additional studies are needed to better understand the mechanisms of synthesis and regulation of these compounds, as well as the mechanisms of action, their role in ecological interactions, and their application in the biological control of diseases in horticultural products.

5. Conclusions

The present study demonstrates that T. longibrachiatum produces phenolic compounds with antifungal activity against phytopathogenic fungi such as A. alternata and F. oxysporum. The results also indicate that the extraction method and solvent used are crucial for obtaining phenolic compounds. Extraction with 70% ethanol turned out to be the most effective for the extraction of phenols and flavonoids from the culture extract. The chromatographic profile of T. longibrachiatum revealed the presence of several phenolic compounds, some of which have not been previously identified in studies on Trichoderma. The identified phenolic acids, especially ferulic, chlorogenic, and p-coumaric acid, showed a clear ability to reduce mycelial growth and spore germination of A. alternata and F. oxysporum. Furthermore, these phenolic acids affected the initial development of spores, suggesting fungistatic and fungicidal effects at different concentrations. These results underline the potential of this Trichoderma species and its secondary metabolites as promising alternatives for disease control in horticultural products.