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. 2026 Sep 18;7:1920680. doi: 10.3389/ffunb.2026.1920680

Trichoderma controls bacterial blight caused by Xanthomonas axonopodis pv. passiflorae in Passiflora edulis

Natália Deniz Brito 1, Quelmo Silva de Novaes 1, Rosa Hermosa 2, M Belén Rubio 2,*
PMCID: PMC13630755  PMID: 42827814

Abstract

Introduction

Several Trichoderma species are recognized as biocontrol agents that can enhance plant defenses against a wide range of biotic and abiotic stresses. However, few studies have investigated Trichoderma activity against bacterial pathogens, and the Xanthomonas axonopodis pv. passiflorae (Xap)-Passiflora edulis pathosystem remains largely unexplored.

Methods

We analyzed the effects of cell-free filtrates from Trichoderma cultures on the growth of Xap. We also investigated the potential of T. asperellum T25 and T. harzianum T146, when applied to the plant growth substrate, to control bacterial blight caused by Xap in P. edulis, as well as the mechanisms underlying systemic defense responses in the host plant. Fifty-day-old plants grown in substrate treated or untreated with a Trichoderma strain were inoculated or not with Xap, and changes in the expression of marker genes representative of jasmonic acid (JA) biosynthesis (LOX2), salicylic acid (SA)-dependent (PR1), and ethylene (ET)-dependent (PDF1.2) defense pathways were analyzed at 48 h post-inoculation (hpi) and 14 days post-inoculation (dpi). At 14 dpi, H₂O₂ content and the activities of three antioxidant enzymes -superoxide dismutase, peroxidase, and catalase- were also evaluated.

Results and discussion

Trichoderma filtrates showed antimicrobial activity against Xap. In the absence of the pathogen, strain T25 promoted the growth of P. edulis plants. Both Trichoderma strains controlled disease symptoms with similar efficacy. However, at the early stage of the P. edulis-Xap interaction, differential expression of SA- and JA-related genes was detected between T25-pretreated and T146-pretreated plants. Furthermore, at the later stage of the interaction (14 dpi), pretreatment with strain T146 increased ROS levels (measured as H₂O₂ content) in response to Xap, whereas the lowest oxidative levels were observed in plants inoculated with the pathogen alone. Our results demonstrate that T. asperellum and T. harzianum effectively control bacterial blight caused by a virulent Xap strain in P. edulis.

Keywords: antibacterial activity, antioxidant enzymes, indirect biocontrol, passion fruit, reactive oxygen species, systemic defense responses

Introduction

Passion fruit (Passiflora edulis Sims) cultivation is of high economic importance in tropical and subtropical regions, with its fruits being widely used for fresh consumption and industrial applications, including production of juices, pulps, and other processed products (Arshad et al., 2025). Beyond the food sector, bioactive compounds present in P. edulis have attracted interest from pharmaceutical, nutraceutical, and cosmetic industries because of their antioxidant, anti-inflammatory, and anxiolytic properties (Deng et al., 2010; Barbosa Santos et al., 2021). The global passion fruit market generated approximately US$ 3.9 billion in 2024, reflecting the growing demand for natural products and plant-based ingredients (DBMR, 2024). Brazil remains the world’s leading producer of passion fruit, accounting for nearly 70% of global production (IBGE, 2024).

Bacterial blight, caused by Xanthomonas axonopodis pv. passiflorae (Xap), is one of the most important diseases affecting passion fruit crops (Munhoz et al., 2015; Monzani et al., 2018). Favored by high humidity and temperature, the disease is characterized by chlorotic and necrotic lesions on leaves, branches, and fruits, frequently associated with severe defoliation, and may reduce productivity by 20-30% (Boro et al., 2011). The bacterium may also invade the vascular system and spread throughout the plant, leading to wilt and even death in susceptible cultivars (Monzani et al., 2018).

Infection by Xanthomonas has been shown to trigger extensive transcriptional reprogramming in rice plants, activating defense mechanisms that limit bacterial growth and disease progression (Grewal et al., 2012; Wang et al., 2019). As documented for a variety of pathogens (Torres et al., 2006), the initial plant response involves the recognition of pathogen-associated molecular patterns, which activate signaling pathways mediated by calcium fluxes, kinase cascades, and reactive oxygen species (ROS). These signals induce transcription factors that regulate defense-related genes, involving plant hormones such as jasmonic acid (JA), ethylene (ET), and salicylic acid (SA), with resistant genotypes generally displaying stronger and faster responses than susceptible plants (Grewal et al., 2012; Wang et al., 2019). A pioneering study in rice demonstrated that SA application induces systemic resistance against Xanthomonas oryzae pv. oryzae (Xoo) (Mohan Babu et al., 2003). Genes involved in cell wall reinforcement and antimicrobial metabolite production further contribute to resistance (Grewal et al., 2012; Wang et al., 2019). In addition, Xanthomonas secretes transcription activator-like effectors that modify host gene expression. Depending on the plant genotype, these effectors may either promote susceptibility or activate resistance genes, resulting in effective defense responses (Timilsina et al., 2020).

However, molecular studies on P. edulis-Xap interaction remain limited. An analysis of subtractive cDNA libraries revealed that LOX2 is the P. edulis gene showing the strongest transcriptional response to Xap infection (Munhoz et al., 2015). Furthermore, most genes associated with canonical pathogen-recognition signaling pathways were repressed following Xap infection (Munhoz et al., 2015).

Management of this disease in P. edulis relies mainly on the use of copper-based compounds and antibiotics such as streptomycin. The effectiveness of these strategies is often temporary and limited due to the emergence of antibiotic resistance and environmental concerns (Ishida and Halfeld-Vieira, 2009; Lamichhane et al., 2018). Although breeding for host resistance remains important, pathogen variability and the instability of field resistance limit the effectiveness of this approach, highlighting the need for sustainable biocontrol alternatives (Silva et al., 2023).

Species of the genus Trichoderma are widely recognized as multifunctional plant-beneficial microorganisms (Woo et al., 2023). Following introduction into the soil, Trichoderma spp. can directly control phytopathogenic fungi through mechanisms such as mycoparasitism, competition, and antibiosis (Harman et al., 2004), as well as plant-parasitic nematodes (Martínez-Medina et al., 2017; Medeiros et al., 2017), thereby contributing to the modulation of crop-associated microbiomes and improving plant health (Illescas et al., 2020). Moreover, some Trichoderma strains colonize the rhizosphere (Mendoza-Mendoza et al., 2018), promoting plant growth while activating immune responses (Rivera-Méndez et al., 2020) and enhancing tolerance to abiotic stresses (Ascaso et al., 2025). Moreover, Trichoderma metabolites have been reported to inhibit bacterial growth (Xiao-Yan et al., 2006; Baazeem et al., 2021). Although several studies have demonstrated the biocontrol abilities of Trichoderma against bacterial pathogens in plants (Brotman et al., 2012; Morán-Diez et al., 2020), information regarding its biocontrol potential against Xanthomonas in woody plants remains scarce. Specifically, Xap causes severe economic losses in P. edulis, a high-demand crop in tropical and subtropical regions.

In the present study, we investigated the potential of two Trichoderma strains to control Xap in P. edulis, by analyzing the effects of fungal filtrates on bacterial growth in vitro and on disease severity in planta. Application of Trichoderma spores to the plant growth substrate enabled the assessment of systemic defense responses induced by Trichoderma in P. edulis following challenge with this bacterial pathogen. The expression of defense-related genes and the oxidative metabolism parameters were analyzed to determine the contribution of plant-mediated signals originating from Trichoderma-colonized roots to the priming of defense responses against Xap.

Materials and methods

Microorganisms and passion fruit seeds

Two Trichoderma strains, belonging to different species, were obtained from the CIALE Culture Collection (Salamanca, Spain) and used in this study: T. asperellum T25 (Morán-Diez et al., 2020; Pedrero-Méndez et al., 2025), and T. harzianum T146, isolated from the wheat rhizosphere. The strains were routinely cultured and sporulated on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) at 28 °C in the dark. Conidia from 7-day-old PDA cultures were harvested, and conidial concentrations were determined as previously described by Rubio et al. (2017) for subsequent use.

The strain Xanthomonas axonopodis pv. passiflorae IBSBF2775, hereafter referred to as Xap, was provided by the Biological Institute of São Paulo (São Paulo, Brazil) and used as the target pathogen. Xap was streaked onto Luria-Bertani (LB; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) agar plates and incubated at 30 °C for 48 h before use.

Passion fruit (Passiflora edulis Sims) seeds were surface-disinfected by vigorous sequential shaking in 70% ethanol and 2% sodium hypochlorite solutions for 10 min each, thoroughly washed three times with sterile distilled water, and then air-dried on a sterile gauze sheet.

Antimicrobial in vitro assay

This assay was conducted to evaluate antibacterial activity of Trichoderma culture filtrates against Xap. Trichoderma strains were inoculated into potato dextrose broth (PDB; Difco Laboratories, Detroit, MI, USA) at a final concentration of 1 x 105 spores mL−1 and incubated at 28 °C on a rotary shaker at 180 rpm for 72 h. The Trichoderma mycelium was removed by centrifugation, and the resulting supernatant was subsequently filtered through 0.22-µm pore-size membrane filters. In parallel, Xap was inoculated into LB medium and incubated at 30 °C and 100 rpm for 48 h. The bacterial suspension was then adjusted to a final concentration of 5 x 105 cells mL−1.

This assay was performed in sterile 96-well microtiter plates with a final volume of 150 µL per well. Trichoderma cell-free filtrates were added at final concentrations of 0, 5, 10, 20 and 40% (v/v). Subsequently, 10 µL of bacterial suspension was added to each well, and the final volume was adjusted to 150 µL with LB medium. In parallel, Xap grown in LB medium without Trichoderma filtrate was included as a positive control (C+), whereas LB medium alone served as a negative control (C-).

Microtiter plates were incubated at 30 °C and 80 rpm for 24 h, and bacterial growth was determined by measuring absorbance at 620 nm (A620) using a Sunrise microtiter plate reader (Tecan Ibérica, Barcelona, Spain) after 5 s of shaking. To normalize background absorbance, the mean absorbance of the negative control (C−) was subtracted from the absorbance values of all treatments. Antibacterial activity was expressed as growth inhibition (%) relative to the positive control (C+) using the following equation: Growth inhibition (%) = [1 − (A620 (Treatment) − A620 (C−))/(A620 (C+) − A620 (C−))] × 100.

Each treatment consisted of three technical replicates using four independent fungal filtrates. Subsequently, 10 µL from each well was spread onto LB agar plates and incubated at 30 °C for 24–48 h to qualitatively confirm bacterial viability.

Greenhouse assay

A greenhouse trial was conducted to evaluate the ability of Trichoderma strains T25 and T146 to protect passion fruit plants against Xap infection and to analyze plant defense responses.

The plant growth substrate consisted of a mixture of sandy soil, vermiculite, and peat at a 1:1:1 ratio (v/v/v), which was autoclaved twice at 121 °C and 1 atm for 60 min, with 24 h interval between the two sterilization cycles. A total of 240 pots (110 mL capacity), each containing 220 g of substrate, were prepared. Trichoderma conidia obtained from PDA cultures, as described above, were applied to the substrate at a final concentration of 5 x 106 conidia g−1 substrate in a final volume of 20 mL per pot. Eighty pots were inoculated with strain T25, another 80 with strain T146, and 80 were left uninoculated as controls. Subsequently, one passion fruit seed was sown per pot, and seedlings were maintained in a growth chamber at 70% relative humidity, 28 °C, and a 12-h light/12-h dark photoperiod.

After 50 days, plant height was measured and expressed in centimeters (cm). Plants showing uniform growth (10–15 cm in height and 3–6 fully expanded leaves) were selected from each treatment. Thirty-two plants from each treatment (control, T25, and T146) were divided into two groups: 16 plants were inoculated with Xap, whereas the remaining 16 plants were left uninoculated. For Xap inoculation, the bacterium was grown in LB medium for 48 h, and the bacterial suspension was adjusted spectrophotometrically at 600 nm to an approximate concentration of 1 x 106 CFU mL−1 using sterile 0.85% NaCl solution. Tween 20 was added at a final concentration of 0.01% (v/v) to facilitate uniform distribution on the leaf surface, and 150 mL of the resulting bacterial suspension used to spray-inoculate 48 plants.

The following treatments were established: untreated, non-inoculated control plants (C); T25-treated plants (T25); T146-treated plants (T146); plants inoculated with Xap alone (Xap); T25-treated inoculated with Xap (T25+Xap); and T146-treated inoculated with Xap (T146+Xap) plants.

At 48 h post-inoculation (hpi) with Xap, leaves were collected from eight plants per treatment for gene expression analysis. Four biological replicates per treatment, each consisting of pooled material from two plants, were used. Plant material was immediately frozen in liquid nitrogen and stored at -80 °C until analysis.

The remaining eight plants per treatment were maintained for disease evaluation. At 14 days post-inoculation (dpi) with Xap, disease incidence (DI) and disease severity (DS) were assessed using four biological replicates, each consisting of two plants. DI was calculated as the percentage of symptomatic leaves [DI = (DP × 100)/TP], where DP represents the number of leaves showing bacterial blight symptoms and TP represents the total number of evaluated leaves. DS was visually estimated according to the diagrammatic scale for bacterial leaf spot of passion fruit described by Monzani et al. (2018), based on the percentage of damaged leaf area. Following disease assessment, leaves were collected, frozen in liquid nitrogen, and stored at -80 °C for subsequent biochemical and gene expression analyses.

Biochemical analyses of plants

H2O2 content was determined in duplicate using 50 mg of frozen plant material, following the potassium iodide method described by Velikova et al. (2000) and Illescas et al. (2021), with absorbance monitored at 390 nm. The results were expressed as µmol g−1 fresh weight (FW). Four biological replicates were analyzed per treatment.

Three antioxidant enzyme activities, namely superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were determined in duplicate as previously described by Nawaz et al. (2020) and Illescas et al. (2021). Proteins were extracted by homogenizing 50 mg of frozen plant material in 1 mL of 50 mM potassium phosphate buffer (pH 7.8) followed by centrifugation at 10,000 rpm for 20 min at 4°C. The resulting supernatant was collected and protein concentration was determined according to Bradford (1976). The activity of SOD was measured according to Illescas et al. (2021) using a mixture reaction containing 2 mL of 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 80 μM nitro blue tetrazolium chloride (NBT), 15 μM riboflavin, and 50 μL of protein extract. One unit of SOD activity was defined as the amount of enzyme needed to inhibit the photochemical reduction of NBT by 50%. CAT and POD activities were determined spectrophotometrically as previously described by Nawaz et al. (2020), and one unit defined as the change of 0.01 absorbance unit per min. The specific activities of SOD, POD, and CAT were expressed as units min−1 mg−1 protein. Four biological replicates per treatment were analyzed.

Quantitative real-time PCR

Total RNA was extracted from frozen plant material using TRIzol™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA treated with DNase I (Sigma-Aldrich, St. Louis, MO, USA) and subsequently reverse-transcribed using an oligo(dT) primer and the Transcriptor First Strand cDNA Synthesis Kit (Takara, Inc., Tokyo, Japan), following the manufacturer’s protocols.

PCR reactions were performed in triplicate on a StepOnePlus thermocycler (Applied Biosystems, Foster City, CA, USA) in a total volume of 10 µL using TB Green Premix Ex Taq II (Takara) and 100 nM of each primer. The thermal cycling conditions consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s.

The expression levels of the LOX2, PR1, and PDF1.2 genes were analyzed. Primer sequences, previously described by Munhoz et al. (2015); Yan et al. (2021), and Wang et al. (2023), are listed in Supplementary Table 1.

Ct values were normalized to those of the P. edulis EF1 gene, and relative gene expression was calculated using the 2-ΔΔCT method (Schmittgen and Livak, 2008). Four biological replicates were performed for each treatment.

Statistical analysis

All statistical analyses were performed using R software version 4.4.2 (R Core Team, Vienna, Austria). Data were initially evaluated for normality of residuals and homogeneity of variances. DI, DS, and LOX2 gene expression data were log(x + 1)-transformed prior to analysis. Experiments were conducted using a completely randomized design. Data were subjected to one-way analysis of variance (ANOVA), considering treatment as the single factor. For the in vitro assays with culture filtrates, each Trichoderma strain was analyzed separately, with filtrate concentration considered as the single factor. For the in vivo experiment, the six experimental treatments were considered independent levels of a single treatment factor. When significant differences were detected by ANOVA (p < 0.05), means were compared using Duncan’s multiple range test at the 5% significance level.

Results

Antimicrobial activity of Trichoderma against X. axonopodis pv. passiflorae

The antimicrobial activity of Trichoderma strains T25 and T146 against Xap was evaluated using an in vitro inhibition assay with increasing concentrations (0, 5, 10, 20, and 40%) of fungal culture filtrates (Figure 1). Both Trichoderma filtrates inhibited Xap growth in a concentration-dependent manner, although differences between strains were observed. Culture filtrates from strain T25 significantly reduced bacterial growth at concentrations ≥10%, whereas complete inhibition was observed at 40% (Figure 1A). By contrast, culture filtrates from strain T146 significantly reduced Xap growth at concentrations of 20% and higher, with complete inhibition also observed at 40% (Figure 1B). These results were further supported by qualitative growth assays on LB agar plates, in which reduced bacterial viability was observed with increasing concentrations of Trichoderma culture filtrates (Figures 1C, D). Overall, strain T25 appeared to exhibit higher direct biocontrol activity than T146.

Figure 1.

Panel A shows a bar chart labeled T25 with growth inhibition (%) on the y-axis and filtrate concentration (%) on the x-axis, displaying increasing inhibition with higher concentrations. Panel B presents a similar bar chart labeled T146, also showing increased inhibition at higher filtrate concentrations, but with lower inhibition at intermediate concentrations. Panel C contains six petri dishes labeled 0%, 5%, 10%, 20%, 40%, C+, and C-, illustrating decreasing microbial growth as filtrate concentration increases. Panel D shows a similar series of plates with the same labels, also indicating reduced microbial growth at higher concentrations.

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Antibacterial activity of cell-free culture filtrates from T. asperellum T25 (A) and T. harzianum T146 (B) against X. axonopodis pv. passiflorae (Xap), expressed as percentage of bacterial growth inhibition (%), and bacterial viability following incubation with T25 (C) and T146 (D) filtrates. Bars represent the mean ± standard deviation of four biological replicates for each treatment (n = 4). Different letters above the bars indicate significant differences according to analysis of variance (ANOVA) followed by Duncan’s test (p < 0.05).

Growth-promoting effects of T. asperellum on P. edulis plants

Significant differences in plant height among T25-treated, T146-treated, and control plants were detected before inoculation with Xap. T25-treated plants exhibited the highest mean plant height (10.90 cm), differing significantly from both T146-treated plants and control plants (p < 0.05). By contrast, T146-treated and control plants showed mean heights of 8.97 and 8.43 cm, respectively, with no significant differences between them.

Indirect biocontrol of Trichoderma against bacterial blight in P. edulis plants

A greenhouse assay was conducted to evaluate the ability of Trichoderma strains T25 and T146 to reduce bacterial blight caused by Xap in passion fruit plants. DS and DI were determined at 14 dpi with Xap in plants previously exposed to Trichoderma. T25- or T146-treated plants inoculated with Xap showed lower DI percentages (15.4 ± 5.0 and 18.6 ± 5.8, respectively) than plants inoculated only with the pathogen (65.7 ± 8.4), although no significant differences were observed between the two Trichoderma strains (Table 1). By contrast, plants treated with Trichoderma and not inoculated with Xap showed values comparable to those of control plants. Regarding DS, both Trichoderma strains reduced bacterial blight symptoms in P. edulis plants, resulting in DS values of 1.3 ± 0.5% and 2.0 ± 0.6% for T25+Xap and T146+Xap, respectively, compared with 11.6 ± 3.3% in plants inoculated with Xap alone. These severity percentages in T25+Xap and T146+Xap plants were comparable to those observed in control plants, indicating almost complete suppression of disease symptoms (Table 1). These results were consistent with the visual evaluation of leaves from each treatment (Figure 2), in which necrotic symptoms were observed only in plants inoculated with Xap alone.

Table 1.

Severity and incidence of bacterial disease 14 days after X. axonopodis pv. passiflorae (Xap) inoculation in P. edulis plants pretreated with Trichoderma strains (T. asperellum T25 and T. harzianum T146), compared with non-pretreated, inoculated only with Xap, and untreated and uninoculated control plants (C).

Treatment Incidence (%) Severity (%)
C 0.0 ± 0.0 c 0.0 ± 0.0 b
Xap 65.7 ± 8.4 a 11.6 ± 3.3 a
T25 0.0 ± 0.0 c 0.0 ± 0.0 b
T146 0.0 ± 0.0 c 0.0 ± 0.0 b
T25+Xap 15.4 ± 5.0 b 1.3 ± 0.5 b
T146+Xap 18.6 ± 5.8 b 2.0 ± 0.6 b
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Different letters within the same column indicate significant differences among treatments according to Duncan’s multiple range test (p < 0.05).

Figure 2.

Six green leaves are arranged side by side on a black background, each labeled below as C, Xap, T25, T146, T25+Xap, and T146+Xap. The Xap leaf displays visible brown damage, while the other leaves appear healthy and vary slightly in color and texture.

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Pretreatment with Trichoderma strains T25 or T146 reduced bacterial blight symptoms in P. edulis plants. Representative leaves collected at 14 dpi with Xap from T25+Xap and T146+Xap plants; plants inoculated with Xap alone (Xap); plants treated with T25 or T146 without pathogen inoculation (T25 and T146); and untreated, non-inoculated control plants (C).

Differential expression of SA- and JA pathway-related genes in Trichoderma-treated P. edulis plants in response to X. axonopodis pv. passiflorae

To evaluate the effects of Trichoderma strains on P. edulis defense responses against Xap, the expression of genes encoding lipoxygenase 2 (LOX2), pathogenesis-related protein 1 (PR1), and plant defensin 1.2 (PDF1.2) was analyzed at two sampling time points after pathogen inoculation in both Trichoderma-treated and untreated plants (Figures 3, 4).

Figure 3.

Bar graph panel A shows relative expression of LOX2 across six treatments with T25 + Xap having the highest expression, followed by Xap, T146 + Xap, T25, T146, and C. Panel B displays relative expression of PR1, with T25 + Xap being notably higher than all other treatments, which are similar to each other. Error bars indicate standard error and different letters above bars denote statistically significant differences.

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Relative expression levels of LOX2 (A) and PR1 (B) at 48 h post-inoculation (hpi) with X. axonopodis pv. passiflorae (Xap) in 60-day-old P. edulis plants subjected to different treatments: T25+Xap, T146+Xap, Xap, T25, T146, and untreated, uninoculated controls (C). Values for PR1 represent relative expression calculated by the 2-ΔΔCT method, whereas LOX2 values are presented on a log10 scale. Bars represent the mean ± standard deviation of four biological replicates (n = 4). Different letters above the bars indicate significant differences according to analysis of variance (ANOVA) followed by Duncan’s test (p < 0.05).

Figure 4.

Two bar graphs display relative gene expression under six treatments: C, Xap, T25, T146, T25 + Xap, and T146 + Xap. Panel A shows LOX2 expression, with Xap treatment resulting in the highest expression, significantly greater than other treatments as indicated by distinct letters above bars. Panel B shows PR1 expression, where Xap also yields the highest expression. Error bars represent variability. Both graphs indicate significant differences among treatments.

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Relative expression levels of LOX2 (A) and PR1 (B) at 14 days post-inoculation (dpi) with X. axonopodis pv. passiflorae (Xap) in 60-day-old P. edulis plants subjected to different treatments: T25+Xap, T146+Xap, Xap, T25, T146, and untreated, uninoculated controls (C). Values represent relative expression calculated by the 2-ΔΔCT method. Bars represent the mean ± standard deviation of four biological replicates (n = 4). Different letters above the bars indicate significant differences according to analysis of variance (ANOVA) followed by Duncan’s test (p < 0.05).

At 48 hpi with the pathogen, plants from the Xap, T25+Xap, and T146+Xap treatments showed significantly higher LOX2 expression levels than control plants, with the highest expression observed in plants from the T25+Xap treatment (Figure 3A). Regarding PR1 expression, upregulation gene was detected only in plants from the T25+Xap treatment compared with control plants (Figure 3B). At 14 dpi with the pathogen, plants from the Xap treatment exhibited upregulation of LOX2 (Figure 4A) and PR1 (Figure 4B), whereas no differences in gene expression were observed in plants from the remaining treatments compared with control plants. No PDF1.2 expression was detected in plants from any of the treatments considered at either sampling time point (data not shown).

Influence of Trichoderma strains on the oxidative status of P. edulis in response to X. axonopodis pv. passiflorae

The effect of Trichoderma strains on oxidative stress-related responses in P. edulis plants, either inoculated or uninoculated with Xap, was evaluated by quantifying endogenous H2O2 levels and the antioxidant enzyme activities of SOD, POD, and CAT (Figure 5).

Figure 5.

Figure contains four bar graphs labeled A through D, each displaying mean values with error bars for different treatments: C, Xap, T25, T146, T25 + Xap, and T146 + Xap. Graph A shows H2O2 levels, with T146 + Xap having the highest value. Graph B represents SOD activity, with Xap displaying the highest bar. Graph C shows POD activity, peaking for Xap, while other treatments are comparably lower. Graph D depicts CAT activity, also highest for Xap. Different letters above bars indicate significant differences among treatments. All axes are labeled, and units are provided for each variable.

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Effect of Trichoderma (T. asperellum T25 and T. harzianum T146) on (A) H2O2 content, (B) superoxide dismutase (SOD), (C) peroxidase (POD), and (D) catalase (CAT) activities in 64-day-old P. edulis plants subjected to different treatments and evaluated at 14 days of inoculation with X. axonopodis pv. passiflorae (Xap), when applicable. Bars represent the mean ± standard deviation of four biological replicates (n = 4). Different letters above the bars indicate significant differences according to analysis of variance (ANOVA) followed by Duncan’s test (p < 0.05).

Plants from the Xap treatment showed a significant decrease in H2O2 content (0.16 ± 0.11 μmol g-1 FW) compared with control plants (0.24 ± 0.02 μmol g-1 FW), whereas plants from the T146+Xap treatment exhibited the highest levels (0.38 ± 0.05 μmol g-1 FW) (Figure 5A). In parallel, the highest SOD (1,999 ± 389.88 units min-1 mg-1 protein), POD (12.40 ± 2.94 units min-1 mg-1 protein), and CAT (22.99 ± 5.63 units min-1 mg-1 protein) activity levels were detected in plants from the Xap treatment (Figures 5B–D). Moreover, only plants from the T146 (1.06 ± 0.59 units min-1 mg-1 protein), T25+Xap (0.86 ± 0.64 units min-1 mg-1 protein), and T146+Xap (0.87 ± 1.09 units min-1 mg-1 protein), treatments had significantly lower POD activity than control plants (3.57 ± 1.86 units min-1 mg-1 protein) (Figure 5C).

Discussion

Managing bacterial blight in tropical and subtropical crops remains challenging under the current scenario of reducing use of fungicides and agricultural antibiotics in favor of more sustainable practices, and studies on the P. edulis-Xap pathosystem focusing on eco-friendly control alternatives are scarce. The present study demonstrated the potential of T. asperellum T25 and T. harzianum T146 as effective biocontrol agents against Xap in passion fruit plants, as reductions in both the incidence and severity of bacterial blight were observed in Trichoderma-treated plants under greenhouse conditions. Trichoderma asperellum and T. harzianum are species frequently cited in biological control studies (Woo et al., 2023), and the biocontrol potential of strain T25 against biotrophic fungi has been previously reported (Carrero-Carrón et al., 2016; Pedrero-Méndez et al., 2025). However, few studies have evaluated biocontrol activity of Trichoderma against bacterial pathogens, with most focusing on other plant-bacterium interactions involving phytopathogenic bacteria (Brotman et al., 2012; Morán-Diez et al., 2020; Islam et al., 2023).

Antibiosis is one of the mechanisms involved in the direct biocontrol activity of Trichoderma against phytopathogenic fungi (Harman et al., 2004; Woo et al., 2023). A pioneering study demonstrated that peptaibols, terpenoids, and pyrones produced by T. koningii exhibited antibacterial activity against a range of Gram-positive bacteria (Xiao-Yan et al., 2006). In the present study, both Trichoderma strains showed activity against a Gram-negative bacterium such as Xanthomonas, although the inhibitory effect observed with the T25 culture filtrate was markedly greater than that observed with T146. This finding is consistent with the antibacterial activity against X. campestris and X. citri reported for T. hamatum extracts (Baazeem et al., 2021), which was associated with the presence of 6-pentyl-α-pyrone (6-PP) as the major component. The metabolite 6-PP is a volatile organic compound frequently detected in culture extracts of Trichoderma spp., including T. harzianum (Rubio et al., 2009). Because bacterial blight caused by Xap is associated with severe defoliation, a reduction in pathogen inoculum in the soil resulting from the antibacterial activity of Trichoderma could also contribute to the effective management of this disease in passion fruit crops.

Trichoderma is a common soil fungus and a natural root colonizer with a recognized ability to enhance plant defenses against a wide range of pathogens (Woo et al., 2023). In the absence of the pathogen, T. asperellum T25 promoted the growth of P. edulis plants. This finding agrees with its growth-promoting effect previously reported for “Picual” olive plants (Carrero-Carrón et al., 2016). In the present study, Trichoderma was applied to the growth substrate of P. edulis, whereas Xap was sprayed onto the leaves. Therefore, the reduced disease incidence and severity observed in T25+Xap and T146+Xap plants appear to result from indirect biocontrol exerted by T. asperellum T25 and T. harzianum T146.

Currently, little is known about the activation defense responses by Trichoderma in passion fruit plants against Xanthomonas. At 48 hpi with the virulent bacterial strain, we observed upregulation of the LOX2 gene in all Xap-inoculated plants, indicating activation of the JA-dependent pathway. Such observation is consistent with a previous report showing that the P. edulis gene most strongly induced in response to Xap attack encodes lipoxygenase 2 (Munhoz et al., 2015). Stomatal openings and wounds are important infection sites for Xap, and increased accumulation of transcripts encoding lipoxygenases has also been reported in passion fruit plants in response to insect herbivory (Jardim et al., 2010). In this study, T25+Xap plants exhibited the highest LOX2 expression levels, accompanied by upregulation of the PR1 gene, indicating activation of the SA-dependent pathway by T. asperellum T25, which has also been reported in onions challenged with a necrotrophic fungal pathogen (Rivera-Méndez et al., 2020). SA-mediated defenses play a critical role in immunity against Xanthomonas spp., a genus of hemibiotrophic bacterial pathogens (Durrant and Dong, 2004; Song et al., 2022).

Our findings are also consistent with a previous study demonstrating increased expression of LOX2 and PR1 following inoculation with Pseudomonas syringae pv. tomato (Pst) DC3000 in T. asperelloides-pretreated Arabidopsis plants (Brotman et al., 2012), which was associated with induction of systemic resistance against Pst. Similarly, T. asperellum has been shown to activate both SA- and JA-dependent pathways and induce resistance against Xoo in rice plants (Islam et al., 2023). No significant changes in PR1 expression were detected in T146+Xap plants at 48 hpi with the bacterial pathogen. Nevertheless, similar reductions in disease severity were achieved in both T146+Xap and T25+Xap plants at later stages of the interaction when disease symptoms became visible. Differences among Trichoderma species in their capacity to confer protection against Pst in tomato plants have also been documented (Morán-Diez et al., 2020).

The complex role of ROS in plant defense mechanisms and their crosstalk with phytohormone signaling pathways is well established. Likewise, ROS-scavenging systems play a key role in regulating ROS generated in response to pathogen attack (Torres et al., 2006). A limitation of our study is that ROS accumulation and ROS-scavenging activities were not evaluated at early stages following pathogen challenge in passion fruit plants. To further clarify the molecular differences observed at 48 hpi in T25+Xap and T146+Xap plants compared with control plants, we analyzed expression levels of LOX2 and PR1, H2O2 content, and three antioxidant enzyme activities at 14 dpi with Xap, when disease symptoms were already visible.

Plants inoculated only with the pathogen exhibited significantly lower H2O2 content, accompanied by significantly higher SOD, POD, and CAT activities. Interestingly, at 14 dpi, plants inoculated solely with Xap exhibited remarkably low H2O2 concentrations alongside the highest activities of antioxidant enzymes (SOD, CAT, and POD). This metabolic response could be explained as a survival strategy employed by the plant during advanced infection stages. Under severe and prolonged bacterial pressure, excessive ROS accumulation poses a direct threat to integrity of host cells. Thus, the host plant might shift its physiological priority toward cell survival by deploying a robust enzymatic system to actively decompose toxic H2O2, thereby preventing self-inflicted tissue necrosis at the expense of early ROS-mediated signaling. Moreover, these plants showed significantly higher expression levels of LOX2 and PR1. Xanthomonas is known for using effector proteins to suppress basal immune responses, establish infection, and reprogram host transcription (Kay and Bonas, 2009; Timilsina et al., 2020). However, Trichoderma is also able to reprogram plant immunity. In this regard, Trichoderma has been reported to modulate JA- and SA-dependent defenses according to the nature of the pathogen infection (Martínez-Medina et al., 2017; Medeiros et al., 2017).

Enhanced transcript levels of lipoxygenase-related genes have also been reported in passion fruit plants at 5 and 9 dpi with Xap, together with increased SOD, CAT, and POD activities (Munhoz et al., 2015). In our study, the significantly lower H2O2 content detected in Xap-inoculated plants may be explained by the higher antioxidant enzyme activities observed in these plants. The discrepancy between our results and those previously reported for passion fruit plants may be attributable to differences in plant age or in the sampling time points used after Xap inoculation. In any case, Xanthomonas spp. can employ a variety of strategies to evade host defense responses (Timilsina et al., 2020).

At present, we do not have a clear explanation for the elevated expression levels of the SA-dependent defense marker gene PR1 observed in Xap-inoculated plants at 14 dpi. Our results showed no significant changes in the expression of LOX2 and PR1 or in SOD, POD, and CAT activities in T25- and T146-treated plants compared with control plants. This may be explained by the fact that, after 2 months, Trichoderma colonized roots were likely already well established, and only minor systemic transcriptional changes would be expected. This interpretation is consistent with observations reported for the T. harzianum-tomato interaction after 3 weeks of colonization (Domínguez et al., 2016).

Likewise, neither gene expression nor antioxidant enzyme activities differed significantly in T25+Xap or T146+Xap plants relative to control plants or to plants treated only with T25 or T146. Increased phenylalanine ammonia-lyase (PAL), POD, and CAT activities have been reported following seed priming with Trichoderma at 72 hpi with Xoo, and these responses were associated with reduced disease severity in rice (Islam et al., 2023). Increased antioxidant enzyme activities and enhanced expression of PAL genes have also been observed in wheat plants treated with T. asperellum T25, which exhibited increased tolerance to drought stress (Ascaso et al., 2025).

In the absence of studies on defense regulation by Trichoderma in Xap-challenged passion fruit plants, we can only speculate that the limited defense responses observed in Trichoderma-treated plants are associated with indirect biocontrol. Given the low disease severity observed in Trichoderma-treated plants, additional defense responses may have been unnecessary. Our results demonstrate that both Trichoderma strains reduced bacterial disease in passion fruit plants, although differences were observed between T25+Xap and T146+Xap plants. Notably, T146+Xap plants differed significantly from Xap-inoculated plants only in H2O2 content. By contrast, activation of both SA- and JA-dependent pathways appears to contribute to defense response in T. asperellum-treated plants. This pattern is consistent with the undulating profile of Trichoderma-triggered plant defenses previously described by Rubio et al. (2014) and Woo et al. (2023). Our findings support previous suggestions that the potential of Trichoderma for managing bacterial diseases has been underappreciated (Islam et al., 2023; Shafi et al., 2026).

Conclusions

Our study demonstrates that T. asperellum T25 and T. harzianum T146 exhibit direct antibacterial activity against Xap in vitro and effectively suppress bacterial blight development in passion fruit plants. The application of Trichoderma spores to the plant growth substrate significantly reduced both disease incidence and severity upon foliar challenge with Xap, confirming that the observed protection is mediated by the induction of systemic defense responses. At early infection stages (48 hpi), distinct expression profiles of the defense-related genes LOX2 and PR1 in T25-treated plants suggest that different strain-specific signaling pathways contribute to this indirect biocontrol efficacy. At later stages (14 dpi), when disease symptoms were fully developed, plants inoculated with Xap alone exhibited the lowest H2O2 levels alongside the highest antioxidant enzymatic activities (SOD, POD, CAT) and the strongest upregulation of defense genes (LOX2, PR1). This indicates a higher physiological demand for late-stage defense activation in plants lacking Trichoderma priming. Overall, our findings highlight the strong potential of Trichoderma as an eco-friendly biocontrol agent against Xap in tropical and subtropical fruit crops. Although field trials remain necessary to validate these greenhouse findings, application of Trichoderma to plant substrate represents a promising and sustainable management tool that aligns with societal demands for environmentally friendly agricultural practices.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the Spanish Government R+D+i project (PID2021-126575OB-I00) funded by Spanish MCIN/AEI/10.13039/501100011033, by “FEDER a way of making Europe”, and by the “Escalera de Excelencia” CLU-2025-2–04 program of the Regional Government of Castilla y León, co-funded by the Castilla y León 2021–2027 Operational Programme (FEDER), Spain. N.D.B. received financial support from the Research and Graduate Studies Office of the Southwest Bahia State University (UESB) through the Institutional Scholarship Program for stricto sensu graduate students under the Doctoral Sandwich Program Abroad (Call No. 301/2024). N.D.B. also acknowledges the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) for its support of the Graduate Program in Agronomy at UESB.

Footnotes

Edited by: Massimo Reverberi, Sapienza University of Rome, Italy

Reviewed by: Olusola Luke Oyesola, Covenant University, Nigeria

Srishti Singh, University of Allahabad, India

Data availability statement

The original contributions presented in the study are publicly available. This data can be found here: GREDOS repository (University of Salamanca, Spain), http://hdl.handle.net/10366/172651.

Author contributions

NDB: Writing – original draft, Formal analysis, Investigation, Data curation, Methodology. QSN: Writing – review & editing, Supervision, Conceptualization. RH: Writing – original draft, Conceptualization, Formal analysis, Funding acquisition, Writing – review & editing, Supervision. MBR: Data curation, Methodology, Investigation, Formal analysis, Writing – original draft.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author RH declared that she was an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ffunb.2026.1920680/full#supplementary-material

Supplementary Table 1

Primers used for quantitative real-time PCR (qPCR) analysis.

Table1.docx (13.7KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1

Primers used for quantitative real-time PCR (qPCR) analysis.

Table1.docx (13.7KB, docx)

Data Availability Statement

The original contributions presented in the study are publicly available. This data can be found here: GREDOS repository (University of Salamanca, Spain), http://hdl.handle.net/10366/172651.

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