For the efficiency of biocontrol, there is generally a search for antagonistic microorganisms for the management of phytopathogens. Microorganisms isolated from roots or the rhizosphere of a specific crop can be more effective due to their higher adaptability in disease control. In this context, Trichoderma shows vast biotechnological potential, with its efficiency being related to the physical, chemical, and biological conditions of the soil [25].

The Plackett-Burman statistical experimental design enables the identification of the main effects on efficiency to provide a fermentative medium with favorable conditions for Trichoderma culture, considering factors with a reduced number of experimental trials [26]. The assays that presented the highest biomass production were assay 6 for MMBF 58/09 and assay 4 for URM 6997/160,821 and IB 19/17, with both conditions corresponding to a ratio of 15 g L− 1 glucose to 8 g L− 1 protein hydrolysate. It is worth noting that the difference in assay 4 lies solely in the strain used (URM 6997/160821 and IB 19/17), since all parameters were otherwise identical. For strain MMBF 58/09, in addition to AS and pH not showing statistical significance at p < 0.10, there was an increase of 10 g L− 1 of Su.

Although the strains belong to the same genus, Trichoderma, they differ in their growth capacity when exposed to the same carbon and nitrogen sources [27]. In biomass and lactic acid production by Rhizopus oryzae, when varying the nitrogen source using chicken feather protein hydrolysate (with a high protein and ash content, 55.8 g (100 g)−1 and 42.1 g (100 g)−1, respectively, and essential amino acids for microbial growth), yeast extract, and ammonium sulfate, with beet molasses as the carbon source, the highest lactic acid yield corresponded to the hydrolysate at an optimal concentration of 7 g L− 1. To achieve this optimal hydrolysate concentration, it was reported that the C/N ratio in the medium can significantly affect microbial growth [28].

A high number of conidia contributes to accelerated proliferation and biomass synthesis. However, after a certain time, nutrient scarcity occurs, leading to a consequent reduction in metabolic activity. Determining the appropriate number of conidia can achieve a balance between nutrient availability and biomass accumulation required for enzymatic production [29]. In fermentations with T. harzianum, biomass increased in accordance with higher carbon availability over time and decreased linearly as the carbon source was depleted, regardless of the C/N ratio [30]. Nevertheless, in this study, biomass showed statistical significance for Gl and HP from 48 h onward in strains MMBF 58/09, URM 6997/160,821, and IB 19/17, with a C/N ratio of 15/8 g L− 1.

Similarly, conidial production (conidia mL− 1) was observed in assays 6 and 10 (96 h) and 10 (72 h) for MMBF 58/09, 1 (96 h) and 6 (24 h) for URM 6997/160,821, and 6 (48 h and 96 h) and 10 (24 h and 96 h) for IB 19/17, with maximum glucose (15 g L− 1), minimum hydrolyzed protein (2 g L− 1), absence of ammonium sulfate (0 g L− 1), and varying amounts of hydrolyzed protein. However, the main significant factors for the assays described were generally pH, followed by hydrolyzed protein, with conidial counts ranging from 967 to 1.25 × 10⁵ conidia mL− 1.

Low-carbon sources, such as sucrose in cultivation media with a C/N ratio close to 5/1 (soy peptone), were ideal for the growth of T. atroviride, allowing faster germination and higher bioactivity. In contrast, higher carbohydrate concentrations may trigger catabolite repression and reduce conidial production [31]. Assays such as 4 and 8 in the three studied strains, with low sucrose and high hydrolyzed protein and ammonium sulfate concentrations, showed considerable conidial production in the individual strains. Among the strains, MMBF 58/09 showed the highest conidial quantification, followed by IB 19/17, while URM 6997/160,821 exhibited the lowest production.

Propagules of filamentous fungi in T. atroviride remain metabolically active for up to 10 weeks of maturation, which confers characteristics similar to those of chemoautotrophic microorganisms. Furthermore, the transition from vegetative mycelium to conidia involves the formation of fruiting structures. In this process, the conidia formed enter a dormant state (this dormant form being a characteristic of ascospore-forming fungi) after the maturation stage. In T. reesei, it was discovered that conidia accumulate transcripts of several hydrolytic enzymes during their maturation [32]. The ability of microorganisms to absorb amino acids supports their growth [33].

In this context, microorganisms first utilize preferential or primary nutrients, repressing the use of secondary sources. When primary nutrients are depleted, metabolism shifts to secondary nutrient sources. This catabolite repression effect has been observed in Trichoderma spp. grown in nutrient-rich media. Additionally, primary nitrogen sources promoted conidiation induced by light more strongly than secondary sources. Thus, conidial production in T. atroviride depends on the carbon source in coordination with light induction, mediated by the expression of the blr1 and blr2 genes.

In T. asperellum and T. pleuroticola, conidiation was triggered by nitrogen catabolite repression, which in turn induced photoconidiation [31]. In a study with T. harzianum, submerged conidia were produced by conidiogenous cells (phialides) attached to hyphae at early growth stages on the second day of fermentation in low-carbon medium. As nitrogen concentrations decreased and carbon concentrations increased, conidial production also increased (7 days, 3.9–9.7 × 104 conidia mL− 1), accompanied by improved vegetative growth [30].

Fungal propagules exhibit physiological differences regarding their production, stability, and microbial activity. Although aerial mycelium produces conidia and can adapt to adverse environments, propagules obtained from solid-state fermentations are more costly and present challenges for scale-up [34]. Liquid fermentation allows high yields of propagules with efficiency and stability through Trichoderma, provided strict quality control is maintained. However, these propagules are often produced with low soil persistence and storage instability. Thus, fungal microsclerotia are preferred for soil application due to their resistance structures [30], offering greater storage stability and higher tolerance to desiccation. Studies report that microsclerotia formation occurs under appropriate nutritional and/or environmental conditions. For example, T. harzianum T-22 forms microsclerotia over a wide range of C/N ratios with a carbon source concentration of 36 g L− 1, while other studies using nutrient-rich media with a lower C/N ratio and no vitamin supplementation utilized T. asperellum [34].

The highest microsclerotia production (microsclerotia mL− 1) for strain MMBF 58/09 was observed in assays 6 (48 h) and 4 (72 h and 96 h), for URM 6997/160,821 in assay 7 (24 h), and for IB 19/17 in assay 4 (72 h and 96 h), considering maximum glucose and minimum ammonium sulfate for assay 4. Both glucose and hydrolyzed protein were significant for MMBF 58/09, while only hydrolyzed protein was significant in assay 7, and hydrolyzed protein and ammonium sulfate in assay 1. Maximum hydrolyzed protein and ammonium sulfate were observed in URM 6997/160,821 and IB 19/17, with quantifications ranging from 0.25 to 28 × 105 and 0.50–150 × 105 microsclerotia mL− 1, respectively.

The formation of microsclerotia occurred in submerged media with T. harzianum under high carbon source conditions, and no submerged conidia were produced. In addition to the culture medium, the fermentation time impacted microsclerotia production, as growth began at 48 h, with the highest production (2.6–4.8 × 104 microsclerotia mL− 1) and compactness observed on day 4 of fermentation, while more melanized and lower production was seen at day 7. Carbon sources (glucose and molasses) and nitrogen sources (soybean meal, cottonseed meal, yeast extract, corn steep liquor, and acid-hydrolyzed casein) supported submerged conidia production, whereas for microsclerotia, cottonseed meal combined with glucose yielded the highest production (C/N ratio 50/1), and molasses did not contribute to microsclerotia formation. The use of a 3-day submerged conidia pre-inoculum increased biomass and microsclerotia production compared to conidia taken from BDA plates [30].

Sucrose is a disaccharide that is easily assimilated compared to complex sugars, such as sorghum flour (used with T. asperellum strains), and is cheaper than glucose for use in submerged fermentations with filamentous fungi as biocontrol agents. Lower concentrations of carbon and nitrogen, in a 10:1 ratio (sucrose and autolyzed yeast), during seven days of submerged fermentation with T. asperellum yielded 2.5 × 104 microsclerotia mL− 1 and 3.5 × 107 CFU mL− 1 (colony-forming units representing total viable propagules) without vitamin supplementation [34].

Fermentations of Trichoderma spp. (T. viride, T. hamatum, and T. harzianum) in different media (corn mash molasses, sucrose nitrate, and glucose tartrate) resulted in the production of both conidia and chlamydospores while the pH fluctuations remained minimal. However, conidia production was more pronounced in static cultures, whereas chlamydospore formation predominated in liquid fermentations. Thus, propagule production depends on the isolate type, nutrient sources, and pH variation between 4 and 7 (Lnwts and Papavizas). Similarly, fermentations of different T. viride strains producing the antibiotic viridin exhibited microbial sporulation at intermediate to high pH, with high concentrations of glucose and nitrogen, and the strain showed enhanced sporulation when three or more trace elements, including iron, were added to the medium. Another strain performed better with intermediate glucose and low nitrogen concentrations [35].

LaeA is a global regulator whose loss leads to downregulation of aflRA, genetically identified in Aspergillus nidulans, and is known as a phylogenetically conserved methyltransferase in various filamentous fungi. It regulates secondary metabolism in Trichoderma spp., as well as conidia production, particularly in T. longibrachiatum. Propagule production, such as chlamydospores, which are thick-walled hyphal structures, occurs under adverse conditions, including significant pH changes, low temperature, and nutrient scarcity, as reported in T. harzianum [36]. Similarly, T. asperellum has been reported to enhance biomass production in water spinach (Ipomoea aquatica) [37].

Assays 1, 4, 8, 11, and 12 of the three studied strains showed significant values predominantly at the later fermentation stages (72 h and 96 h) with p < 0.10. The nutritional composition of the culture medium can affect fungal traits, including mycelial germination and conidia production. Therefore, careful optimization of carbon, nitrogen, and mineral sources is necessary to ensure optimal development, viability, and efficacy of the fungi selected as biological control agents. Achieving an appropriate carbon-to-nitrogen ratio is fundamental for high-quality conidia production [31].

The results of this study show that the strains MMBF 58/09, URM 6997/160,821, and IB 19/17 produced siderophores at all times, preferably at 48 h, with the highest production in assays 4, 5, and 11, respectively, although only strain MMBF 58/09 presented significant results at 96 h. Iron (Fe) is recognized as a critical factor affecting agricultural production due to its involvement in various essential biochemical and physiological processes in plants. Fe exists in two oxidation states, Fe²⁺ and Fe³⁺, which enable its roles in photosynthesis, respiration, nitrogen assimilation, and detoxification of reactive oxygen species. However, its concentration in soil is generally low and can be influenced by physical and chemical factors [38], and it is usually found in soluble, oxidized, or precipitated forms [39]. Siderophores are high-affinity ferric iron-chelating compounds produced by microorganisms and some plants under Fe deficiency, contributing to plant growth promotion and disease suppression [40].

Iron uptake in plants can be mediated by volatile organic compounds released by Trichoderma. For instance, the strains T. harzianum T78 and T. asperellum T34 promoted the expression of IRT1 and FRO2 in Arabidopsis thaliana. In soybean roots, T. afroharzianum T22 is capable of inducing the expression of citrate synthase (GmCs) and malate synthase (GmMs), which are responsible for the production of organic acids that lower soil pH and enhance Fe chelation following Fe deficiency, as soil acidification is a key mechanism for Fe solubilization. Trichoderma is also able to produce metabolites that function as siderophores, which are essential for fungal Fe homeostasis and related activities [41].

Enzymatic activity quantification revealed chitinase, β-1,3-glucanase, and protease production, with the highest chitinase activity observed in assays 6 (MMBF 58/09) and 1 (IB 19/17) at 24 h; β-1,3-glucanase in assays 2 (48 h), 8 (72 h), 9 (24 h and 96 h), and 10 (24 h) for MMBF 58/09, as well as assay 2 (URM 6997/160821) at 24 h; and protease in assay 6 for all three strains at 24 h (MMBF 58/09) and 96 h (URM 6997/160821 and IB 19/17). Regarding enzymatic activities, chitinases are responsible for hydrolyzing chitin, a linear polymer composed of N-acetyl-2-amino-deoxy-D-glucopyranose or N-acetylglucosamine units linked by β(1→4)-glycosidic bonds [29]. In a study cultivating Trichoderma isolates in a minimal synthetic buffered medium using 1% colloidal chitin as a carbon source, the isolates showed maximum activity equivalent to 62.12 pka mL− 1 (3.73 × 10− 3 mg mL− 1) of chitinase in T. harzianum and 9.94 nkat mL− 1 (5.96 × 10− 1 mg mL− 1) of β-1,3-glucanase in T. viride when 1% laminarin was used as the carbon source. These activities were analyzed with respect to pH and temperature: chitinase activity increased within a pH range of 3.0–6.0 and temperature of 15–30 °C, while β-1,3-glucanase activity varied within pH 3.0-5.5 and 15–25 °C. Another effect was observed when other carbon sources, such as glucose and sucrose, were used, which suppressed both chitinase and β-1,3-glucanase activities compared to colloidal chitin and laminarin, respectively. As a nitrogen source, ammonium nitrate resulted in the highest enzymatic activity [25].

In the present study, no supplementation with colloidal chitin or laminarin was used, and the enzymatic activity values were similar. Assay 6 showed the highest chitinase quantification at 24 h for the MMBF 58/09 strain, and assay 1 at 72 h for IB 19/17, both conducted at 28 °C with 15 g L− 1 glucose, without ammonium sulfate as the nitrogen source but with 8 g L− 1 of hydrolyzed protein (HP). Another study using solid-state fermentation with Trichoderma and colloidal chitin as the carbon source reported increased chitinase production compared to other sources, such as chitin powder or flakes, due to the colloidal conformation facilitating microbial metabolization. For nitrogen sources, yeast extract yielded the highest enzymatic activity compared to peptone, urea, corn steep liquor, ammonium chloride, and ammonium sulfate [29].

In a study on T. harzianum cultivation for β-1,3-glucanase production, an increase was observed at 24 h, with maximum activity reached at 96 h, and the optimal pH range was 4.0–6.0 [42]. The production of chitinase and β-1,3-glucanase is influenced by pH, with acidic conditions reported as critical for enzymatic production in T. harzianum, showing an optimum pH of 6. Another important factor is the carbon source; high enzymatic activity requires induction by fungal cell wall components [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43], which contain linear ordered chitin and amorphous β-1,3-glucan [44]. In contrast, carbon catabolite repression can occur with sugars such as glucose and fructose. Another study reported that glucose can act as a catabolic repressor in enzyme production, while sucrose may be associated with lower protein synthesis [45]. Assays 2 (48 h), 8 (72 h), and 9 (24 h, 72 h, and 96 h) showed the highest β-1,3-glucanase activity for strain MMBF 58/09, and assay 2 at 24 h for strain URM 6997/160,821.

The determination of β-1,3-glucanase enzymatic activity depends on several factors, including the strain used, culture conditions, and the type of substrate/carbon source. In a liquid fermentation using TLE medium with T. asperellum, varying substrates such as glucose, starch, chitosan, chitin, laminarin, CWRS (cell wall of Rhizoctonia solani), and cellulose were tested for β-1,3-glucanase production. The highest enzyme activity was obtained with CWRS and starch, while the other polysaccharides showed lower activity, and no activity was detected with glucose as the carbon source. The activity levels varied according to the type of glycosidic linkage and the carbohydrate structure. Additionally, β-1,3-glucanase activity was observed in T. harzianum grown with the cell walls of phytopathogens such as R. solani, Sclerotium rolfsii, and Pythium spp. The optimal pH was in the range of 4.0–6.0, precisely 3.6, and the optimal temperature was 45 °C [44].

Among the sugars tested, the highest β-1,3-glucanase activity was observed with galactose, and the lowest with sucrose. For chitinase, the highest activity was observed with glucose and the lowest with maltose. Indirectly, T. viride exhibited maximum biomass production when ammonium sulfate was used as the nitrogen source [25]. In this study, significance for ammonium sulfate was observed at p < 0.10 in assay 2 (48 h) and 9 (96 h) for strain MMBF 58/09, and at 24 h for strain URM 6997/160,821, regarding β-1,3-glucanase activity, with the highest concentration in assay 2. Chitinase activity reached its maximum in assays 1 and 6 for strains IB 19/17 and MMBF 58/09, respectively. Ammonium sulfate did not show significance for strains MMBF 58/09 and URM 6997/160,821, only for IB 19/17 at 72 h, with maximum production observed in assay 6 for strain MMBF 58/09 and assay 4 for strains URM 6997/160,821 and IB 19/17, at the highest hydrolyzed protein (HP) concentration of 8 g L− 1. The addition of glucose to a fermentation medium for β-1,3-glucanase production may be advantageous when using T. harzianum, but for other species, enzyme synthesis can be inhibited in the presence of high levels of glucose or readily fermentable carbon sources [46].

IAA is a plant hormone that supports plant growth, similar to auxins, promoting root development during early plant stages. Trichoderma species are capable of producing the metabolite IAA [1]. Studies with Trichoderma in submerged fermentation using Tryptic Soy Broth reported IAA production ranging from 7.19 to 21.14 × 10− 3 mg mL− 1, which was increased by the addition of L-tryptophan [18]. Similarly, the addition of L-tryptophan in Potato Dextrose broth fermented with Diaporthe terebinthifolli resulted in a maximum IAA production of 121.21 × 10− 3 mg. mL− 1, and higher pH values can lead to elevated IAA production in T. harzianum. This effect may be due to alterations in protein conformation, causing enzyme denaturation and modifying the metabolite profile. A similar effect can occur with increased temperature, as temperatures between 30 and 35 °C in Trichoderma promote higher IAA production [47].

IAA production is not a universal trait among all Trichoderma species. Nevertheless, the genus can promote plant growth regardless of the environment from which strains are isolated. In fungi, IAA biosynthesis is tryptophan-dependent, and adding amino acids to the culture medium enhances its production. Also, factors such as pH, temperature, and carbon and nitrogen sources significantly influence IAA biosynthesis [48–49].

In this study, the assays showing the highest protease quantification and significance at p < 0.10 were assays 1 and 8 for strain MMBF 58/09, 1 and 6 for strain URM 6997/160,821 at 24 h, and 3 at 24 h and 6 at 48 h for strain IB 19/17. All three strains showed significance for hydrolyzed protein (HP). According to Metwally et al. [50], who used organic residues (fish waste) on onion seeds (Allium cepa L.) together with mycorrhizal fungi and Trichoderma, an increase in onion shoot growth was observed when co-inoculated with mycorrhizal fungi and T. viride, as well as enhanced root surface area and nutrient accessibility, more when the fungi were applied individually.