Trichoderma endophyticum , T. asperellum, and T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina

Trichoderma endophyticum , T. asperellum, and T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Trichoderma endophyticum , T. asperellum, and T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina Jayne Deboni da Veiga, Juliane Ludwig, Samuel Francisco Chitolina, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6751408/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract Soybean is one of the most important commodities in the world, and is affected by several phytopathogens, such as the fungus Macrophomina phaseolina , which can occur at all stages of development, causing significant losses in production. The use of different cultural, chemical and genetic control strategies has shown low efficiency against this phytopathogen. Thus, the objective of this study was to evaluate the use of Trichoderma asperellum , T. endophyticum and T. harzianum in seed treatment to control M. phaseolina and promote plant growth. Healthy seeds inoculated with M. phaseolina were treated with the three Trichoderma species, the plant growth regulator Stimulate®, an insecticide based on Thiamethoxam and combinations of Trichoderma species with Stimulate® or Thiamethoxam. The antagonistic capacity of Trichoderma species against M. phaseolina and their compatibility with Stimulate® and Thiamethoxam, germination and seedling vigor, growth and dry biomass of plants were evaluated. Trichoderma species showed antagonism against M. phaseolina , especially T. endophyticum . In vitro , Stimulate® reduced the growth of T. endophyticum and T. harzianum , while Thiamethoxam reduced the growth of T. harzianum . M. phaseolina reduced approximately 80% of soybean seed germination. The three Trichoderma species increased germination and vigor of infected seeds. In healthy seeds, T. harzianum reduced germination, while T. endophyticum combined with Stimulate® increased seedling vigor. These results show that T. endophyticum , T. harzianum and T. asperellum are effective in treating seeds infected with M. phaseolina , and the use of T. harzianum isolate CCT 7589 is not recommended for the treatment of healthy soybean seeds. Biological control Glycine max Growth promoters Thiamethoxam Stimulate® Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Soybean ( Glycine max [L.] Merrill) is one of the most important food and economic crops globally (Chen et al., 2023 ), with a total cultivated area of 139.85 million hectares and a production of 394.73 million tons (USDA, 2024 ). This is attributed to its high nutritional value, with over a thousand products derived from its grains (Kamolovna & Zavkiddinova, 2023 ), making it vital for ensuring global food security (Inglada et al., 2015 ). However, the crop can be affected by various pathogens that may occur from seed germination to grain filling. Among these pathogens is the fungus Macrophomina phaseolina (Tassi) Goid (Goidanish, 1947 ). It is a generalist soil inhabitant found worldwide, affecting at least 500 plant species across more than 100 families (Marquez et al., 2021 ). In soybeans, it causes charcoal rot and is one of the main diseases of the crop, capable of occurring at all stages of plant development and leading to production losses of 30–50% (Šućur Elez et al., 2023). Infected plants exhibit reduced leaf and seed size and may wilt and die prematurely (Wrather et al., 2008 ). This fungus produces microsclerotia as a resistant structure, surviving in seeds, crop residues, living plants (Dhingra & Sinclair, 1978 ; Abawi & Pastor Corrales, 1990), and soil for up to 15 years (Gupta et al., 2012 ). Due to being a polyphagous pathogen and forming resistant structures, crop rotation as a control strategy does not have a direct effect on reducing the disease in the field (Short; Wyllie & Bristow, 1980 ). On the other hand, some consecutively planted crops may even increase pathogen levels in the field. In Brazil, it is common to plant second-season corn after soybean harvest. The consecutive cultivation of these two host crops facilitates the increased incidence of the disease in the field and the perpetuation of the pathogen in the area (Seixas et al., 2020 ; Short; Wyllie & Bristow ,1980). Furthermore, in recent years, there has been an increase in the incidence and severity of this disease in cultivated areas, attributed to climate change (Cohen et al., 2022 ). The severity of charcoal rot is associated with two predominant factors: water deficit and high soil temperatures (Šućur Elez et al., 2023). Under high temperatures (30–35°C) and soil moisture below 60%, this fungus can cause substantial yield losses in various crops, including soybeans, impacting farmers' yields (Kaur et al., 2012 ; Gupta et al., 2012 ; Abawi & Pastor Corrales, 1990). Strategies for managing charcoal rot using fungicides have not been successful (Bellaloui et al., 2023 ), and in Brazil, there are no fungicides registered for controlling the disease in soybeans (Agrofit, 2024 ). An economically more viable management strategy would be the use of resistant cultivars. However, there are no resistant soybean cultivars available on the market (Bellaloui et al., 2023 ). Therefore, various cultural, chemical, and genetic control strategies against M. phaseolina in the field have shown low or no efficiency, making the management of charcoal rot in soybean crops a challenge (Marquez et al., 2021 ). Biological control, including the use of different species of Trichoderma , has emerged as an important alternative for controlling various soil-dwelling pathogens, as observed with M. phaseolina in different hosts (Bastakoti et al., 2017 ; Marquez et al., 2021 ). In soybeans, isolates of T. harzianum have proven effective in controlling M. phaseolina (Larran et al., 2023 ). However, few studies have tested isolates of T. asperellum and T. endophyticum for controlling charcoal rot in soybeans, as well as their compatibility with growth regulators and insecticides used in soybean seed treatment. Therefore, the objectives of this study were: (i) to evaluate the potential for controlling charcoal rot and promoting growth in soybean plants using T. asperellum , T. endophyticum , and T. harzianum ; (ii) to assess the compatibility of the three Trichoderma species with the plant growth regulator Stimulate® and the insecticide Thiamethoxam used in soybean seed treatment; and (iii) to evaluate the combination of the three Trichoderma species with the growth regulator Stimulate® and the insecticide Thiamethoxam in treating soybean seeds infected with M. phaseolina . Materials and methods Fungal strains The Trichoderma isolates were obtained from commercial products: Lalnix Resist® ( T. endophyticum isolate IBCB 56/12, Lallemand®), Organic WP® ( T. asperellum isolate URM 5911, Lallemand®), and Stimucontrol® ( T. harzianum isolate CCT 7589, Simbiose®). An aliquot of each product was spread evenly across a Petri dish containing PDA (Potato Dextrose Agar at 4%). The fungus M. phaseolina (isolate 231) was obtained from the collection of the Agris Institute. Fragments of the fungus were placed at the center of a Petri dish containing PDA. The Petri dishes were then incubated in a growth chamber at 25 ± 1°C with a photoperiod of 12 hours at 40 µmol s − 1 m − 2 for seven days. Antagonism in dual cultures The dual culture technique was applied to evaluate the antagonism of the three Trichoderma species against M. phaseolina , following the method of Bell, Wells & Markham ( 1982 ). Mycelial discs of M. phaseolina and the three Trichoderma isolates, each with a diameter of 4 mm, were taken from colonies after seven days of growth. The mycelial discs of M. phaseolina and the individual Trichoderma isolates were placed on a PDA culture medium 1 cm from the inner edge of 90 mm diameter Petri dishes, opposite each other. The control treatment consisted solely of a mycelial disc of M. phaseolina . No fungicide was used as a positive control due to the lack of registered products for controlling M. phaseolina in soybeans. The Petri dishes were incubated in a growth chamber at 25 ± 1°C with a photoperiod of 12 hours at 40 µmol s − 1 m − 2 for eight days. Afterward, the antagonistic ability of the Trichoderma isolates against M. phaseolina was assessed using a scale proposed by Bell, Wells & Markham ( 1982 ) with some adaptations (Supplementary material 1), as well as by measuring the inhibition of mycelial growth of M. phaseolina and the Trichoderma species. To achieve this, the diameters of the colonies of M. phaseolina and the Trichoderma species were measured with a digital caliper (Vonder®, China) at two equidistant points, and the averages were subjected to the formula used by Tomah et al., (2024). The experiment was conducted in a completely randomized design (CRD) with six repetitions, with each Petri dish serving as a repetition. Compatibility test The compatibility of Trichoderma species with the plant growth regulator (Stimulate®, Stoller) and the insecticide Thiamethoxam (Cruiser 350 FS®, Syngenta) was evaluated. Both the minimum and maximum recommended doses of the growth regulator were tested for foliar application on soybeans (250 and 500 mL of the active ingredient per 150 L of solution) and seed treatment (5 and 7.5 mL of the active ingredient per kg of seeds), as well as for Thiamethoxam in seed treatment (0.5 and 3 mL of the active ingredient per kg of seeds) (Agrofit 2024 ). The recommended seed treatment doses were added to the culture medium in the amount specified by the manufacturer to treat 2 g of seeds per plate. The products were mixed in Erlenmeyer flasks containing a PDA medium. For each 90 mm diameter Petri dish, 15 mL of the solution was poured in. After the culture medium solidified, a 5 mm diameter mycelial disc of each Trichoderma species was placed at the center of each Petri dish. The plates were then incubated in a growth chamber at 25 ± 1°C with a photoperiod of 12 hours at 40 µmol s − 1 m − 2 . After 72 hours of incubation, two orthogonal measurements of the colony diameters (mm) were taken using a digital caliper. The experiment was conducted in a completely randomized design (CRD) with six repetitions per treatment. Inoculation of soybean seeds with M. phaseolina Soybean seeds of the cultivar Intacta Xtend M6110 i2x® were used for this study. Initially, the seeds underwent a germination and health test to assess the quality of the batch, following the Seed Analysis Rules (BRASIL, 2009). Although the seeds exhibited good health and were free of M. phaseolina , they were disinfected in 1% sodium hypochlorite for 1 minute, then rinsed three times with sterile distilled water and air-dried for 24 hours. A portion of the disinfected seeds was inoculated with M. phaseolina . The osmotic restriction method using mannitol was employed to achieve an osmotic potential of − 1.0 MPa (Machado et al., 2001 ). The amount of mannitol added to the PDA culture medium was calculated based on the osmotic potential formula proposed by Van’t Hoff (Taiz & Zeiger, 2017 ). Ten mL portions of the sterilized PDA medium were transferred to 90 mm Petri dishes and allowed to solidify. A 5 mm diameter disc containing M. phaseolina mycelium was placed in the center of each Petri dish, which was then incubated in a growth chamber at 28 ± 1°C with a photoperiod of 12 hours at 40 µmol s − 1 m − 2 for seven days. Following this incubation, a total of 100 seeds were placed in each Petri dish, remaining in contact with the fungus for 72 hours (adapted from Cruciol & Costa, 2018 ). Seed treatments The doses of the plant growth regulator and the insecticide, recommended by the manufacturers (Agrofit, 2024 ), and which demonstrated the lowest percentage of inhibition of growth among the different species of Trichoderma , were used in the treatments. The treatments included: T. asperellum , T. endophyticum , T. harzianum , Stimulate®, Thiamethoxam, Stimulate® + T. asperellum , Stimulate® + T. endophyticum , Stimulate® + T. harzianum , Thiamethoxam + T. asperellum , Thiamethoxam + T. endophyticum , and Thiamethoxam + T. harzianum . For the treatment of seeds with the biocontrol agents, the fungi were previously cultured on a PDA medium in a growth chamber at 28 ± 1°C with a 12-hour photoperiod at 40 µmol s − 1 m − 2 for seven days. After this period, an inoculum suspension was prepared by adding sterile water containing 1% Tween 20 to the plates. The surface of the colonies was scraped with a sterile Drigalski spatula, and the spore suspension was filtered using a double layer of gauze, adjusting the concentration to 1 × 10 8 spores/mL using a hemocytometer. The following amounts were used: 0.5, 2, and 5 mL.kg − 1 of seeds for the insecticide (Thiamethoxam), the spore suspension, and the plant growth regulator (Stimulate®), respectively. Control treatments consisted of inoculated and non-inoculated seeds treated with 2 mL.kg − 1 of distilled water containing 1% Tween 20. The seeds were placed in plastic bags with the treatments, agitated manually for 1 minute, and then left open to dry and for the adhesion of the different treatments to the seeds. Seed germination test The standard germination test (roll paper method) outlined in the MAPA manual (2009) was employed to assess germination rates and the germination speed index (IVG) of the seeds. A total of 300 seeds were used per treatment, divided into six repetitions of 50 seeds each. The seeds were incubated in a growth chamber at 25 ± 1°C with a 12-hour photoperiod at 40 µmol s − 1 m − 2 . Daily counts of the number of germinated seeds were conducted to determine the IVG, following Maguire ( 1962 ) methodology. On the eighth day of incubation, the germination rate and dry biomass of both the aerial parts (DBAP) and roots (DBRP) were evaluated. Seeds exhibiting a root protrusion greater than 2 mm were considered germinated. From each repetition, ten seedlings were randomly selected for measuring shoot and root lengths using a digital caliper. For dry biomass determination, ten seedlings were cut at the root insertion height, separating the aerial parts from the roots, which were then placed in kraft paper bags and dried in a forced-air oven at 60 ± 5°C until a constant weight was achieved. The experiment was conducted in a completely randomized design (CRD). Emergence and development of soybean seedlings in pots under controlled conditions Inoculated and treated seeds were germinated in plastic pots, with three seeds per pot, each containing 900 g of sterile substrate (Red Latosol:sand:manure; 2:1:1 by weight). The substrate was sterilized by autoclaving three times at 120°C to ensure complete disinfection. Non-inoculated and treated seeds were sown in a substrate previously infested with M. phaseolina . For this, rice grains with husks were placed in Erlenmeyer flasks and moistened with distilled water for 10 minutes. After removing the water, the grains were autoclaved for 20 minutes at 120°C. Once cooled, 20 discs of M. phaseolina mycelium (5 mm in diameter) were added, and the flasks were incubated in a growth chamber at 28 ± 1°C with a 12-hour photoperiod for 14 days. The flasks were shaken daily to ensure uniform colonization. Subsequently, nine rice grains were deposited in each pot at a depth of 5 cm and maintained in a germination room at 25 ± 3°C for 7 days (adapted from Cruciol & Costa 2018 ). After this period, the seeds were sown in the substrate. The pots were kept in a controlled environment room with an average temperature of 25°C and relative air humidity around 80%. Control treatments consisted of inoculated and non-inoculated seeds treated with 2 mL.kg − 1 of distilled water and sown in either sterile or infested substrate. Following seedling emergence, thinning was performed to leave one seedling per pot. On day 24 post-sowing, symptoms of gray rot were assessed using the rating scale proposed by Abawi and Pastor Corrales (1990), adapted by the authors (Supplementary material 2), and the disease severity index (ISD) was calculated according to McKinney ( 1923 ). The dry biomass of the aerial parts (DBAP*) and roots (DBRP*) was also evaluated. Plants were cut at the base, and the roots were rinsed with running water to avoid damaging the root system. Both aerial parts and roots were placed in kraft paper bags and dried in a forced-air oven at 60 ± 5°C until constant weight was achieved. The experiments were conducted in a completely randomized design (CRD) with six repetitions. Statistical analysis Data were subjected to an analysis of variance (ANOVA) to assess differences among treatments. Normality and homogeneity of variance were evaluated through the inspection of residual plots. Disease severity data and the biomass of aerial and root parts were transformed using \(\:\sqrt{Y}\:+\:0.5\) procedure to achieve normality. Following transformation, ANOVA and other statistical inference procedures were conducted. The Scott-Knott test (α < 0.05) was applied, where appropriate, to determine the significance of differences between mean values, using the Sisvar 5.6 software. The standard error was also calculated for the reported means. Results Antagonism in dual cultures All species of Trichoderma inhibited the growth of M. phaseolina (Fig. 1 ). T. endophyticum exhibited the highest growth in culture media and inhibited more than 66% of the mycelial growth of M. phaseolina (Fig. 1 A–C), receiving a score of 2 according to the Bell, Wells & Markham ( 1982 ) scale. This was followed by T. harzianum and T. asperellum , which showed 59% and 57% inhibition, respectively, and received a score of 3 (Fig. 1 A and B). Although T. harzianum inhibited the pathogen more effectively, it exhibited lower overall growth compared to T. asperellum (Fig. 1 A–C). Hyperparasitism activity was also observed on the M. phaseolina colony, particularly by T. endophyticum (Fig. 1 A). Other control mechanisms against M. phaseolina , such as antibiosis evidenced by the formation of an inhibition halo and competition for space and nutrients in the Petri dish, were noted in all three Trichoderma species (Fig. 1 A). Compatibility test results Trichoderma asperellum showed the highest mycelial growth in the presence of the insecticide Thiamethoxam and the growth regulator (Stimulate®), when added to the culture medium at both tested seed treatment doses compared to the control treatment (Fig. 2 A–D). However, Stimulate® only induced growth of T. asperellum at the lower dose indicated for foliar treatment (Fig. 2 A and C). In contrast, the growth of T. harzianum was inhibited in the presence of both (insecticide and growth regulator), particularly at the higher doses (Fig. 2 A–D). T. endophyticum did not show significant differences in growth whether in the presence or absence of the insecticide (Fig. 2 A and B). However, in the presence of Stimulate®, T. endophyticum experienced growth inhibition at both the foliar and seed treatment doses (Fig. 2 A, C, and D). Seed germination test Macrophomina phaseolina reduced the germination of infected and untreated soybean seeds by 80% (Fig. 3 A). The germination of infected seeds was higher when treated with the three species of Trichoderma and their combinations with Stimulate® and the insecticide Thiamethoxam, with the application of the three species of Trichoderma promoting higher germination than the other treatments. In the infected seeds, Thiamethoxam and Stimulate® did not promote an increase in seed germination compared to the control. In the non-infected seeds, the treatments did not increase seed germination; however, T. harzianum and its combinations with Stimulate® and Thiamethoxam caused a reduction in the germination rate of these seeds. When Thiamethoxam and Stimulate® were applied without T. harzianum , they did not reduce the germination of these seeds. Infected and treated seeds compared to healthy and treated seeds had a lower germination rate, except for the treatments involving T. harzianum and its combination with Stimulate®, where there was no significant difference. The presence of M. phaseolina also reduced the germination velocity index (IVG) of the seeds (Fig. 3 B). Infected and treated seeds had a lower IVG compared to healthy seeds, except for the treatment with isolated T. harzianum , which did not differ between healthy and inoculated seeds. When the seeds were infected and treated with Trichoderma , regardless of the species, and their combinations with Thiametoxam and Stimulate®, a higher IVG was observed compared to the infected and untreated seeds. A lower IVG was observed in infected seeds treated with Stimulate® and Thiametoxam, which did not differ from the infected and untreated seeds. In healthy seeds, the treatments did not provide a higher IVG; however, T. harzianum and its combinations with Thiametoxam and Stimulate® caused a reduction in IVG, impairing the germination speed of the healthy seeds. The dry biomass of the aerial part (DBAP) of seedlings originating from infected and treated seeds was higher than that of non-treated seeds in the treatments with the combination of T. endophyticum with Stimulate® or with T. asperellum and T. harzianum , both combined with Thiamethoxam (Table 2 ). Among these, the combination of T. endophyticum with Stimulate® was the treatment that induced the highest accumulation of DBAP. In healthy seeds, only T. endophyticum provided a greater accumulation of DBAP, while T. harzianum and its combinations with Thiamethoxam and Stimulate® as well as Thiamethoxam alone negatively influenced this variable. The presence of M. phaseolina in the seeds, across all treatments, affected the accumulation of DBAP, reducing the growth of the aerial part of the seedlings compared to those originating from healthy seeds. In infected seeds, all treatments induced greater accumulation of dry root biomass (DBRP), except for Stimulate® and Thiamethoxam (Table 2 ). None of the treatments induced accumulation of DBRP in seedlings from healthy seeds. The presence of M. phaseolina in the seeds negatively affected the accumulation of DBRP in all treatments when compared to the DBRP of seedlings from healthy and treated seeds. Emergence and development of soybean seedlings in pots under controlled conditions The disease severity index (DSI) was influenced by the inoculation methods of M. phaseolina and the treatments applied to the soybean seeds (Fig. 4 ). When the pathogen was inoculated on the seeds, the severity of charcoal rot was reduced with the application of T. harzianum and T. endophyticum , both alone and in combination with Stimulate®. The other treatments did not reduce the severity of charcoal rot when compared to the inoculated, untreated seeds. When healthy seeds were sown in an infested substrate, all treatments reduced the severity of charcoal rot compared to untreated seeds. The application of the three species of Trichoderma and T. endophyticum combined with Stimulate® were the treatments that most significantly reduced the symptoms of charcoal rot in these seeds. The severity of charcoal rot was greater when the pathogen was infecting the seeds than when it was infesting the substrate, except for the applications of Thiamethoxam and T. endophyticum and its combination with Stimulate®. The dry biomass of aerial parts (DBAP*) and root biomass (DBRP*) of the plants was also influenced by the inoculation methods and the treatments applied to the seeds (Table 2 ). In the infected seeds, all treatments resulted in a greater accumulation of DBAP*, except for T. endophyticum combined with Thiamethoxam or with T. harzianum . The application of the three species of Trichoderma , Thiamethoxam alone, and the combinations of Thiamethoxam with T. asperellum and T. endophyticum with Stimulate® were the treatments that induced the highest accumulation of DBAP* in the inoculated seeds. No treatment induced an accumulation of DBAP* in plants derived from healthy seeds sown in an infested substrate. When comparing the inoculation methods, treatments with Stimulate® and its combinations with the three species of Trichoderma , as well as Thiamethoxam combined with T. endophyticum or T. harzianum , resulted in a lower accumulation of DBAP* in plants originating from infected seeds, compared to those in an infested substrate. All treatments promoted a greater accumulation of root biomass (DBRP*), except for T. endophyticum combined with Thiamethoxam or T. harzianum in the inoculated seeds (Table 2 ). The three species of Trichoderma and T. endophyticum combined with Stimulate® were the treatments that most induced DBRP*. No treatment resulted in a greater accumulation of DBRP* when the substrate was infested by the pathogen. Comparing the inoculation methods, the seeds that were inoculated and treated with Stimulate® and its combinations with T. asperellum or T. harzianum , as well as Thiamethoxam combined with T. endophyticum or T. harzianum , showed the highest accumulations of DBRP*, in comparison to the healthy seeds that received the same treatments and were sown in an infested substrate. Discussion Trichoderma endophyticum , T. asperellum , and T. harzianum , as well as the combination of T. endophyticum with Stimulate®, showed promising results in the treatment of soybean seeds for controlling charcoal rot. In the antagonism in dual cultures test, all species of Trichoderma demonstrated antagonistic potential, forming inhibition halos, reducing the colony, and parasitizing M. phaseolina . This effect is attributed to the combined action of various control mechanisms employed by the antagonists, such as competition, hyperparasitism, the ability to produce antimicrobial compounds (Chua, Soung & Ting, 2024; Kumari et al., 2024 ), and enzymes that degrade the cell walls of hyphae, like chitinase and β-1,3-glucanase (Kumari et al., 2024 ). In the compatibility test among the three species of Trichoderma with the plant growth regulator Stimulate® and the insecticide Thiamethoxam used in the treatment of soybean seeds, T. asperellum showed compatibility with both products, exhibiting greater mycelial growth when the products were present in the culture medium. This is likely because T. asperellum utilizes ingredients present in both formulations for its growth (Alves, Moino Júnior & Almeida, 1998). Conversely, T. endophyticum experienced inhibited mycelial growth with Stimulate®, while T. harzianum was inhibited in the presence of both products. In contrast to the findings of this study, Dwivedi & Vishunavat ( 2018 ) demonstrated that T. harzianum was compatible with Thiamethoxam. This divergence in results indicates that the use of different species of Trichoderma or even different isolates of the same species can yield varied compatibility outcomes with chemical products applied in seed treatment. Although this is the first study testing the compatibility between Trichoderma species and plant growth regulators, these products should not inhibit the growth and multiplication of biocontrol agents, as they do not possess fungitoxic or fungistatic actions (Medeiros et al., 2019 ), which could interfere with their efficacy. In the inoculated and untreated seeds, M. phaseolina was able to reduce soybean seed germination by 80%. However, it is important to note that the artificial inoculation method used provided high exposure of the seeds to the pathogen, which does not occur naturally, resulting in seeds with a high infection rate. Despite this, the three species of Trichoderma and the combination of T. endophyticum with the growth regulator Stimulate® increased the germination rate of the highly infected seeds. In contrast, Stimulate® and Thiamethoxam were unable to increase the germination of the infected seeds, and thus are not effective in controlling M. phaseolina in soybean seeds, as expected since they are a growth regulator and an insecticide, respectively. In healthy seeds that were treated, no increase in germination was observed in any of the treatments. This may be related to the high quality of the seed lot used, which had a 98% germination rate, as seeds with medium and low vigor are more responsive to different seed treatments (Carvalho & Nakagawa, 2012 ). However, the germination of healthy seeds treated with T. harzianum and its combinations with Thiamethoxam or the growth regulator Stimulate® was reduced, whereas Thiamethoxam and the growth regulator applied individually did not cause this reduction. It is known that Thiamethoxam does not have a toxic effect on soybean seed germination (Catão et al., 2024 ). Thus, this demonstrates that T. harzianum was the causal agent of the reduction in seed germination, which may be due to the tested dose. It is known that applying doses above or below the recommended level for Trichoderma species can alter their effectiveness or even hinder seed germination by acting as a saprophytic agent, utilizing the seed as a substrate (Woo et al., 2023 ). Higher germination rates of the infected seeds were also observed when treated with the three species of Trichoderma , the combination of T. endophyticum with Stimulate®, and Thiamethoxam combined with T. harzianum . However, in vitro, Stimulate® inhibited the mycelial growth of T. endophyticum , while Thiamethoxam inhibited the growth of T. harzianum . What may have occurred is that, upon seed germination, part of the growth regulator and Thiamethoxam was absorbed, and another part was diluted in the substrate, resulting in lower concentrations than in the culture medium, which did not hinder T. endophyticum and T. harzianum from controlling M. phaseolina in the seeds. Regarding the dry biomass of both shoots and roots, plants from infected seeds showed that all treatments, except for Thiamethoxam and Stimulate® applied alone, were effective. T. endophyticum and its combination with Stimulate® stood out as the best treatments, also inducing growth in the shoots and roots of plants from healthy seeds. These results demonstrate the ability of the three species of Trichoderma to control M. phaseolina , which leads to greater development of plants from infected seeds, and T. endophyticum in combination with Stimulate® promotes growth in the shoots and roots of soybeans. It is known that Trichoderma species can act as stimulants, promoting plant development through phytohormones, improving nutrient assimilation (especially phosphorus), and inducing plant resistance (Harman et al., 2004 ). The literature did not reveal studies using the combination of T. endophyticum and T. asperellum with Stimulate® to promote growth or for seed treatment to control charcoal rot in soybeans. Some studies have demonstrated the ability of Thiamethoxam to induce growth in cotton plants (Lauxen; Villela & Soares, 2010 ), stimulate the physiological performance of carrot seeds (Almeida et al., 2012 ), activate genes of enzymes related to secondary plant metabolism, producing precursors of plant hormones (Castro, 2006 ), and at low doses, increase leaf area and root growth in soybean plants (Godói et al., 2023 ). In this study, Thiamethoxam did not promote growth in either the shoots or roots of Intacta Xtend M6110 i2x® soybean seeds. In fact, the active ingredient caused a reduction in the root growth of uninfected seedlings. This divergence in results may be due to the fact that different genotypes and plant species may respond differently to the use of Thiamethoxam as well as to the growth regulator Stimulate®. In pots under controlled conditions, although M. phaseolina negatively affected soybean seeds, most biocontrol treatments, except for the combination of Thiamethoxam with T. endophyticum or T. harzianum , resulted in greater plant growth compared to inoculated and untreated seeds. It's crucial to highlight that seeds severely infected with M. phaseolina often do not germinate or even emerge (Abawi & Pastor Corrales, 1990). This can be attributed to the direct contact of M. phaseolina with the seeds, causing physical damage to the seed coat, leading to rotting. An important characteristic of M. phaseolina is its high capacity to produce hydrolytic enzymes and lignocelluloses to penetrate host tissues (Marquez et al., 2021 ). Therefore, when seeds are inoculated with the pathogen, the damage is more severe, hindering physiological processes related to water and nutrient absorption, photosynthesis, and growth (Cruciol & Costa, 2018 ). However, the application of T. harzianum and T. endophyticum , both alone and in combination with Stimulate®, drastically reduced the severity of charcoal rot, demonstrating the biocontrol potential of these antagonists in suppressing symptoms of charcoal rot even in severe infections, starting from the early stages of plant development. These results may be attributed to a complex and simultaneous action of various mechanisms, such as competition due to rapid and intense colonization of the seed and root zones (Bucio; Flores & Estrella, 2015 ), which makes it difficult for the pathogen to establish itself. Other mechanisms include the production of antimicrobial compounds, hyperparasitism, and disease escape during pre- and post-emergence due to induced greater root growth, aiding in nutrient and water acquisition as well as shoot development (Melo, 1991 ). It is important to note that plant-pathogen-antagonist-environment interactions are complex, with both biotic and abiotic factors directly influencing these interactions. M. phaseolina is a pathogen whose microsclerotia germination and disease severity in the field are increased by temperatures between 30°C and 33°C (Viana & Souza, 2002 ). Even under conditions favorable for M. phaseolina infection and colonization imposed in this study, the three Trichoderma species managed to mitigate the destructive effects of the pathogen on soybean seeds with high infection rates. This indicates that Trichoderma species can alleviate stress caused by high temperatures in soybean plants, making them less susceptible to disease occurrence. Therefore, selecting antagonists should take this factor into account. Within the Macrophomina genus, there is a wide range of morphological, pathogenic, physiological, and genetic variability among its isolates (Reznikov et al., 2018 ). This diversity allows the fungus to adapt to various environmental conditions, hosts, and control measures implemented in its management. This scenario further underscores the importance of implementing biological control agents as a strategy for disease management, since these agents exhibit different mechanisms of action that can work independently or in concert against M. phaseolina and the host plant. Consequently, the selection of pathogen populations resistant to biocontrol agents becomes less of a challenge. This study demonstrated that the three Trichoderma species that were evaluated have the potential to protect and control M. phaseolina when applied as seed treatments. Notably, T. endophyticum and T. harzianum are capable of controlling charcoal rot even in severe infections, as well as stimulating the growth of both the aerial and root parts of infected soybean plants. This highlights the relevance of biocontrol agents in the search for sustainable and effective strategies for soybean management. Conclusion Trichoderma asperellum , T. endophyticum , and T. harzianum exhibit antagonistic effects against M. phaseolina , acting through different mechanisms to suppress the pathogen. The biocontrol agents are compatible with the insecticide Thiamethoxam, and the growth regulator Stimulate®, except for T. harzianum with both products and T. endophyticum with Stimulate®. For the soybean cultivar Intacta Xtend M6110 i2x®, in the absence of the pathogen, only T. endophyticum and its combination with Stimulate® enhance seedling vigor, while T. harzianum at a concentration of 1 × 10 8 spores mL − 1 negatively affects seed germination and seedling growth. The greatest growth of soybean seedlings, originating from seeds infected with M. phaseolina , is induced by all three Trichoderma species and T. endophyticum combined with Stimulate®. Thiamethoxam and Stimulate® do not promote increased germination or growth of Intacta Xtend M6110 i2x® soybean seedlings, regardless of whether the seeds are infected or healthy. In seeds with a high rate of infection, all three Trichoderma species help control charcoal rot, particularly T. harzianum , T. endophyticum , and their combination with Stimulate®, which significantly reduce the damage caused by M. phaseolina . Meanwhile, Thiamethoxam and Stimulate® do not have fungitoxic effects and do not control charcoal rot. Declarations Author contributions All authors contributed to the study's conception and design . JDV conceived and designed the study, performed the sampling, processed and analyzed the data, and wrote the manuscript. ACS and JL reviewed drafts and contributed to writing the manuscript. SFC, JCJCC, EFS, and TFM implemented and monitored the experiment and compiled the data. All authors read and approved the final manuscript. Project funding This study was supported by the Brazilian Federal Agency for Support and Evaluation of Graduate Education [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)]. Data Availability The datasets generated and/or analyzed during the current study are available in the Zenodo repository (Deboni da Veiga et al. 2024–2025) at https://doi.org/10.5281/zenodo.14245506. Conflict of interest The authors declare no competing interests. References Abawi, G. S., Pastor Corrales, M. A. (1990) Root Rots of Beans in Latin America and Africa: Diagnosis, Research Methodologies, and Management Strategies. Centro Internacional de Agricultura Tropical (CIAT), Cali, CO, pp.114 (publicação CIAT nº 35). https://hdl.handle.net/10568/54258. AGROFIT (2024) Ministry of Agriculture and Livestock . https://www.gov.br/agricultura/pt-br/assuntos/insumos-agropecuarios/insumos-agricolas/agrotoxicos/agrofit. Almeida, A. S., Villela, F., Tillmann, M. A. A., Meneghello, G. E. (2012) Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds ( Daucus carota L.). In: Soloneski S, Larramendy M. Insecticides: Basic and Other Applications . pp. 3–16. https://books.google.com.br/books?hl=pt-BR&lr=lang_en&id=_-GdDwAAQBAJ&oi=fnd&pg= PA3&dq=Thiametoxam+tomate&ots=8YHyQk7WpB&sig=rCsoKkkFxIiXurXbmn5mgGWX29E#v=onepage&q&f=false. Alves, S. B., Moino Jr, A., Almeida, J. E. M. (1998) Phytosanitary products and entomopathogens. In: Alves SB (Ed.) Microbial Control of Insects , Piracicaba: FEALQ, pp. 217–238. Bastakoti, S., Belbase, S., Manandhar, S., Arjyal, C. (2017) Trichoderma species as Biocontrol agent against soil borne fungal pathogens. Nepal J Biotechnol 5:39–45. https://doi.org/10.3126/njb.v5i1.18492. Battaglia, M. E., Martinez, S. I., Covacevich, F., Consolo, V. F. (2024). Trichoderma harzianum enhances root biomass production and promotes lateral root growth of soybean and common bean under drought stress. Annals of Applied Biology . https://doi.org/10.1111/aab.12909. Bell, D. K., Wells, H. D., Markham, C. R. (1982) In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology 72 (4): 379–382. 10.1094/Phyto-72-379. Bellaloui, N., Mengistu, A., Smith, J. R., Abbas, H.K., Accinelli, C., Shier, W. T. (2023) Soybean Seed Sugars: A role in the mechanism of resistance to charcoal rot and potential use as biomarkers in selection. Plants 12 , 392. 10.3390/plants12020392. Bucio, J. L., Flores, R. P., Estrella, A. H. (2015) Trichoderma as biostimulant: exploiting the multilevel properties of a plant beneficial fungus. Sci Hortic 196 :109–123. https://doi.org/10.1016/j.scienta.2015.08.043. Carvalho, N. M., Nakagawa, J. (2012). Seeds: science, technology, and production . Funep, Jaboticabal. Castro, P. R. C. (2006) Hormonal agrochemicals in tropical agriculture . Piracicaba: ESALq. 46 pp. (Rural Producer Series, 32). https://www.esalq.usp.br/biblioteca/sites/default/files/publicacoes-a-venda/pdf/SPR32.pdf. Catão, H. C. R. M., Pontes, B. S., Pinheiro, D. T., Filho, M. A. O., Santos, A. L. C., Zolla, M. C. (2024) Chemical treatment and mobilization of reserves of soybean seeds under water déficit . J Seed Sci 46 . https://doi.org/10.1590/2317-1545v46278828. Chen, H., Li, H., Liu, Z., Zhang, C., Zhang, S., Atkinson, P. M. (2023) A novel Greenness and Water Content Composite Index (GWCCI) for soybean mapping from single remotely sensed multispectral images. Remote Sens Environ 295 . https://doi.org/10.1016/j.rse.2023.113679. Chua, R. W., Song, K. P., Ting, A. S. Y. (2024) Characterization and antimicrobial activities of bioactive compounds from endophytic Trichoderma asperellum isolated from Dendrobium orchids. Biol 79 :569–584. https://doi.org/10.1007/s11756-023-01562-9. Cohen, R., Elkabetz, M., Paris, H. S., Gur, A., Dai, N., Rabinovitz, O., Freeman, S. (2022) Occurrence of Macrophomina phaseolina in Israel: Challenges for Disease Management and Crop Germplasm Enhancement. Plant Dis 106 :15–25. https://doi.org/10.1094/PDIS-07-21-1390-FE. Cruciol, G. C. D., Costa, M. L. N. (2018) Influence of inoculation methodologies of Macrophomina phaseolina on the performance of soybean cultivars. Summa Phytopathology 44 :32–37. https://www.scielo.br/j/sp/a/KtXhXXgVbntCNf6nspgJHBC/?format=pdf&lang=pt. Deboni da Veiga, J. (2024–2025) Trichoderma endophyticum , T. asperellum e T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina [Dataset]. Zenodo. https://doi.org/10.5281/zenodo.14245506. Dhingra, O. D., Sinclair, J. B. (1978) Biology and Pathology of Macrophimina phaseolina . Australasian Plant Pathology 7 , 25. https://doi.org/10.1007/BF03212337. Dwivedi, M., Vishunavat, C. (2018) Compatibility of Trichoderma strains and their mutants with common agrochemicals. J Pharmacogn Phytochem 7 (5):2744–2747. https://www.phytojournal.com/archives/2018/vol7issue5/PartAT/7-4-561-108.pdf. Gajera, H., Bambharolia, R., Patel, S., Khatrani, T., Goalkiya, B. (2012) Antagonism of Trichoderma spp. against Macrophomina phaseolina : evaluation of coiling and cell wall degrading enzymatic activities. J Plant Pathol Microbiol 3 :149. 10.4172/2157-7471.1000149. Godói, C. T. D., Campos, S. O., Monteiro, S. H., Ronchi, C. P., Silva, A. A., Guedes, R. N. C. (2023) Thiamethoxam in soybean seed treatment: Plant bioactivation and hormesis, besides whitefly control? Sci Total Environ 857 (3):20. https://doi.org/10.1016/j.scitotenv.2022.159443. Goidanish, G. (1947) Revisione del genere Macrophomina Petrak. Species tipica: Macrophomina phaseolina (Tass.) Goid. nov. comb. nec. M. phaseoli . Ashby Annali della Sperimentazione Agraria 1 : 449–461. Gupta, G. K., Sharma, S. K., Ramteke, R. (2012) Biology, epidemiology and management of the pathogenic fungus Macrophomina phaseolina (Tassi) goid with special reference to charcoal rot of soybean ( Glycine max (L.) Merrill). J. Phytopathol 160 :167–180. 10.1111/j.1439-0434.2012.01884.x. Harman, G. E., Howell, C. R., Viterbo, A., Chet, I., Lorito, M. (2004) Trichoderma species--opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2 (1):43–56. 10.1038/nrmicro797. Inglada, J., Arias, M., Tardy, B., Hagolle, O., Valero, S., Morin, D., Dedieu, G., Sepulcre, G., Bontemps, S., Defourny, P., Koetz, B. (2015) Assessment of an operational system for crop type map production using high temporal and spatial resolution satellite optical imagery. Remote Sens 7 :12356–12379. https://doi.org/10.3390/rs70912356. Iqbal, U., Mukhtar, T. (2014) Morphological and pathogenic variability among Macrophomina phaseolina isolates associated with mungbean ( Vigna radiata L.) Wilczek from Pakistan. Sci World J 1 . 10.1155/2014/950175. Kamolovna, K. N., Zavkiddinova, A. S. (2023) 11th -ICARHSE International Conference on Advance Research in Humanities, Applied Sciences and Education . https://conferencea.org. Kaur, S., Dhillon, G. S., Brar, S. K., Vallad, G. E., Chand, R., Chauhan, V. B. (2012) Emerging phytopathogen Macrophomina phaseolina : biology, economic importance and current diagnostic trends. Crit Rev Microbiol 38 (2):136–151 . 10.3109/1040841x.2011.640977. Kumari, R., Kumar, V., Arukha, A. P., Rabbee, M. F., Ameen, F., Koul, B. (2024) Screening of the Biocontrol Efficacy of Potent Trichoderma Strains against Fusarium oxysporum f.sp. cicero and Sclerotium rolfsii Causing Wilt and Collar Rot in Chickpea. Microorg 12 :1280. https://doi.org/10.3390/microorganisms12071280. Larran, S., Simón, M. R., Santamarina, M. P., Caselles, J. R., Consolo, V. F., Perelló, A. (2023) Endophytic Trichoderma strains increase soya bean growth and promote charcoal rot control. J Saudi Soc Agric Sci 22 (7):395–406. https://doi.org/10.1016/j.jssas.2023.03.005. Lauxen, L. R., Villela, F. A., Soares, R. C. (2010) Physiological performance of cotton seeds treated with thiamethoxam. Braz J Seeds 32 (3):061–068. https://doi.org/10.1590/S0101-31222010000300007. Maguire, J. D. (1962) Speed of germination aid in selection and evaluation for seedling emergence and vigor. Crop Sci 2 (2):176–177. Machado, J., Oliveira, J., Vieira, M., Alves, M. (2001) Inoculação artificial de sementes de soja por fungos, utilizando solução de manitol. Rev Bras Semente 23:95–101. Marquez, N., Giachero, M. L., Declerck, S., Ducasse, D. A. (2021) Macrophomina phaseolina : General Characteristics of Pathogenicity and Methods of Control. Front Plant Sci 12 : 634397. https://doi.org/10.3389/fpls.2021.634397. Mckinney, H. H. (1923) Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium sativum . J Agric Res 26 (5):195–218. https://handle.nal.usda.gov/10113/IND43966679. Medeiros, F. H. V, Guimarães, R. A., Silva, J. C. P., Magalhães, V. C., Souza, J. T. (2019) Trichoderma: interactions and strategies. In: Meyer, M. C., Mazaro, S. M., Silva, J. C. Trichoderma: use in agriculture . Melo, I. S. (1991) Potential of using Trichoderma spp. in the biological control of plant diseases. In: Bettiol, W. Biological control of plant diseases . EMBRAPA, pp. 154. https://www.alice.cnptia.embrapa.br/handle/doc/10080. Ons, L., Bylemans, D., Thevissen, K., Cammue, B. P. A. (2020) Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control . Microorg 8 (12):1930. https://doi.org/10.3390/microorganisms8121930. Reznikov, S., Chiesa, M. A., Pardo, E. M., Lisi, V., Bogado, N., González, V., Ledesma, F., Morandi, E. N., Ploper, L. D., Castagnaro, A. P. (2018) Soybean- Macrophomina phaseolina -Specific Interations and indentification of a Novel Source of Resistance. Phytopathol 109 :1. https://doi.org/10.1094/PHYTO-08-17-0287-R. Seixas, C. D. S., Soares, R. M., Godoy, C. V., Mayer, M. C., Constamilan, L. M., Dias, W. P., Almeida, A. M. R. (2020) Disease management. In: Seixas CDS, Neumaier N, Balbinot Junior AA, Krzyzanowski FC, Leite RMVBC. Soybean production technologies . EMBRAPA, Londrina. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/223209/1/SP-17-2020-online-1.pdf. Short, G. E., Wyllie, T. D., Bristow, P. R. (1980) Survival of Macrophomina phaseolina . In soil and in residue of soybean. Ecol Epidemiol 70 (1):13–17. https://www.apsnet.org/publications/phytopathology/backissues/Documents/1980Abstracts/Phyto70_13.htm. Šućur, E. J., Petrović, K., Crnković, M., Krsmanović, S., Rajković, M., Kaitović, Ž., Malenčić, Đ. (2023) Susceptibility of the Most Popular Soybean Cultivars in South-East Europe to Macrophomina phaseolina (Tassi) Goid. Plants 12 :2467. https://doi.org/10.3390/plants12132467. Taiz, L., Zeiger, E. (2017) Plant Physiology . 6 ed. Porto Alegre: Artmed. 10.3923/ppj.2012.73.76 USDA (2024). World agricultural production . https://fas.usda.gov/data/world-agricultural-production-11082024. Viana, F. M. P., Souza, N. L. (2002) Effect of temperature-water tension interaction on the germination of micro-sclerotia of Macrophomina phaseolina . Braz Phytopathol 27 :268–272. https://doi.org/10.1590/S0100-41582002000300005. Woo, S. L., Hermosa, R., Lorito, M., Monte, E. (2023) Trichoderma: a multipurpose, plant beneficial microorganism for eco-sustainable agriculture. Nat Rev Microbiol 21 :312–326. https://doi.org/10.1038/s41579-022-00819-5. Wrather, J. A., Shannon, J. G., Carter, T. E., Bond, J. P., Rupe, J. C., Almeida, A. M. R. (2008) Reaction of drought-tolerant soybean genotypes to Macrophomina phaseolina . Plant Health Prog 9 :16. https://doi.org/10.1094/PHP-2008-0618-01-RS. Tables Tables 1 and 2 are available in the Supplementary Files section. Supplementary Files Tables.docx Supplementarymaterial1.docx Supplementarymaterial2.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in European Journal of Plant Pathology → Version 1 posted Editorial decision: Major revisions 12 Oct, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 10 Jul, 2025 Editor invited by journal 03 Jun, 2025 Editor assigned by journal 28 May, 2025 First submitted to journal 26 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6751408","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483507812,"identity":"1df0ac40-0823-4142-901f-b4174cb05ff5","order_by":0,"name":"Jayne Deboni da Veiga","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0001-5265-3340","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Jayne","middleName":"Deboni da","lastName":"Veiga","suffix":""},{"id":483507813,"identity":"ff4a5d86-4c03-40c1-ad78-e97187d33fed","order_by":1,"name":"Juliane Ludwig","email":"","orcid":"","institution":"UFFS: Universidade Federal da Fronteira Sul","correspondingAuthor":false,"prefix":"","firstName":"Juliane","middleName":"","lastName":"Ludwig","suffix":""},{"id":483507814,"identity":"2792a3f0-b041-40ad-aeef-fc2aa68509fe","order_by":2,"name":"Samuel Francisco Chitolina","email":"","orcid":"","institution":"UFFS: Universidade Federal da Fronteira Sul","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"Francisco","lastName":"Chitolina","suffix":""},{"id":483507815,"identity":"8d17e887-89c9-4623-9424-34640f3ea3eb","order_by":3,"name":"José Carlos Júnior da Cruz de Camargo","email":"","orcid":"","institution":"UFFS: Universidade Federal da Fronteira Sul","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Carlos Júnior da Cruz","lastName":"de Camargo","suffix":""},{"id":483507816,"identity":"652b3cda-1726-4091-b4bd-9837be87fd75","order_by":4,"name":"Eveline Ferreira Soares","email":"","orcid":"","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Eveline","middleName":"Ferreira","lastName":"Soares","suffix":""},{"id":483507817,"identity":"654355bd-cca7-4c2f-a3ba-1269bfb55c00","order_by":5,"name":"Thainá Fogliatto Moreira","email":"","orcid":"","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Thainá","middleName":"Fogliatto","lastName":"Moreira","suffix":""},{"id":483507818,"identity":"61700517-ed2c-455f-97fd-2f0eca407095","order_by":6,"name":"André Costa da Silva","email":"","orcid":"https://orcid.org/0000-0001-5505-7678","institution":"UFRGS: Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Costa da","lastName":"Silva","suffix":""}],"badges":[],"createdAt":"2025-05-26 13:44:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6751408/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6751408/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-026-03192-8","type":"published","date":"2026-02-27T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86709799,"identity":"96fd3353-0602-40c0-8082-44b9e1d6865b","added_by":"auto","created_at":"2025-07-14 18:16:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198205,"visible":true,"origin":"","legend":"\u003cp\u003eAntagonism of \u003cem\u003eTrichoderma asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, and \u003cem\u003eT.harzianum\u003c/em\u003eon \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e after 8 days of incubation in culture medium. (a) Dual culture technique demonstrating the antagonism of the three \u003cem\u003eTrichoderma\u003c/em\u003especies against \u003cem\u003eM. phaseolina\u003c/em\u003e, where red lines indicate the inhibition halo, and red arrows highlight the growth of \u003cem\u003eTrichoderma\u003c/em\u003e species over the \u003cem\u003eM. phaseolina \u003c/em\u003ecolony. (b) Percentage of mycelial growth inhibition. (c) Average colony size of the three \u003cem\u003eTrichoderma\u003c/em\u003e species. *Values are presented as the mean of six replicates. Bars with the same letter above did not differ according to the Scott-Knott test (P \u0026lt; 0.001). Corresponding error bars represents standard deviation (S.D.)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/d3fe02fa0421de251643896e.png"},{"id":86709803,"identity":"23bf621c-f104-4104-af56-45ab87451cd4","added_by":"auto","created_at":"2025-07-14 18:16:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258993,"visible":true,"origin":"","legend":"\u003cp\u003eThis figure illustrates the compatibility between doses of the insecticide Thiamethoxam/Cruiser 350 FS® (a and b) and the plant growth regulator/Stimulate® (a, c, and d), recommended for seed treatment (TS) and foliar application (TF) with different \u003cem\u003eTrichoderma\u003c/em\u003e species. Values are presented as means calculated using six replicates. Different uppercase letters indicate significant differences between doses for each \u003cem\u003eTrichoderma\u003c/em\u003especies. Different lowercase letters indicate differences between \u003cem\u003eTrichoderma\u003c/em\u003especies at the same tested dose according to the Scott-Knott test, P \u0026lt; 0.001. Corresponding error bars represents standard deviation (S.D.)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/90f1fc6c65120e6f16c8a004.png"},{"id":86710184,"identity":"3148ec0b-6f49-4b65-bb6c-9935fcc3776d","added_by":"auto","created_at":"2025-07-14 18:24:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105344,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of germination (a) and germination velocity index (IVG) (b) of soybean seeds inoculated or not with \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e and subjected to treatments with \u003cem\u003eTrichoderma asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, \u003cem\u003eT. harzianum\u003c/em\u003e, the plant growth regulator (Stimulate®), the insecticide Thiamethoxam (Cruiser 350 FS), and their combinations. Seeds that had 2 mm of root protrusion were considered germinated. Values are presented as the mean calculated using six replicates. Different uppercase letters indicate significant differences between treatments, and different lowercase letters indicate differences based on inoculation status, according to the Scott-Knott test, P \u0026lt; 0.001. Corresponding error bars represents standard deviation (S.D.)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/f0c4ed4b5e5a717f2ee3c90c.png"},{"id":86710188,"identity":"45f05350-b9c3-43dc-9aca-8cde5d2b51d9","added_by":"auto","created_at":"2025-07-14 18:24:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34364,"visible":true,"origin":"","legend":"\u003cp\u003eDisease Severity Index (DSI) (%) at 24 days after sowing of seeds infected by \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e or healthy seeds sown in substrate infested with the pathogen and subjected to different treatments with \u003cem\u003eTrichoderma endophyticum\u003c/em\u003e, \u003cem\u003eT. asperellum\u003c/em\u003e, \u003cem\u003eT. harzianum\u003c/em\u003e, the plant growth regulator (Stimulate®), Thiamethoxam, and their combinations. Values are presented as the mean calculated using six replicates. The data were transformed to meet normality assumptions (√Y + 0.5). The results are presented in the original scale to facilitate interpretation. Different uppercase letters indicate significant differences between treatments, while different lowercase letters indicate differences between inoculation methods, according to the Scott-Knott test, P \u0026lt; 0.001. Corresponding error bars represents standard deviation (S.D.)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/95b0577fa5f1b991be559d2f.png"},{"id":103765537,"identity":"0973c9a8-5563-48f0-9d6a-f2d9d9a550b0","added_by":"auto","created_at":"2026-03-02 16:03:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1370283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/2b53cc3f-3fe5-4732-92b6-78ab71ad7f34.pdf"},{"id":86710186,"identity":"4df6904d-004e-411d-9a8f-24c3a8392d8d","added_by":"auto","created_at":"2025-07-14 18:24:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21281,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/e1986d076d7f076d881757fa.docx"},{"id":86709808,"identity":"e35fc9d8-cbe8-4562-876c-c7826b364fbd","added_by":"auto","created_at":"2025-07-14 18:16:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":330139,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/fef062d99e8db855e22d48f3.docx"},{"id":86709806,"identity":"301001d3-3bb5-4a11-8f9b-b8222c96ac86","added_by":"auto","created_at":"2025-07-14 18:16:50","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":143721,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6751408/v1/4ef88c5aaf48c5a8f5a045ef.docx"}],"financialInterests":"","formattedTitle":"Trichoderma endophyticum , T. asperellum, and T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoybean (\u003cem\u003eGlycine max\u003c/em\u003e [L.] Merrill) is one of the most important food and economic crops globally (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with a total cultivated area of 139.85\u0026nbsp;million hectares and a production of 394.73\u0026nbsp;million tons (USDA, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is attributed to its high nutritional value, with over a thousand products derived from its grains (Kamolovna \u0026amp; Zavkiddinova, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), making it vital for ensuring global food security (Inglada et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the crop can be affected by various pathogens that may occur from seed germination to grain filling. Among these pathogens is the fungus \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Tassi) Goid (Goidanish, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1947\u003c/span\u003e). It is a generalist soil inhabitant found worldwide, affecting at least 500 plant species across more than 100 families (Marquez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In soybeans, it causes charcoal rot and is one of the main diseases of the crop, capable of occurring at all stages of plant development and leading to production losses of 30\u0026ndash;50% (Šućur Elez et al., 2023). Infected plants exhibit reduced leaf and seed size and may wilt and die prematurely (Wrather et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This fungus produces microsclerotia as a resistant structure, surviving in seeds, crop residues, living plants (Dhingra \u0026amp; Sinclair, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Abawi \u0026amp; Pastor Corrales, 1990), and soil for up to 15 years (Gupta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Due to being a polyphagous pathogen and forming resistant structures, crop rotation as a control strategy does not have a direct effect on reducing the disease in the field (Short; Wyllie \u0026amp; Bristow, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). On the other hand, some consecutively planted crops may even increase pathogen levels in the field. In Brazil, it is common to plant second-season corn after soybean harvest. The consecutive cultivation of these two host crops facilitates the increased incidence of the disease in the field and the perpetuation of the pathogen in the area (Seixas et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Short; Wyllie \u0026amp; Bristow ,1980). Furthermore, in recent years, there has been an increase in the incidence and severity of this disease in cultivated areas, attributed to climate change (Cohen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The severity of charcoal rot is associated with two predominant factors: water deficit and high soil temperatures (Šućur Elez et al., 2023). Under high temperatures (30\u0026ndash;35\u0026deg;C) and soil moisture below 60%, this fungus can cause substantial yield losses in various crops, including soybeans, impacting farmers' yields (Kaur et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gupta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Abawi \u0026amp; Pastor Corrales, 1990). Strategies for managing charcoal rot using fungicides have not been successful (Bellaloui et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and in Brazil, there are no fungicides registered for controlling the disease in soybeans (Agrofit, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). An economically more viable management strategy would be the use of resistant cultivars. However, there are no resistant soybean cultivars available on the market (Bellaloui et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, various cultural, chemical, and genetic control strategies against \u003cem\u003eM. phaseolina\u003c/em\u003e in the field have shown low or no efficiency, making the management of charcoal rot in soybean crops a challenge (Marquez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Biological control, including the use of different species of \u003cem\u003eTrichoderma\u003c/em\u003e, has emerged as an important alternative for controlling various soil-dwelling pathogens, as observed with \u003cem\u003eM. phaseolina\u003c/em\u003e in different hosts (Bastakoti et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Marquez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In soybeans, isolates of \u003cem\u003eT. harzianum\u003c/em\u003e have proven effective in controlling \u003cem\u003eM. phaseolina\u003c/em\u003e (Larran et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, few studies have tested isolates of \u003cem\u003eT. asperellum\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e for controlling charcoal rot in soybeans, as well as their compatibility with growth regulators and insecticides used in soybean seed treatment. Therefore, the objectives of this study were: (i) to evaluate the potential for controlling charcoal rot and promoting growth in soybean plants using \u003cem\u003eT. asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, and \u003cem\u003eT. harzianum\u003c/em\u003e; (ii) to assess the compatibility of the three \u003cem\u003eTrichoderma\u003c/em\u003e species with the plant growth regulator Stimulate\u0026reg; and the insecticide Thiamethoxam used in soybean seed treatment; and (iii) to evaluate the combination of the three \u003cem\u003eTrichoderma\u003c/em\u003e species with the growth regulator Stimulate\u0026reg; and the insecticide Thiamethoxam in treating soybean seeds infected with \u003cem\u003eM. phaseolina\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eFungal strains\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eTrichoderma\u003c/em\u003e isolates were obtained from commercial products: Lalnix Resist\u0026reg; (\u003cem\u003eT. endophyticum\u003c/em\u003e isolate IBCB 56/12, Lallemand\u0026reg;), Organic WP\u0026reg; (\u003cem\u003eT. asperellum\u003c/em\u003e isolate URM 5911, Lallemand\u0026reg;), and Stimucontrol\u0026reg; (\u003cem\u003eT. harzianum\u003c/em\u003e isolate CCT 7589, Simbiose\u0026reg;). An aliquot of each product was spread evenly across a Petri dish containing PDA (Potato Dextrose Agar at 4%). The fungus \u003cem\u003eM. phaseolina\u003c/em\u003e (isolate 231) was obtained from the collection of the Agris Institute. Fragments of the fungus were placed at the center of a Petri dish containing PDA. The Petri dishes were then incubated in a growth chamber at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a photoperiod of 12 hours at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for seven days.\u003c/p\u003e\u003cp\u003eAntagonism in dual cultures\u003c/p\u003e\u003cp\u003eThe dual culture technique was applied to evaluate the antagonism of the three \u003cem\u003eTrichoderma\u003c/em\u003e species against \u003cem\u003eM. phaseolina\u003c/em\u003e, following the method of Bell, Wells \u0026amp; Markham (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Mycelial discs of \u003cem\u003eM. phaseolina\u003c/em\u003e and the three \u003cem\u003eTrichoderma\u003c/em\u003e isolates, each with a diameter of 4 mm, were taken from colonies after seven days of growth. The mycelial discs of \u003cem\u003eM. phaseolina\u003c/em\u003e and the individual \u003cem\u003eTrichoderma\u003c/em\u003e isolates were placed on a PDA culture medium 1 cm from the inner edge of 90 mm diameter Petri dishes, opposite each other. The control treatment consisted solely of a mycelial disc of \u003cem\u003eM. phaseolina\u003c/em\u003e. No fungicide was used as a positive control due to the lack of registered products for controlling \u003cem\u003eM. phaseolina\u003c/em\u003e in soybeans. The Petri dishes were incubated in a growth chamber at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a photoperiod of 12 hours at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for eight days. Afterward, the antagonistic ability of the \u003cem\u003eTrichoderma\u003c/em\u003e isolates against \u003cem\u003eM. phaseolina\u003c/em\u003e was assessed using a scale proposed by Bell, Wells \u0026amp; Markham (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) with some adaptations (Supplementary material 1), as well as by measuring the inhibition of mycelial growth of \u003cem\u003eM. phaseolina\u003c/em\u003e and the \u003cem\u003eTrichoderma\u003c/em\u003e species. To achieve this, the diameters of the colonies of \u003cem\u003eM. phaseolina\u003c/em\u003e and the \u003cem\u003eTrichoderma\u003c/em\u003e species were measured with a digital caliper (Vonder\u0026reg;, China) at two equidistant points, and the averages were subjected to the formula used by Tomah et al., (2024). The experiment was conducted in a completely randomized design (CRD) with six repetitions, with each Petri dish serving as a repetition.\u003c/p\u003e\u003cp\u003eCompatibility test\u003c/p\u003e\u003cp\u003eThe compatibility of \u003cem\u003eTrichoderma\u003c/em\u003e species with the plant growth regulator (Stimulate\u0026reg;, Stoller) and the insecticide Thiamethoxam (Cruiser 350 FS\u0026reg;, Syngenta) was evaluated. Both the minimum and maximum recommended doses of the growth regulator were tested for foliar application on soybeans (250 and 500 mL of the active ingredient per 150 L of solution) and seed treatment (5 and 7.5 mL of the active ingredient per kg of seeds), as well as for Thiamethoxam in seed treatment (0.5 and 3 mL of the active ingredient per kg of seeds) (Agrofit \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The recommended seed treatment doses were added to the culture medium in the amount specified by the manufacturer to treat 2 g of seeds per plate. The products were mixed in Erlenmeyer flasks containing a PDA medium. For each 90 mm diameter Petri dish, 15 mL of the solution was poured in. After the culture medium solidified, a 5 mm diameter mycelial disc of each \u003cem\u003eTrichoderma\u003c/em\u003e species was placed at the center of each Petri dish. The plates were then incubated in a growth chamber at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a photoperiod of 12 hours at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. After 72 hours of incubation, two orthogonal measurements of the colony diameters (mm) were taken using a digital caliper. The experiment was conducted in a completely randomized design (CRD) with six repetitions per treatment.\u003c/p\u003e\u003cp\u003eInoculation of soybean seeds with \u003cem\u003eM. phaseolina\u003c/em\u003e\u003c/p\u003e\u003cp\u003eSoybean seeds of the cultivar Intacta Xtend M6110 i2x\u0026reg; were used for this study. Initially, the seeds underwent a germination and health test to assess the quality of the batch, following the Seed Analysis Rules (BRASIL, 2009). Although the seeds exhibited good health and were free of \u003cem\u003eM. phaseolina\u003c/em\u003e, they were disinfected in 1% sodium hypochlorite for 1 minute, then rinsed three times with sterile distilled water and air-dried for 24 hours. A portion of the disinfected seeds was inoculated with \u003cem\u003eM. phaseolina\u003c/em\u003e. The osmotic restriction method using mannitol was employed to achieve an osmotic potential of \u0026minus;\u0026thinsp;1.0 MPa (Machado et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The amount of mannitol added to the PDA culture medium was calculated based on the osmotic potential formula proposed by Van\u0026rsquo;t Hoff (Taiz \u0026amp; Zeiger, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ten mL portions of the sterilized PDA medium were transferred to 90 mm Petri dishes and allowed to solidify. A 5 mm diameter disc containing \u003cem\u003eM. phaseolina\u003c/em\u003e mycelium was placed in the center of each Petri dish, which was then incubated in a growth chamber at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a photoperiod of 12 hours at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for seven days. Following this incubation, a total of 100 seeds were placed in each Petri dish, remaining in contact with the fungus for 72 hours (adapted from Cruciol \u0026amp; Costa, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeed treatments\u003c/p\u003e\u003cp\u003eThe doses of the plant growth regulator and the insecticide, recommended by the manufacturers (Agrofit, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and which demonstrated the lowest percentage of inhibition of growth among the different species of \u003cem\u003eTrichoderma\u003c/em\u003e, were used in the treatments. The treatments included: \u003cem\u003eT. asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, \u003cem\u003eT. harzianum\u003c/em\u003e, Stimulate\u0026reg;, Thiamethoxam, Stimulate\u0026reg; + \u003cem\u003eT. asperellum\u003c/em\u003e, Stimulate\u0026reg; + \u003cem\u003eT. endophyticum\u003c/em\u003e, Stimulate\u0026reg; + \u003cem\u003eT. harzianum\u003c/em\u003e, Thiamethoxam\u0026thinsp;+\u0026thinsp;\u003cem\u003eT. asperellum\u003c/em\u003e, Thiamethoxam\u0026thinsp;+\u0026thinsp;\u003cem\u003eT. endophyticum\u003c/em\u003e, and Thiamethoxam\u0026thinsp;+\u0026thinsp;\u003cem\u003eT. harzianum\u003c/em\u003e. For the treatment of seeds with the biocontrol agents, the fungi were previously cultured on a PDA medium in a growth chamber at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12-hour photoperiod at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for seven days. After this period, an inoculum suspension was prepared by adding sterile water containing 1% Tween 20 to the plates. The surface of the colonies was scraped with a sterile Drigalski spatula, and the spore suspension was filtered using a double layer of gauze, adjusting the concentration to 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e spores/mL using a hemocytometer. The following amounts were used: 0.5, 2, and 5 mL.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of seeds for the insecticide (Thiamethoxam), the spore suspension, and the plant growth regulator (Stimulate\u0026reg;), respectively. Control treatments consisted of inoculated and non-inoculated seeds treated with 2 mL.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of distilled water containing 1% Tween 20. The seeds were placed in plastic bags with the treatments, agitated manually for 1 minute, and then left open to dry and for the adhesion of the different treatments to the seeds.\u003c/p\u003e\u003cp\u003eSeed germination test\u003c/p\u003e\u003cp\u003eThe standard germination test (roll paper method) outlined in the MAPA manual (2009) was employed to assess germination rates and the germination speed index (IVG) of the seeds. A total of 300 seeds were used per treatment, divided into six repetitions of 50 seeds each. The seeds were incubated in a growth chamber at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12-hour photoperiod at 40 \u0026micro;mol s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. Daily counts of the number of germinated seeds were conducted to determine the IVG, following Maguire (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) methodology. On the eighth day of incubation, the germination rate and dry biomass of both the aerial parts (DBAP) and roots (DBRP) were evaluated. Seeds exhibiting a root protrusion greater than 2 mm were considered germinated. From each repetition, ten seedlings were randomly selected for measuring shoot and root lengths using a digital caliper. For dry biomass determination, ten seedlings were cut at the root insertion height, separating the aerial parts from the roots, which were then placed in kraft paper bags and dried in a forced-air oven at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C until a constant weight was achieved. The experiment was conducted in a completely randomized design (CRD).\u003c/p\u003e\u003cp\u003eEmergence and development of soybean seedlings in pots under controlled conditions\u003c/p\u003e\u003cp\u003eInoculated and treated seeds were germinated in plastic pots, with three seeds per pot, each containing 900 g of sterile substrate (Red Latosol:sand:manure; 2:1:1 by weight). The substrate was sterilized by autoclaving three times at 120\u0026deg;C to ensure complete disinfection. Non-inoculated and treated seeds were sown in a substrate previously infested with \u003cem\u003eM. phaseolina\u003c/em\u003e. For this, rice grains with husks were placed in Erlenmeyer flasks and moistened with distilled water for 10 minutes. After removing the water, the grains were autoclaved for 20 minutes at 120\u0026deg;C. Once cooled, 20 discs of \u003cem\u003eM. phaseolina\u003c/em\u003e mycelium (5 mm in diameter) were added, and the flasks were incubated in a growth chamber at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12-hour photoperiod for 14 days. The flasks were shaken daily to ensure uniform colonization. Subsequently, nine rice grains were deposited in each pot at a depth of 5 cm and maintained in a germination room at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C for 7 days (adapted from Cruciol \u0026amp; Costa \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After this period, the seeds were sown in the substrate. The pots were kept in a controlled environment room with an average temperature of 25\u0026deg;C and relative air humidity around 80%. Control treatments consisted of inoculated and non-inoculated seeds treated with 2 mL.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of distilled water and sown in either sterile or infested substrate. Following seedling emergence, thinning was performed to leave one seedling per pot. On day 24 post-sowing, symptoms of gray rot were assessed using the rating scale proposed by Abawi and Pastor Corrales (1990), adapted by the authors (Supplementary material 2), and the disease severity index (ISD) was calculated according to McKinney (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1923\u003c/span\u003e). The dry biomass of the aerial parts (DBAP*) and roots (DBRP*) was also evaluated. Plants were cut at the base, and the roots were rinsed with running water to avoid damaging the root system. Both aerial parts and roots were placed in kraft paper bags and dried in a forced-air oven at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C until constant weight was achieved. The experiments were conducted in a completely randomized design (CRD) with six repetitions.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were subjected to an analysis of variance (ANOVA) to assess differences among treatments. Normality and homogeneity of variance were evaluated through the inspection of residual plots. Disease severity data and the biomass of aerial and root parts were transformed using \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sqrt{Y}\\:+\\:0.5\\)\u003c/span\u003e\u003c/span\u003e procedure to achieve normality. Following transformation, ANOVA and other statistical inference procedures were conducted. The Scott-Knott test (α\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was applied, where appropriate, to determine the significance of differences between mean values, using the Sisvar 5.6 software. The standard error was also calculated for the reported means.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAntagonism in dual cultures\u003c/p\u003e\n\u003cp\u003eAll species of \u003cem\u003eTrichoderma\u003c/em\u003e inhibited the growth of \u003cem\u003eM. phaseolina\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eT. endophyticum\u003c/em\u003e exhibited the highest growth in culture media and inhibited more than 66% of the mycelial growth of \u003cem\u003eM. phaseolina\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C), receiving a score of 2 according to the Bell, Wells \u0026amp; Markham (\u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e) scale. This was followed by \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. asperellum\u003c/em\u003e, which showed 59% and 57% inhibition, respectively, and received a score of 3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Although \u003cem\u003eT. harzianum\u003c/em\u003e inhibited the pathogen more effectively, it exhibited lower overall growth compared to \u003cem\u003eT. asperellum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C). Hyperparasitism activity was also observed on the \u003cem\u003eM. phaseolina\u003c/em\u003e colony, particularly by \u003cem\u003eT. endophyticum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Other control mechanisms against \u003cem\u003eM. phaseolina\u003c/em\u003e, such as antibiosis evidenced by the formation of an inhibition halo and competition for space and nutrients in the Petri dish, were noted in all three \u003cem\u003eTrichoderma\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eCompatibility test results\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrichoderma asperellum\u003c/em\u003e showed the highest mycelial growth in the presence of the insecticide Thiamethoxam and the growth regulator (Stimulate\u0026reg;), when added to the culture medium at both tested seed treatment doses compared to the control treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). However, Stimulate\u0026reg; only induced growth of \u003cem\u003eT. asperellum\u003c/em\u003e at the lower dose indicated for foliar treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and C). In contrast, the growth of \u003cem\u003eT. harzianum\u003c/em\u003e was inhibited in the presence of both (insecticide and growth regulator), particularly at the higher doses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). \u003cem\u003eT. endophyticum\u003c/em\u003e did not show significant differences in growth whether in the presence or absence of the insecticide (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). However, in the presence of Stimulate\u0026reg;, \u003cem\u003eT. endophyticum\u003c/em\u003e experienced growth inhibition at both the foliar and seed treatment doses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, C, and D).\u003c/p\u003e\n\u003cp\u003eSeed germination test\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMacrophomina phaseolina\u003c/em\u003e reduced the germination of infected and untreated soybean seeds by 80% (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The germination of infected seeds was higher when treated with the three species of \u003cem\u003eTrichoderma\u003c/em\u003e and their combinations with Stimulate\u0026reg; and the insecticide Thiamethoxam, with the application of the three species of \u003cem\u003eTrichoderma\u003c/em\u003e promoting higher germination than the other treatments. In the infected seeds, Thiamethoxam and Stimulate\u0026reg; did not promote an increase in seed germination compared to the control. In the non-infected seeds, the treatments did not increase seed germination; however, \u003cem\u003eT. harzianum\u003c/em\u003e and its combinations with Stimulate\u0026reg; and Thiamethoxam caused a reduction in the germination rate of these seeds. When Thiamethoxam and Stimulate\u0026reg; were applied without \u003cem\u003eT. harzianum\u003c/em\u003e, they did not reduce the germination of these seeds. Infected and treated \u003cem\u003eseeds\u003c/em\u003e compared to healthy and treated seeds had a lower germination rate, except for the treatments involving \u003cem\u003eT. harzianum\u003c/em\u003e and its combination with Stimulate\u0026reg;, where there was no significant difference.\u003c/p\u003e\n\u003cp\u003eThe presence of \u003cem\u003eM. phaseolina\u003c/em\u003e also reduced the germination velocity index (IVG) of the seeds (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Infected and treated seeds had a lower IVG compared to healthy seeds, except for the treatment with isolated \u003cem\u003eT. harzianum\u003c/em\u003e, which did not differ between healthy and inoculated seeds. When the seeds were infected and treated with \u003cem\u003eTrichoderma\u003c/em\u003e, regardless of the species, and their combinations with Thiametoxam and Stimulate\u0026reg;, a higher IVG was observed compared to the infected and untreated seeds. A lower IVG was observed in infected seeds treated with Stimulate\u0026reg; and Thiametoxam, which did not differ from the infected and untreated seeds. In healthy seeds, the treatments did not provide a higher IVG; however, \u003cem\u003eT. harzianum\u003c/em\u003e and its combinations with Thiametoxam and Stimulate\u0026reg; caused a reduction in IVG, impairing the germination speed of the healthy seeds.\u003c/p\u003e\n\u003cp\u003eThe dry biomass of the aerial part (DBAP) of seedlings originating from infected and treated seeds was higher than that of non-treated seeds in the treatments with the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg; or with \u003cem\u003eT. asperellum\u003c/em\u003e and \u003cem\u003eT. harzianum\u003c/em\u003e, both combined with Thiamethoxam (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Among these, the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg; was the treatment that induced the highest accumulation of DBAP. In healthy seeds, only \u003cem\u003eT. endophyticum\u003c/em\u003e provided a greater accumulation of DBAP, while \u003cem\u003eT. harzianum\u003c/em\u003e and its combinations with Thiamethoxam and Stimulate\u0026reg; as well as Thiamethoxam alone negatively influenced this variable. The presence of \u003cem\u003eM. phaseolina\u003c/em\u003e in the seeds, across all treatments, affected the accumulation of DBAP, reducing the growth of the aerial part of the seedlings compared to those originating from healthy seeds. In infected seeds, all treatments induced greater accumulation of dry root biomass (DBRP), except for Stimulate\u0026reg; and Thiamethoxam (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). None of the treatments induced accumulation of DBRP in seedlings from healthy seeds. The presence of \u003cem\u003eM. phaseolina\u003c/em\u003e in the seeds negatively affected the accumulation of DBRP in all treatments when compared to the DBRP of seedlings from healthy and treated seeds.\u003c/p\u003e\n\u003cp\u003eEmergence and development of soybean seedlings in pots under controlled conditions\u003c/p\u003e\n\u003cp\u003eThe disease severity index (DSI) was influenced by the inoculation methods of \u003cem\u003eM. phaseolina\u003c/em\u003e and the treatments applied to the soybean seeds (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). When the pathogen was inoculated on the seeds, the severity of charcoal rot was reduced with the application of \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e, both alone and in combination with Stimulate\u0026reg;. The other treatments did not reduce the severity of charcoal rot when compared to the inoculated, untreated seeds. When healthy seeds were sown in an infested substrate, all treatments reduced the severity of charcoal rot compared to untreated seeds. The application of the three species of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Stimulate\u0026reg; were the treatments that most significantly reduced the symptoms of charcoal rot in these seeds. The severity of charcoal rot was greater when the pathogen was infecting the seeds than when it was infesting the substrate, except for the applications of Thiamethoxam and \u003cem\u003eT. endophyticum\u003c/em\u003e and its combination with Stimulate\u0026reg;.\u003c/p\u003e\n\u003cp\u003eThe dry biomass of aerial parts (DBAP*) and root biomass (DBRP*) of the plants was also influenced by the inoculation methods and the treatments applied to the seeds (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the infected seeds, all treatments resulted in a greater accumulation of DBAP*, except for \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Thiamethoxam or with \u003cem\u003eT. harzianum\u003c/em\u003e. The application of the three species of \u003cem\u003eTrichoderma\u003c/em\u003e, Thiamethoxam alone, and the combinations of Thiamethoxam with \u003cem\u003eT. asperellum\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg; were the treatments that induced the highest accumulation of DBAP* in the inoculated seeds. No treatment induced an accumulation of DBAP* in plants derived from healthy seeds sown in an infested substrate. When comparing the inoculation methods, treatments with Stimulate\u0026reg; and its combinations with the three species of \u003cem\u003eTrichoderma\u003c/em\u003e, as well as Thiamethoxam combined with \u003cem\u003eT. endophyticum\u003c/em\u003e or \u003cem\u003eT. harzianum\u003c/em\u003e, resulted in a lower accumulation of DBAP* in plants originating from infected seeds, compared to those in an infested substrate.\u003c/p\u003e\n\u003cp\u003eAll treatments promoted a greater accumulation of root biomass (DBRP*), except for \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Thiamethoxam or \u003cem\u003eT. harzianum\u003c/em\u003e in the inoculated seeds (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The three species of \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Stimulate\u0026reg; were the treatments that most induced DBRP*. No treatment resulted in a greater accumulation of DBRP* when the substrate was infested by the pathogen. Comparing the inoculation methods, the seeds that were inoculated and treated with Stimulate\u0026reg; and its combinations with \u003cem\u003eT. asperellum\u003c/em\u003e or \u003cem\u003eT. harzianum\u003c/em\u003e, as well as Thiamethoxam combined with \u003cem\u003eT. endophyticum\u003c/em\u003e or \u003cem\u003eT. harzianum\u003c/em\u003e, showed the highest accumulations of DBRP*, in comparison to the healthy seeds that received the same treatments and were sown in an infested substrate.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eTrichoderma endophyticum\u003c/em\u003e, \u003cem\u003eT. asperellum\u003c/em\u003e, and \u003cem\u003eT. harzianum\u003c/em\u003e, as well as the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg;, showed promising results in the treatment of soybean seeds for controlling charcoal rot. In the antagonism in dual cultures test, all species of \u003cem\u003eTrichoderma\u003c/em\u003e demonstrated antagonistic potential, forming inhibition halos, reducing the colony, and parasitizing \u003cem\u003eM. phaseolina\u003c/em\u003e. This effect is attributed to the combined action of various control mechanisms employed by the antagonists, such as competition, hyperparasitism, the ability to produce antimicrobial compounds (Chua, Soung \u0026amp; Ting, 2024; Kumari et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and enzymes that degrade the cell walls of hyphae, like chitinase and β-1,3-glucanase (Kumari et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the compatibility test among the three species of \u003cem\u003eTrichoderma\u003c/em\u003e with the plant growth regulator Stimulate\u0026reg; and the insecticide Thiamethoxam used in the treatment of soybean seeds, \u003cem\u003eT. asperellum\u003c/em\u003e showed compatibility with both products, exhibiting greater mycelial growth when the products were present in the culture medium. This is likely because \u003cem\u003eT. asperellum\u003c/em\u003e utilizes ingredients present in both formulations for its growth (Alves, Moino J\u0026uacute;nior \u0026amp; Almeida, 1998). Conversely, \u003cem\u003eT. endophyticum\u003c/em\u003e experienced inhibited mycelial growth with Stimulate\u0026reg;, while \u003cem\u003eT. harzianum\u003c/em\u003e was inhibited in the presence of both products. In contrast to the findings of this study, Dwivedi \u0026amp; Vishunavat (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) demonstrated that \u003cem\u003eT. harzianum\u003c/em\u003e was compatible with Thiamethoxam. This divergence in results indicates that the use of different species of \u003cem\u003eTrichoderma\u003c/em\u003e or even different isolates of the same species can yield varied compatibility outcomes with chemical products applied in seed treatment. Although this is the first study testing the compatibility between \u003cem\u003eTrichoderma\u003c/em\u003e species and plant growth regulators, these products should not inhibit the growth and multiplication of biocontrol agents, as they do not possess fungitoxic or fungistatic actions (Medeiros et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which could interfere with their efficacy.\u003c/p\u003e\u003cp\u003eIn the inoculated and untreated seeds, \u003cem\u003eM. phaseolina\u003c/em\u003e was able to reduce soybean seed germination by 80%. However, it is important to note that the artificial inoculation method used provided high exposure of the seeds to the pathogen, which does not occur naturally, resulting in seeds with a high infection rate. Despite this, the three species of \u003cem\u003eTrichoderma\u003c/em\u003e and the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e with the growth regulator Stimulate\u0026reg; increased the germination rate of the highly infected seeds. In contrast, Stimulate\u0026reg; and Thiamethoxam were unable to increase the germination of the infected seeds, and thus are not effective in controlling \u003cem\u003eM. phaseolina\u003c/em\u003e in soybean seeds, as expected since they are a growth regulator and an insecticide, respectively. In healthy seeds that were treated, no increase in germination was observed in any of the treatments. This may be related to the high quality of the seed lot used, which had a 98% germination rate, as seeds with medium and low vigor are more responsive to different seed treatments (Carvalho \u0026amp; Nakagawa, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the germination of healthy seeds treated with \u003cem\u003eT. harzianum\u003c/em\u003e and its combinations with Thiamethoxam or the growth regulator Stimulate\u0026reg; was reduced, whereas Thiamethoxam and the growth regulator applied individually did not cause this reduction. It is known that Thiamethoxam does not have a toxic effect on soybean seed germination (Cat\u0026atilde;o et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, this demonstrates that \u003cem\u003eT. harzianum\u003c/em\u003e was the causal agent of the reduction in seed germination, which may be due to the tested dose. It is known that applying doses above or below the recommended level for \u003cem\u003eTrichoderma\u003c/em\u003e species can alter their effectiveness or even hinder seed germination by acting as a saprophytic agent, utilizing the seed as a substrate (Woo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHigher germination rates of the infected seeds were also observed when treated with the three species of \u003cem\u003eTrichoderma\u003c/em\u003e, the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg;, and Thiamethoxam combined with \u003cem\u003eT. harzianum\u003c/em\u003e. However, in vitro, Stimulate\u0026reg; inhibited the mycelial growth of \u003cem\u003eT. endophyticum\u003c/em\u003e, while Thiamethoxam inhibited the growth of \u003cem\u003eT. harzianum\u003c/em\u003e. What may have occurred is that, upon seed germination, part of the growth regulator and Thiamethoxam was absorbed, and another part was diluted in the substrate, resulting in lower concentrations than in the culture medium, which did not hinder \u003cem\u003eT. endophyticum\u003c/em\u003e and \u003cem\u003eT. harzianum\u003c/em\u003e from controlling \u003cem\u003eM. phaseolina\u003c/em\u003e in the seeds. Regarding the dry biomass of both shoots and roots, plants from infected seeds showed that all treatments, except for Thiamethoxam and Stimulate\u0026reg; applied alone, were effective. \u003cem\u003eT. endophyticum\u003c/em\u003e and its combination with Stimulate\u0026reg; stood out as the best treatments, also inducing growth in the shoots and roots of plants from healthy seeds. These results demonstrate the ability of the three species of \u003cem\u003eTrichoderma\u003c/em\u003e to control \u003cem\u003eM. phaseolina\u003c/em\u003e, which leads to greater development of plants from infected seeds, and \u003cem\u003eT. endophyticum\u003c/em\u003e in combination with Stimulate\u0026reg; promotes growth in the shoots and roots of soybeans. It is known that \u003cem\u003eTrichoderma\u003c/em\u003e species can act as stimulants, promoting plant development through phytohormones, improving nutrient assimilation (especially phosphorus), and inducing plant resistance (Harman et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The literature did not reveal studies using the combination of \u003cem\u003eT. endophyticum\u003c/em\u003e and \u003cem\u003eT. asperellum\u003c/em\u003e with Stimulate\u0026reg; to promote growth or for seed treatment to control charcoal rot in soybeans. Some studies have demonstrated the ability of Thiamethoxam to induce growth in cotton plants (Lauxen; Villela \u0026amp; Soares, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), stimulate the physiological performance of carrot seeds (Almeida et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), activate genes of enzymes related to secondary plant metabolism, producing precursors of plant hormones (Castro, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and at low doses, increase leaf area and root growth in soybean plants (God\u0026oacute;i et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, Thiamethoxam did not promote growth in either the shoots or roots of Intacta Xtend M6110 i2x\u0026reg; soybean seeds. In fact, the active ingredient caused a reduction in the root growth of uninfected seedlings. This divergence in results may be due to the fact that different genotypes and plant species may respond differently to the use of Thiamethoxam as well as to the growth regulator Stimulate\u0026reg;.\u003c/p\u003e\u003cp\u003eIn pots under controlled conditions, although \u003cem\u003eM. phaseolina\u003c/em\u003e negatively affected soybean seeds, most biocontrol treatments, except for the combination of Thiamethoxam with \u003cem\u003eT. endophyticum\u003c/em\u003e or \u003cem\u003eT. harzianum\u003c/em\u003e, resulted in greater plant growth compared to inoculated and untreated seeds. It's crucial to highlight that seeds severely infected with \u003cem\u003eM. phaseolina\u003c/em\u003e often do not germinate or even emerge (Abawi \u0026amp; Pastor Corrales, 1990). This can be attributed to the direct contact of \u003cem\u003eM. phaseolina\u003c/em\u003e with the seeds, causing physical damage to the seed coat, leading to rotting. An important characteristic of \u003cem\u003eM. phaseolina\u003c/em\u003e is its high capacity to produce hydrolytic enzymes and lignocelluloses to penetrate host tissues (Marquez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, when seeds are inoculated with the pathogen, the damage is more severe, hindering physiological processes related to water and nutrient absorption, photosynthesis, and growth (Cruciol \u0026amp; Costa, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the application of \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. endophyticum\u003c/em\u003e, both alone and in combination with Stimulate\u0026reg;, drastically reduced the severity of charcoal rot, demonstrating the biocontrol potential of these antagonists in suppressing symptoms of charcoal rot even in severe infections, starting from the early stages of plant development. These results may be attributed to a complex and simultaneous action of various mechanisms, such as competition due to rapid and intense colonization of the seed and root zones (Bucio; Flores \u0026amp; Estrella, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which makes it difficult for the pathogen to establish itself. Other mechanisms include the production of antimicrobial compounds, hyperparasitism, and disease escape during pre- and post-emergence due to induced greater root growth, aiding in nutrient and water acquisition as well as shoot development (Melo, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is important to note that plant-pathogen-antagonist-environment interactions are complex, with both biotic and abiotic factors directly influencing these interactions. \u003cem\u003eM. phaseolina\u003c/em\u003e is a pathogen whose microsclerotia germination and disease severity in the field are increased by temperatures between 30\u0026deg;C and 33\u0026deg;C (Viana \u0026amp; Souza, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Even under conditions favorable for \u003cem\u003eM. phaseolina\u003c/em\u003e infection and colonization imposed in this study, the three \u003cem\u003eTrichoderma\u003c/em\u003e species managed to mitigate the destructive effects of the pathogen on soybean seeds with high infection rates. This indicates that \u003cem\u003eTrichoderma\u003c/em\u003e species can alleviate stress caused by high temperatures in soybean plants, making them less susceptible to disease occurrence. Therefore, selecting antagonists should take this factor into account.\u003c/p\u003e\u003cp\u003eWithin the \u003cem\u003eMacrophomina\u003c/em\u003e genus, there is a wide range of morphological, pathogenic, physiological, and genetic variability among its isolates (Reznikov et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This diversity allows the fungus to adapt to various environmental conditions, hosts, and control measures implemented in its management. This scenario further underscores the importance of implementing biological control agents as a strategy for disease management, since these agents exhibit different mechanisms of action that can work independently or in concert against \u003cem\u003eM. phaseolina\u003c/em\u003e and the host plant. Consequently, the selection of pathogen populations resistant to biocontrol agents becomes less of a challenge. This study demonstrated that the three \u003cem\u003eTrichoderma\u003c/em\u003e species that were evaluated have the potential to protect and control \u003cem\u003eM. phaseolina\u003c/em\u003e when applied as seed treatments. Notably, \u003cem\u003eT. endophyticum\u003c/em\u003e and \u003cem\u003eT. harzianum\u003c/em\u003e are capable of controlling charcoal rot even in severe infections, as well as stimulating the growth of both the aerial and root parts of infected soybean plants. This highlights the relevance of biocontrol agents in the search for sustainable and effective strategies for soybean management.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eTrichoderma asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, and \u003cem\u003eT. harzianum\u003c/em\u003e exhibit antagonistic effects against \u003cem\u003eM. phaseolina\u003c/em\u003e, acting through different mechanisms to suppress the pathogen. The biocontrol agents are compatible with the insecticide Thiamethoxam, and the growth regulator Stimulate\u0026reg;, except for \u003cem\u003eT. harzianum\u003c/em\u003e with both products and \u003cem\u003eT. endophyticum\u003c/em\u003e with Stimulate\u0026reg;. For the soybean cultivar Intacta Xtend M6110 i2x\u0026reg;, in the absence of the pathogen, only \u003cem\u003eT. endophyticum\u003c/em\u003e and its combination with Stimulate\u0026reg; enhance seedling vigor, while \u003cem\u003eT. harzianum\u003c/em\u003e at a concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e spores mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e negatively affects seed germination and seedling growth. The greatest growth of soybean seedlings, originating from seeds infected with \u003cem\u003eM. phaseolina\u003c/em\u003e, is induced by all three \u003cem\u003eTrichoderma\u003c/em\u003e species and \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Stimulate\u0026reg;. Thiamethoxam and Stimulate\u0026reg; do not promote increased germination or growth of Intacta Xtend M6110 i2x\u0026reg; soybean seedlings, regardless of whether the seeds are infected or healthy. In seeds with a high rate of infection, all three \u003cem\u003eTrichoderma\u003c/em\u003e species help control charcoal rot, particularly \u003cem\u003eT. harzianum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e, and their combination with Stimulate\u0026reg;, which significantly reduce the damage caused by \u003cem\u003eM. phaseolina\u003c/em\u003e. Meanwhile, Thiamethoxam and Stimulate\u0026reg; do not have fungitoxic effects and do not control charcoal rot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/em\u003eAll authors contributed to the study\u0026apos;s conception and design\u003cstrong\u003e. \u0026nbsp;\u003c/strong\u003eJDV conceived and designed the study, performed the sampling, processed and analyzed the data, and wrote the manuscript. ACS and JL reviewed drafts and contributed to writing the manuscript. SFC, JCJCC, EFS, and TFM implemented and monitored the experiment and compiled the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject funding\u0026nbsp;\u003c/strong\u003eThis study was supported by the Brazilian Federal Agency for Support and Evaluation of Graduate Education [Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The datasets generated and/or analyzed during the current study are available in the Zenodo repository (Deboni da Veiga et al. 2024\u0026ndash;2025) at https://doi.org/10.5281/zenodo.14245506.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbawi, G. S., Pastor Corrales, M. A. (1990) \u003cem\u003eRoot Rots of Beans in Latin America and Africa: Diagnosis, Research Methodologies, and Management Strategies.\u003c/em\u003e Centro Internacional de Agricultura Tropical (CIAT), Cali, CO, pp.114 (publica\u0026ccedil;\u0026atilde;o CIAT n\u0026ordm; 35). https://hdl.handle.net/10568/54258.\u003c/li\u003e\n \u003cli\u003eAGROFIT (2024) \u003cem\u003eMinistry of Agriculture and Livestock\u003c/em\u003e. https://www.gov.br/agricultura/pt-br/assuntos/insumos-agropecuarios/insumos-agricolas/agrotoxicos/agrofit.\u003c/li\u003e\n \u003cli\u003eAlmeida, A. S., Villela, F., Tillmann, M. A. A., Meneghello, G. E. (2012) Insecticide Thiamethoxam: A Bioactive Action on Carrot Seeds (\u003cem\u003eDaucus carota\u003c/em\u003e L.). In: Soloneski S, Larramendy M. \u003cem\u003eInsecticides: Basic and Other Applications\u003c/em\u003e. pp. 3\u0026ndash;16. https://books.google.com.br/books?hl=pt-BR\u0026amp;lr=lang_en\u0026amp;id=_-GdDwAAQBAJ\u0026amp;oi=fnd\u0026amp;pg=\u003cbr\u003ePA3\u0026amp;dq=Thiametoxam+tomate\u0026amp;ots=8YHyQk7WpB\u0026amp;sig=rCsoKkkFxIiXurXbmn5mgGWX29E#v=onepage\u0026amp;q\u0026amp;f=false.\u003c/li\u003e\n \u003cli\u003eAlves, S. B., Moino Jr, A., Almeida, J. E. M. (1998) Phytosanitary products and entomopathogens. In: Alves SB (Ed.) \u003cem\u003eMicrobial Control of Insects\u003c/em\u003e, Piracicaba: FEALQ, pp. 217\u0026ndash;238.\u003c/li\u003e\n \u003cli\u003eBastakoti, S., Belbase, S., Manandhar, S., Arjyal, C. (2017) \u003cem\u003eTrichoderma\u003c/em\u003e species as Biocontrol agent against soil borne fungal pathogens. \u003cem\u003eNepal J Biotechnol\u003c/em\u003e 5:39\u0026ndash;45. https://doi.org/10.3126/njb.v5i1.18492.\u003c/li\u003e\n \u003cli\u003eBattaglia, M. E., Martinez, S. I., Covacevich, F., Consolo, V. F. (2024). \u003cem\u003eTrichoderma harzianum\u003c/em\u003e enhances root biomass production and promotes lateral root growth of soybean and common bean under drought stress. \u003cem\u003eAnnals of Applied Biology\u003c/em\u003e. https://doi.org/10.1111/aab.12909.\u003c/li\u003e\n \u003cli\u003eBell, D. K., Wells, H. D., Markham, C. R. (1982) \u003cem\u003eIn vitro\u003c/em\u003e antagonism of \u003cem\u003eTrichoderma\u0026nbsp;\u003c/em\u003especies against six fungal plant pathogens. \u003cem\u003ePhytopathology\u003c/em\u003e \u003cem\u003e72\u003c/em\u003e(4): 379\u0026ndash;382. 10.1094/Phyto-72-379.\u003c/li\u003e\n \u003cli\u003eBellaloui, N., Mengistu, A., Smith, J. R., Abbas, H.K., Accinelli, C., Shier, W. T. (2023) Soybean Seed Sugars: A role in the mechanism of resistance to charcoal rot and potential use as biomarkers in selection. \u003cem\u003ePlants\u003c/em\u003e \u003cem\u003e12\u003c/em\u003e, 392. 10.3390/plants12020392.\u003c/li\u003e\n \u003cli\u003eBucio, J. L., Flores, R. P., Estrella, A. H. (2015) \u003cem\u003eTrichoderma\u003c/em\u003e as biostimulant: exploiting the multilevel properties of a plant beneficial fungus. \u003cem\u003eSci Hortic\u003c/em\u003e \u003cem\u003e196\u003c/em\u003e:109\u0026ndash;123. https://doi.org/10.1016/j.scienta.2015.08.043.\u003c/li\u003e\n \u003cli\u003eCarvalho, N. M., Nakagawa, J. (2012). \u003cem\u003eSeeds: science, technology, and production\u003c/em\u003e. Funep, Jaboticabal.\u003c/li\u003e\n \u003cli\u003eCastro, P. R. C. (2006) \u003cem\u003eHormonal agrochemicals in tropical agriculture\u003c/em\u003e. Piracicaba: ESALq. 46 pp. (Rural Producer Series, 32). https://www.esalq.usp.br/biblioteca/sites/default/files/publicacoes-a-venda/pdf/SPR32.pdf.\u003c/li\u003e\n \u003cli\u003eCat\u0026atilde;o, H. C. R. M., Pontes, B. S., Pinheiro, D. T., Filho, M. A. O., Santos, A. L. C., Zolla, M. C. (2024) Chemical treatment and mobilization of reserves of soybean seeds under water d\u0026eacute;ficit\u003cem\u003e. J Seed Sci\u003c/em\u003e \u003cem\u003e46\u003c/em\u003e. https://doi.org/10.1590/2317-1545v46278828.\u003c/li\u003e\n \u003cli\u003eChen, H., Li, H., Liu, Z., Zhang, C., Zhang, S., Atkinson, P. M. (2023) A novel Greenness and Water Content Composite Index (GWCCI) for soybean mapping from single remotely sensed multispectral images. \u003cem\u003eRemote Sens Environ\u003c/em\u003e \u003cem\u003e295\u003c/em\u003e. https://doi.org/10.1016/j.rse.2023.113679.\u003c/li\u003e\n \u003cli\u003eChua, R. W., Song, K. P., Ting, A. S. Y. (2024) Characterization and antimicrobial activities of bioactive compounds from endophytic \u003cem\u003eTrichoderma asperellum\u003c/em\u003e isolated from \u003cem\u003eDendrobium\u003c/em\u003e orchids. \u003cem\u003eBiol 79\u003c/em\u003e:569\u0026ndash;584. https://doi.org/10.1007/s11756-023-01562-9.\u003c/li\u003e\n \u003cli\u003eCohen, R., Elkabetz, M., Paris, H. S., Gur, A., Dai, N., Rabinovitz, O., Freeman, S. (2022) Occurrence of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e in Israel: Challenges for Disease Management and Crop Germplasm Enhancement. \u003cem\u003ePlant Dis\u003c/em\u003e \u003cem\u003e106\u003c/em\u003e:15\u0026ndash;25. https://doi.org/10.1094/PDIS-07-21-1390-FE.\u003c/li\u003e\n \u003cli\u003eCruciol, G. C. D., Costa, M. L. N. (2018) Influence of inoculation methodologies of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e on the performance of soybean cultivars. \u003cem\u003eSumma Phytopathology\u003c/em\u003e \u003cem\u003e44\u003c/em\u003e:32\u0026ndash;37. https://www.scielo.br/j/sp/a/KtXhXXgVbntCNf6nspgJHBC/?format=pdf\u0026amp;lang=pt.\u003c/li\u003e\n \u003cli\u003eDeboni da Veiga, J. (2024\u0026ndash;2025) \u003cem\u003eTrichoderma endophyticum\u003c/em\u003e, \u003cem\u003eT. asperellum\u003c/em\u003e e \u003cem\u003eT. harzianum\u003c/em\u003e suppress charcoal rot in soybean seeds infected by \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e [Dataset]. Zenodo. https://doi.org/10.5281/zenodo.14245506.\u003c/li\u003e\n \u003cli\u003eDhingra, O. D., Sinclair, J. B. (1978) Biology and Pathology of \u003cem\u003eMacrophimina phaseolina\u003c/em\u003e. Australasian \u003cem\u003ePlant Pathology\u003c/em\u003e \u003cem\u003e7\u003c/em\u003e, 25. https://doi.org/10.1007/BF03212337.\u003c/li\u003e\n \u003cli\u003eDwivedi, M., Vishunavat, C. (2018) Compatibility of \u003cem\u003eTrichoderma\u003c/em\u003e strains and their mutants with common agrochemicals. \u003cem\u003eJ Pharmacogn Phytochem\u003c/em\u003e \u003cem\u003e7\u003c/em\u003e(5):2744\u0026ndash;2747. https://www.phytojournal.com/archives/2018/vol7issue5/PartAT/7-4-561-108.pdf.\u003c/li\u003e\n \u003cli\u003eGajera, H., Bambharolia, R., Patel, S., Khatrani, T., Goalkiya, B. (2012) Antagonism of \u003cem\u003eTrichoderma\u003c/em\u003e spp. against \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e: evaluation of coiling and cell wall degrading enzymatic activities. J \u003cem\u003ePlant Pathol Microbiol\u003c/em\u003e \u003cem\u003e3\u003c/em\u003e:149. 10.4172/2157-7471.1000149.\u003c/li\u003e\n \u003cli\u003eGod\u0026oacute;i, C. T. D., Campos, S. O., Monteiro, S. H., Ronchi, C. P., Silva, A. A., Guedes, R. N. C. (2023) Thiamethoxam in soybean seed treatment: Plant bioactivation and hormesis, besides whitefly control? \u003cem\u003eSci Total Environ\u003c/em\u003e \u003cem\u003e857\u003c/em\u003e(3):20. https://doi.org/10.1016/j.scitotenv.2022.159443.\u003c/li\u003e\n \u003cli\u003eGoidanish, G. (1947) Revisione del genere \u003cem\u003eMacrophomina\u003c/em\u003e Petrak. Species tipica: \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Tass.) Goid. nov. comb. nec. \u003cem\u003eM. phaseoli\u003c/em\u003e. \u003cem\u003eAshby Annali della Sperimentazione Agraria 1\u003c/em\u003e: 449\u0026ndash;461.\u003c/li\u003e\n \u003cli\u003eGupta, G. K., Sharma, S. K., Ramteke, R. (2012) Biology, epidemiology and management of the pathogenic fungus \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Tassi) goid with special reference to charcoal rot of soybean (\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merrill). \u003cem\u003eJ. Phytopathol\u003c/em\u003e \u003cem\u003e160\u003c/em\u003e:167\u0026ndash;180. 10.1111/j.1439-0434.2012.01884.x.\u003c/li\u003e\n \u003cli\u003eHarman, G. E., Howell, C. R., Viterbo, A., Chet, I., Lorito, M. (2004) \u003cem\u003eTrichoderma\u003c/em\u003e species--opportunistic, avirulent plant symbionts. \u003cem\u003eNat Rev Microbiol 2\u003c/em\u003e(1):43\u0026ndash;56. 10.1038/nrmicro797.\u003c/li\u003e\n \u003cli\u003eInglada, J., Arias, M., Tardy, B., Hagolle, O., Valero, S., Morin, D., Dedieu, G., Sepulcre, G., Bontemps, S., Defourny, P., Koetz, B. (2015) Assessment of an operational system for crop type map production using high temporal and spatial resolution satellite optical imagery. \u003cem\u003eRemote Sens\u003c/em\u003e \u003cem\u003e7\u003c/em\u003e:12356\u0026ndash;12379. https://doi.org/10.3390/rs70912356.\u003c/li\u003e\n \u003cli\u003eIqbal, U., Mukhtar, T. (2014) Morphological and pathogenic variability among \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e isolates associated with mungbean (\u003cem\u003eVigna radiata\u003c/em\u003e L.) Wilczek from Pakistan. \u003cem\u003eSci World J 1\u003c/em\u003e. 10.1155/2014/950175.\u003c/li\u003e\n \u003cli\u003eKamolovna, K. N., Zavkiddinova, A. S. (2023) 11th -ICARHSE International Conference on Advance Research in Humanities, \u003cem\u003eApplied Sciences and Education\u003c/em\u003e. https://conferencea.org.\u003c/li\u003e\n \u003cli\u003eKaur, S., Dhillon, G. S., Brar, S. K., Vallad, G. E., Chand, R., Chauhan, V. B. (2012) Emerging phytopathogen \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e: biology, economic importance and current diagnostic trends. \u003cem\u003eCrit Rev Microbiol 38\u003c/em\u003e(2):136\u0026ndash;151\u003cem\u003e.\u003c/em\u003e 10.3109/1040841x.2011.640977.\u003c/li\u003e\n \u003cli\u003eKumari, R., Kumar, V., Arukha, A. P., Rabbee, M. F., Ameen, F., Koul, B. (2024) Screening of the Biocontrol Efficacy of Potent \u003cem\u003eTrichoderma\u003c/em\u003e Strains against \u003cem\u003eFusarium oxysporum\u003c/em\u003e f.sp. \u003cem\u003ecicero\u003c/em\u003e and \u003cem\u003eSclerotium rolfsii\u003c/em\u003e Causing Wilt and Collar Rot in Chickpea. \u003cem\u003eMicroorg\u0026nbsp;\u003c/em\u003e\u003cem\u003e12\u003c/em\u003e:1280. https://doi.org/10.3390/microorganisms12071280.\u003c/li\u003e\n \u003cli\u003eLarran, S., Sim\u0026oacute;n, M. R., Santamarina, M. P., Caselles, J. R., Consolo, V. F., Perell\u0026oacute;, A. (2023) Endophytic \u003cem\u003eTrichoderma\u003c/em\u003e strains increase soya bean growth and promote charcoal rot control. J Saudi Soc Agric Sci \u003cem\u003e22\u003c/em\u003e(7):395\u0026ndash;406. https://doi.org/10.1016/j.jssas.2023.03.005.\u003c/li\u003e\n \u003cli\u003eLauxen, L. R., Villela, F. A., Soares, R. C. (2010) Physiological performance of cotton seeds treated with thiamethoxam. \u003cem\u003eBraz J Seeds\u003c/em\u003e \u003cem\u003e32\u003c/em\u003e(3):061\u0026ndash;068. https://doi.org/10.1590/S0101-31222010000300007.\u003c/li\u003e\n \u003cli\u003eMaguire, J. D. (1962) Speed of germination aid in selection and evaluation for seedling emergence and vigor. \u003cem\u003eCrop Sci\u003c/em\u003e \u003cem\u003e2\u003c/em\u003e(2):176\u0026ndash;177.\u003c/li\u003e\n \u003cli\u003eMachado, J., Oliveira, J., Vieira, M., Alves, M. (2001) Inocula\u0026ccedil;\u0026atilde;o artificial de sementes de soja por fungos, utilizando solu\u0026ccedil;\u0026atilde;o de manitol. Rev Bras Semente 23:95\u0026ndash;101.\u003c/li\u003e\n \u003cli\u003eMarquez, N., Giachero, M. L., Declerck, S., Ducasse, D. A. (2021) \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e: General Characteristics of Pathogenicity and Methods of Control. \u003cem\u003eFront Plant Sci\u003c/em\u003e \u003cem\u003e12\u003c/em\u003e: 634397. https://doi.org/10.3389/fpls.2021.634397.\u003c/li\u003e\n \u003cli\u003eMckinney, H. H. (1923) Influence of soil temperature and moisture on infection of wheat seedlings by \u003cem\u003eHelminthosporium sativum\u003c/em\u003e. \u003cem\u003eJ Agric Res\u003c/em\u003e \u003cem\u003e26\u003c/em\u003e(5):195\u0026ndash;218. https://handle.nal.usda.gov/10113/IND43966679.\u003c/li\u003e\n \u003cli\u003eMedeiros, F. H. V, Guimar\u0026atilde;es, R. A., Silva, J. C. P., Magalh\u0026atilde;es, V. C., Souza, J. T. (2019) Trichoderma: interactions and strategies. In: Meyer, M. C., Mazaro, S. M., Silva, J. C. \u003cem\u003eTrichoderma: use in agriculture\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eMelo, I. S. (1991) Potential of using Trichoderma spp. in the biological control of plant diseases. In: Bettiol, W. \u003cem\u003eBiological control of plant diseases\u003c/em\u003e. EMBRAPA, pp. 154. https://www.alice.cnptia.embrapa.br/handle/doc/10080.\u003c/li\u003e\n \u003cli\u003eOns, L., Bylemans, D., Thevissen, K., Cammue, B. P. A. (2020) Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control\u003cem\u003e. Microorg\u003c/em\u003e \u003cem\u003e8\u003c/em\u003e(12):1930. https://doi.org/10.3390/microorganisms8121930.\u003c/li\u003e\n \u003cli\u003eReznikov, S., Chiesa, M. A., Pardo, E. M., Lisi, V., Bogado, N., Gonz\u0026aacute;lez, V., Ledesma, F., Morandi, E. N., Ploper, L. D., Castagnaro, A. P. (2018) Soybean- \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e-Specific Interations and indentification of a Novel Source of Resistance. \u003cem\u003ePhytopathol\u003c/em\u003e \u003cem\u003e109\u003c/em\u003e:1. https://doi.org/10.1094/PHYTO-08-17-0287-R.\u003c/li\u003e\n \u003cli\u003eSeixas, C. D. S., Soares, R. M., Godoy, C. V., Mayer, M. C., Constamilan, L. M., Dias, W. P., Almeida, A. M. R. (2020) Disease management. In: Seixas CDS, Neumaier N, Balbinot Junior AA, Krzyzanowski FC, Leite RMVBC. \u003cem\u003eSoybean production technologies\u003c/em\u003e. EMBRAPA, Londrina. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/223209/1/SP-17-2020-online-1.pdf.\u003c/li\u003e\n \u003cli\u003eShort, G. E., Wyllie, T. D., Bristow, P. R. (1980) Survival of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e. In soil and in residue of soybean. \u003cem\u003eEcol Epidemiol\u003c/em\u003e \u003cem\u003e70\u003c/em\u003e(1):13\u0026ndash;17. https://www.apsnet.org/publications/phytopathology/backissues/Documents/1980Abstracts/Phyto70_13.htm.\u003c/li\u003e\n \u003cli\u003e\u0026Scaron;ućur, E. J., Petrović, K., Crnković, M., Krsmanović, S., Rajković, M., Kaitović, Ž., Malenčić, Đ. (2023) Susceptibility of the Most Popular Soybean Cultivars in South-East Europe to \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Tassi) Goid. \u003cem\u003ePlants\u0026nbsp;\u003c/em\u003e\u003cem\u003e12\u003c/em\u003e:2467. https://doi.org/10.3390/plants12132467.\u003c/li\u003e\n \u003cli\u003eTaiz, L., Zeiger, E. (2017) \u003cem\u003ePlant Physiology\u003c/em\u003e. 6 ed. Porto Alegre: Artmed. 10.3923/ppj.2012.73.76\u003c/li\u003e\n \u003cli\u003eUSDA (2024). \u003cem\u003eWorld agricultural production\u003c/em\u003e. https://fas.usda.gov/data/world-agricultural-production-11082024.\u003c/li\u003e\n \u003cli\u003eViana, F. M. P., Souza, N. L. (2002) Effect of temperature-water tension interaction on the germination of micro-sclerotia of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e. \u003cem\u003eBraz Phytopathol\u003c/em\u003e \u003cem\u003e27\u003c/em\u003e:268\u0026ndash;272. https://doi.org/10.1590/S0100-41582002000300005.\u003c/li\u003e\n \u003cli\u003eWoo, S. L., Hermosa, R., Lorito, M., Monte, E. (2023) Trichoderma: a multipurpose, plant beneficial microorganism for eco-sustainable agriculture. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cem\u003e21\u003c/em\u003e:312\u0026ndash;326. https://doi.org/10.1038/s41579-022-00819-5.\u003c/li\u003e\n \u003cli\u003eWrather, J. A., Shannon, J. G., Carter, T. E., Bond, J. P., Rupe, J. C., Almeida, A. M. R. (2008) Reaction of drought-tolerant soybean genotypes to \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e. \u003cem\u003ePlant Health Prog\u003c/em\u003e \u003cem\u003e9\u003c/em\u003e:16. https://doi.org/10.1094/PHP-2008-0618-01-RS.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biological control, Glycine max, Growth promoters, Thiamethoxam, Stimulate®","lastPublishedDoi":"10.21203/rs.3.rs-6751408/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6751408/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoybean is one of the most important commodities in the world, and is affected by several phytopathogens, such as the fungus \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, which can occur at all stages of development, causing significant losses in production. The use of different cultural, chemical and genetic control strategies has shown low efficiency against this phytopathogen. Thus, the objective of this study was to evaluate the use of \u003cem\u003eTrichoderma asperellum\u003c/em\u003e, \u003cem\u003eT. endophyticum\u003c/em\u003e and \u003cem\u003eT. harzianum\u003c/em\u003e in seed treatment to control \u003cem\u003eM. phaseolina\u003c/em\u003e and promote plant growth. Healthy seeds inoculated with \u003cem\u003eM. phaseolina\u003c/em\u003e were treated with the three \u003cem\u003eTrichoderma\u003c/em\u003e species, the plant growth regulator Stimulate\u0026reg;, an insecticide based on Thiamethoxam and combinations of \u003cem\u003eTrichoderma\u003c/em\u003e species with Stimulate\u0026reg; or Thiamethoxam. The antagonistic capacity of \u003cem\u003eTrichoderma\u003c/em\u003e species against \u003cem\u003eM. phaseolina\u003c/em\u003e and their compatibility with Stimulate\u0026reg; and Thiamethoxam, germination and seedling vigor, growth and dry biomass of plants were evaluated. \u003cem\u003eTrichoderma\u003c/em\u003e species showed antagonism against \u003cem\u003eM. phaseolina\u003c/em\u003e, especially \u003cem\u003eT. endophyticum\u003c/em\u003e. \u003cem\u003eIn vitro\u003c/em\u003e, Stimulate\u0026reg; reduced the growth of \u003cem\u003eT. endophyticum\u003c/em\u003e and \u003cem\u003eT. harzianum\u003c/em\u003e, while Thiamethoxam reduced the growth of \u003cem\u003eT. harzianum\u003c/em\u003e. \u003cem\u003eM. phaseolina\u003c/em\u003e reduced approximately 80% of soybean seed germination. The three \u003cem\u003eTrichoderma\u003c/em\u003e species increased germination and vigor of infected seeds. In healthy seeds, \u003cem\u003eT. harzianum\u003c/em\u003e reduced germination, while \u003cem\u003eT. endophyticum\u003c/em\u003e combined with Stimulate\u0026reg; increased seedling vigor. These results show that \u003cem\u003eT. endophyticum\u003c/em\u003e, \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. asperellum\u003c/em\u003e are effective in treating seeds infected with \u003cem\u003eM. phaseolina\u003c/em\u003e, and the use of \u003cem\u003eT. harzianum\u003c/em\u003e isolate CCT 7589 is not recommended for the treatment of healthy soybean seeds.\u003c/p\u003e","manuscriptTitle":"Trichoderma endophyticum , T. asperellum, and T. harzianum suppress charcoal rot in soybean seeds infected by Macrophomina phaseolina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 18:16:45","doi":"10.21203/rs.3.rs-6751408/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-10-13T02:54:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-14T10:59:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T12:26:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2025-06-03T12:35:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-29T03:57:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2025-05-26T09:42:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d8e6a1bf-ca8f-472f-b054-3cf77ad38b5a","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-6751408","link":"https://doi.org/10.1007/s10658-026-03192-8","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2026-02-27 15:58:04","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-07-14 18:16:45","video":"","vorDoi":"10.1007/s10658-026-03192-8","vorDoiUrl":"https://doi.org/10.1007/s10658-026-03192-8","workflowStages":[]},"version":"v1","identity":"rs-6751408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6751408","identity":"rs-6751408","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}