Antibacterial Activity of an Ethyl Acetate Extract from Cladobotryum virescens Against Plant-Pathogenic Bacteria and Putative Metabolite Annotation by UHPLC-ESI-HRMS/MS

1. Introduction

Bacterial diseases represent a major constraint to agricultural productivity worldwide, particularly in high-value perennial and horticultural crops. Among the most economically relevant bacterial phytopathogens, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. actinidiae, and Xanthomonas campestris pv. campestris are responsible for severe yield losses in fruit trees, kiwifruit, and Brassicaceae crops, respectively [1,2]. These pathogens are widely distributed and thrive under humid, low temperatures, and the presence of wounds, which facilitate infection and disease development under field conditions [2].

These bacterial pathogens represent a significant phytosanitary challenge for economically important crops. P. syringae pv. syringae is prevalent in temperate fruit-growing regions and has been associated with bacterial canker, bud necrosis, and dieback in cherry, grapevine, pear, and other Prunus species. Disease outbreaks are particularly severe under climatic conditions characterized by frequent rainfall, winter frosts, and pruning-related wounds [3]. P. syringae pv. actinidiae is one of the most destructive pathogens affecting kiwifruit production. It causes bacterial canker in Actinidia spp., leading to vine decline and substantial economic losses, which has prompted the implementation of strict phytosanitary measures [1]. In addition, X. campestris pv. campestris, the causal agent of black rot, constitutes a major constraint in the production of Brassicaceae crops such as cabbage, broccoli, and cauliflower [2]. This disease is particularly problematic in vegetable-producing areas where seed transmission and favorable environmental conditions facilitate outbreaks [2,3,4].

The management of bacterial plant diseases remains challenging, as effective control options are limited. Current strategies rely primarily on cultural practices, the use of resistant cultivars, and the application of copper-based compounds or antibiotics. However, their efficacy is often inconsistent, and repeated use raises concerns regarding environmental impact, phytotoxicity, and the emergence of resistant bacterial populations [5]. Consequently, there is growing interest in the identification of alternative and sustainable disease management strategies based on natural products and biologically derived compounds [6].

Fungi are recognized as prolific producers of bioactive secondary metabolites with diverse antimicrobial properties. Fungal extracts have demonstrated promising antibacterial and antifungal activities against a wide range of plant pathogens, highlighting their potential as sources of novel biocontrol agents [6,7]. Cladobotryum virescens is a filamentous fungus of the family Hypocreaceae with a saprotrophic or mycoparasitic lifestyle and a strong capacity for microbial competition. Its ability to produce antimicrobial secondary metabolites makes this species a promising source of natural compounds for the sustainable management of plant pathogens [8].

The complexity of metabolite composition in natural extracts has driven the use of advanced analytical techniques for their characterization. Electrospray ionization tandem mass spectrometry in positive and negative modes has been extensively applied to the structural characterization of natural products [9]. In this context, ultra-high-performance liquid chromatography coupled with tandem mass spectrometry using electrospray ionization (UHPLC-ESI-MS/MS) has established itself as a reference technique for qualitative and quantitative analysis in complex matrices [10,11]. This technique allows for the detection of compounds at trace levels, provides precise information on molecular fragmentation, and, under specific conditions, discriminates structural isomers, thereby facilitating structural elucidation [12,13].The high sensitivity and selectivity of the UHPLC-ESI-MS/MS system are key elements for investigating complex extracts, optimizing extraction processes, and establishing correlations between chemical composition and biological activity.

In this study, the ethyl acetate extract obtained from Cladobotryum virescens was first evaluated its antibacterial effect under in vitro conditions against Pseudomonas syringae pv. syringae, P. syringae pv. actinidiae, and Xanthomonas campestris pv. campestris. Subsequently, was chemically characterized in order to identify major components potentially associated with antibacterial activity. The results of this work contribute to the understanding of fungal-derived ethyl acetate extracts as alternative tools for the sustainable management of bacterial diseases affecting agricultural crops.

2. Materials and Methods

2.1. Fungal Strain, Growth Media and Conditions

C. virescens (INIFAT C10/110), preserved in the World Federation for Culture Collections (WFCC 853) at the Institute of Fundamental Research in Tropical Agriculture “Alexander von Humboldt” (INIFAT, Cuba), was used in this study. The strain was routinely maintained on oat agar (OA) plates and incubated for 10 days at 25 ± 2 °C, following the culture conditions described by Hernández et al. (2023)[14].

2.2. Preparation of the Ethyl Acetate Crude Extract

A mycelial plug of C. virescens was transferred to oatmeal peptone agar (OPA) plates (10 g oatmeal, 2.5 g peptone, and 15 g agar per 1000 mL deionized water) and periodically subcultured. For large-scale metabolite production, semisolid cultures were prepared in 20 Erlenmeyer flasks (1 L each), containing 1.5 g of sterile cotton wool and 250 mL of oatmeal peptone medium (2.5 g oatmeal and 0.625 g peptone per 250 mL deionized water), yielding a total volume of 5 L. Each flask was inoculated with a 10 × 10 mm agar plug of actively growing mycelium and incubated at room temperature (≈25 °C) for 28 days without agitation.

After incubation, the mycelia were separated from the culture broth by vacuum filtration, frozen, and extracted with ethyl acetate (3 × 1 L). The remaining culture broth was subsequently extracted with ethyl acetate (3 × 1 L). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure to obtain a brown crude extract (1.2 g). The extract was stored at −20 °C until use.

2.3. Microbial Strains and Culture Conditions

Three phytopathogenic bacterial strains were used to evaluate the antimicrobial activity of the fungal extract: Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. actinidiae, and Xanthomonas campestris pv. campestris. All bacterial strains were obtained from the laboratory culture collection and maintained on nutrient agar (NA) at 4 °C. Prior to each assay, cultures were reactivated on Luria–Bertani (LB) agar plates and incubated at 28 °C for 24 h.

2.4. Antimicrobial Assay

The antimicrobial activity of C. virescens ethyl acetate extract was evaluated using the agar disk diffusion method. Briefly, 100 µL of a standardized bacterial suspension (0.5 McFarland) was evenly spread onto Luria–Bertani (LB) agar plates using sterile cotton swabs previously dipped into the bacterial suspension. Sterile Watman 1 paper disks (6 mm diameter) were placed on the inoculated surface and impregnated with 20 µL of the extract at concentrations of 250, 125, 62.5, and 31.25 mg mL⁻¹. Sterile distilled water and ethyl acetate were used as negative controls. Plates were incubated at 28 °C for 24 h, and antimicrobial activity was determined by measuring the inhibition zones around each disk. The diameter of each inhibition halo was measured perpendicularly (length and width) using a digital caliper. The results were analyzed to determine the median concentration dose (EC₅₀), which was calculated from dose–response curves fitted to the inhibition data.

2.5. Statistical Analysis

Data for inhibition zone diameters were expressed as mean ± standard deviation (SD) of three experimental runs, each performed in triplicate as technical replicates using the same extract. Differences in inhibition halo diameters among extract concentrations were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test (p < 0.05). Dose–response relationships between extract concentration and antibacterial activity were fitted to a four-parameter logistic (4PL) model to estimate the median concentration dose (EC₅₀) for each bacterial strain. Nonlinear regression analyses were performed using GraphPad Prism v10.0. The goodness of fit of each curve was assessed by the coefficient of determination (R²) and residual distribution.

2.6. UHPLC-ESI-HRMS/MS Conditions and Data Analysis

For the qualitative composition study of the ethyl acetate crude, 4 μL of the sample with a concentration of 50 µg/mL was injected into an ultra-high-performance liquid chromatograph (Elute, Germany) equipped with a Kinetex C18 column (1.7 μm; 2.1 mm x 100 mm). A gradient system was used starting with H₂O (solvent A; Milli-Q, Merck Millipore) and CH₃CN (solvent B; Chormasolv LC-MS, Riedel-de-Haen, Honeywell), 88:12 (each containing 0.1% (v/v) formic acid (eluent additive for LC-MS, Honeywell Fluka), decreased to 60:40 (during 1 min), decreased to 1:99 in 10 min, isocratic 1:99 in another 3 min and finally increased to 1:10 (during 1 min), to 88:12 in 0.5 min and then hold 88:12 for another minute. The sample was eluted at 40°C and a flow rate of 400 μL mL⁻¹. Total ion spectra over the m/z range of 50–1300 in positive and negative modes were obtained on a Compact mass spectrometer (Bruker, Germany) equipped with an electrospray ionization source and a time-of-flight analyzer. For MS² analyses, the collision energy for each fragmentation was 30%. The optimized instrumental parameters were capillary temperature 230°C, capillary voltage 13 V, aerosol voltage 4.5 kV, main gas flow rate 35 (nitrogen, arbitrary units), pressure 1.8 bar, and auxiliary gas flow rate 8.0 L min⁻¹ assisted by nitrogen as the drying gas. Compass DataAnalysis 4.4 software (Bruker) was used to visualize the chromatograms and mass spectra in negative and positive ionization modes.

3. Results

3.1. Qualitative Assessment of Antibacterial Activity of C virescens

The antibacterial activity of the ethyl acetate extract obtained from C. virescens was evaluated against three phytopathogenic bacteria: P. syringae pv. syringae, P. syringae pv. actinidiae, and X. campestris pv. campestris.

Representative inhibition zones exhibited a clear dose-dependent increase in halo diameter for all three bacterial species (Figure 1). At higher extract concentrations (250 and 125 mg mL⁻¹), well-defined and homogeneous inhibition halos were observed in all pathogens. The antibacterial effect decreased progressively with the reduction of extract concentration, as lower doses (≤ 31.25 mg mL⁻¹) produced smaller or diffuse inhibition halos, reflecting a dose-dependent decline in activity. Notably, in X. campestris pv. campestris, inhibition halos were larger and more uniform compared with both pathovars of P. syringae. No inhibition was observed around the paper disks containing water or ethyl acetate solvent, confirming that the antimicrobial effect was exclusively attributable to the fungal extract.

3.2. Quantitative Analysis and Dose–Response Curves

To complement the qualitative observations, the antibacterial activity of the C. virescens ethyl acetate extract was quantitatively assessed by measuring the inhibition zone diameters at different extract concentrations. The extract exhibited a clear concentration-dependent antibacterial effect against all tested phytopathogenic bacteria (Table 1). Xanthomonas campestris pv. campestris showed the highest susceptibility to the extract, with inhibition zones increasing from 13.46 mm at 31.25 mg mL⁻¹ to 25.12 mm at 250 mg mL⁻¹. P. syringae pv. syringae displayed an intermediate level of susceptibility, with inhibition zones ranging from 7.84 to 23.08 mm across the concentration range. In contrast, P. syringae pv. actinidiae was the least susceptible strain, producing inhibition zones between 2.18 and 13.91 mm. In agreement with these results, statistical analysis revealed significant differences among the bacterial strains in their susceptibility to the fungal extract. The mean inhibition zone was significantly lower for P. syringae pv. actinidiae (7.67 mm; a) compared with X. campestris pv. campestris (19.08 mm; b) and P. syringae pv. syringae (14.58 mm; b), whereas no significant difference was found between the latter two bacterial strains. In agreement with these results, statistical analysis revealed significant differences among the bacterial strains in their susceptibility to the fungal extract. The extract produced stronger inhibition in X. campestris pv. campestris and P. syringae pv. syringae compared with P. syringae pv. actinidiae. In contrast, no significant difference was found between P. syringae pv. syringae and X. campestris pv. campestris, indicating a similar level of susceptibility to the fungal extract. Notably, the two Pseudomonas strains differed significantly in their susceptibility to the extract, despite belonging to the same genus, with P. syringae pv. syringae showing greater susceptibility than P. syringae pv. actinidiae.

Normalized inhibition values allowed the fitting of dose–response curves, which clearly differentiated the antibacterial behavior of the extract from that of the water and solvent controls (Figure). In accordance with the observed data, X. campestris pv. campestris was the only strain exhibiting a pronounced concentration-dependent response, characterized by a gradual and consistent increase in inhibition at higher extract concentrations. In contrast, both P syringae pv. syringae and P. syringae pv. actinidiae displayed nearly flat curves with minimal inhibition, indicating a lack of meaningful dose responsiveness. As shown in the controls, neither water nor ethyl acetate produced any detectable antibacterial effect, confirming that the inhibitory activity observed in X. campestris pv. campestris was exclusively attributable to the bioactive metabolites present in the extract.

The median effective concentration (EC₅₀) values were estimated from the dose–response curves obtained using the logarithm of extract concentration (Figure 2). X. campestris pv. campestris exhibited the highest sensitivity to the extract, with the lowest EC₅₀ value (102 mg mL⁻¹). In contrast, P. syringae pv. actinidiae and P. syringae pv. syringae showed higher EC₅₀ values of 121 and 136 mg mL⁻¹, respectively, indicating lower sensitivity according to this parameter. Statistical analysis confirmed significant differences among bacterial strains (p < 0.05), with X. campestris pv. campestris showing significantly greater inhibition than both Pseudomonas strains. No inhibitory effect on bacterial growth was observed for either water or the extraction solvent, ethyl acetate, confirming that the observed antibacterial activity was associated with the fungal extract rather than the solvents used in the assay.

3.3. Analysis of Ethyl Acetate Extract of C. virescens Strain Culture Broth by UHPLC-ESI-MS/MS

The chemical profile of the ethyl acetate crude extract was investigated using UHPLC-ESI-MS/MS. The total ion chromatogram (Figure S1 A and C) shows the chromatographic profile of the metabolites with elution time from 0.86 to 16 min and the complexity of the crude. The total ion spectrum in m/z range from 50-1140 Da (Figure S1 B and D) shows the pseudomolecular ions in the negative and positive ionization modes of each peak in both chromatograms. A total of 21 compounds were identified in the extract (Figure 3, Table 2). The study conducted in the present investigation is the first report of secondary metabolites identification in ethyl acetate of C. virescens (C10/110) strain.

In negative ionization mode, three fatty acids were identified with pseudo molecular ions [M-H]^- at m/z 117.0283, m/z 131.0799 and m/z 295.2473 respectively. Compound 1 was tentatively assigned as 3-hydroxypentanoic acid, compound 2 as 2-hydroxyhexanoic acid, and compound 11 as (10E,12E)-9-hydroxyoctadeca-10,12-dienoic acid. Additionally, three phenolic acids were detected. Compound 3 at m/z 153.0339 was identified as protocatechuic acid, compounds 4 and 7 as 3-phenyllactic acid and dihydro p-coumaric acid, respectively. Additionally, the methylated phenolic acid compound 6 as p-methoxybenzoic acid was also identified.

Compound 8 with pseudomolecular ion [M-H]^- at m/z 195.0808 showed in the MS² spectra, fragment ions at m/z 177.0707 [M-H-H₂O]^- m/z 151.0880 and m/z 107.0948 corresponding to the loss of water, carbon dioxide and combined loses of -(2CH₂O+CO) respectively. The structure was tentatively assigned as (E)-4,5-bis(hydroxymethyl)-6-(prop-1-en-1-yl)-2H-pyran-2-one, an α-pyranone derivative of cladobotrin V, metabolite isolated and identified from Cladobotryum varium by Tezuka et al 1994 [19].

Compound 9 with pseudomolecular ion [M-H]^- at m/z 315.0744 was identified as isorhamnetin according to their fragmentation pattern [20] and compound 10 with pseudomolecular ion [M-H]^- at m/z 329.2549 was tentatively assigned as 2-(4,5,6,8-tetrahydroxy-9,10-dioxo-9,10-dihydroantracen-2-yl) acetic acid due to the fragmentation pattern associated with anthraquinone structures.

In the second-order spectra of ion at m/z 419.1604 (Figure S2), a prominent peak at m/z 387.1345 [M-H-32]^- was detected, associated with the loss of a neutral molecule of CH₄O. The diagnostic fragment ion at m/z 179.0144 arises from the Retro Diels-Alder (RDA) ^1,2A^- cleavage, is characteristic of flavonols with substituents at the 3' and 4' positions [21]. The peak at m/z 299.0404 can be attributed to the loss of CH₄ from 315.0641 (Y₀ ^-), and the peak at m/z 285.0592 [Y₀-H-30]^- is associated with the loss of a neutral molecule of CH₂O characteristic of flavonoids with a methoxyl substituent on the aromatic ring. Additionally, other diagnostic fragment ions were observed at m/z 151.0160 [^1,2A^--CO]^- and m/z 107.0229 [^1,2A^--CO-CO₂]^-, indicative of the OMe substituent is located on ring B, and the low-intensity fragment ion at m/z 243.0541 confirms the position of this substituent at the 3' position. Based on these observations, isorhamnetin is proposed as the aglycone. Additionally, fragment ions were detected at m/z 315.0641[Y₀-H-104]^- associated with the loss of benzoic acid and m/z 314.0668[Y₀-H-104]^{- ●} because of the loss of this substituent by homolytic cleavage. The relative intensity of [Y₀-H]^{- ●} > Y₀^- suggests that the ester bond is formed with the hydroxyl group at position 3 of the aglycone [22]. Compound 10 was tentatively identified as: 3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxo-4H-chromen-7-yl-benzoate, an isorhamnetin derivative. All compounds were detected for the first time in Cladobotryum genus.

Similar study was performed in positive ionization mode. A total of 11 compounds were identified by analysis of their MS² spectra. Compounds 12 and 13 with pseudomolecular ions [M+H]⁺ at m/z 211.1450 and m/z 245.1340, respectively, were assigned as a diketopiperacine cyclo (L-Leu-L-Pro) and cyclo (L-Pro-L-Phe) according to the fragmentation pattern observed on their MS² mass spectra and correlation with the reported by Furtado et al [15]. This is the first report of compound 13 in Cladobotryum genus.

Six destruxins were detected (compounds 14-19 and 21) with pseudomolecular ions at m/z 624.4019, m/z 630.3644, m/z 564.3672, m/z 578.3864, m/z 580.4065, m/z 594.4232, and m/z 608.4357, respectively (Table 1). In all cases fragment ion at m/z 157 (^4-5b₂) were observed due to the loss of hydroxy acids and undergo the same consecutive losses of 113 Da, 28 Da, 85 Da, 28 Da, and 69 Da to give ^4-5 b₃ ion after 4-5 ring opening. Loss of 99 Da in compound 20, suggesting the presence of valine as AA⁴; however, 113 Da in cases of compounds 16,17,18,19 and 21 suggested the presence of N-methyl valine in the same position. Fragment ions in the MS² spectra were compared with a previously published report by Arroyo –Manzanares et al. [17] Compound 21 showed the same pseudomolecular ion and therefore molecular formula than Destruxin B₁ however, the fragmentation pattern did not match with Destruxin B₁. Metabolite 21 showed the same fragment ions as Destruxin B, ^4-5b₅, ^4-5a₅,^4-5 b₄,^4-5a₅,^4-5b₃, ^4-5b₂, ^4-5b₁, D⁺, and D₂⁺ except ^3-4b₂ ion, but has a difference of 14 Da (CH₂) with respect to Destruxin B. It suggested that the difference between both destruxins is at the level of AA⁴, and the N-methyl valine residue (AA⁴) of Destruxin B was substituted by N-methyl isoleucine in compound 21. After extensive analysis of the MS² spectra, a fragmentation pathway was proposed for metabolite 21 (Figure S3) in which was corroborated that this compound has a methyl isoleucine at position 4 instead of methyl valine present in Destruxin B due to the presence of fragment ion at m/z 255.1773 formed because of open ring 3-4. Compound 21 was assigned as homoDestruxin B.

Destruxins identified in the present study, eluted in the sequence: D (5.8 min) < Cl (5.9 min) <A2 (6.5 min) <A (7.0 min) < DesmB (7.5 min) < B (8.0 min) <Homodestruxin B (8.7 min). This sequence is in agreement with the proposed by Arroyo –Manzanares et al. [17]

Compound 20 with pseudomolecular ion [M+H]⁺ at m/z 373.1473, was assigned as a pentamethoxy flavone sinensetin. Fragment ions in the second order spectra were compared with the literature and corroborates the proposal structure [23].

4. Discussion

The results of the present study explore the potential of the ethyl acetate extract of C. virescens as a source of antibacterial compounds against P. syringae pv. syringae, P. syringae pv. actinidiae, and X. campestris pv. campestris. This study focused on the use of ethyl acetate extract produced by the fungus C. virescens (C10/110). Results regarding the effect of organic extract on bacterial growth in vitro demonstrated significant biocontrol potential against those bacterial strains. These findings were supported by EC₅₀ tests. The observed inhibition zones in the disc diffusion assay underscore the potential of C. virescens as a source of antimicrobial compounds that may prove valuable in biocontrol strategies against Gram-negative phytobacteria.

The ethyl acetate extract showed a maximum inhibition zone of 25 mm against X. campestris pv. campestris. A similar result was obtained in antibacterial assays of the ethyl acetate extract of Trichoderma harzianum against X. campestris, which produced inhibition zones of 24.5 ± 1.06 mm [19]. The comparable antibacterial activity observed in both studies suggests that fungal metabolites extracted with ethyl acetate may represent an important source of bioactive compounds effective against phytopathogenic bacteria. Moreover, the inhibition zones observed in the present study were greater than those reported for several plant-derived ethyl acetate extracts tested against Xanthomonas species, which generally produced inhibition zones ranging from 10 to 20 mm[24]. The strong antibacterial activity displayed by C. virescens therefore places this fungus among the most promising microbial sources of antibacterial metabolites active against phytopathogenic bacteria. In the present study, the antibacterial activity of the extract was concentration dependent, with inhibition zones increasing progressively as extract concentration increased. Similar dose-dependent responses have been reported for natural antimicrobial products against phytopathogenic bacteria. The susceptibility of P. syringae pv. actinidiae to natural antimicrobial compounds has been previously demonstrated, with inhibition halos reaching approximately 21 mm and minimum inhibitory concentrations of 1 mg mL⁻¹.In addition to inhibiting bacterial growth, these extracts were shown to reduce motility and other virulence-associated traits, suggesting that natural metabolites can interfere with multiple physiological processes involved in bacterial pathogenicity[25].

Interestingly, the extract also exhibited a strong inhibitory effect against P. syringae pv. syringae, producing inhibition zones of up to 23 mm at the highest concentration tested. Comparable antibacterial activities have been reported for fungal metabolites obtained from species of Trichoderma, Penicillium and Aspergillus, which have shown inhibitory effects against several Gram-negative phytopathogenic bacteria, including species of Pseudomonas and Xanthomonas [26,27]. The similarity between these reports and the results obtained in the present study suggests that C. virescens may produce bioactive secondary metabolites with broad-spectrum antibacterial properties. Such metabolites are frequently associated with polyketides, peptaibols, terpenoids and other low-molecular-weight compounds involved in microbial competition and antagonism [28]. In contrast, P. syringae pv. actinidiae exhibited lower sensitivity, with maximum inhibition zones of approximately 14 mm. Nevertheless, growth inhibition was consistently observed across all concentrations tested, indicating that the pathogen remains susceptible to the fungal metabolites.

Differences in susceptibility among the evaluated Gram-negative phytopathogenic bacteria may be associated with variations in outer membrane composition, lipopolysaccharide structure, membrane permeability, efflux pump activity, and other physiological mechanisms involved in resistance to antimicrobial compounds. Although both Pseudomonas and Xanthomonas belong to the Gram-negative group, species-specific structural and metabolic characteristics can strongly influence their sensitivity to natural products and fungal-derived metabolites [29]. In the present study, these differences were reflected in the EC₅₀ values, with X. campestris pv. campestris exhibiting the lowest EC₅₀ and, therefore, the highest overall susceptibility to the extract. In contrast, both P. syringae pathovars displayed lower sensitivity, which may be related to the intrinsic resistance mechanisms commonly reported for members of this genus, including reduced membrane permeability and the presence of multidrug efflux systems that limit the intracellular accumulation of antimicrobial compounds [30]. Consequently, the reduced responsiveness observed in P. syringae may reflect a greater capacity to tolerate, detoxify, or actively exclude the bioactive metabolites present in the C. virescens extract.

The antibacterial activity observed in this study is likely related to secondary metabolites extracted by ethyl acetate. This solvent has been widely employed for the recovery of antimicrobial compounds from fungi because of its ability to extract metabolites of intermediate polarity, including polyketides, lactones, terpenoids and aromatic compounds. The complete absence of inhibition in the solvent and water controls confirms that the antibacterial effect was exclusively attributable to fungal-derived metabolites.

The analysis of ethyl acetate extract of C. virescens using UHPLC-ESI-MS/MS, resulted in the identification for the first time of 21 secondary metabolites. The present investigation is the first report of compounds 1-11 and 13-21 in the genus. Some of these were flavonoids and cyclic peptides as destruxins. Cyclo (L-Leu-L-Pro) (compound 20) previously reported in Cladobotryum genus by Tezuka et al. [19], is an important chemical and bioassay standard for dereplication of crude microbial extracts and is a useful chemo-taxonomic marker for bacteria, actinomycetes and fungi. The compound has inhibitory effect against E. coli ATCC 35218 and S. aureus CPCC 140594 [31]. Similarly showed antibacterial properties against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. In addition, cyclo (L-Pro-L-Phe) found to disrupt Quorum Sensing Activity [32]. Furthermore, this metabolite decreases the viability of Staphylococcus aureus biofilm and inhibit the synthesis of extracellular polymeric substances[33]. In general, diketopiperacines contain two cis-amide bonds and possess two H-bond acceptors and two H-bond donors. As a result of this chemical structure, they have the ability to bind to a wide range of receptors and enzymes [34] The presence of both diketopiperacines that have been already shown to inhibit the bacterial growth could help explain the antibacterial activity of the ethyl acetate extract.

Although direct microbiological data are limited, the biochemical mechanisms of destruxins suggest how they could exert antibacterial effects if demonstrated in an in vitro system. These compounds act as ionophores, altering gradients of Ca²⁺ and other ions, causing membrane depolarization and ion channel dysfunction. In insect cells, this causes cell damage and death, so a similar ionophoric mechanism could destabilize bacterial plasma membranes, especially in bacteria with an inner membrane rich in anionic phospholipids. However, membrane integrity assays or standard MICs against Gram-positive and Gram-negative collections are required to validate this effect. Therefore, the lack of MIC studies, bacterial death kinetics, or specific membrane analyses in Gram-positive and Gram-negative bacteria with purified destruxins opens a field for future research.

Phenolic compounds exhibit great structural diversity, such as the presence of hydroxyl groups, as well as their substitution position, which confers antibacterial activity. Isorhamnetin (compound 8) is associated with this bioactivity. It has been reported inhibition of bacterial growth and the ability to permeate the bacterial cell membrane through oxidative stress [35]. In addition, the growth inhibition effect of this compound against five bacterial strains (E. coli, K. pneumoniae, P. mirabilis, S. aureus, and S. bacillus) has been documented [36]. Furthemore, it has been identified as one of the compounds responsible for antibacterial activity in stingless bee honey, with efficacy against multidrug-resistant S. aureus [37]. Polymethoxylated flavonoids such as sinensetin (compound 20), identified in this study, possess potent antimicrobial activity. In an in vitro assay, sinensetin at 100 μM significantly inhibits the motility halos of E. coli O157:H7 at 24 h [38].

Nevertheless, the rigid planar structure of anthraquinones given by the central skeleton is necessary for antibacterial activity through a mechanism that affects bacterial DNA. Furthermore, the different substituents in the structure affect bioactivity [39]. These results highlight the potential of C. virescens ethyl acetate extract against Gram-negative phytopathogens.

The antibacterial activity displayed by the ethyl acetate extract of C. virescens is particularly noteworthy considering the diversity of secondary metabolites identified in this study. Although the contribution of individual metabolites remains to be elucidated, the broad inhibitory response observed against both Pseudomonas and Xanthomonas species suggest that the antibacterial activity is likely the result of a complex interaction among multiple bioactive compounds. Taken together, these findings not only expand the current knowledge of the secondary metabolite profile of C. virescens but also highlight this fungus as a promising source of natural antibacterial compounds with potential application in the management of economically important bacterial diseases of plants.

5. Conclusions

Several secondary metabolites were identified in the ethyl acetate extract of C. virescens (C10/110), including cyclo(L-Phe-L-Pro), cyclo(L-Pro-L-Val), sinensetin, and isorhamnetin, compounds previously associated with antimicrobial activity. The extract exhibited antibacterial activity against P. syringae pv. syringae, P. syringae pv. actinidiae, and X. campestris pv. campestris, with the latter showing the highest susceptibility. Collectively, these findings demonstrate that C. virescens produce metabolites capable of suppressing the growth of economically important phytopathogenic bacteria and highlight its potential as a source of antibacterial compounds for sustainable disease management. However, further studies are required to identify the metabolites primarily responsible for the observed bioactivity and to evaluate their efficacy under in vivo conditions and across different pathosystems.