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Amazonian Trichoderma spp. as Biocontrol Agents Against Agroathelia rolfsii and Plant Growth Promoters in Tomato

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08 September 2026

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09 September 2026

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Abstract
Sclerotium rot, caused by Agroathelia rolfsii, is one of the main soilborne diseases that limit plant growth and tomato yield. This study evaluated the efficacy of Amazonian Trichoderma species to control sclerotium rot, and promote plant growth under field conditions, in Manaus, Brazil. We used six species (isolates) of Trichoderma based on their previously assessed performance in field trials. The incidence of sclerotium rot, tomato fruit yield and the production of indoleacetic acid (IAA) and siderophores were evaluated. The isolates reduced the incidence of sclerotium rot by up to 75% and also increased fruit yield. T. rugulosum had the best performance, as it greatly reduced sclerotium rot incidence and improved fruit yield. Although no differences were found in biomass allocation to plant organs, T. endophyticum, T. agriamazonicum, and T. asperelloides showed high production of IAA. The results highlight the potential of Amazonian Trichoderma isolates as bio fungicides to be used in sustainable cultivation of tomato. Furthermore, we show the potential of Amazonian species of Trichoderma to both reduce the incidence of sclerotium rot, and to increase the production of bioactive compounds that stimulate plant growth. By reducing the use of synthetic agrochemicals, the use of Trichoderma promotes healthier and more sustainable agricultural practices.
Keywords: 
sustainable agriculture
;  
plant growth promotion
;  
microbial bioinputs
Subject: 
Biology and Life Sciences  -   Agricultural Science and Agronomy

1. Introduction

Brazil occupies the ninth position on worldwide production of tomatoes (Solanum lycopersicum), with an annual production of 4.1 million tons [1]. The production of the state of Amazonas in 2025 was 692 tons [2,3] with an average productivity of 14,1 t/ha.
The low tomato productivity in the state of Amazonas is due to environmental, genetic and management factors. The temperatures that occur in the region between 25 and 35 °C and the high air and soil moisture favor the incidence of several pests and diseases, resulting in low productivity [4,5].
Among the main pathogens affecting tomato crop is Agroathelia rolfsii (Sclerotium rolfsii) which causes sclerotium rot. The symptoms of the disease generally appear in the early stages of plant development, as a necrosis of the root collar and/or the base of the stem (Figure 1), which may evolve to wilting of the aerial part, and ultimately the disease may lead to plant death (Figure 2) [6].
In Amazonas, other important diseases of tomato crop are bacterial wilt (Ralstonia solanacearum), a serious vascular disease that leads to plant collapse; bacterial leaf spot (Xanthomonas spp.), which causes necrotic lesions with chlorotic halo on leaves, stems and fruits; the damping-off of seedlings, caused by Pythium spp., common in nurseries under high humidity; rhizoctonia (Rhizoctonia solani), which attacks the root system and the base of the stems, promoting brown lesions and rot; early blight (Phoma destructiva), which causes dark lesions on leaves and fruits, reducing the quality of production; hollow stalk, a pathophysiological disorder generally associated with nutritional deficiencies and environmental stresses, but which can also be favored by infections; brown eye spot (Cercospora spp.), characterized by grayish leaf spots with defined edges; and target spot (Corynespora cassiicola), which forms necrotic concentric lesions on leaves, affecting the photosynthetic capacity of the plant [5].
Besides its occurrence in tomato, in humid tropical regions, A. rolfsii also causes diseases in other species, such as bell pepper (Capsicum annuum L.), and cubiu (Solanum sessiliflorum Dunal.), among others. Losses caused by sclerotium rot in tomato can reach more than 50% in the Amazon [7]. The fungus directly infects plant tissues with the production of oxalic acid and pectinolytic enzymes that degrade the plant tissue, favoring plant colonization by the pathogen [8]. The pathogen A. rolfsii has a wide range of host plants that makes its management even more challenging due to its high capacity for survival and dissemination in different crops, making it difficult to adopt specific and effective control strategies [9].
Several fungicides can be used to control sclerotium rot in tomato plants, one of them is based on fluxapyroxad and pyraclostrobin. The use of fungicides, however, chemical fungicides can be ineffective to control sclerotium rot [11]. For instance, it has been reported that despite the treatment with fungicides, the viability of the sclerotia was not significantly reduced, evidencing the resistance of these structures to the evaluated fungicides [12].
Biological control using agents such as Trichoderma can be an ecological and economical strategy for the management of plant diseases [14]. This effect occurs through mechanisms such as competition for space and nutrients, mycoparasitism, and antibiosis [11]. Forms of control based on Trichoderma are efficient in reducing the incidence of plant damping-off, and also to reduce the severity of diseases caused by soil-dwelling pathogens, such as Fusarium, Phytophthora, Phytium, Rhizoctonia, Agroathelia and Sclerotininia [15].
The use of beneficial Trichoderma strains is an alternative to chemical fungicides for the control sclerotium rot [16]. In Brazil, this fungus has been widely used in the biological management of soil diseases, especially for the control of pathogens such as A. rolfsii, Rhizoctonia solani and Fusarium spp. [17]. In this country, some important use of Trichoderma include its application in crops such as soybean (Glycine max), corn (Zea mays), cowpea (Vigna unguiculata), tomato and vegetables, with a focus on promoting plant growth, inducing resistance and suppressing phytopathogens [18,19].
To be used against sclerotium rot, currently there are no public or commercial biofungicides registered at the Brazilian Ministry of Agriculture and Livestock, based on Trichoderma strains native to the Amazon. Most of the species of Trichoderma used in commercial biofungicide are based on T. harzianum and T. asperellum, obtained from other regions of Brazil or abroad [20]. The lack of Trichoderma strains native to the Amazon in commercial biofungicides makes this research relevant from a technological point of view, not to mention the importance of finding highly efficient native isolates of Trichoderma to control of A. rolfsii for sustainable horticulture in the Amazon region. It is important to note, however, that although several species of Trichodema can be useful as biocontrol agent of plant pathogens some species of Trichoderma can be pathogenic to cultivated plants.
The beneficial strains of Trichoderma affect phytopathogenic fungi by several mechanisms, such as mycoparasitism, competition for space and nutrients, antibiosis, and induction of resistance in plants, being promising alternatives for the sustainable management of sclerotium rot and other soil diseases [21,22]. In addition to suppressing phytopathogens, beneficial species of Trichoderma have the ability to stimulate plant growth, through direct and indirect mechanisms, which involve biochemical and hormonal interactions and improvements of soil and rhizosphere conditions, such as phytohormone production, increased nutrient availability (e.g., via siderophore production), whose final effect is the stimulation of plant growth [23,24].
To address the global challenges for food supply in the current scenario, that include, but not only, climate change and demographic growth, it is imperative to ensure environment conservation and promote sustainable agriculture, which among several other practices include the use of biological agents to control plant pathogens, and in this manner these practices contribute to improve crop yield. In this respect, the use of beneficial microorganisms, such as Trichoderma strains native to the Amazon, are a promising approach for sustainable horticulture, as some Trichoderma spp. besides its potential to control plant diseases they may also promote plant growth.
In this study, we aimed to evaluate the efficiency of Amazonian isolates of Trichoderma in the control of sclerotium rot, as well as to assess their potential to promote the plant growth of tomato of under field conditions. The Trichoderma isolates used in this study were selected taken into account their performance in previous evaluation

2. Materials and Methods

In this study three experiments were carried out between May 2023 and June 2024, being the first two experiments conducted under field conditions at the Alejo von der Pahlen Experimental Station – INPA (02° 59’S, 60° 01’W), in Manaus, State of Amazonas, Brazil). The soil of the experimental area had a pH of 6.7 (in water), clay texture and mineral nutrient content as described in Table A1, in the appendix. In the first experiment (May-August 2023) we evaluated the efficacy of Trichoderma isolates to control sclerotium rot, while in the second experiment (March-June, 2024) we investigated the potential of these isolates to promote plant growth. The third experiment (September, 2023) was carried out under laboratory conditions (Phytopathology Laboratory, INPA) to evaluate the ability of Trichoderma isolates to synthetize indoleacetic acid (IAA) and siderophores.

2.1. Biological Material

Six strains (hereafter referred to as isolates) of Trichoderma (INPA 2473, INPA 2951, INPA 2957, INPA 2959, INPA 2961 and INPA 2475) and the phytopathogenic isolate A. rolfsii (INPA 3014) were used in the study, they were obtained from the Collection of Microorganisms of Agrosilvicultural Interest–INPA and maintained at 4 °C, in a potato-dextrose-agar medium (Table 1). The isolates INPA 2959 and INPA 2961 belong to the same species but they have different characteristics, not described here for the sake of simplicity.

2.2. Experiment 1: Effect of Trichoderma on the Incidence of Sclerotium Rot and Tomato Yield

The Experiment 1 was carried out under field conditions, between May and August of 2023, using tomato plants (cv. Yoshimatsu) resistant to bacterial wilt caused by Ralstonia solanacearum. The seedlings were produced from seeds which were germinated (in about one week) under greenhouse conditions in a substrate containing vermiculite and rice husk. After germination, the seedlings were transplanted to the field after 25 days after emergence (on April 22 of 2023). The treatments were four isolates of Trichoderma (INPA: 2951, 2957, 2959, 2475, Table 1); a microbiological fungicide based on Trichoderma asperellum (hereafter referred to as external biofungicide or ext-biofung.), a fungicide based on pyraclostrobin and fluxapyroxad (here referred to as chemical fungicide or chem-fung.), and a control treatment without chemical or biological application (control), totaling seven treatments with four replications. The experiment was a complete randomized block design with four replications per treatment. The experimental plot consisted of an area of 15 m2 (2.5m × 6m), in which we planted three rows of tomato plants, 1 m apart and 2.5 m long (i.e., 7.5 m2 per useful plot), and within each row, five tomato plants per row with 0.5 m between plants were planted. During the experimental period, the standard agronomic practices for tomato cultivation were adopted.
During the experimental period (May-August, 2023) mean temperature was 29.0 °C and mean relative humidity 71.9%. Mean daily solar radiation was 13.1 MJm2 day–1 while monthly rainfall was 67.8 mm/month, month (Table 2, data compiled from the local –Manaus weather station of the Instituto Nacional de Meteorologia - INMET, Brazil).

2.2.1. Soil Inoculation and Application of Treatments

To ensure the pathogen (Agroathelia rolfsii) was present in the soil, it was infested with the pathogen 10 days before the transplantation of tomato seedlings. The inoculum was produced on sterilized rice grains, then along each of three rows in the experimental plot 35 g/m of infested rice grain was incorporated into the soil.
The Trichoderma isolate was prepared as suspensions adjusted to 2.6 × 107 conidia ml−1, following the concentration of the external biofungide, and for each application, a 10-ml aliquot of the conidia suspension was applied to the soil, around the base of the plant. Overall, four applications of Trichoderma were made on tomato plants, at 20, 32, 42, and 52 day after emergence. The same is true for the external biofungicide and the chemical fungicide.
The first application of Trichoderma was made five days before transplantation and then, three times at 10-day-intervals, as mentioned above. The applications were made in the morning (07:00 am), due to the high temperatures (> 30 °C, Table 2) that prevailed during the experimental period.
For the treatment with external biofungicide, four applications of 10 ml plant-1 (2.6 × 107 conidia ml−1) were also made, as mentioned above. Regarding the chemical fungicide, four applications (each with 50 mg/m2 active ingredient) were made after transplantation, by spraying the fungicide solution (300 ml/plot) to the soil surface. The chemical fungicide was applied the same day and time, as the Trichoderma isolates.

2.2.2. Variables Evaluated

The following variables were evaluated: the incidence of the disease; tomato yield (number and mass of fresh fruits).
The fruits were harvested weekly, over a period of 30 days, beginning at 60 days after transplantion. During this period, the number and mass of fresh fruits produced were recorded. In addition, the incidence of the disease was evaluated by visually observing the symptoms of sclerotium rot in the plants. The disease progress was monitored during the experiment by assessing the disease incidence (percentage of infected plant, taking the original plant population as the base line) at regular intervals (~twice a week). To quantify the development of the disease, the Area Under the Disease Progress Curve (AUDPC, in relative unit) was computed, as it represents the integral of the disease severity as a function of time [25], with the higher values indicating more severe disease symptoms. The AUDPC was calculated as follows:
Preprints 232363 i001
where: n denotes the number of evaluations; yi + yi+1: the severity of disease observed at two consecutive evaluation dates; and (ti+1 – ti): the day intervals between two evaluations.
Disease severity was assessed at regular intervals throughout the experimental period using a standardized disease visually severity scale. The same plants were evaluated at each assessment, and the area under the disease progress curve (AUDPC) was calculated from the successive severity values according.

2.3. Experiment 2: Effect of Trichoderma on Promoting Plant Growth of Tomato

To evaluate the potential of Trichoderma in promoting plant growth of tomato we used six isolates of Trichoderma (INPA 2959; INPA 2951; INPA 2961; INPA 2475; INPA 2957; INPA 2473), the external biofungicide and a control without chemical or biological application (control). The experiment was conducted in a randomized block design, with eight treatments and four replications per treatment. The useful plot comprised three rows (each 2.5 m long and 1.0 m apart, i.e., 7.5 m2), with five tomato plants/row of the Yoshimatsu cultivar, as previously described. During the experimental period (March-June, 2024), mean temperature was 27.9 °C and mean RH 80.9%. Mean solar radiation was 10.8 MJm2 day–1 and monthly rainfall 268.7 mm (Table 2).

2.3.1. Application of Treatments

Tomato seeds were treated with a suspension of Trichoderma isolates conidia (2.6 × 107 conidia ml−1) for 24 hours [microbiolization] and placed to germinate in a substrate based on vermiculite and rice husk; the seeds geminate in about one week. At 30 days after emergence the seedlings were transplanted to the field plots, previously infested with sclerotium rot, as described above. Overall, after emergence, the seedlings received two applications of Trichoderma, at 20 and 36 days after emergence (i.e the last application six days after transplantation). At each Trichoderma application, the seedlings received 10 mL of a suspension of conidia (2.6 × 107 conidia ml−1), except for the control [without treatment], as previously described. In the field, the plants were cultivated using wooden sticks for staking (Figure 3), while drip irrigation was used for watering. The standard agronomic practices for tomato cultivation, such as fertilization, weed and crop management, were adopted.

2.3.2. Variables Evaluated

During the crop cycle we measured plant height, stem diameter, and the number and mass of fruits, total fruit production, root length, fresh and dry mass (biomass) of the shoot and roots, and root volume, as previously described [26].

2.4. Experiment 3: Evaluation of the Ability of Trichoderma to Synthetize Indoleacetic Acid and Siderophores

2.4.1. Production of Siderophores

Six isolates of Trichoderma (INPA 2959; INPA 2951; INPA 2961; INPA 2475; INPA 2957; INPA 2473) were cultured in potato dextrose agar (PDA) medium for five days, then a mycelium sample of two 5-mm diameter discs was transferred to an Erlenmeyer containing 50 ml of potato dextrose (PD) medium. The Trichoderma culture was incubated for seven days in darkness at 30 °C and constant agitation (150 rpm). After incubation, the Erlenmeyer content was centrifuged at 10,000 rpm for 15 minutes at low temperature (4-5 °C) . In 2-ml microtubes, 1 ml of the supernatant and 1 ml of Cromoazurol S (CAS) solution were mixed; then the mixture was kept in darkness for two hours at 30 °C. Thereafter, the absorbance was measured at 630 nm with a spectrophotometer (Kasvi, K23-UV, Labor Import Ltd.a, São Paulo, Brazil) [27]. The experiment was a completely randomized design, with seven treatments (six isolates and the control) and three replications per treatment.

2.4.2. Production of Indoleacetic Acid

To assess the production of indoleacetic acid (IAA), six Trichoderma isolates (INPA 2959; INPA 2951; INPA 2961; INPA 2475; INPA 2957; INPA 2473) were cultured for five days in PDA medium. Then, a sample of two 5-mm mycelium discs were transferred to an Erlenmeyer containing 50 ml of PD medium with tryptophan (150 mg L−1), and the culture incubated for seven days in darkness at 30 °C, and constant agitation (150 rpm). The supernatant was obtained by centrifugation (10,000 rpm, 15 min). In the next step, 1 ml of the supernatant was mixed with 1 ml of Salkowski’s solution, and in darkness incubated for 60 minutes at 30 °C. Then, the absorbance was measured at 595 nm with a spectrophotometer (K23-UV, Kasvi). The control was a mixture of PD and tryptophan, without the Trichoderma isolates. The experiment was a completely randomized design, with seven treatments (six isolates plus the control) and three replications per treatment [28]. Since tryptophan is a common precursor in the biosynthesis of indoleacetic acid (IAA), and considering that Trichoderma species usually produce this amino acid in very low concentrations, IAA was added to the medium in order to better evaluate the efficiency of the fungus on the production of IAA.

2.5. Data Analysis

The data were analyzed for normality (Shapiro-Wilk test) and homogeneity of variance (Levene test), and when necessary the data were log transformed. Then they were subjected to ANOVA. For mean separation the Scott-Knott test was used. Data statistical analyses were conducted using R software (version 4.1.3) and Microsoft Excel (Office 2010), considering a significance level of p ≤ 0.05).

3. Results

3.1. Experiment 1- Effect of Trichoderma on the Incidence of Sclerotium Rot and Tomato Yield

3.1.1. Incidence of Sclerotium Rot in Tomato Plants

The application of Amazonian isolates of Trichoderma resulted in significant dif-ferences in the incidence of sclerotium rot between the treatments (Table 3). The iso-late INPA 2957 highlighted with only 12% of infected plants, statistically similar to the chemical fungicide, which did not present incidence of the disease. The isolates INPA 2951 (20%), INPA 2959 (27%), INPA 2475 (23%), and external biofungicide (28%) showed intermediate performance, with no significant differences between them. These treatments were significantly more effective than the no-treatment group, which had a 48% incidence. On average, taking the control as the base line, the Trichoderma isolates reduced the incidence of the disease by 28%. Furthermore, the isolate INPA 2957 was better than the other isolates, as the disease incidence was re-duced down to 12%, being its ability to control sclerotium to comparable to that of the chemical fungicide (Table 3). In fact, the isolate 2957 (T. rugulosum) reduced the incidence of sclerotium rot by up to 75%, and also increased fruit yield, as shown in next sections.

3.1.2. Temporal Dynamics of Sclerotium Rot Incidence and the Effect of Treatments

Overall, the evolution of the incidence of sclerotium rot ranged from 0 to 48% in those plots no treated with Trichodema, chemical or biological funcigicides (Figure 4, red circle). It can be seen in this Figure that disregarding the treatment that received the chemical fungicide (no disease incidence), the disease incidence in plots treated with the INPA 2957 isolate remainded very low except in last six evaluations, as shown in Figure 4 (green square). Taking the external biofungicide as a referente to compare the INPA’s Trichoderma isolate, it can be seen that the INPA’s isolates out-performed the external biofungicide (brown star in Figure 4), and in this manner evi-dencing the potential of these isolates as biocontrol agents. Since the untreated con-trol had the largest disease incidence, it also showed the greatest variability, as shown in the box-plots of Figure 5. Overall, these results reinforce the feasibility of using Amazonian Trichoderma as bioinputs in the sustainable management of sclerotium rot in tomato plants.
The area under the disease progress curve (AUDPC) did not detect differences among the effects of the Amazonian isolates on the development of sclerotium rot (Table 2). This result may reflect the limitations of AUDPC in assessing soilborne diseases, particularly those affecting roots and the stem base, where symptom development is often less gradual and more difficult to quantify than in foliar diseases. In contrast, AUDPC has been more widely and successfully applied to monitor the temporal pro-gression of foliar diseases, in which disease severity can be assessed more consistently over time.

3.1.3. Fruit Yields

In addition to its efficiency to control sclerotium rot, the INPA 2957 isolate also enhanced fruit production, as it promoted the highest fruit yield (1,436.7 g m−2) and fruit number (30.53 fruits m−2). In fact, it surpassed even the treatment that received the chemical fungicide. All treatments with Trichoderma isolates, except INPA 2475, resulted in higher yields than the control treatment, indicating the potential of Amazonian isolates for both disease management and improving fruit production.
The isolates INPA 2957 and INPA 2959 had similar performance than the chemical treatment and the external biofungicide, which highlights the potential of these isolates for their potential to be used as biological agents to control sclerotium rot in tomato and to improve productivity in this part of the Amazon region.

3.2. Experiment 2: Effect of Trichoderma on Promoting the Growth of Tomato Plants

In comparison with the weather conditions that prevailed during Experiment 1, rainfall intensity, temperature, and air humidity were quite different during the course of Experiment 2. Rainfall intensity increased from a mean of 68 to 269 mm month–1. While the mean temperature decreased from 29.0 to 27.9 °C, but perhaps the most important was the increase in air relative humidity from 72% in 2023 to 81% in 2024. As a result of these variations in weather conditions during this experimental period there was a remarkable increase in the incidence of target spot (Figure 6), a foliar disease caused by Corynespora cassiicola, which seems to be favored by high humidity and mild temperatures, and as a result the productivity and plant growth of tomato were negatively affected.

3.2.1. Biometric and Productive Parameters of Tomato Plants

After the flowering period, tomato plants showed symptoms of target spot disease, caused by the leaf fungus Corynespora cassiicola (Figure 6) which resulted in poor plant development and low productivity.
No significant differences were observed between the growth variables in the plants treated with Trichoderma spp. isolates. The lack of a significant effect of Trichoderma on plant growth can be attributed to several factors such as a poor adaptation of the isolate to the experimental environment, which does not seem much likely as the experimental area was similar to that of Experiment 1. On the other hand, the lack of an effect of treatments on plant growth and yield can be ascribed to the incidence of the of target spot which have prevented the tomato plants of expressing its maxi-mum yield potential, as mentioned above. However, this result is important as it shows that for the beneficial effect of Trichoderma strains on plant growth and de-velopment of tomato, the physical environment should meet the minimum requirement for the plant to growth and at the same time incidence of pathogen should not be too severe to reduce plant growth.
Hence, the severe incidence of the target spot can be the reason for the absence of significant differences in plant growth of tomato presented in Table 4. On the other hand, it is important to observe that regarding the use of Trichoderma, it is still necessary to conduct additional studies aimed at optimizing the conditions of use, adjustments of the form of inoculation, dose, time of application, and also to proceed with the evaluation of other isolates, aiming to enhance the beneficial effects of Trichoderma strains on plant growth of horticulture crops in the Amazon. One limitation of this study is that plant growth promotion was evaluated under high disease pressure and within a single growing condition. Therefore, future studies should investigate the performance of these isolates under lower disease pressure and across different growing seasons to assess the consistency of their effects under diverse environmental and edaphoclimatic conditions.

3.2.2. Experiment 3: Analysis of Physiological Traits Linked to Plant Growth Promotion Mechanisms

The production of indoleacetic acid (IAA) varied significantly among the isolates of Trichoderma spp. The isolates INPA 2473, INPA 2475 and INPA 2951 showed the highest productions of IAA (60.72, 60.53 and 59.70 μg ml-1, respectively), forming a statistically superior group. The isolate INPA 2957 showed intermediate production (35.18 μg ml-1), while the isolates INPA 2959 and INPA 2961 showed the lowest val-ues (9.27 and 8.99 μg ml-1), statistically similar to the control (Table 5).
Regarding the production of siderophores, all Trichoderma isolates differed significantly from the control, which did not show siderophores production (0.00 μg ml-1). The values ranged from 55.01 μg ml-1 (INPA 2957) to 65.03 μg ml-1 (INPA 2473), with no statistically significant differences between the treatments with isolates, in-dicating similar performance in this variable (Table 5).

4. Discussion

The results obtained in this study highlight the agronomic and biotechnological potential of Amazonian isolates of Trichoderma spp. in the control of sclerotium rot (A. rolfsii) in tomato plants. Among the isolates tested, INPA 2957 (T. rugulosum) was particularly effective, presenting control levels statistically similar to the chemical fungicide. In addition, several isolates demonstrated the ability to produce bioactive metabolites, such as indoleacetic acid (IAA) and siderophores, with positive implications for root growth and crop health. The approach of combining isolates with diferent characteristics offers promising potential for the development of biological fungicides adapted to the edaphoclimatic conditions of the Amazon region.

4.1. Control of Sclerotium Rot

Table 3 reveals that the Amazonian isolates tested, especially INPA 2957, promoted a significant reduction in the incidence of the disease, with a performance statistically similar to that of the chemical treatment. These findings reinforce the efficacy of Trichoderma isolates as biocontrol agents against soilborne pathogens. The ability of isolates to suppress A. rolfsii is associated with mechanisms of action, such as mycoparasitism, production of lytic enzymes, and competition for ecological niches and nutrients [29]. The efficacy of Trichoderma isolates is generally superior when applied in regions where they originate, since they are adapted to specific environmental conditions and soil microbiota, favoring their colonization and biocontrol activity [30,31]. The performance of species of Trichoderma also relates to the method of application, since it directly influences its survival, colonization capacity, and interaction with the host [32,33]. In addition, not all isolates have the same potential for promoting plant growth, as this activity is associated with the specific metabolic profile of each lineage, including the production of phytohormones, enzymes, and bioactive compounds [34].

4.2. Temporal Dynamics of Biocontrol

The disease evolution of sclerotium rot (Figure 4) reinforce the importance of considering not only the final incidence of the pathology, but also the temporal dynamics of the effect of Trichoderma strains over time. Isolates such as INPA 2957 and INPA 2951 demonstrated persistence in the inhibitory effect on sclerotium rot, which is desirable in sustainable production systems. This behavior allows reducing the frequency of reapplications, increasing the operational and economic efficiency of biological control.
Results similar to those obtained in this study were reported by [35] who evaluated the use of T. harzianum and organic compost in the management of sclerotium rot in crops with mulch. Although mulch increased disease intensity by 35.5%, expressed as AUDPC, the application of T. harzianum and 3 L of mulch compound promoted significant reductions of 38.1% and 37.5%, respectively, in disease severity. The integration of these practices was even more effective with the combination of T. harzianum with mulch reduced AUDPC by 52.8% when compared to the use of mulch alone.

4.3. Fruit Yield

The results presented in Table 3 show that overall treatments with Trichoderma isolates improved tomato fruit yield. The isolates INPA 2957 and INPA 2959 provided the highest average yields per plant, with a significant increase in relation to the control, indicating the efficiency of these microorganisms in promoting crop yield.
This effect can be attributed to the combined biological control action of sclerotium rot. By reducing biotic stress, plants are able to direct more metabolic resources towards growth and fruiting. Such mechanisms contribute synergistically to both the health and agronomic performance of the crop, which reinforces the potential of using native isolates adapted to the edaphoclimatic conditions of the region. [36] also observed an increase in fruit yield in tomato plants treated with T. asperellum and effectively decreased the incidence of bacterial wilt of tomato plants caused by Ralstonia solanacearum, highlighting the role of these species in inducing growth and phytosanitary protection. The application of Trichoderma asperellum resulted in a significant increase in fruit yield and reduction of fusarium disease, evidencing the dual mechanism of growth promotion by nutritional and physiological improvement, and biological control via induction of systemic and acquired resistance and direct inhibition of pathogens [37].
In addition, the use of native isolates may have favored adaptation to local edaphoclimatic conditions, which contributes to greater efficiency in the field when compared to commercial products imported from other regions [38]

4.4. Indoleacetic Acid (IAA) Production and Growth Promotion

As shown in Table 5, the isolates INPA 2473 (T. endophyticum), INPA 2475 (T. agriamazonicum) and INPA 2951 (T. asperelloides) excelled in IAA production, with IAA concentrations, reaching up to 60.72 μg ml−1. These values exceed those reported in previous studies [39], which ranged between 11.35 and 52.42 μg ml−1, and indicates the ability of these isolates to stimulate root growth by inducing morphological changes, such as increased root length and higher root hair density [40,41]. In studies carried out by [42], Trichoderma isolates evaluated did not demonstrate the ability to produce indoleacetic acid (IAA) in vitro, even in the presence of its precursor, L-tryptophan. This result suggests that there are metabolic variations among different Trichoderma isolates, influencing its ability to synthesize phytohormones and highlights the functional diversity within the genus Trichoderma, with distinct mechanisms.
Although no statistical differences were observed in plant growth parameters between the treatments (Table 4), it was possible to identify positive trends in the isolates producing IAA, especially in fresh root mass and root volume. This suggests that the isolates tested may not have, in fact, expressive potential to promote plant growth or induce significant changes in root morphology [43]. For example, [44] reported marked differences between lineages in the production of secondary metabolites and phyto-hormones. Studies indicate that certain isolates of T. harzianum did not influence the emergence of tomato seedlings, despite increasing their subsequent growth [45]. This demonstrates that the mechanisms of action are specific and depend on factors such as substrate, nutrients, and associated microbiota.

4.5. Production of Siderophores: Absence of Significant Differentiation

Table 5 shows no significant effect on siderophore production among Trichoderma spp. isolates, with values ranging from 55.01 to 65.03 μg ml−1. This homogeneity may be related to the characteristics of Amazonian soils, which are naturally rich in iron [46], which reduces selective pressure by highly efficient mechanisms for capturing this micronutrient. This scenario contrasts with that observed in soils with low iron availabil-ity, where the production of siderophores is usually differential [47].

4.6. Interference of Foliar Diseases and Abiotic Factors

Despite the efficacy of Trichoderma isolates in suppressing sclerotium rot, the occurrence of target spot (Corynespora cassiicola) after flowering negatively affected the final crop performance. This foliar pathogen reduced the active photosynthetic area, impacting plant metabolism and partially masking the benefits of root biocontrol. This shows a limitation of Trichoderma isolates against pathogens that attack the leaves, as discussed by [48] who pointed out to the low adhesion and persistence of these microrganisms on the leaf surface as a limiting factor.
The high temperatures (> 30 °C) observed during the crop cycle probably negatively affected the physiological development of tomato plants. The optimum temperature range for the crop is between 18 °C and 27 °C, and values above this range compromise processes such as pollination, photosynthesis and fruit set, resulting in lower productivity [49,50]. In tomato plants, a significant reduction in diseases such as Fusarium wilt (up to 78%) was observed, as well as improved nutrient absorption and biomass accumulation [51]. The association between T. asperellum and Funneliformis mosseae has been shown to be safe and beneficial, promoting plant growth without restraining mycorrhizal colonization [52].
The variation in temperature attributed to the existence of Trichoderma species in specific niches affects growth, metabolic activity, enzyme production, and volatile antibiotic production. Reduced soil moisture or increased soil temperature have made it very difficult for Trichoderma colonies to establish in soils, reducing hyphal growth, germination, and spore production [33].
The results obtained with Amazonian isolates, especially INPA 2957 and INPA 2951, show that the selection of strains adapted to the region can be an effective strat-egy both for disease control and for increasing agricultural productivity. These isolates represent a sustainable alternative to conventional inputs, with potential for application in ecologically based agricultural systems.
A limitation of this study is that the evaluation of the efficacy of the isolates was restricted to one crop cycle, which limits the analysis of seasonal or longterm effects. Furthermore, possible interactions between Trichoderma isolates and the native soil microbiota were not considered, a factor that can influence its efficacy. On the other hand, the study clearly shows the benefit of using Amazonian Trichoderma isolates on controlling an important disease of tomato plants, which highlight its potential to promote sustainable horticulture in the Amazon region.

5. Conclusions

Amazonian Trichoderma spp. isolates showed potential for suppressing southern blight in tomato plants and promoting plant growth, possibly through the production of bioactive metabolites. These findings suggest that these microorganisms may represent a promising alternative to synthetic agrochemicals for disease management, particularly in agricultural systems of the humid tropics. However, further studies are needed to validate their efficacy under different cultivation conditions, assess the consistency of their performance over time, and elucidate the mechanisms underlying the interactions among the isolates, the pathogen, and the host plant. As future perspectives, studies will be conducted to investigate the synergistic interactions between these Trichoderma isolates and the native soil microbiota across different growing seasons, as well as to develop more stable formulations and validate their efficacy under field conditions. Furthermore, compliance with regulatory requirements will be essential to facilitate the transfer of this technology to the agricultural sector and expand the prac-tical application of these isolates in sustainable agriculture.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org, Table S1: Raw data of disease incidence and severity in tomato plants; Table S2: Raw data of plant growth variables.

Author Contributions

ADPRB: Investigation, writing and editing, data analysis and interpretation; ALW: Methodology, data analysis and interpretation, writing and editing; LAGA, LNA, JSM: Investigation; CAQ: Investigation, Data analysis and interpretation; GFS: Data analysis and interpretation, Resources; RACN: Conceptualization and Methodology; REH: Conceptualization, Methodology, Data analysis and interpretation, Funding acquisition.

Funding

This research was funded by FAPEAM (Call for Proposals 008/2021 - PROSPAM/FAPEAM. Project: Trichoderma spp. isolates for disease control in economically important crops in the state of Amazonas; Call for Proposals no. 17/2024 - Divulga CT&I/FAPEAM and POSGRAD/FAPEAM) and the Coordination for the Improvement of Higher Education Personnel (CAPES) (finance code 001 and consolidation project PDPG 3 and 4, process No. 88887.707273/2022-01.

Data Availability Statement

We encourage all authors of articles published in MDPI journals to share their research data. In this section, please provide details regarding where data supporting reported results can be found, including links to publicly archived datasets analyzed or generated during the study. Where no new data were created, or where data is unavailable due to privacy or ethical restrictions, a statement is still required. Suggested Data Availability Statements are available in section “MDPI Research Data Policies” at https://www.mdpi.com/ethics.

Acknowledgments

To the Instituto Nacional de Pesquisas da Amazônia (INPA) for making available the isolates of Trichoderma, to Embrapa, for technical and institutional support. This research was funded by FAPEAM (Call for Proposals 008/2021 - PROSPAM/FAPEAM. Project: Trichoderma spp. isolates for disease control in economically important crops in the state of Amazonas; Call for Proposals no. 17/2024 - Divulga CT&I/FAPEAM and POSGRAD/FAPEAM) and the Coordination for the Improvement of Higher Education Personnel (CAPES) (finance code 001 and consolidation project PDPG 3 and 4, process No. 88887.707273/2022-01. ADPRB would also like to thank the student Jennifer Salgado da Fonseca for her support and dedication during the execution of the experimental activities.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

CMIA Collection of microorganisms of interest to agroforestry.
IAA Indole-3-acetic acid
INPA National Institute for Research in the Amazon
AUDPC area under the disease progress curve
NVMs Non-Volatile Metabolites
p p-value (statistical significance)
rpm Revolutions per minute

Appendix

Table A1. Chemical content and granulometry of the soil in the experimental area used in this study. Soil sample for analyses were collected at a depth of 0-20 cm, before the beginning of the experiments. Manaus-AM, 2023.
Table A1. Chemical content and granulometry of the soil in the experimental area used in this study. Soil sample for analyses were collected at a depth of 0-20 cm, before the beginning of the experiments. Manaus-AM, 2023.
pH pH C SOM N P K K Ca Mg Al H+Al Sand Clay Silt
(water) (KCl) -------------g kg-1------------ --------mg kg-1-------- --------------------Cmolc kg-1-------------------- --------------g kg-1--------------
6.66 6.17 10.88 17.75 1.03 37.4 49.20 0.13 3.13 1.24 0.06 1.82 812 76 113
pH: pH in water; C: organic carbon; SOM: soil organic matter; N: total nitrogen; Ca: exchangeable calcium (Ca2+); Mg: exchangeable magnesium (Mg 2+); Al: exchangeable aluminum (Al3+); H +Al: potential acidity at pH 7; K: exchangeable potassium (K+); P: Mehlich-1 available phosphorus. Note: The P value is from Lima [53].

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Figure 1. Base of the stem of a tomato plant (Solanum lycopersicum) infected by Agroathelia rolfsii, showing characteristic signs of the pathogen, such as abundant white mycelium and brown sclerotia, as well as typical symptoms of sclerotium rot, such as tissue necrosis and girdling of the stem near the ground. Photo of Antonia D.P.R. Batista.
Figure 1. Base of the stem of a tomato plant (Solanum lycopersicum) infected by Agroathelia rolfsii, showing characteristic signs of the pathogen, such as abundant white mycelium and brown sclerotia, as well as typical symptoms of sclerotium rot, such as tissue necrosis and girdling of the stem near the ground. Photo of Antonia D.P.R. Batista.
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Figure 2. Tomato plants (Solanum lycopersicum) in staked system, with wooden sticks tied to the stem. A) Initial symptoms of sclerotium rot; B) Plant with severe symptoms of the disease. Photo: courtesy of Luiz A. G. Assis.
Figure 2. Tomato plants (Solanum lycopersicum) in staked system, with wooden sticks tied to the stem. A) Initial symptoms of sclerotium rot; B) Plant with severe symptoms of the disease. Photo: courtesy of Luiz A. G. Assis.
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Figure 3. Tomato plants showing the staking with wooden sticks. It can be seen that the plants had a good vegetative growth and development. Photo of Antonia D.P.R. Batista.
Figure 3. Tomato plants showing the staking with wooden sticks. It can be seen that the plants had a good vegetative growth and development. Photo of Antonia D.P.R. Batista.
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Figure 4. Disease incidence of sclerotium rot (Agroathelia rolfsii) in tomato plants during the cultivation period. The seedlings were transplanted on April 22 of 2023 (day of year, DOY 132), 25 days after emergence, which occurred on DOY 107. The first evaluation was made on June 9, 2023 (DOY 160). The plants were treated as de-scribed in the text. In parenthesis is shown the number of plants per treatment of the original population (60 plants/treatment) that had been survived up to at the last evaluation.
Figure 4. Disease incidence of sclerotium rot (Agroathelia rolfsii) in tomato plants during the cultivation period. The seedlings were transplanted on April 22 of 2023 (day of year, DOY 132), 25 days after emergence, which occurred on DOY 107. The first evaluation was made on June 9, 2023 (DOY 160). The plants were treated as de-scribed in the text. In parenthesis is shown the number of plants per treatment of the original population (60 plants/treatment) that had been survived up to at the last evaluation.
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Figure 5. Box plots of the incidence of sclerotium rot in tomato plants treated with Trichoderma, an external biofungicide or a chemical fungicide. Abbreviations: T1: INPA 2951, T2: INPA 2957, T3: INPA 2959, T4: INPA 2475, T5: external biofungicide, T6: chemical fungicide and T7: untreated control (T7- control).
Figure 5. Box plots of the incidence of sclerotium rot in tomato plants treated with Trichoderma, an external biofungicide or a chemical fungicide. Abbreviations: T1: INPA 2951, T2: INPA 2957, T3: INPA 2959, T4: INPA 2475, T5: external biofungicide, T6: chemical fungicide and T7: untreated control (T7- control).
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Figure 6. Symptoms of target spot (Corynespora cassiicola) on tomato leaves, characterized by circular necrotic lesions with a brown center, defined margins and yellowish halos around it, conferring the concentric aspect typical of the disease. Photo: courtesy of Luiz A. G. Assis.
Figure 6. Symptoms of target spot (Corynespora cassiicola) on tomato leaves, characterized by circular necrotic lesions with a brown center, defined margins and yellowish halos around it, conferring the concentric aspect typical of the disease. Photo: courtesy of Luiz A. G. Assis.
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Table 1. Trichoderma isolates from the Collection of Microorganisms of Agrosilvicultural Interest–INPA used in the experiments.
Table 1. Trichoderma isolates from the Collection of Microorganisms of Agrosilvicultural Interest–INPA used in the experiments.
Isolate Species Origin (coordinates)
INPA 2473 Trichoderma endophyticum 03° 05’ 29”S, 59° 59’ 35”W
INPA 2951 Trichoderma asperelloides 03° 05’ 87”S, 59° 58’ 40”W
INPA 2957 Trichoderma rugulosum 03° 05’ 87”S, 59° 58’ 40”W
INPA 2959 Trichoderma asperellum 03° 05’ 87”S, 59° 58’ 40”W
INPA 2961 Trichoderma asperellum 03° 05’ 87”S, 59° 58’ 40”W
INPA 2475 Trichoderma agriamazonicum Not available
INPA 3014 Agroathelia rolfsii Not available
all of them were collected in the municipality of Manaus, State of Amazonas, Brazil. Isolates INPA: 2951, 2957, 2959, and 2961 were collected from soil under fallow vegetation, while the isolates INPA 2473 and 2475 from the tree Scleronema micranthum Ducke. INPA 3014 (the pathogen) was isolated from tomato.
Table 2. Climate data recorded during the field experiments.
Table 2. Climate data recorded during the field experiments.
Year Month Tmean
(°C)		 Tmax
(°C)		 Tmin
(°C)		 RHmean
(%)		 RHmax
(%)		 RHmin
(%)		 SR(MJ m–2 day–1) Rainfall
(mm month–1)
2023 May 27.8 33.8 23.1 80.1 98.0 53.0 12.02 135.0
2023 June 28.1 34.3 20.2 75.3 97.0 44.0 11.35 48.4
2023 July 29.4 35.5 22.3 68.2 97.0 36.0 13.41 69.8
2023 August 30.5 37.7 24.1 64.1 96.0 34.0 15.75 18.0
M-A Mean 29.0 35.3 22.4 71.9 97.0 41.8 13.10 67.8
2024 March 27.8 35.1 22.4 82.0 98.0 50.0 12.41 353.8
2024 April 27.8 34.7 23.5 82.0 98.0 45.0 11.72 219.2
2024 May 27.5 34.1 22.8 83.6 98.0 49.0 8.97 386.6
2024 June 28.6 34.8 23.6 76.0 98.0 40.0 9.96 115.2
M-J Mean 27.9 34.7 23.1 80.9 98.0 46.0 10.8 268.7
Abbreviation: T: temperature, min: absolute minimum recorded, max: absolute maximum recorded during the indicated month; SR: solar radiation. Data compiled from the INMET meteorological station of Manaus (https://tempo.inmet.gov.br/TabelaEstacoes/A101).
Table 3. Agronomic traits and sclerotium rot incidence of tomato plants treated with Trichoderma and chemical fungicide.
Table 3. Agronomic traits and sclerotium rot incidence of tomato plants treated with Trichoderma and chemical fungicide.
Treatment Disease incidence (%) Fresh fruit (g m–2) Fresh fruits (unit m–2) Fresh fruits
(g fruits–1)		 AUDPC
(rel. un.)
1-INPA 2951 20.0b 781.53b 17.9b 43.66a 71b
2-INPA 2957 11.7c 1,436.7a 30.5a 47.06a 45b
3-INPA 2959 26.7b 1,199.7a 27.1a 44.27a 106b
4-INPA 2475 23.3b 626.2b 15.2b 41.19a 88b
5-Ext-biofung. 28.3b 1,019.6a 24.4a 41.73a 96b
6-Chem-Fung. 0.0c 1,177.8a 27.2a 43.30a 0c
7-Control 48.3a 828.1 b 17.6b 46.93a 172a
CV (%) 64 32 29 11 55
Abbreviation: AUDPC: Area under the disease progress curve (in relative unit, rel. un.), Ext-biofung.: external biofungicide, Chem-Fung: Chemical Fungicide ; max: maximal. Fresh fruits were collected for 30 days, from 60 to 90 days after transplantation. Means followed by a different letter differ significantly according to the Scott-Knott test (p ≤ 0.05).
Table 4. Tomato plants traits treated with Trichoderma isolates.
Table 4. Tomato plants traits treated with Trichoderma isolates.
Treatment Fruit production SDM RDM SFM RFM RL D FRV H
No.m-2 Mass (g m-2) ---------------------- (g/m2) --------- cm/plant mm cm3/plant m
INPA 2961 32.8a 990.3a 28.94a 1.92a 156.4a 9.0a 15.08a 8.64a 9.50a 1.45a
INPA 2473 29.0a 834.4a 30.14a 2.25a 169.7a 13.9a 15.50a 8.97a 10.50a 1.37a
Ext-biofung. 27.2a 914.8a 24.82a 1.90a 132.1a 8.7a 15.08a 8.77a 10.92a 1.39a
INPA 2475 26.7a 844.2a 22.45a 1.90a 125.0a 10.5a 14.33a 8.21a 9.75a 1.33a
INPA 2951 26.6a 775.8a 31.67a 1.89a 157.7a 10.3a 14.83a 8.64a 11.50a 1.41a
Control 26.6a 866.2a 20.69a 1.82a 116.1a 8.7a 14.67a 7.56a 10.08a 1.34a
INPA 2959 22.4a 698.8a 24,01a 1.62a 130.9a 9.1a 13.42a 8.19a 8.83a 1.33a
INPA 2957 18.8a 659.6a 23.12a 1.96a 140.3a 12.7a 14.92a 7.68a 10.33a 1.31a
CV (%) 22 25 28 27 27 27 11 7 30 5
Means followed by the same letter in the column do not differ significantly ac-cording to the Scott-Knott test at 5% probability. SDM: shoot dry mass. RDM: root dry mass, SFM: shoot fresh mass, RL: root length, D: stem diameter, FRV: fresh root vol-ume, H: plant height. Ext-biofung: External biofungicide based on Trichoderma asperellum; Control: no management of the disease. .
Table 5. In vitro production of indoleacetic acid (IAA) and siderophores by isolates of Trichoderma isolates.
Table 5. In vitro production of indoleacetic acid (IAA) and siderophores by isolates of Trichoderma isolates.
Treatments IAA (μg ml-1) Siderophores (μg ml-1)
INPA 2473 60.72 a 65.03 a
INPA 2475 60.53 a 59.74 a
INPA 2951 59.70 a 61.12 a
INPA 2957 35.18 b 55.01 a
INPA 2959 9.27 c 61.17 a
INPA 2961 8.99 c 61.23 a
No treatment 0.00 c 0.00 b
CV (%) 11 11
Means followed by the same letter do not differ significantly according to the Scott–Knott test at 5% probability. CV (%): Coefficient of variation in percentage.
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