Characterization of novel Trichoderma spp. isolates: phytostimulant and biocontrol properties in the model plant Arabidopsis thaliana

Abstract

Among microbials, Trichoderma spp. have been the focus of significant research due to their capacity to promote plant growth and suppress a wide range of phytopathogens. A total of 27 native isolates of Trichoderma spp. were obtained from diverse agroecological regions of the province of Tucumán, Argentina, and were characterized by their antagonistic activity and phytostimulant potential. In vitro assays revealed the ability of the isolates to combat fungal phytopathogens that affect various economically important crops, such as Penicillium digitatum, Alternaria alternata, Botrytis cinerea, Phytophthora capsici, and Fusarium oxysporum. Furthermore, it was determined that diffusible compounds secreted by Trichoderma isolates did not induce the elongation of the primary root of Arabidopsis thaliana in vitro; however, volatile compounds released by certain isolates not only induced this process but also increased the area occupied by the lateral roots of the plant. Based on this screening, five isolates were selected for further analyses: T. longibrachiatum (CP-1), T. breve (HM-1), T. scalesiae (L1-03), T. yunnanense (M4Ar-05), and T. atrobrunneum (M5Ar-03). The strains were evaluated for their ability to promote the growth of A. thaliana under greenhouse conditions and modulate the expression of growth and defense-related genes. All treated plants exhibited increased biomass and leaf area, accompanied by isolate-dependent changes in the expression of genes involved in plant development and immune responses. These results suggest that the selected Trichoderma spp. possess multiple beneficial traits, with valuable agricultural properties, and provide a basis for the development of bioformulations adapted to local production systems.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00425-026-04988-y.

Introduction

The increasing global demand for sustainable agricultural practices has led to significant interest in biological alternatives to traditional chemical inputs. Among these alternatives, Trichoderma spp. have emerged as extensively investigated microorganisms, which are widely employed in agriculture as biocontrol agents (Hermosa et al. 2012; Tyśkiewicz et al. 2022; Xue et al. 2021). These fungi are key components of biopesticides, biofertilizers, and products enhancing plant growth and stimulating natural defense mechanisms (Debnath & Saha 2020; Liu et al. 2022; Woo et al. 2014). Their widespread application is attributed to their remarkable ability to protect crops, suppress pathogenic populations in diverse agricultural environments, and improve soil health over time. Additionally, Trichoderma spp. serve as effective soil inoculants by enhancing nutrient availability, facilitating decomposition processes, and supporting biodegradation activities (Visconti et al. 2023; Zhang et al. 2018). These features make Trichoderma strains attractive candidates for eco-friendly agricultural applications.

Trichoderma strains act as biological control agents, employing a variety of complex direct and indirect mechanisms to combat pathogenic fungi (Mukherjee et al. 2013). These strategies often operate synergistically, contributing collectively to the biocontrol process. Direct mechanisms include the secretion of cell wall-degrading enzymes, the production of antibiotics, competition for key nutrients, such as carbon, nitrogen, and iron, and the establishment of direct mycoparasitic interactions with target pathogens (Benítez et al. 2004). Indirectly, Trichoderma stimulates plant defense mechanisms, either locally or systemically, by releasing elicitors derived from the cell walls of the host plant or the pathogen itself (Ghorbanpour et al. 2018). The specific mechanisms employed vary among strains and depend on the nature of the interaction between the Trichoderma strain, the pathogen, and the host plant. These multifaceted biocontrol mechanisms have established Trichoderma as a key component of integrated pest management strategies.

In addition to biocontrol, Trichoderma also acts as a potent plant growth-promoting agent. These fungi are able to synthesize phytohormones, such as auxins, gibberellins, and cytokinins, which directly stimulate root elongation and branching, thereby increasing nutrient and water uptake efficiency (Contreras-Cornejo et al. 2009). They also improve nutrient availability by facilitating nitrogen and phosphorus uptake and producing extracellular enzymes that enhance nutrient solubilization (Subramaniam et al. 2022). In addition, Trichoderma promotes plant growth by inducing systemic resistance. This process involves the activation of defense signaling pathways, particularly those mediated by jasmonic acid and salicylic acid, which strengthen plant responses under biotic and abiotic stress conditions (Harman et al. 2004). The combined effects of these mechanisms lead to improved root architecture, increased biomass accumulation, and enhanced crop yields, highlighting the key role of Trichoderma as a phytostimulant in sustainable agriculture.

Considering the lack of biological alternatives based on native Trichoderma strains adapted to the northern Argentinian conditions, this study aimed at isolating and characterizing novel Trichoderma strains by evaluating their biocontrol efficiency and growth-promoting effects. The isolates were initially characterized in vitro to determine their antagonistic activity against various fungal phytopathogens. In vitro and greenhouse assays were then conducted using the model plant Arabidopsis thaliana to assess its growth-promoting capacity. A gene expression analysis was also performed on A. thaliana plants exposed to Trichoderma isolates. This comprehensive evaluation supports the integration of these novel isolates into a sustainable agriculture.

Materials and methods

Model plant: Arabidopsis thaliana

In this study, seeds of the ecotype Columbia-0 [Col-0, Nottingham Arabidopsis Stock Centre, Nottingham, UK] were used. Surface disinfection was performed by washing the seeds three times with 70% (v/v) ethanol, followed by two washes with absolute ethanol, with a 30 s shaking interval for each wash. After this process, the seeds were allowed to dry and stored at 4 °C until further use (Romero-Contreras et al. 2019). Seeds of A. thaliana were germinated and grown on 0.2X Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), adjusted to pH 5.7, and supplemented with 0.5% (w/v) sucrose and 0.8% (w/v) agar.

Fungal phytopathogens

Penicillium digitatum CSM/Pd-01 and Alternaria alternata ISIBMMA/F-Alsp19-S were obtained from the Phytopathology Laboratory of the citrus company San Miguel S.A. and from the strain collection of INSIBIO-CONICET (Higher Institute of Biological Research) in Tucumán, Argentina, respectively. Botrytis cinerea BC0510, Phytophthora capsici, and Fusarium oxysporum belong to the Center of Genomic Sciences of the National Autonomous University of Mexico, and the Center for Research in Biotechnology of the Autonomous University of the State of Morelos in Morelos, Mexico. All phytopathogens were grown on Potato Dextrose Agar medium (PDA; 4 g/L potato extract, 20 g/L glucose, 15 g/L agar; Biokar, Allonne, France) at 25 °C for 7–10 days. Spores were harvested from the plates using a sterile saline solution containing 0.1% Tween 80 and adjusted to a final concentration of 10⁶ spores/mL using a Neubauer cell counting chamber. This spore suspension was used in subsequent assays.

Isolation of native Trichoderma strains

Trichoderma isolates were obtained from four different sampling sites in the province of Tucumán, Argentina. These sites included an organic farm, a nature reserve, an organic lemon field, and an organic blueberry field. A quadrant sampling system was employed, whereby five equidistant samples were collected from each site. Sample processing was conducted in accordance with the methodology outlined by Siddiquee (2017). Briefly, soil samples were taken at a depth of 15–20 cm and stored in sterile collection bags. 5 g of soil from each sample were suspended in 50 mL of sterile distilled water and shaken for 20 min at 150 rpm using an orbital shaker (Bioamerican Science, BS 875). After settling for 10 min, 200 µL of the supernatant were plated on Rose Bengal Agar (RBA) (Martin 1950) and PDA. Plates were incubated at 28 °C for 4–7 days. During this time, green fungal colonies, which are indicative of the Trichoderma genus, were observed. Potential Trichoderma colonies were then transferred to PDA medium. Pure cultures were obtained from a single spore, as described by Covacevich and Consolo (2014). Preliminary identification was based on colony morphology and microscopic characteristics of conidiophores. Molecular identification was subsequently performed to confirm their taxonomic classification as follows.

Molecular identification

Genomic DNA was extracted from 7 day-old actively growing cultures of Trichoderma isolates on PDA medium. Mycelium was scraped from the plates, and 0.5–1 mg of fungal biomass was collected in sterile microtubes. Samples were suspended in 50 µL of sterile distilled water and treated with 2 µL of lyticase (5 mg/mL, Sigma-Aldrich) for 30 min at 37 °C to facilitate cell lysis. Following enzymatic treatment, DNA extraction was performed using the Epicentre MasterPure™ Complete DNA and RNA Purification Kit (Biosearch Technologies), according to the manufacturer’s protocol. Taxonomic identification was performed by PCR amplification of the ITS1/ITS2 region using primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (Siddiquee 2017). The PCR reaction mixture (final volume, 50 μL) contained: 50–100 ng/μL of purified genomic DNA, 0.5 μM of each primer, 200 μM of deoxyribonucleoside triphosphate (dNTPs), 1X Phusion High-Fidelity Buffer, and 0.02 U/μL of Phusion DNA polymerase. The PCR conditions were as follows: initial denaturation at 98 °C for 5 min, 30 cycles of denaturation at 98 °C for 10 s, annealing at 59 °C for 30 s, extension at 72 °C for 30 s; followed by a final extension at 72 °C for 5 min. Amplified PCR products were analyzed by electrophoresis on a 0.8% (w/v) agarose gel. The resulting bands were purified using the Gel and PCR clean-up kit (NucleoSpin, Düren, Germany).

Sequencing of the purified PCR products was performed by Microsynth Seqlab (Göttingen, Germany). Sequences were edited using Clone Manager 9 Software (Cary, NC, USA), and similarity searches were conducted using the NCBI BLAST service (http://www.ncbi.nlm.nih.gov/BLAST). Species identification was based on the highest sequence identity with a maximum query coverage of 100%.

Phylogenetic analyses were performed using MEGA 12 software. Phylogenetic relationships were inferred using the Maximum-Likelihood method based on the Tamura–Nei model (Tamura and Nei 1993), which produced the tree with the highest log likelihood (−2,217.08). Bootstrap support was assessed using 1000 replicates. Ambiguous positions were excluded by applying the pairwise deletion option, resulting in a final dataset of 699 nucleotide positions from 45 sequences. The phylogenetic tree was rooted with Nectria eustromatica CBS 121896 as the outgroup and visualized using the same software.

In vitro antagonism assay against fungal phytopathogens

The antagonistic activity of Trichoderma isolates against various fungal phytopathogens was evaluated using a dual-culture technique on Petri dishes, as described by Pereyra et al. (2021), with slight modifications. Petri dishes (60 mm diameter) containing PDA medium were inoculated with 3 µL of a Trichoderma spore suspension at one end, and an equal volume of the pathogen suspension at the opposite end, maintaining 3 cm between the two inoculation points. Control plates were inoculated with the pathogen suspension alone. Plates were incubated at 28 °C for 7 days. After incubation, the radius of the pathogen colony facing the Trichoderma colony (R2) was measured. The colony radius in the control plates (R1) was also recorded in parallel. Each treatment was performed in triplicate, and the entire experiment was repeated twice to ensure reproducibility.

The antagonistic effect of Trichoderma isolates was quantified as the percentage of inhibition of radial growth (PIRG) of the pathogen using the following formula (Rahman et al. 2009):

$$PIRG=\frac{\left(R1-R2\right)}{R1}\times 100$$

Trichoderma spp.: Arabidopsis thaliana interactions

In vitro phytostimulant effect of Trichoderma isolates

The assay was conducted as described by Romero-Contreras et al. (2024) with slight modifications. A. thaliana Col-0 seeds were surface disinfected and vernalized for 48 h before being placed on plates containing 0.2X MS medium. Ten seeds were sown on the top of each plate, which were then incubated vertically at 24 °C in a plant growth chamber (MRClab, model PGI-500 H) under a 16 h light/8 h dark photoperiod.

After 4 days of germination, 5 μL of a Trichoderma spore suspension (1 × 10⁶ spores/mL) was inoculated at the bottom of each plate. Before any physical contact between the fungal hyphae and plant roots, the plates were examined under a stereomicroscope (Zeiss Discovery V8 with an AxioCam MRc camera, Jena, Germany) on the fourth day post-inoculation. Each treatment was performed in triplicate, and an additional replicate was reserved for RNA extraction. Plants exposed to Trichoderma and corresponding non-exposed controls were collected in microtubes and stored at − 80 °C until further analysis.

The phytostimulant effect of volatile organic compounds (VOCs) produced by Trichoderma isolates was also evaluated using divided Petri dishes. Five A. thaliana Col-0 seeds were placed on the MS medium-containing side of the plate. After 4 days of germination under the above-described conditions, 5 μL of each Trichoderma spore suspension was inoculated into the opposite compartment containing PDA medium. Plates were incubated under identical conditions, and root development was observed under a stereomicroscope at day 7.

In both assays, the phytostimulatory effect of Trichoderma isolates was evaluated by measuring the primary root length and the lateral root area of A. thaliana plants. Control plates were prepared by growing A. thaliana seeds without Trichoderma inoculation. Each treatment was carried out in triplicate, and the experiment was repeated twice to ensure reproducibility.

Phytostimulatory effect of Trichoderma on A. thaliana under greenhouse conditions

For pot inoculation assays, 1-week-old A. thaliana Col-0 seedlings germinated in a 3:1 (v/v) mixture of soil and vermiculite, and maintained in a greenhouse under controlled conditions (22 ± 2 °C, 60% relative humidity, and a 16 h light/8 h dark photoperiod), were individually transferred to germination trays containing the same soil mixture and incubated for 7 days. Inoculation with Trichoderma suspensions (1 × 10⁶ spores/mL) was conducted once per week for four consecutive weeks. Control plants were maintained under identical conditions, but no Trichoderma treatment was applied. Throughout the experiment, irrigation was carried out every three days using non-sterile water. Each treatment consisted of five pots and was performed in triplicate. To assess the phytostimulatory effect of the Trichoderma isolates, fresh weight and leaf area of A. thaliana plants were measured 7 days after the final inoculation.

RT-qPCR analysis

To evaluate the expression of genes associated with the growth-promoting effect of Trichoderma isolates on A. thaliana, plant material was collected from the in vitro phytostimulation assay previously described.

Frozen plants (–80 °C) were used for total RNA extraction with TRIzol™ reagent, following the manufacturer’s protocol (Invitrogen). RNA concentration and purity were determined using a NanoDrop spectrophotometer (Implen NP80, Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was confirmed by 1% agarose gel electrophoresis. To remove genomic DNA contamination, 1 µg of total RNA was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Subsequently, cDNA was synthesized from the DNase-treated RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Primers sequences used for the RT-qPCR analysis are listed in Table S1. Reactions were conducted following the methodology outlined by Aragón et al. (2021).

Statistical analysis

Primary root length, lateral root area, and leaf area of A. thaliana were quantified using ImageJ2 software (Schneider et al. 2012). Fresh weight (g) of individual plantlets was measured with an analytical balance, with each value representing one biological replicate (n = 30).

For in vitro assays, normality and homogeneity of variance were evaluated using the Shapiro–Wilk and Levene’s tests, respectively. Primary root length data did not meet parametric assumptions and were, therefore, analyzed using non-parametric methods. Differences among treatments were first assessed using the Kruskal–Wallis test (Kruskal and Wallis 1952), followed by pairwise comparisons against the control group using the Wilcoxon rank-sum test with Benjamini–Hochberg correction for multiple testing.

In contrast, lateral root area data satisfied normality and homogeneity of variance assumptions and were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons among treatments.

For greenhouse experiments (fresh weight and leaf area), normality and homogeneity of variance were evaluated prior to analysis. Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test to compare each treatment against the untreated control.

For gene expression analysis, relative transcript levels were quantified by qPCR, normalized to reference genes, and analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test.

Significance was set at p < 0.05. All statistical analyses were performed using RStudio and GraphPad Prism version 9.4.0 (GraphPad Software, San Diego, CA, USA).

Results

Isolation and identification of native Trichoderma spp.

A total of 27 native Trichoderma isolates were obtained from four distinct sampling sites in the province of Tucuman, Argentina. The organic blueberry field yielded the highest number of isolates (16), followed by the organic lemon field (7), the organic farm (2), and the nature reserve (2).

A comprehensive analysis of the isolates revealed macroscopic and microscopic characteristics consistent with the genus Trichoderma. Macroscopic evaluation of the colonies revealed characteristic features of the genus, such as rapid radial growth, with most isolates fully colonizing 90 mm Petri dishes within 3–4 days. Most colonies exhibited a flocculent texture and pigmentation ranging from bluish-green to yellowish-green; however, only a few isolates lacked pigmentation. In some cases, concentric rings of pigment were observed, an attribute previously reported for specific Trichoderma species (Fig. S1a) (Siddiquee 2017). Microscopic observation revealed key features of the genus, including the tendency of conidiophores to aggregate in fascicles or pustules, a strong tendency to branch in an irregular pattern, and abundant sporulation characterized by globose conidia (Fig. S1b).

For molecular identification, genomic DNA was extracted from all Trichoderma isolates, and the ITS1–ITS2 ribosomal region was amplified and sequenced. A single PCR product of approximately 500–600 bp was obtained for each isolate. Sequence analysis revealed > 99% identity with reference sequences of the NCBI database, allowing provisional assignment of the isolates to ten distinct Trichoderma species (Table 1). The most frequently identified species were Trichoderma longibrachiatum (5 isolates), T. dorotheae (4 isolates), T. crassum (4 isolates), and T. atrobrunneum (4 isolates).

Table 1.

Identification of Trichoderma spp. isolates based on sequence analysis of the ITS region. The table shows the isolate code, source of isolation, molecular identification at the species level, percentage of sequence identity, and corresponding GenBank accession numbers

Isolate ID	Source	Species	Identity (%)	GenBank accession number
CP-1	Organic farm	Trichoderma longibrachiatum	99.45	PV822512
CP-2		Trichoderma longibrachiatum	99.07	PV822513
HM-1	Nature reserve	Trichoderma breve	99.28	PV822514
HM-2		Trichoderma hamatum	99.63	PV822515
L1-01	Lemon field	Trichoderma dorotheae	100	PV822516
L1-02		Trichoderma dorotheae	100	PV822517
L1-03		Trichoderma scalesiae	99.81	PV822518
L2-01		Trichoderma dorotheae	99.81	PV822519
L3-01		Trichoderma dorotheae	100	PV822520
L3-02		Trichoderma breve	99.64	PV822521
L3-03		Trichoderma breve	99.64	PV822522
M1Ar-01	Blueberry field	Trichoderma hamatum	99.81	PV822523
M2Ar-01		Trichoderma yunnanense	100	PV822524
M2Ar-02		Trichoderma crassum	99.82	PV822525
M4Ar-01		Trichoderma crassum	99.82	PV822526
M4Ar-02		Trichoderma crassum	99.82	PV822527
M4Ar-03		Trichoderma longibrachiatum	100	PV822528
M4Ar-04		Trichoderma crassum	99.64	PV822529
M4Ar-05		Trichoderma yunnanense	100	PV822530
M4Ar-06		Trichoderma longibrachiatum	99.82	PV822531
M4Ar-07		Trichoderma longibrachiatum	100	PV822532
M4Ar-08		Trichoderma rifaii	100	PV822533
M4Ar-09		Trichoderma rugulosum	99.45	PV822534
M5Ar-01		Trichoderma atrobrunneum	100	PV822535
M5Ar-02		Trichoderma atrobrunneum	100	PV822536
M5Ar-03		Trichoderma atrobrunneum	100	PV822537
M5Ar-04		Trichoderma atrobrunneum	100	PV822538

A phylogenetic placement was conducted using the Maximum-Likelihood method based on ITS sequences to further validate these assignments. The resulting phylogenetic tree (Fig. 1) revealed that all isolates clustered within well-supported clades corresponding to their respective species. Notably, isolates assigned to T. longibrachiatum, T. dorotheae, and T. crassum formed strongly supported monophyletic groups, with bootstrap values exceeding 90%.

Fig. 1.

Phylogenetic relationships of the Trichoderma isolates. The tree was constructed using a Maximum-Likelihood analysis based on ITS sequences in MEGA. Bootstrap values based on 1000 replications are shown above the node. The tree was rooted with Nectria eustromatica CBS 121896 as the outgroup

In vitro biocontrol activity of Trichoderma isolates

All Trichoderma isolates obtained from different sampling sites were subjected to an in vitro antagonism test in order to evaluate their efficacy as biocontrol agents against P. digitatum, F. oxysporum, B. cinerea, P. capsici, and A. alternata. Remarkably, all isolates demonstrated significant antagonistic activity against the tested pathogens, highlighting their potential as broad-spectrum biocontrol agents.

The results revealed that P. digitatum exhibited a higher degree of resistance to Trichoderma-mediated competition, with isolate CP-1 showing the highest inhibitory effect of 76% (Fig. 2a). F. oxysporum and P. capsici displayed similar susceptibility patterns when confronted with Trichoderma, with inhibition values ranging from 67 to 89% (Fig. 2b and d). The most effective inhibition for each pathogen was observed with isolates L1-01 and M4Ar-06, respectively. Regarding B. cinerea, isolates from L1-01 to L3-01 showed the greatest antagonistic potential (Fig. 2c), with inhibition rates ranging from 96 to 98%. A. alternata demonstrated the highest degree of variability in response to Trichoderma isolates: the inhibition percentages ranged from 51 to 96%, with isolates M1Ar-01, L1-03, and L2-01 being most effective (Fig. 2e).

Fig. 2.

Percentage of mycelial growth inhibition by Trichoderma isolates of P. digitatum a, F. oxysporum b, B. cinerea c, P. capsici d, and A. alternata e. Plates were obtained using the dual-culture technique, after 7 days of incubation at 28 °C. Significance based on Tukey’s HSD test. Different letters indicate significant differences (p < 0.05). Control: without Trichoderma

A significant proportion of the Trichoderma isolates tested were capable of producing low-molecular-weight diffusible compounds that inhibited the growth of phytopathogenic fungi. Visible inhibition zones were observed for isolates M5Ar-01, CP-1, and M5Ar-03 against P. digitatum, F. oxysporum, and B. cinerea, respectively (Fig. S2). In addition, distinct levels of mycoparasitic interaction were observed. According to the scale proposed by Ezziyyani et al. (2004), T. atrobrunneum M4Ar-05 exhibited the highest degree of mycoparasitism (grade 4) against P. digitatum, B. cinerea, and P. capsici, which implied overgrowth and sporulation of the fungi on the pathogen. In contrast, T. scalesiae L1-03 exhibited grade 4 mycoparasitism against B. cinerea, and grade 3 against A. alternata, achieving complete invasion of the plate with and without sporulation, respectively (Fig. S2).

In vitro phytostimulant effect of Trichoderma on A. thaliana

The phytostimulant effect of Trichoderma isolates on A. thaliana was initially evaluated in vitro by assessing the production of diffusible and volatile compounds with potential phytostimulant effects. The results indicated that plants exposed to diffusible compounds exhibited primary root length values that were comparable to or lower than those of the control group. Several isolates, particularly those obtained from blueberry fields, significantly reduced root length compared to the untreated control (Fig. 3a). Regarding VOCs, the results revealed significant differences between treatments. Isolates M4Ar-06 and M4Ar-08 induced the most substantial increase in root length, significantly exceeding that of the control group (Fig. 3b).

Fig. 3.

Effect of Trichoderma spp. diffusible compounds a and VOCs b on primary root length of A. thaliana. Boxplots represent the distribution of individual primary root length values (cm) for each treatment. Colored bars indicate the origin of each isolate. Data were analyzed using non-parametric methods due to violation of normality and homogeneity of variance assumptions (Shapiro–Wilk and Levene’s tests). Differences among isolates were first evaluated using the Kruskal–Wallis test, followed by pairwise comparisons against the untreated control using the Wilcoxon rank-sum test with Benjamini–Hochberg correction for multiple testing. Asterisks denote significant differences compared to the untreated control (p < 0.05). The experiment was conducted in Petri dishes under controlled conditions, and the results were assessed 4 and 7 days after inoculation, respectively

Stereomicroscopic analysis revealed that A. thaliana plants exposed to diffusible compounds from Trichoderma spp. exhibited an increase in the area occupied by lateral roots. However, this increase was not considered to be significant when compared to the control group, except for isolate CP-1 (Fig. 4a). In contrast, plants exposed to VOCs released by Trichoderma exhibited a significant augmentation in lateral root area in comparison to the control plants. Some examples of these results include isolates CP-1, HM-1, M4Ar-06, and M4Ar-08 (Fig. 4b). The development of both the primary root and the area occupied by lateral roots in plants exposed to diffusible and volatile compounds in a split-plate assay is illustrated in Fig. 5, along with the corresponding stereomicrograph.

Fig. 4.

Effect of Trichoderma spp. diffusible compounds a and VOCs b on the total projected lateral root area of A. thaliana. Boxplots represent the distribution of individual lateral root area values (mm.²) for each treatment. Data were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test. Different letters indicate statistically significant differences among treatments (p < 0.05)

Fig. 5.

Representative images of A. thaliana root development when exposed to Trichoderma diffusible compounds a or VOCs b. Top: Petri dishes showing the growth of A. thaliana in the presence of Trichoderma compounds, compared to a control (left side of the plates). Bottom: stereomicroscope images of lateral root development under each treatment, illustrating differences in lateral root area. Scale bars = 1000 μm

Phytostimulant effect of Trichoderma on A. thaliana under greenhouse conditions

From the 27 native Trichoderma isolates obtained from diverse agroecological regions, five representative strains were selected to encompass both ecological diversity and the strongest phytostimulant responses: T. longibrachiatum CP-1, T. breve HM-1, T. scalesiae L1-03, T. yunnanense M4Ar-05, and T. atrobrunneum M5Ar-03. The five selected strains were evaluated under greenhouse conditions.

The inoculation of soil with these isolates exerted a significant effect on the fresh weight of 7 week-old A. thaliana plants (Fig. 6a). Plants treated with CP-1, HM-1, M4Ar-05, and M5Ar-03 showed a significant increase in biomass, indicating a strong growth-promoting effect. The M4Ar-05 isolate produced the highest average biomass; in contrast, treatment with L1-03 showed no statistical difference compared to the control, emphasizing the functional variability among the selected Trichoderma isolates.

Fig. 6.

Effect of Trichoderma isolates on fresh weight a and leaf area b of 7 week-old A. thaliana plants under greenhouse conditions. Data represent mean ± SD (n = 30). Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. Asterisks indicate statistically significant differences compared to the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001). Representative images of plants from each treatment group are shown below the graph

Leaf area analysis further supported the previous result, as plants inoculated with CP-1, HM-1, M4Ar-05, and M5Ar-03 showed the highest values (Fig. 6b). In comparison, isolate L1-03 produced a more modest increase.

Gene expression profiling in A. thaliana in response to Trichoderma isolates

The interaction between Trichoderma and plants involves multiple mechanisms that synergistically enhance immune responses and/or promote growth (Yao et al. 2023). The expression levels of key genes related to plant defense and growth regulation were quantified to explore the mechanisms induced by the Trichoderma isolates. Defense-related genes included AOS (jasmonic acid biosynthesis), PDF1.2 (defensin-like protein), PR1 and PR4 (pathogenesis-related proteins), and ZAT12 (a zinc finger protein involved in stress responses). Additionally, genes related to growth regulation were analyzed, including ABI5 (abscisic acid signaling), At-IAA1 (auxin response), and SAUR9 (auxin-responsive growth regulator).

A. thaliana seedlings were exposed to selected Trichoderma isolates in a dual-culture system, allowing the assessment of gene expression changes triggered by fungal metabolites. After incubation, treated plants were compared with non-exposed controls to identify transcriptional changes (Fig. 7). Among the genes analyzed, SAUR9 and PR4 did not show statistically significant expression changes relative to the control. In contrast, ABI5 was significantly upregulated in response to all isolates except CP-1, suggesting activation of ABA-mediated stress responses. Growth-related genes showed more variable expression patterns. For instance, At-IAA1 was either significantly downregulated or unchanged, depending on the isolate, indicating differential modulation of auxin signaling.

Fig. 7.

Relative expression levels of growth- and defense-related genes in A. thaliana plants treated with native Trichoderma isolates (CP-1, HM-1, L1-03, M4Ar-05, and M5Ar-03) compared to untreated control plants. Gene expression was quantified by qPCR and normalized to reference genes. Bars represent mean ± SD (n = 3). Different letters indicate statistically significant differences between treatments according to Tukey’s HSD test (p < 0.05)

Interestingly, some isolates induced a statistically significant increase in the expression of at least one defense-related gene, despite Trichoderma being characterized as a non-pathogenic fungus. In this sense, PR1, a key marker of salicylic acid-mediated defense, was strongly upregulated in response to isolates L1-03 and M4Ar-05.

Discussion

Fungi belonging to the genus Trichoderma are among the most extensively studied microorganisms in agricultural biotechnology due to their remarkable capacity to act as biocontrol agents and plant growth promoters. These properties make them versatile symbionts with significant potential for sustainable crop management and environmental protection.

The isolation method employed in this study yielded 27 native Trichoderma spp. isolates from four distinct sampling sites. Initial identification was performed using classical morphological methods, based on both micro- and macroscopic characteristics of the colonies, as described by Siddiquee (2017). These characteristics align with classical taxonomic descriptions and provide a reliable basis for preliminary identification. Interestingly, contrary to findings reported by Elad et al. (1981) and Küçük and Kivanç (2003), the use of RBA medium did not facilitate the recovery of Trichoderma colonies in this study. Successful isolation and growth were achieved exclusively on PDA medium, in accordance with the previous reports that have highlighted PDA as a favorable medium for enhancing pigmentation and mycelial development of Trichoderma spp. (Samuels et al. 2002).

A molecular approach was employed to support morphological identification and propose a tentative species-level assignment of the isolates. Sequence analysis revealed that all isolates shared more than 99% identity—some even reaching 100%—with reference strains, indicating a close genetic affiliation with previously described Trichoderma taxa. Although certain clustering patterns were consistent with the site of isolation, phylogenetic relationships did not strictly correspond to geographic origin, suggesting the coexistence of diverse Trichoderma lineages within each environment. However, given the limited resolution of the ITS region in discriminating closely related Trichoderma species, particularly within species complexes, further confirmation through multi-locus phylogenetic analyses will be necessary (Cai and Druzhinina 2021).

Members of the genus Trichoderma are recognized as potent biocontrol agents capable of antagonizing a broad spectrum of phytopathogenic fungi through multiple mechanisms, including mycoparasitism, antibiosis, and competition for nutrients and space. Their effectiveness has been demonstrated against both soilborne and postharvest pathogens, highlighting their versatility across diverse agroecosystems (Nakkeeran et al. 2016; Li et al. 2025). In this study, in vitro experiments were conducted to assess the capacity of the 27 isolates to impede the proliferation of a diverse array of fungal phytopathogens. The pathogens selected were: P. digitatum, a postharvest citrus pathogen causing green mold disease (Palou 2014); F. oxysporum, a well-known soilborne rhizosphere pathogen (Di Pietro et al. 2003); B. cinerea, responsible for gray mold disease in over 200 plant species (Williamson et al. 2007); P. capsici, a destructive pathogen affecting vegetable crops, such as tomato and pepper (Lamour et al. 2012); and A. alternata, the causal agent of Alternaria rot primarily affecting berries (Wang et al. 2021).

The suppressive capacity of Trichoderma, as evidenced in the assays, can be partially ascribed to its rapid colonization and growth rate, a factor that confers upon it a significant advantage over competing pathogenic fungi. Variations in susceptibility were also observed among the pathogens, which could be due to differences in their cell wall composition and/or structural characteristics, as well as to intrinsic resistance mechanisms to the antifungal metabolites produced by Trichoderma spp. The ability of Trichoderma isolates to inhibit pathogen growth through the production of diffusible metabolites has been documented in preceding studies investigating the secretion of peptaiboles, polyketides, and other secondary metabolites with antimicrobial activity (Benítez et al. 2004; Howell 2007; Sood et al. 2020). The inhibition halos observed in this study serve to reinforce the role of diffusible bioactive compounds as one of the primary mechanisms involved in the suppression of pathogens.

Additionally, strong mycoparasitic properties were demonstrated, as indicated by the overgrowth and sporulation of Trichoderma on the colonies of pathogenic fungi. Some authors also based their results on the scale proposed by Ezziyyani et al. (2004). Ruiz-Gómez and Miguel-Rojas (2021) observed comparable mycoparasitic behavior against Rhizoctonia solani. They found that eight of the ten Trichoderma isolates selected for their analysis presented the highest degree at the mycoparasitism scale (4), while the remaining two presented a grade 3 of mycoparasitism. In this context, the strong mycoparasitic response of certain isolates, including T. atrobrunneum M4Ar-05 and T. scalesiae L1-03, underscores their potential as effective biocontrol agents, given that mycoparasitism involves the degradation of the conidia and reproductive structures of the pathogen, thereby impeding its proliferation (Mathys et al. 2012).

In order to ascertain the phytostimulant potential of Trichoderma isolates on A. thaliana, in vitro assays were initially conducted focusing on the production of diffusible and volatile compounds associated with plant growth promotion. Concerning diffusible compounds, the results obtained in the treatments demonstrated that primary root development in the plant was less than or equal to that of the control. In this assay, the interaction period between Trichoderma and A. thaliana was constrained due to the fungus’s rapid growth, with physical contact occurring in no more than 4 days. Two non-mutually exclusive mechanisms may explain the effects observed in the primary root of A. thaliana: firstly, Trichoderma isolates could be inhibiting root elongation by competing with the plant for space and nutrients; or second, they could be producing auxins that act as signaling molecules regulating microbial gene expression and triggering morphological change in the host (Rao et al. 2010). This observation has also been recorded by Maruri-López et al. (2024), in experiments with the yeast Hanseniaspora opuntiae. The authors reported that the yeast caused substantial changes in A. thaliana root development, resulting in shorter but more branched roots. The stereomicroscope images obtained in this study are consistent with the aforementioned findings, particularly in the volatile’s exposure assay, due to the absence of spatial limitations, nutrient requirements, or incubation duration constraints. In this case, the area occupied by the lateral roots of the exposed plants was significantly greater than that of the unexposed control, consistent with increased branching of the primary root. Notably, the compounds produced by CP-1 and HM-1 isolates consistently demonstrated the greatest stimulation in both assays. The development of lateral roots is of particular significance in this context, as they play a crucial role in nutrient and water uptake, as well as in communication with surrounding microorganisms (Carol and Dolan 2002). González-Pérez et al. (2018) reported analogous findings, having tested the growth-promoting effect of Trichoderma virens and Trichoderma atroviride on A. thaliana by direct contact (diffusible compounds) or in a split-plate assay (volatile compounds). Three days after inoculation, no differences in the primary root length of A. thaliana were observed in either assay, while the major difference in lateral root length was observed in the split-plate assay, where the number of lateral roots doubled. As in the present work, the authors reported that 5 days post-inoculation, primary root development was inhibited when plants interacted directly with Trichoderma strains; however, its increase in the split-plate assay with T. virens. Concurrently, the lateral roots of A. thaliana exhibited a substantial increase in both the direct contact and split-plate assays. Other authors also reported that volatile mixtures emitted by Trichoderma viride stimulated the growth of A. thaliana in the absence of physical contact with the plant. These observations revealed increases in plant size, fresh weight, chlorophyll content, lateral root development, and flower number (Hung et al. 2013).

Greenhouse trials and the induction of the transcriptional response of A. thaliana were performed with selected Trichoderma isolates. The selection aimed to combine well-studied species with promising under-explored strains, also taking into account the environmental and geographic diversity of the isolates and based on the parameters studied here. T. longibrachiatum (CP-1) and T. atrobrunneum (M5Ar-03) are widely recognized for their strong antagonistic activity against soilborne phytopathogens, as well as their ability to promote plant growth and enhance stress tolerance (Liu et al. 2023; Natsiopoulos et al. 2022). T. yunnanense (M4Ar-05), although less studied, has been reported to exhibit broad-spectrum antifungal activity and beneficial effects on plant growth-promoting (Karmakar et al. 2021). There is a lack of reports regarding the agricultural applications of T. scalesiae (L1-03) and T. breve (HM-1), highlighting the novelty of these isolates.

Each Trichoderma isolate elicited a distinct transcriptional response in A. thaliana, consistent with the previous studies highlighting the strain-dependent nature of Trichoderma–plant interactions (Sun et al. 2025; Guzmán-Guzmán et al. 2025). The observed variability underscores the need to carefully select isolates for specific biotechnological applications. The ability of each strain to modulate distinct signaling pathways suggests that the efficacy of Trichoderma as a biocontrol agent and plant growth promoter is highly context dependent (Hermosa et al. 2013). Consistent with Mathys et al. (2012), who observed early induction of PR1 but no activation of PDF1.2 in A. thaliana exposed to T. hamatum T382, our results showed a similar pattern, with transcriptional activation of PR1 in response to isolates L1-03 and M4Ar-05. However, our data also revealed isolate-dependent regulation of other defense, growth, and stress-related genes, highlighting the functional diversity among native Trichoderma strains. These findings suggest that Trichoderma could activate both systemic acquired resistance (SAR) and induced systemic resistance (ISR) pathways, depending on the isolate and metabolite profile.

These findings support the hypothesis that the biocontrol activity of Trichoderma spp. arises from a multifactorial mode of action, with individual strains prioritizing different mechanisms (Asad 2022; Monfil and Casas-Flores 2014). Moreover, the use of isolates from ecologically diverse environments emphasizes the importance of environmental adaptation and genetic diversity in identifying strains with optimal agricultural performance.

The observation that certain isolates induced the expression of pathogenesis-related genes even in the absence of pathogen challenge suggests that Trichoderma may precondition plants for enhanced resistance through a “priming” effect. From a biocontrol perspective, future research should evaluate whether this transcriptional activation translates into improved protection under pathogen pressure, providing deeper insight into the ecological and agronomic significance of these responses.

Conclusion

A total of 27 native Trichoderma strains were isolated from agricultural soils in the province of Tucumán, Argentina, representing probably ten distinct species. Morphological and molecular identification confirmed significant genetic and phenotypic diversity among the isolates. All strains exhibited in vitro antagonistic activity against economically important phytopathogens, such as Penicillium digitatum, Fusarium oxysporum, Botrytis cinerea, Phytophthora capsici, and Alternaria alternata, with inhibition rates reaching up to 98%. This showed their strong biocontrol potential mediated by mechanisms, such as antibiosis, nutrient competition, or mycoparasitism. Although not all strains promoted primary root elongation in Arabidopsis thaliana under in vitro conditions, an increase in the density and length of lateral roots was observed, underscoring the complexity and specificity of plant–microbe interactions. Under greenhouse conditions, four of the five selected strains (CP-1, HM-1, M4Ar-05, and M5Ar-03) significantly enhanced biomass accumulation and leaf area of A. thaliana, confirming their phytostimulatory effect in a setting closer to field conditions. All evaluated strains modulated the expression of genes related to plant development and defense; however, no common expression pattern was identified, suggesting that transcriptional responses are highly strain-dependent and likely influenced by their individual metabolic profiles. These findings emphasize the importance of rational strain selection based on functional performance and agroecological adaptability, aligned with specific biotechnological objectives. In this context, any of the four selected strains that demonstrated consistent improvements in plant biomass and foliar development represent promising candidates for implementation as biofertilizers or plant biostimulants in sustainable agriculture.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

M.M.P., Y.J.R.C., M.S., and J.R.D. conceived and designed the research. M.M.P., Y.J.R.C., F.I.C., and I.G. conducted experiments. M.M.P. and D.D.M. analyzed data. M.M.P., Y.J.R.C., D.D.M., F.I.C., I.G., M.S., J.R.D., and R.D. wrote and revised the manuscript. All authors read and approved the manuscript.

Funding

Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, N203023, Mario Serrano, Alexander von Humboldt Foundation.

Data availability

None.

Declarations

Conflict of interest

The authors declare that the research was conducted without any commercial or financial relationships that could be perceived as a potential conflict of interest.