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Article

Combined Soil Inoculation with Mycorrhizae and Trichoderma Alleviates Nematode-Induced Decline in Mycorrhizal Diversity

by
Fernanda Covacevich
1,*,†,
Gabriela Fernandez-Gnecco
1,*,†,
Verónica F. Consolo
1,
Pablo L. Burges
2,
Gonzalo F. Calo
1 and
Eduardo A. Mondino
3
1
Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET), Fundación para las Investigaciones Biológicas Aplicadas (FIBA), Mar del Plata 7602, Argentina
2
Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Balcarce 7620, Argentina
3
Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (IPADS; CONICET-INTA), Balcarce 7620, Argentina
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2025, 17(5), 334; https://doi.org/10.3390/d17050334
Submission received: 30 March 2025 / Revised: 28 April 2025 / Accepted: 1 May 2025 / Published: 5 May 2025
(This article belongs to the Special Issue Ecology and Diversity of Soil Microbial Communities in an Era of Global Change)
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Abstract

:
Arbuscular mycorrhizal fungi (AMF) and Trichoderma spp. (T) are known as plant-beneficial fungi effective against root-knot nematodes, but their interactions in the rhizosphere are not well understood. This study examined how Meloidogyne javanica influences AMF colonization and community diversity at the root-soil interface of tomato plants. A 60-day growth chamber experiment was conducted with tomato plants grown in non-sterile agricultural soil, either infected or not with M. javanica, that received a single inoculation with AMF or Trichoderma (strains T363 or TJ15), combined AMF + T inoculations, or no inoculation (Control). Both single and combined inoculations significantly reduced root galls, eggs, and soil nematode larvae. An AMF community analysis via single-strand conformation polymorphism of the D1 region of 28S rDNA gene (Glomeraceae family) revealed that M. javanica decreased AMF diversity and altered community structure, in plants single-inoculated with AMF. However, a combined inoculation with Trichoderma appears to prevent this reduction and maintain AMF diversity. While M. javanica reduced root mycorrhizal colonization, it did not affect Trichoderma abundance. These results suggest that Trichoderma may be more resilient to nematode infection, helping stabilize AMF communities and enhance biocontrol. Thus, combining AMF and Trichoderma inoculations could better preserve root health and improve biological control effectiveness against M. javanica.

Graphical Abstract

1. Introduction

Agricultural practices, including fertilization, pesticide application, and crop management can significantly impact the diversity and abundance of soil beneficial fungi, particularly arbuscular mycorrhizal fungi (AMF), which play a crucial role in nutrient uptake, host plant health, and soil structure [1]. Fertilization, especially with high phosphorus inputs, can reduce AMF diversity and abundance by altering soil nutrient balances and decreasing plant reliance on symbiotic relationships [2]. Pesticides, particularly broad-spectrum types used to control weeds and minimize pest damage, can also harm fungal communities and disrupt the soil microbiome, interfering with beneficial interactions between AMF and plants [1]. Additionally, root pathogens—especially fungi and plant-parasitic nematodes—can further alter the structure and function of beneficial root-associated fungal communities. Studies suggest that certain soil-borne pathogens may reduce AMF diversity, limiting their biocontrol capacity. For example, nematodes can damage plant roots, weakening AMF colonization and impairing their role in promoting plant health and nutrient uptake [3]. Overall, the interactions between agricultural practices, root pathogens, and mycorrhizal fungi are complex, and further research is needed to develop sustainable management practices.
Root-knot nematodes (RKNs), particularly those from the Meloidogyne genus, are destructive pathogens that cause root galls, reduce nutrient and water absorption, and increase susceptibility to other root pathogens [4,5]. In tomato (Solanum lycopersicum), a globally important crop, RKN infections lead to significant yield losses, especially in greenhouse production areas such as Buenos Aires (Pampas region), Argentina, where M. incognita, M. javanica, and M. arenaria are prevalent [6,7]. Traditional control methods, such as nematicides and soil fumigation, can damage soil health and beneficial organisms, leading to increased interest in biological control as a more sustainable alternative [8,9].
AMF and Trichoderma are recognized as plant growth-promoting microorganisms (PGPMs) that enhance soil health and plant resilience. AMF form symbiotic relationships with plant roots, improving nutrient uptake and soil structure, while Trichoderma, a cosmopolitan fungus associated with plant roots, is known for protecting plants from pests and improving plant health and nutrition [10,11]. Together, these microorganisms contribute to maintaining soil biodiversity and promoting crop productivity. Regarding biocontrol, mycorrhizal establishment generally enhances the suppression of endoparasitic nematodes, improves plant host tolerance, and increases overall disease resistance [10]. However, AMF have been reported to be ineffective against ectoparasitic nematodes (e.g., Criconematidae family), with nematode populations increasing in the presence of the fungus [12]. Although these nematodes are plant parasites, it has been suggested that AMF may extend the root surface area, providing more feeding sites and consequently supporting higher nematode densities. Competition for root space between AMF and RKNs may limit nematode development, as both organisms inhabit plant roots, with mycorrhizal arbuscules forming in the root cortex where nematodes feed [11]. Mondino et al. [13] found that AMF isolated from Argentine Pampas soils reduced M. incognita infection in tomato roots but did not improve plant development. In the case of Trichoderma, nematode control is achieved through egg parasitism and the production of enzymes such as chitinase and proteases, which degrade nematode egg walls and bodies [14].
Effective biological control requires a thorough understanding of the ecological relationships involved and how they can be leveraged for sustainable crop protection [11]. Combined inoculation with AMF and Trichoderma spp. has been shown to enhance plant growth and help control root pathogens [10,15], although effectiveness depends on factors such as strain type and plant nutritional status. For instance, Fernández-Gnecco et al. [16] found no significant improvement in maize performance with combined inoculation of AMF and Trichoderma strains from Germany. However, to our knowledge, interactions between these two microorganisms in the biocontrol of RKNs remain largely unknown. Given the complex interactions between biological controllers, RKNs, and host plants, further research is needed to determine whether these interactions are synergistic, neutral, or antagonistic. While much research has focused on pathogen biocontrol, fewer studies have explored how PGPMs such as AMF and Trichoderma are affected by RKNs in tomato roots.
This study investigates the potential of co-inoculation with AMF and Trichoderma to mitigate tomato disease induced by M. javanica, as well as the possible changes in AMF abundance and diversity associated with nematode-infected tomato roots. Understanding these relationships could contribute to developing sustainable strategies for nematode control in agriculture.

2. Materials and Methods

2.1. Inocula Preparation

The AMF inoculum consisted of colonized root segments, extraradical hyphae, spores, and native microbiota multiplied from soil collected from five agricultural sites in the southeastern Argentine Pampas. Composite samples were collected from each site, close to the roots (1.5 cm from the main stem), at a depth of 0–20 cm. Initial identification of AMF taxa in the inoculum was based on spore morphology and characterization according to the International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi (INVAM) database [17]. The identified AMF genera included Glomus, Funneliformis, Rhizophagus, Acaulospora, Scutellospora, and Gigaspora. The inoculum was propagated on ryegrass (Lolium multiflorum) for 120 days, achieving ≥60% total arbuscular mycorrhizal colonization (AMC) and ≥40% arbuscule content on roots, as quantified according to the method described by Brundrett [18]. For this, the presence of arbuscules, vesicles, and external mycelium associated with the roots were considered indicators of AMC, prior to root processing and staining using the trypan blue technique [17]. Additionally, 80 spores per 100 g of soil were quantified, as detailed by Mondino et al. [13].
Two Trichoderma harzianum strains (T363 and TJ15), isolated from soils in southeastern Buenos Aires Province, Argentina, and previously characterized by Bader et al. [19], were used in this study. For inoculum multiplication, both strains were separately re-cultured on Potato Dextrose Agar at 24 °C for 7 days prior to use.
Meloidogyne javanica was isolated from roots of Smallanthus sonchifolius (Peruvian ground apple) in Tucumán Province, Argentina, and maintained on tomato (Lycopersicon esculentum Mill cv. Platense). Egg masses were collected from galls on infected tomato roots, treated with 0.5% NaOCl for 4 min, sieved (100- and 500-mesh), washed, and incubated in sterile water at 28 °C for 3 days to obtain second-stage juveniles (J2) [20,21].

2.2. In Vitro and in Plant Experiments

Prior to the in-plant experiment, an in vitro confrontation test was conducted to assess the ability of Trichoderma strains to control M. javanica, using 24-well culture plates [22]. The experimental in vitro test treatments included the following: Control (M. javanica alone), M. javanica + T363 (1 × 103 conidia/mL), M. javanica + TJ15 (1 × 103 conidia/mL), M. javanica + T363 (1 × 104 conidia/mL), and M. javanica + TJ15 (1 × 104 conidia/mL). In each well, 450 µL of a suspension containing 90 ± 3 M. javanica eggs was added, followed by 500 µL of the respective Trichoderma suspension or sterile water for the control. The plates were incubated at 24 °C in the dark for 10 days, and the number of unhatched eggs, as well as the presence of J2 larvae, were recorded daily using a light microscope. The percentage of egg hatching was then calculated. The bioassay was performed twice, with five replicates per treatment.
For the in-plant experiment, Trichoderma conidia were coated (separately for the T363 and TJ15 strains) onto tomato seeds following the method of Cordo et al. [23]. Briefly, seeds were surface sterilized by immersion in 10% NaClO for 1 min, followed by 10% alcohol for 1 min, and then rinsed with sterile distilled water. The seeds were then shaken for 30 min in 90 mL of 0.25% agar, 10 mL of Trichoderma aqueous suspension (1 × 108 conidia/mL), and a drop of Tween 20® surfactant. After air-drying overnight, the seeds were germinated on wet paper at 28 °C in the dark for three days. Following this, the previously inoculated tomato seeds were pre-germinated for 96 h at 26 °C with a 12 h light/12 h dark cycle. Afterward, two germinated plantlets were transferred to speedling trays (55 mL per well) containing non-sterile agricultural soil from the southeastern Argentine Pampas (37°45′ S, 58°18′ W; pH 5.63; organic matter 5.51%). Six treatments, consisting of single and combined inoculations, were established in a randomized complete block design with ten replications: Control (non-inoculated), AMF (single inoculation with AMF), T363 (single inoculation with Trichoderma T363), TJ15 (single inoculation with Trichoderma TJ15), AMF + T363 (combined inoculation of AMF and T363), and AMF + TJ15 (combined inoculation of AMF and TJ15). For the treatments containing AMF inoculation, 5 g of AMF inocula was mixed with the soil. Control treatments received 5 mL of AMF filtrate (22 μM). Plants were maintained in a growth chamber at 26 °C with a 12 h light/12 h dark photoperiod for 4 weeks. After this period, AMC and arbuscule content on roots was estimated as described [17,18] at 32 days post-inoculation, with two replicates per treatment.
Following the confirmation of mycorrhizal colonization, eight replicates from each treatment were transplanted into 500 mL pots filled with a soil–substrate mixture consisting of non-sterile soil, sterile sand, and perlite (2:1:1 v/v), simulating standard tomato transplanting practices. Three days after transplanting, an aqueous suspension of M. javanica eggs and juveniles (2000 ± 100 individuals per pot) was inoculated into four replicates per treatment, while the remaining four replicates were not inoculated with M. javanica (Control plants, which only received water), that resulted in twelve treatments. The nematodes were placed into two 1 cm deep holes around the tomato roots, which were then covered with the soil–substrate. Plants were grown under controlled conditions at 26 °C with a 12 h light/12 h dark photoperiod.

2.3. Root and Soil Sampling

At 63 days post-inoculation, soil, root, and aerial plant material were harvested. Roots from each treatment and replication were split into five sub-samples of known weight, and the following analyses were performed: (i) about 3 g of root material was processed to evaluate AMC and arbuscule content as described previously; (ii) 150 mg of roots were stored at −80 °C for molecular fingerprinting analysis; (iii) about 500 mg was used to quantify Trichoderma abundance in roots; (iv) about 5 g was used to quantify galls and egg masses of M. javanica, as described in Hussey and Barker [20]; (v) the remaining root material was used to determine fresh and dry weights by gravimetry.

2.4. Molecular Fingerprinting of AMF Diversity from Root DNA

Since the majority of representatives (>65%) in the inocula used in this study were identified as belonging to the Glomeraceae family, the genetic diversity within this AMF family was further assessed using PCR–Single-Strand Conformation Polymorphism (SSCP). Genomic DNA was extracted from tomato roots using the PowerSoil® DNA Extraction Kit (MoBio, Carlsbad, CA, USA), following the manufacturer’s instructions. A composite sample of root tissues (approximately 600 mg, consisting of a mixture of 150 mg of roots from each treatment replicate) was frozen in liquid nitrogen and macerated with the kit’s first extraction buffer using a drill. The remaining procedure followed the manufacturer’s guidelines. The extracted DNA was stored at −20 °C. DNA quality and yield were assessed by electrophoresis on a 1% agarose gel using 1× TBE buffer and stained with Gel Red®.
A nested PCR was used to amplify a fragment of the large subunit of the 28S rDNA gene of AMF. The first PCR reaction was performed using the fungal-specific primer pair LSU0061/LSU0599 (5′-AGCATATCAATAAGCGGAGGA-3′/5′-TGGTCCGTGTTTCAAGACG-3′; [24]), followed by a nested PCR with the LSUrk4f/LSUrk7r primers (5′-GGGAGGTAAATTTCTCCTAAGGC-3′/5′-ATCGAAGCTACATTCCTCC-3′; [24,25]). PCR protocols followed those described by Covacevich et al. [26]. All PCR products were verified by electrophoresis on a 1% agarose gel as previously described.
The PCR amplicons generated by the LSUrk4f and LSUrk7r primers were analyzed by SSCP. Briefly, the amplicons were denatured and electrophoresed on an 8% acrylamide gel. The SSCP gels were then silver-stained [27], scanned, and analyzed. The protocols followed those used in previous studies [25,26].
Defined individual bands in the SSCP gel were excised, re-amplified, and purified for subsequent sequencing as described by Covacevich et al. [26]. All sequences were deposited in the NCBI GenBank (EMBL Nucleotide Sequence Database) under accession numbers OQ980485.1 to OQ980496.1.

2.5. Trichoderma Abundance

Per treatment and replicate, ten root segments, each 1 cm in length, were immersed in a 0.5% sodium hypochlorite solution for 1 min, followed by a 5 min rinse with sterile water through shaking. Segments were then placed in Petri dishes with Trichoderma Selective Medium [28] and incubated in the dark at 24 °C for 6 days. After this period, the presence of Trichoderma growth associated with the roots was confirmed [29]. Additionally, Trichoderma colony-forming units (CFUs) in the soil–substrate were quantified using the serial decimal dilution method on PDA medium plates [30].

2.6. Root Infestation Levels by M. javanica

Roots were submerged in an erioglaucine solution (0.0075 g/L) for 10–15 min, and the number of galls and egg masses was counted using a lighted stereomicroscope (30 x), following the method described by Atamian et al. [31]. M. javanica juveniles were extracted from the soil–substrate of pots using the sucrose flotation–centrifugation method [32] and counted under a stereomicroscope (Zeiss Stemi SV6, Carl Zeiss, Jena, Germany).

2.7. Plant Growth Parameters

After determining shoot and root fresh weights (SFW and RFW, respectively), plant material was oven-dried at 60 °C for 3 days, and the dry weights (SDW and RDW, respectively) were then determined. The inoculation response (IR) was calculated for each treatment according to Cavagnaro et al. [33], using the equation IR = [SDW (i) − X ¯ SDW (Control)]/ X ¯ SDW (Control), where SDW represents the individual SDW of inoculated (i) plants and X ¯ SDW is the mean SDW of Control (non-inoculated) plants. The IR was similarly calculated for RDW.

2.8. Data Analysis

The SSCP banding pattern, including band presence/absence and intensity, was analyzed using Phoretix 1D Pro software (version 1.3, Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK). The resulting matrix was used to calculate the Shannon–Weaver diversity index (H) for AMF (HSSCP), following the method of Zulfarina et al. [34]. Treatment diversity and banding patterns were visualized using a scatter plot generated with the ggplot2 package [35]. Based on the same data, a hierarchical clustering analysis was conducted using the complete linkage method and Euclidean distance to evaluate the similarity among treatments [36]. The resulting dendrogram was visualized with ggplot2 and ggdendro [35,37].
In the experimental design, inoculation was considered a fixed factor, and a statistical analysis was conducted using a one-factor model. Treatment effects were compared using an analysis of variance (ANOVA), and when the inoculation factor was found to be significant (p < 0.05), mean comparisons were performed using Tukey’s Honestly Significant Difference (HSD) post hoc test. In the plant growth experiment, one of the four replicates for each control treatment was excluded from the analysis due to a lack of plant growth.
To explore potential ecological interactions between AMF, Trichoderma, and nematodes, a linear regression model was used to analyze relationships. Furthermore, to further investigate how treatments affected AMF colonization and community structure—both derived from root-associated AMF—principal component analysis (PCA) was conducted. This analysis examined relationships among arbuscule content, Trichoderma CFUs, nematode infestation levels (including galls, eggs, and larval counts), and plant biomass (root and shoot dry weights) in relation to AMC (%) distribution. All data were centered and scaled to ensure comparability. Additionally, pairwise Pearson correlations were calculated to interpret variable associations, and the variables were projected as loading vectors in PCA biplots to visualize their contributions. PCA was carried out using the prcomp function, and visualizations were created with the ggplot2 package.
All analyses were performed in R version 4.4.3 (https://www.r-project.org/, accessed on 1 March 2025) using the specified R packages.

3. Results

3.1. Strain-Specific Effects of AMF and Trichoderma on Tomato Growth Under Nematode Infection

Tomato plant growth responses to fungal inoculation varied significantly depending on nematode infection. In the absence of M. javanica (−Nematodes), inoculation with AMF and/or Trichoderma led to significant increases in the inoculation response (IR) for both shoot dry weight (SDW) and root dry weight (RDW) compared to the Control (p < 0.001; Figure 1). The highest IR for both SDW and RDW was observed in plants inoculated with the Trichoderma strain T363, compared to the Control. Conversely, the lowest IR for SDW was recorded in plants receiving combined inoculation of AMF + T (either TJ15 or T363), relative to the Control. Additionally, treatments with single AMF inoculation exhibited the lowest IR-RDW compared to Control.
Under M. javanica infection (+Nematodes), IR-SDW and IR-RDW were also significantly influenced by the inoculation treatment (both p < 0.001). Single inoculation with the Trichoderma TJ15 strain, followed by plants with combined inoculation of AMF + T (both T strains), showed significantly higher IR-SDW compared to Control. These findings were consistent with visual observations of enhanced shoot growth (Figure S1). However, IR-RDW was significantly lower in treatments involving single-inoculation with Trichoderma (both strains), relative to the Control.

3.2. Trichoderma Exhibits Antagonism Toward Nematodes Independently of Their Abundance

The in vitro confrontation test revealed a strong antagonistic effect of Trichoderma on M. javanica egg hatching and larval development (p = 0.01). Wells inoculated with the T363 strain at both tested concentrations (1 × 103 and 1 × 104 conidia/mL) exhibited the highest number of unhatched eggs and the lowest number of second-stage juveniles (J2) (Table S1). Similarly, the TJ15 strain showed inhibitory effects, though these were significant only at the higher concentration (1 × 104 conidia/mL). The lowest egg hatch rates (43–45%) were observed in T363-treated wells. Additionally, Trichoderma hyphae were seen surrounding nematode eggs, indicating direct fungal antagonism (Figure S2).
In the in planta experiment, all plants under nematode infection developed root galls (Figure S3), but the severity was significantly reduced in treatments involving single and combined AMF and/or Trichoderma inoculation (p < 0.001). Single-AMF inoculation resulted in a lower number of galls compared to the Control (Table 1). Furthermore, both single and combined inoculation with AMF and Trichoderma (both strains) led to the lowest number of egg masses at the roots and larvae in the soil–substrate across all treatments. Single inoculation with TJ15 reduced the number of galls and egg masses at the roots, as well as the number of J2 larvae in the soil–substrate, compared to the Control, though the effect was less pronounced than that of single or combined AMF inoculation. Although inoculation with T363 resulted in similar reductions in the number of egg masses and larvae compared to TJ15, no reduction in the number of root galls was observed compared to the Control. Nematode egg invasion by hyphae was detected, though it was not possible to determine whether the mycelia originated from AMF or Trichoderma (Figure S4). Furthermore, the regression analysis showed a negative relationship between Trichoderma abundance and nematode larvae in the soil-substrate where tomato plants were grown, as presented in the Figure S5 (p = 0.07, R2 = 0.14). However, a small portion of the variability in nematode larvae counts could be explained by the abundance of Trichoderma.

3.3. Trichoderma Mitigates Nematode-Induced Declines in AMF Diversity and Colonization

Single-AMF and combined AMF + T inoculation resulted in highest root colonization levels (≥50%) compared to Control and single inoculation with Trichoderma (≤3%), regardless of nematode infection (Figure 2). Under nematode infection (+Nematodes), significant reductions in AMC levels across all AMF inoculated treatments compared to those without nematode infection (−Nematodes; p < 0.01) were found. Regression analysis revealed a significant negative relationship between AMC and both nematode galls and larvae (both p < 0.001; R2 = 0.61 and 0.5, respectively; Figure 3).
The SSCP fingerprint analysis revealed distinct banding patterns, which reflect AMF diversity and community structure, depending on the presence or absence of nematode infection (−Nem vs. +Nem, respectively; Figure 4). Control treatments exhibited similar AMF community structures, regardless of nematode presence. Likewise, treatments with combined AMF + T inoculations displayed consistent banding patterns, clustering similarly in the dendrogram, irrespective of the Trichoderma strain used (Figure 4A). In the absence of nematode infection (−Nem), single-AMF inoculation resulted in a distinct banding pattern compared to other treatments. This treatment also exhibited the highest diversity index among all treatments (Figure 4B), and it was clearly separated in the similarity dendrogram (Figure 4A). Under nematode infection (+Nem), single-AMF inoculation showed a different banding pattern, which correlated with a reduced AMF diversity (Figure 4A). Control treatments and combined inoculation with AMF + T (both T strains) displayed a similar banding pattern, as reflected in their clustering within the dendrogram (Figure 4A). A sequence analysis of re-amplified bands from polyacrylamide gels revealed a high similarity to Glomus and Rhizophagus species, both belonging to the Glomeraceae family (Table S2).
To further investigate the effects of treatments on AMC and community diversity/structure—both associated with root-colonizing AMF—PCA was performed to explore the multivariate relationships between plant growth, fungal variables, and nematode infection levels in the absence (−Nematodes) and presence (+Nematodes) of M. javanica. The PCA revealed that nematode infection influenced AMC (Figure S6), resulting in two distinct clusters. In the absence of nematode infection, the first two principal components explained 90% of the total variance, with PC1 accounting for 51% and PC2 for 39% (Figure 5A). In contrast, under nematode infection, the AMC pattern shifted, with the first two components explaining a smaller proportion of the variance (PC1 = 48%, PC2 = 20%, totaling 68%; Figure 5B). Among the predictors, RDW (p = 0.03), arbuscule content (p < 0.001), and Trichoderma abundance (p = 0.004) were positively and significantly correlated with AMC in the absence of nematodes, while galls (p = 0.02), eggs (p = 0.04), and larvae numbers (p = 0.002) were negatively and significantly correlated with AMC in the presence of nematodes.

4. Discussion

In this study, we investigated the potential of combined inoculation with AMF and Trichoderma to mitigate tomato disease induced by Meloidogyne javanica, as well as the impact of the phytoparasitic nematode on AMF diversity and root colonization in the host plant. Additionally, we explored how different Trichoderma strains could help alleviate these effects.

4.1. Interactions Between Plant Beneficial Fungi and Nematodes in Soil Ecosystems

In natural and agricultural soil ecosystems, fungal species often coexist with nematodes, forming dynamic interactions that can influence plant health. Nematodes, particularly RKN, and fungi, including both symbiotic and associative species, interact with plant roots through direct contact or chemical signaling. These interactions have significant ecological and economic implications. They may lead to antagonistic effects, especially when AMF and Trichoderma act as biological controllers of RKNs. Such interactions may occur through direct competition for resources such as nutrients and space, or indirectly by enhancing plant defense mechanisms like induced systemic resistance and alterations in root exudation patterns. While much of the focus has been on the biocontrol effects of beneficial fungi against RKN, a deeper understanding of these interactions could lead to the development of ecologically sustainable pest management strategies, enhancing plant resistance to soil-borne pathogens, including nematodes like Meloidogyne spp.
In the current study, we observed that AMC in tomato roots was reduced following nematode infection, likely due to competition for root space and nutrients between AMF and nematodes [38,39]. As expected, AMC was highly responsive to fungal inoculation treatments. Despite the reduction, AMC levels remained within the 30–50% range, consistent with previous findings [39]. It is plausible that nematodes not only directly affect the root cells that contribute to AMF nutrition, but also trigger plant defense responses, which in turn alter root exudation patterns and subsequently affect AMF colonization dynamics. Moreover, the disparity in infection rates between AMF and nematodes may play a role, as nematodes typically establish themselves more rapidly in the root system. A principal component analysis (Figure 5) further illustrated that, under nematode-infection (+Nematodes), AMC was primarily influenced by infection-related factors, while in the absence of nematodes (−Nematodes), host plant growth parameters were the main drivers. These findings suggest important implications for future analyses and the development of effective biocontrol management strategies.

4.2. AMF Diversity: Impact of M. javanica and the Role of Trichoderma

The SSCP analysis, which separates single-stranded DNA based on differences in mobility resulting from their folded secondary structures (heteroduplexes) [40], provided valuable insights into the genetic diversity of AMF community, particularly those in the Glomeraceae family, which is the predominant taxon in agricultural root systems [41]. While acknowledging some limitations of SSCP, such as the potential for the same DNA sequence to produce multiple bands on the gel, this technique has been successfully used in microbial community profiling and phylogenetic studies [42]. Furthermore, it has also been useful in assessing shifts in AMF diversity in response to land-use changes and agricultural practices [26,43].
In our study, the SSCP patterns revealed that Meloidogyne javanica infection altered AMF community composition, reducing diversity in tomato roots, which was consistent with the observed decrease in mycorrhizal colonization. Interestingly, when AMF was co-inoculated with Trichoderma, this negative effect on AMF diversity was mitigated (Figure 4; original image is presented in Figure S7). Although the mechanisms underlying this phenomenon remain unclear and warrant further investigation, we suggest that AMF-Trichoderma combined inoculation could serve as an effective biocontrol strategy against M. javanica while preserving AMF community diversity.
However, given the limitations of the SSCP technique, future research should confirm these findings using more advanced molecular methods. We also do not yet fully understand the mechanisms or factors involved in the reduction in AMF diversity by M. javanica and its mitigation by Trichoderma. These findings open a new avenue for research into biocontrol strategies that can protect plants from nematodes while maintaining beneficial fungal diversity. Moreover, although our study did not examine changes in Trichoderma diversity due to the presence of RKN, the abundance of Trichoderma was not affected by M. javanica. Future studies should explore the potential effects of nematodes on the diversity of Trichoderma strains used in biological control.

4.3. Interaction of AMF and Trichoderma for Plant Growth-Promoting and Nematode Biocontrol

In this study, a consortium containing at least six AMF species along with their associated microflora was inoculated, which likely helped alleviate any potential incompatibility between AMF species and Trichoderma strains. The results suggest a synergistic relationship between AMF and Trichoderma, which, in addition to their biocontrol activity against nematodes, may also contribute to improved plant mineral nutrition—an area that warrants further exploration. The higher growth responses observed in both aerial and root biomass due to single and combined inoculation with AMF and Trichoderma highlight their potential as PGPMs. Although reductions in plant growth under nematode-infection were expected, the PGPM effect of the tested fungi was still evident even in the presence of M. javanica infection. Several Trichoderma strains, including T363 [19], have been identified as phosphorus solubilizers, which could enhance phosphorus uptake in mycorrhizal plants. Although the impact on mineral nutrition was not specifically analyzed in this study, the observed increase in plant growth following inoculation with both AMF and Trichoderma may be reflected in improved nutrient uptake.
Biocontrol by AMF and Trichoderma was evidenced by reductions in galls, eggs, and J2 larvae in both the tomato roots and in the soil-substrate in which the tomato plants were grown, respectively. The biocontrol effect of Trichoderma was first confirmed through an in vitro experiment, where it significantly reduced M. javanica egg hatching and J2 larval mortality, consistent with previous reports on Trichoderma’s ability to parasitize nematode eggs [44,45]. This biocontrol activity is likely due to Trichoderma’s mycoparasitic interactions and the secretion of secondary metabolites, as demonstrated by the fungal colonization of nematode eggs and the formation of an extensive mycelial network [45,46]. However, while the in vitro results showed strong biocontrol by Trichoderma, the in planta experiment revealed a stronger biocontrol effect from AMF inoculation compared to Trichoderma. This aligns with the weak and non-significant correlation between Trichoderma abundance and M. javanica larvae in the soil–substrate in which the tomato plants were grown (Figure S5). Additionally, fungal hyphae (which could not be specifically identified as AMF or Trichoderma) were observed surrounding nematode eggs in tomato roots inoculated with both fungi (Figure S4). Future studies should aim to identify the mycelium in this context.
The biocontrol effect of AMF observed in this study is consistent with previous research by Mondino et al. [13], who reported reduced root infection by M. incognita and a decrease in J2 larval abundance in tomato roots pre-inoculated with AMF. In contrast, although the biocontrol effect of Trichoderma was less pronounced than that of AMF, this study demonstrates its ability to control M. javanica, further supporting the findings of Bader et al. [19] on the multiple beneficial roles of Trichoderma strains (e.g., biocontrol of pathogenic fungi, phosphorus solubilizing). Nevertheless, the two Trichoderma strains tested did not show identical results, either in terms of growth promotion or biocontrol, suggesting that their effectiveness may vary depending on the strain, inoculation combination, and presence of nematodes.
We hypothesize that the stronger biocontrol effect of AMF compared to Trichoderma may be due to differences in their colonization patterns. While Trichoderma predominantly colonizes the root surface, typically limited to the first or second cell layers [47], AMF colonizes root cortical cells and competes directly with nematodes for space and nutrients [38,39], suggesting distinct mechanisms of action between the two organisms. Our study further confirms previous findings [11] that pre-inoculation with AMF can serve as an effective preventive measure for managing root-knot nematodes.

5. Conclusions

Based on the findings of this study, it can be concluded that the combined inoculation of AMF and Trichoderma spp. presents a promising agricultural strategy for mitigating the negative effects of root-knot nematodes on host plants. This combined inoculation not only reduced nematode populations but also seemed to preserve the diversity and structure of AMF communities within the roots. These results suggest that synergistic interactions between AMF and Trichoderma could enhance root health, making this combined inoculation approach a valuable tool for integrated pest management and sustainable agricultural practices. Future research should focus on optimizing biological control strategies, incorporating sensitivity analyses to assess the influence of varying experimental conditions (such as soil type and plant variety), and evaluating the long-term effects of this approach on soil health and plant productivity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d17050334/s1, Figure S1. Growth of tomato plants, either infected (+Nem) or not infected (−Nem) with Meloidogyne javanica, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strains T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. Figure S2. Eggs of Meloidogyne javanica observed under a binocular microscope (63×) in contact with hyphae of Trichoderma strains TJ15 and T363 after 10 days of the in vitro confrontation test. A: TJ15 concentration 103. B: TJ15 concentration 10⁴. C: T363 concentration 103. Black arrow: Trichoderma hyphae. Red arrows: Eggs of M. javanica. Blue arrows: J2 larvae of M. javanica. Figure S3. Tomato root infected with Meloidogyne javanica observed under a stereo microscope (30×). Abbreviations: M: egg mass; A: nematode gall. Figure S4. Eggs of Meloidogyne javanica observed under a binocular microscope (750×), surrounded by fungal hyphae in the roots of tomato plants inoculated with arbuscular mycorrhizal fungi (AMF) and Trichoderma. A: Roots from the treatment with combined inoculation of AMF + T363. B: Roots from the treatment with combined inoculation of AMF + TJ15. Figure S5. Linear correlation analysis between Trichoderma abundance (T363 and TJ15 strains) in roots and J2 larvae of Meloidogyne javanica in the soil-substrate, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strains T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. Figure S6. Principal component (PC) analysis showing the effects of different inoculation treatments with arbuscular mycorrhizal fungi (AMF) and Trichoderma (T) on total arbuscular mycorrhizal colonization (AMC) in tomato plants, either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica. Treatments: Control = non-inoculated; AMF = single inoculation with AMF; T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. Asterisks (*) indicates a significant correlation between AMC and plant growth parameters, nematode infection levels, and/or fungal variables (Pearson correlation, p < 0.05). Figure S7: Original image of the SSCP gel, before being cropped (removal of the positive control, Glomus intraradices amplicon in the first lane on the left, and overall gel cropping) and enhanced (contrast and brightness). The final image result is presented in Figure 4. Table S1. Antagonistic in vitro confrontation test between Trichoderma (T363 and TJ15 strains) and Meloidogyne javanica. Number (n) of eggs and J2 larvae of M. javanica, and egg hatching per well. Treatments: Control: non-inoculated; T363 103 and T363 104: inoculated with Trichoderma strain T363 at concentrations of 103 or 104 conidia/mL, respectively; TJ15 103 and TJ15 104: inoculated with Trichoderma strain TJ15 at concentrations of 103 or 104 conidia/mL, respectively. Mean values are presented ± SE. Different letters indicate significant differences between inoculation treatments (ANOVA–Tukey test, p < 0.05). Table S2. Accession numbers of sequences deposited in the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, accessed on 3 February 2025) for amplicons belonging to the Glomeraceae family, associated with tomato roots, obtained by reamplification of bands electrophoresed using SSCP methodology. Homologies with sequences available in the NCBI and MarrjAM (https://maarjam.ut.ee/, accessed on 3 February 2025) databases are provided.

Author Contributions

Conceptualization of experiment, F.C., E.A.M. and V.F.C.; methodology, F.C., E.A.M., V.F.C., G.F.C., P.L.B. and G.F.-G.; formal analysis, G.F.-G.; investigation, F.C., E.A.M., V.F.C., G.F.C., P.L.B. and G.F.-G.; resources, F.C. and E.A.M.; data curation, F.C. and G.F.-G.; writing—original draft preparation, F.C., E.A.M. and V.F.C.; writing—review and editing, F.C., E.A.M., V.F.C., G.F.C. and G.F.-G.; visualization, F.C. and G.F.-G.; supervision, F.C. and E.A.M.; project administration F.C. and E.A.M.; funding acquisition, F.C. and E.A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Instituto Nacional de Tecnología Agropecuaria (INTA, Argentina) under Grant No. 023-PD-L03-I093, the Fondo para la Investigación Científica y Tecnológica Grant No. PICT-2020-SERIEA-03338, and the National Scientific and Technical Research Council (CONICET, Argentina) under Grant No. PIP 11220200101732CO01.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, and further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AMFArbuscular mycorrhizal fungi
T Trichoderma
RKNRoot-knot nematodes
PGPMPlant growth-promoting microorganisms
AMCArbuscular mycorrhizal colonization
NemNematode
SSCPSingle-strand conformation polymorphism
IRInoculation response
CFUColony-forming units

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Figure 1. Inoculation response of shoot and root dry weight (SDW and RDW, respectively) of tomato plants either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Lowercase letters indicate significant differences for each parameter among inoculation treatments. Capital letters indicate significant differences for each treatment between infection and non-infection with nematodes (ANOVA–Tukey test, p < 0.05).
Figure 1. Inoculation response of shoot and root dry weight (SDW and RDW, respectively) of tomato plants either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Lowercase letters indicate significant differences for each parameter among inoculation treatments. Capital letters indicate significant differences for each treatment between infection and non-infection with nematodes (ANOVA–Tukey test, p < 0.05).
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Figure 2. Root arbuscular mycorrhizal colonization (AMC) and Trichoderma colony-forming units (CFU) in the soil–substrate of tomato plants either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Lowercase letters indicate significant differences for each parameter among inoculation treatments. Capital letters indicate significant differences for each treatment between infection and non-infection with nematodes (ANOVA–Tukey test, p < 0.05).
Figure 2. Root arbuscular mycorrhizal colonization (AMC) and Trichoderma colony-forming units (CFU) in the soil–substrate of tomato plants either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Lowercase letters indicate significant differences for each parameter among inoculation treatments. Capital letters indicate significant differences for each treatment between infection and non-infection with nematodes (ANOVA–Tukey test, p < 0.05).
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Figure 3. Linear regression analysis between arbuscular mycorrhizal colonization (AMC) and (A) Meloidogyne javanica galls in tomato roots, and (B) the M. javanica J2 larvae abundance in the soil–substrate, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strains T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF.
Figure 3. Linear regression analysis between arbuscular mycorrhizal colonization (AMC) and (A) Meloidogyne javanica galls in tomato roots, and (B) the M. javanica J2 larvae abundance in the soil–substrate, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strains T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF.
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Figure 4. Arbuscular mycorrhizal fungi (AMF) communities (Glomeraceae family) associated with tomato plants either infected (+Nem) or not infected (−Nem) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi; T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. (A) Similarity dendrogram based on the Shannon diversity index of SSCP profiles of AMF communities; (B) Shannon diversity index values of SSCP profiles of AMF communities; (C) SSCP profiles of PCR-amplified D1 variable domain of the 28S rDNA region of AMF communities (Glomeraceae family).
Figure 4. Arbuscular mycorrhizal fungi (AMF) communities (Glomeraceae family) associated with tomato plants either infected (+Nem) or not infected (−Nem) with Meloidogyne javanica under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi; T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. (A) Similarity dendrogram based on the Shannon diversity index of SSCP profiles of AMF communities; (B) Shannon diversity index values of SSCP profiles of AMF communities; (C) SSCP profiles of PCR-amplified D1 variable domain of the 28S rDNA region of AMF communities (Glomeraceae family).
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Figure 5. Principal component (PC) analysis showing the effects of different inoculation treatments with arbuscular mycorrhizal fungi (AMF) and Trichoderma (T) on total arbuscular mycorrhizal colonization (AMC) in tomato plants, either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica. Treatments: Control = non-inoculated; AMF = single inoculation with AMF; T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. Asterisks (*) indicates a significant correlation between AMC and plant growth parameters, nematode infection levels, and/or fungal variables (Pearson correlation, p < 0.05).
Figure 5. Principal component (PC) analysis showing the effects of different inoculation treatments with arbuscular mycorrhizal fungi (AMF) and Trichoderma (T) on total arbuscular mycorrhizal colonization (AMC) in tomato plants, either infected (+Nematodes) or not infected (−Nematodes) with Meloidogyne javanica. Treatments: Control = non-inoculated; AMF = single inoculation with AMF; T363 or TJ15 = single inoculation with Trichoderma strain T363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with Trichoderma strain and AMF. Asterisks (*) indicates a significant correlation between AMC and plant growth parameters, nematode infection levels, and/or fungal variables (Pearson correlation, p < 0.05).
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Table 1. Meloidogyne javanica infection levels in tomato plants: number of galls and egg masses at the roots, and J2 larvae in the soil–substrate, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain TJ363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Different letters indicate significant differences between inoculation treatments (ANOVA-Tukey test, p < 0.05).
Table 1. Meloidogyne javanica infection levels in tomato plants: number of galls and egg masses at the roots, and J2 larvae in the soil–substrate, under different inoculation treatments. Treatments: Control = non-inoculated; AMF = single inoculation with arbuscular mycorrhizal fungi (AMF); T363 or TJ15 = single inoculation with Trichoderma strain TJ363 or TJ15, respectively; T363 + AMF or TJ15 + AMF = combined inoculation with each Trichoderma strain and AMF. Mean values are presented ± SE. Different letters indicate significant differences between inoculation treatments (ANOVA-Tukey test, p < 0.05).
TreatmentGalls.g dry root−1Eggs masses.g dry root−1J2 larvae.g dry soil–substrate−1
Control126 ± 15 b147 ± 14 a7 ± 0.2 a
AMF62 ± 15 d69 ± 2 c3 ± 0.5 c
T363147 ± 23 a125 ± 2 b5 ± 0.7 b
AMF + T36373 ± 10 d75 ± 6 c3 ± 0.9 c
TJ15104 ± 10 c121 ± 7 b3 ± 0.7 c
AMF + TJ15102 ± 6 c95 ± 11 c5 ± 0.7 b
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MDPI and ACS Style

Covacevich, F.; Fernandez-Gnecco, G.; Consolo, V.F.; Burges, P.L.; Calo, G.F.; Mondino, E.A. Combined Soil Inoculation with Mycorrhizae and Trichoderma Alleviates Nematode-Induced Decline in Mycorrhizal Diversity. Diversity 2025, 17, 334. https://doi.org/10.3390/d17050334

AMA Style

Covacevich F, Fernandez-Gnecco G, Consolo VF, Burges PL, Calo GF, Mondino EA. Combined Soil Inoculation with Mycorrhizae and Trichoderma Alleviates Nematode-Induced Decline in Mycorrhizal Diversity. Diversity. 2025; 17(5):334. https://doi.org/10.3390/d17050334

Chicago/Turabian Style

Covacevich, Fernanda, Gabriela Fernandez-Gnecco, Verónica F. Consolo, Pablo L. Burges, Gonzalo F. Calo, and Eduardo A. Mondino. 2025. "Combined Soil Inoculation with Mycorrhizae and Trichoderma Alleviates Nematode-Induced Decline in Mycorrhizal Diversity" Diversity 17, no. 5: 334. https://doi.org/10.3390/d17050334

APA Style

Covacevich, F., Fernandez-Gnecco, G., Consolo, V. F., Burges, P. L., Calo, G. F., & Mondino, E. A. (2025). Combined Soil Inoculation with Mycorrhizae and Trichoderma Alleviates Nematode-Induced Decline in Mycorrhizal Diversity. Diversity, 17(5), 334. https://doi.org/10.3390/d17050334

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