Co-Cultivations of Beauveria bassiana, Metarhizium anisopliae, and Trichoderma harzianum to Produce Bioactive Compounds for Application in Agriculture

Abstract

Regenerative agriculture aims to improve soil quality and restore soil biodiversity, re-establishing natural systems in agricultural areas. Among some strategies, it is important to reduce the use of chemical pesticides that affect the productive capacity of the soil and cause problems to the environment. Based on this necessity, we present a strategy of producing a single product with bioinsecticidal and biofungicidal effects by submerged co-cultivations and paired cultivations of Beauveria bassiana, Metarhizium anisopliae, and Trichoderma harzianum using different concentrations of glucose, sucrose, hydrolyzed animal protein (HAP), soybean meal hydrolysate plus organic phosphorus (SMH), and hydrolyzed feathers (HF) as renewable nutrients. The single cultivations and double and triple co-cultivations were carried out for 7 days at 28 °C in orbital agitation at 120 rpm. Most of the highest values of conidia were obtained in the treatments at the central point, in which (g L⁻¹) glucose (20), sucrose (10), HAP (7.5), SMH (2.5), and HF (2.5) were used. The fermented broths were applied to the backs of adult bugs (Euschistus heros), which mostly provided 66–88% mortality. Beauveria bassiana + Metarhizium anisopliae showed approximately 70% inhibition against Sclerotinia sclerotiorum and Macrophomina phaseolina. As a way forward, this product demonstrated integrated bioactivities as insecticide and fungicide and can be optimized to substitute chemical pesticides that have negative impacts on the environment.

1. Introduction

Corn, wheat, and beans are crops widely used in human nutrition. They are also used in animal feed, such as for poultry and swine. According to the Brazilian National Supply Company (CONAB—https://www.conab.gov.br/info-agro/safras/graos/boletim-da-safra-de-graos, accessed on 19 November 2024), in the crop year 2023/2024, Brazil harvested 147,718 tons of soybean in 46,148 hectares with average grain yield of 3201 kg ha⁻¹, which is a production 17.7% larger than the crop year 2021/2022. Bean and corn in the crop year 2023/2024 reached 3243 and 119,813 tons, respectively, while 8096 tons of wheat were produced in the crop year 2024. However, pests compromise the desired growth and development of these and other crops, such as phyllophagous bugs. The damage caused by these insects is usually irreversible due to their attack directly on the grains, causing a severe reduction in quality and productivity. The most abundant phyllophagous bug species in soybean is Euschistus heros (Hemiptera: Pentatomidae), known as the neotropical brown stink bug. It is found in South America, with a higher incidence in regions with higher temperatures [1].

The white mold caused by Sclerotinia sclerotiorum is another problem. It causes a severe reduction in crop yield due to the reduction in the number and weight of grains from the girdling of the stems and the rupture of the xylem and phloem. The sclerotia mixed in the grains can reduce selling prices and the infection by this pathogen inside seed pods can reduce the amount of oil and the germination potential of the seeds [2].

Root rot complex disease caused by the phytopathogenic fungus Macrophomina phaseolina is difficult to control due to the lack of chemical control, the increase in resistant varieties, and the polyphagy of the fungus, even though sustainable strategies of biocontrol have been studied [3]. The first report of this pathogen in Brazil occurred in the common bean (Phaseolus vulgaris (L).) crop, infecting its roots [4]. The damage caused by this phytopathogen was neglected for several years due to its appearance occurring in the final stages of crop development [5]. Anthracnose is another disease that is difficult to eradicate, which is caused by fungi of the genus Colletotrichum. In soybean, for instance, it appears at the end of the cycle, which may affect the initial phase of pod formation favored by conditions of high rainfall and high temperatures [6].

Although the most adopted method for controlling pests and diseases is still chemical control, when used massively and incorrectly, it causes different problems, such as residues and resistance of target insects to the applied insecticide. Biological control is a method that has been demonstrated to be very efficient and less harmful to the environment [7]. Entomopathogenic fungi are important agents due to their broad spectrum of action on the target insect, being able to infect different stages of development, such as eggs, nymphs, larvae, pupae, and adults [8,9]. Considering that direct ingestion of pathogens is not required, it is possible to infect the insect through physical contact [8,9].

Knowing the representativeness of brown stink bug damage and fungal diseases, new studies related to these pathogenicities can contribute as an additional way for their control. Therefore, this study presents the co-cultivation and paired cultivation of the entomopathogenic fungi Beauveria bassiana, Trichoderma harzianum, and Metarhizium anisopliae for the production of metabolites with integrated insecticidal and fungicidal effects for the biological control of the insect pest Euschistus heros and fungi Sclerotinia sclerotiorum, Macrophomina phaseolina, Colletotrichum truncatum, and Colletotrichum gloesporioides. The objective was to provide an effective contribution to integrated pest management, providing alternatives for the phytosanitary management of crops around the world.

2. Materials and Methods

2.1. Sample Acquisition and Preparation

The fungal isolates of Beauveria bassiana, Trichoderma harzianum, and Metarhizium anisopliae were maintained in the Biotec Factory at the Federal University of Santa Maria (UFSM). They were multiplied in a standard basal culture medium adapted from the medium formulated by Schmaltz et al. [10]: monopotassium phosphate—KH₂PO₄ (2 g L⁻¹), iron sulfate heptahydrate—FeSO₄·7H₂O (0.05 g L⁻¹), magnesium sulfate heptahydrate—MgSO₄·7H₂O (0.5 g L⁻¹), manganese sulfate heptahydrate—MnSO₄·7H₂O (0.5 g L⁻¹), zinc sulfate heptahydrate—ZnSO₄·7H₂O (0.2 g L⁻¹), calcium chloride—CaCl₂ (0.5 g L⁻¹), cobalt chloride—CoCl₂ (0.4 g L⁻¹), sodium chloride—NaCl (1.0 g L⁻¹), and PDA (potato dextrose agar; 39.0 g L⁻¹) sterilized. All materials and culture media were acquired form Synth (Diadema, Brazil) and were previously sterilized in an autoclave at 121 ± 1 °C for 30 min, according to Brun et al. [7].

The maintenance of fungal strains was performed as proposed by Aita et al. [11] and Brun et al. [7] using Petri dishes with PDA medium. A disc of approximately 20 mm of medium containing each fungus was transferred to new Petri dishes with PDA medium, which were maintained at 28 °C for 7 days in a microbiological oven (7 Lab, Bio CBi 11L, Rio de Janeiro, Brazil). Thereafter, they were stored in a refrigerator at 4 ± 1 °C. After observing the mycelial growth in the Petri dishes, slants were prepared by transferring the fungus from the plates, with the aid of a platinum loop, to 10 mL Falcon tubes containing PDA medium. They were maintained for 96 h in the microbiological oven (7 Lab, Bio CBi 11L, Rio de Janeiro, Brazil) at 28 °C. In the sequence, the fungal mycelia were scraped from the slants, transferred to 50 mL Falcon tubes containing 10 mL of PD medium, and stored again in the oven at 28 °C for 24 h. The fungal spore suspensions resulting from this process were used as inoculum for co-cultivation processes.

Eggs of Euschistus heros were purchased from Promip^® (Piracicaba, Brazil). The growth of insects was carried out in the Integrated Pest Management Laboratory (LabMIP) at UFSM. After hatching out, the insects were fed with an organic bean pod and were grown in an acclimatized environment with a temperature of 25 ± 2 °C, relative humidity of 60 ± 20%, and a 14 h photoperiod, until reaching the adult phase.

The Macrophomina phaseolina (Tassi) Goid isolate, registered in GenBank MK 450343, was supplied by the Faculty of Agronomy Eliseu Maciel, from the Federal University of Pelotas (UFPel, Brazil). Sclerotinia sclerotiorum, Colletotricum truncatum, and Colletotricum gloesporioides, with their respective records in GenBank MMBF 03/18, MMBF 05/05, and MMBF 02/12, were acquired from the Instituto Biológico de São Paulo—SP.

2.2. Cultivations and Co-Cultivations

The cultivations and co-cultivations were carried out in a submerged medium (

Table 1. Plackett–Burman experimental design with coded variables in parentheses.

Assay	Glucose (g L−1)	Sucrose (g L−1)	HAP (g L−1)	SMH (g L−1)	HF (g L−1)
1	30 (1)	5 (−1)	10 (1)	0 (−1)	0 (−1)
2	30 (1)	15 (1)	5 (−1)	5 (1)	0 (−1)
3	10 (−1)	15 (1)	10 (1)	0 (−1)	5 (1)
4	30 (1)	5 (−1)	10 (1)	5 (1)	0 (−1)
5	30 (1)	15 (1)	5 (−1)	5 (1)	5 (1)
6	30 (1)	15 (1)	10 (1)	0 (−1)	5 (1)
7	10 (−1)	15 (1)	10 (1)	5 (1)	0 (−1)
8	10 (−1)	5 (−1)	10 (1)	5 (1)	5 (1)
9	10 (−1)	5 (−1)	5 (−1)	5 (1)	5 (1)
10	30 (1)	5 (−1)	5 (−1)	0 (−1)	5 (1)
11	10 (−1)	15 (1)	5 (−1)	0 (−1)	0 (−1)
12	10 (−1)	5 (−1)	5 (−1)	0 (−1)	0 (−1)
13	20 (0)	10 (0)	7.5 (0)	2.5 (0)	2.5 (0)
14	20 (0)	10 (0)	7.5 (0)	2.5 (0)	2.5 (0)
15	20 (0)	10 (0)	7.5 (0)	2.5 (0)	2.5 (0)

HAP: hydrolyzed animal protein; SMH: soybean meal hydrolysate + organic phosphorus; HF: hydrolyzed feathers.

), in which the Plackett–Burman experimental design was applied to evaluate the effects of the variables glucose, sucrose, hydrolyzed animal protein (HAP), soybean meal hydrolysate plus organic phosphorus (SMH), and hydrolyzed feathers (HF), based on Aita et al. [11]. The Erlenmeyer flasks (250 mL) for each treatment were filled with 60 mL of cultivation medium. Suspensions of spores of the fungi Beauveria bassiana, Trichoderma harzianum, and Metarhizium anisopliae, and their combinations, were inoculated in the cultivation medium. The single cultivations and double and triple co-cultivations were carried out for 7 days at 28 °C in orbital agitation at 120 rpm, based on Todero et al. [12].

After the cultivations and co-cultivations, the broths were filtered using 1 μm pore Whatman™ Glass microfiber paper with the aid of a vacuum pump (GlassLab, model 820, São Paulo, Brazil). The filter papers were previously dried in an oven at 60 °C for 36 h. After filtering, the papers with the broth biomass were placed again in the oven at 60 °C for 7 days for mass evaluation. The permeated fraction was stored in 50 mL Falcon tubes until the application to control Euschistus heros.

The conidia in the broth were evaluated, which is based on the preparation of a conidial suspension of Trichoderma sp. and counting the number of conidia with the aid of a Neubauer Chamber (hemacytometer) under an optical microscope. The preparation of the diluent consisted of adding 9 g of NaCl 0.1 M and 1 mL of Tween 80 in 1 L of distilled water and, thereafter, the mixture was autoclaved. The broths were maintained at room temperature (25 °C) for 40 min before the dilution process. The dilution consisted of adding 10 mL of each broth into Erlenmeyer flasks and completing with 90 mL of the diluent solution, corresponding to a 10:1 dilution. The Erlenmeyer flasks were placed in an orbital shaker for 60 min at 90 rpm and, in sequence, in an ultrasound bath for 5 min. The contents of the Erlenmeyer flasks were transferred to beakers and stirred for 5 min. After stirring, 1 mL was transferred to test tubes and the dilution was performed 3 times. An aliquot of 0.5 µL from this dilution was added to plates and covered by the coverslip. Counting was performed under an optical microscope (Primo Star, brand Carl Zeiss, São Paulo, Brazil) with a magnification of 400× and a Neubauer camera.

2.3. Insecticidal Effect Against Euschistus heros

The insects (Euschistus heros), 5 per treatment, were put in plastic pots filled with autoclaved peeled peanuts. The permeated fraction (5 μL) was suspended on the back of each insect with a micropipette. In the control treatment, only distilled water was used. A completely randomized design was applied with 5 repetitions for each treatment (105 treatments). The mortality of Euschistus heros was observed daily, for 10 days. The mortality of the insects was attributed when the insects that comprised the repetition of the treatment were considered dead, discounting the mortality of insects in the control [13]. The statistical analysis of data was carried out in Statistica 7.0^® software with a 95% confidence level.

2.4. Fungicidal Effect Against Sclerotinia sclerotiorum, Macrophomina phaseolina, Colletotrichum truncatum, and Colletotrichum gloesporioides

The paired cultivation technique was used in a Petri dish containing autoclaved PDA medium. Streptomycin^® was added at a concentration of 0.2 g mL⁻¹. Sclerotia of pathogenic fungi were transferred, with the aid of a platinum loop, to Petri dishes with PDA medium poured and positioned on the edge of the dishes. The control fungi (F_control) were sown on the opposite side of the dishes and incubated in an oven at 28 °C. The inhibition potential of the F_control was evaluated 15 days after sowing on the dishes, with measurements of the mycelial growth of the phytopathogen (F_pathogen). A completely randomized design was applied, comprising 7 treatments and 3 repetitions for each F_pathogen. The results are presented as a percentage of mycelial growth inhibition (Equation (1)).

Inhibition (%) = ([F_control diameter − F_pathogen diameter]/F_control diameter) × 100

(1)

The data were submitted for the analysis of normality and homogeneity. When they were significant, the analysis of variance (ANOVA, F test at 5% probability) was conducted by comparing the averages with Tukey’s test at 5% probability of error using the statistical package SISVAR 5.0^®.

3. Results

3.1. Conidia and Dry Biomass of Broths

The conidia concentration and dry biomass for the cultivations of Beauveria bassiana (BV), Metarhizium anisopliae (MT), and Trichoderma harzianum (TC), and the co-cultivations of Beauveria bassiana + Metarhizium anisopliae (BVMT), Beauveria bassiana + Trichoderma harzianum (BVTC), Metarhizium anisopliae + Trichoderma harzianum (MTTC), and Beauveria bassiana + Metarhizium anisopliae + Trichoderma harzianum (BVMTTC), are presented in Figure 1.

For conidia, most of the highest values were obtained in the treatments at the central point, in which 20 g/L of glucose, 10 g/L of sucrose, 7.5 g/L of HAP, 2.5 g/L of SMH, and 2.5 g/L of HF were used. Regarding conidia in 1 mL of broth, it was possible to reach 16.4 × 10⁶ with BV, 19.9 × 10⁶ with MT, 16.1 × 10⁶ with TC, 17.5 × 10⁶ with BVMT, 15.2 × 10⁶ with BVTC, 16.3 × 10⁶ with MTTC, and 15.0 × 10⁶ with BVMTTC. Regarding dry biomass, it was possible to reach 1.18 g with BV, 1.46 g with MT, 1.23 g with TC, 1.16 g with BVMT, 0.87 g with BVTC, 1.25 g with MTTC, and 0.69 g with BVMTTC. Statistical analyses indicated that cultivations using TC and co-cultivations using BVMT presented significant differences in the conidia concentrations (Figure 2).

3.2. Insecticidal Effects of Cultivations and Co-Cultivations

The insect mortality on the 10th day (10 DAS) is presented in Figure 3. It was evaluated daily, and the first broth to cause mortality was assay number 11 with MT (cultivation using 10 g L⁻¹ of glucose, 15 g L⁻¹ of sucrose, 5 g L⁻¹ of HAP, 0 g L⁻¹ of SMH, and 0 g L⁻¹ of HF), showing mortality on the first day of evaluation.

3.3. Fungicidal Effects of Paired Cultivation

The evaluation of the paired cultures proceeded 15 days after the inoculation of the fungi in the Petri dishes, and the results are presented in Figure 4. It is observed that the highest inhibition of growth occurred by MT against Macrophomina phaseolina. It was followed by 70% inhibition of BVMT against the Sclerotinia sclerotiorum. BVMT also showed inhibition of Colletotrichum gloesporioides, while TC inhibited Colletotrichum truncatum by approximately 50%. The lowest growth inhibitions occurred with TC, BVMTTC, BV, and BVTC on Colletotrichum gloesporioides, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Colletotrichum truncatum, with respective inhibitions of 10, 10, 20, and 30%. The results of statistical analyses are presented in

Table 2. Comparison of inhibition (%) (n = 3) of F_control on Sclerotinia sclerotiorum, Macrophomina phaseolina, Colletotrichum gloesporioides, and Colletotrichum truncatum.

Fcontrol	S. sclerotiorum *	M. phaseolina *	C. gloesporioides *	C. truncatum *
BVMT	73.3 A	68.9 AB	50.0 A	40.7 A
MT	50.0 AB	91.5 A	31.1 B	41.5 A
BVTC	57.0 AB	33.3 BC	29.6 B	31.5 A
BV	27.8 AB	31.9 BC	40.0 AB	40.0 A
MTTC	42.6 AB	31.9 BC	25.0 B	40.7 A
BVMTTC	35.2 AB	11.9 C	33.3 B	39.3 A
TC	51.9 AB	15.2 C	10.4 C	48.1 A

* Means followed by the same letter in each column do not differ statistically by Tukey’s test at 5% probability of error.

.

4. Discussion

In cultivation with TC, the variable HAP presented a negative significative effect on conidia. This means it is preferable to use lower contents of HAP in this cultivation. In the co-cultivation with BVMT, the variable sucrose presented a negative significant effect, indicating that the highest conidia concentrations are obtained when using lower sucrose contents, such as 5 g L⁻¹, as in the treatments of numbers 4, 8, and 9. In these assays, approximately 17.2–17.5 × 10⁶ conidia mL⁻¹ were obtained. In the scientific literature, an optimization of the culture medium of Beauveria bassiana and spore yield was evaluated using response surface methodology. The authors reported maximum predicted sporulation using the stock medium as 5.55 × 10⁶ mL⁻¹ using a mathematical model and Box–Behnken design. The formula comprised 54.8 g of potato, 77.7 g of wheat bran, and 101.5 g of wheat [14]. Therefore, the necessity of defining the best concentration of nutrients to produce conidia is evident.

No significant effect of variables on dry biomass was observed. This agrees with the findings reported by Li et al. [15]. Higher biological activity cannot necessarily be attributed to higher dry biomass production. Although no significant effect was evidenced, the dry biomass ranged from 1.30 to 1.46 g when using MT at the central point. Otherwise, in the BVMT assay, the dry biomass at the central point ranged from 0.21 to 1.11 g. In the scientific literature, the effects of fermented broths were evaluated on whitefly (Bemisia tabaci Genna dius) and Ceratitis capitata (Diptera: Tephritidae) control. The virulence of fungi is directly affected by the initial amount of conidia that are in contact with the insect [8,16].

The comparisons of insecticidal action of cultivations and co-cultivations were performed based on the control treatment, that is, the mortalities were discounted from the mortality in the control treatment. The evaluation time used in this research is related to the time described by Qu and Wang [17]. Unlike chemical insecticides, which can be lethal in a few hours, biological insecticides from entomopathogenic fungi require 24 to 48 h to infect the insect, with mortality occurring between the second and third DAS.

Most cultivations and co-cultivations presented high insect mortalities. Overall, the assays using MT, TC, and BVMTTC presented the highest control rate, with most of the mean mortalities ranging from 66 to 89%. For all cultivations and co-cultivations, treatment number 4 is favorable for reaching the best mortalities, corroborating the results of conidia. Although different factors may be related to the mortality of insects subjected to control with broth from entomopathogenic fungi, it is worth mentioning that the latent period between conidial contact and insect death will vary according to the strength of the insect’s immune response and the virulence of the fungus [18]. Also, Vega et al. [19] pointed out that fungal efficacy is directly related to the number of propagules that come into contact with the host surface.

Some studies indicate that isolates from a fungal species can vary significantly in their ability to infect and kill a particular insect pest, as is the case of Beauveria bassiana [16]. This inference is corroborated by Nora et al. [13], where differences in virulence between isolates became evident after six days at the highest concentration of conidia and after nine days at the lowest concentration, with mortality occurring more quickly at higher concentrations. The action of fungi such as Beauveria bassiana depends on the conidia production [13]. The outcomes found in this research point out good ways for the use of cultivations and co-cultivations in the biological control of insects, demonstrating the emergence of a new product. Elsewhere, Sala et al. [20] also demonstrated the potential of solid-state fermentation for conidia production. When compared with the tray bioreactor, the packed bed obtained higher conidia production due to its better use of the total reactor volume.

The efficiency of Trichoderma harzianum increases the benefits of using different isolates of Trichoderma in crops by causing the total mortality of most insects. Elsewhere, it was observed that the inoculation of Trichoderma asperellum UFT 201 in seeds of the soybean cultivar 48B32 (BASF^®) increased the characteristics of biomass and productivity [21]. It demonstrated the effectiveness of the fungus in promoting plant growth and, consequently, productivity, contributing to lower production costs [21]. Considering the potential for controlling pests and increasing crop productivity, the multiple benefits of using Trichoderma, observed through the development of studies, are presented as an important incentive for the use of biological inputs in many crops.

The high mortality caused by treatments with Metarhizium anisopliae in this study is also verified elsewhere with pests that infest the sugar beet crop using Metarhizium (Beta vulgaris L.) [22]. The authors found high mortality of adult and juvenile Euschistus heros, between 83 and 100%, caused by Metarhizium brunneum ARSEF 4556. This mortality rate is similar to or higher than the rates of other pathogenic species to other insects, including Nezara viridula and Dichelops melacanthus. The results point out Metarhizium as a strong candidate for further investigation as a control agent for stink bugs and other soybean pests.

The triple co-cultivation of Beauveria bassiana + Metarhizium anisopliae + Trichoderma harzianum is highlighted, which provided percentages of insect mortality near to 90% in many experimental assays (7 from 15). Although the rate of growth of each fungus was not measured in most of the conditions, the consumption of nutrients and their mechanism of interaction were favorable to biosynthesizing metabolites that could kill the targeted insect. Regarding the double co-cultures, mortalities near to 90% were also reached, which are important outcomes to control this predominant stink bug that affects many crops. Co-cultivations of Beauveria bassiana + Metarhizium anisopliae and Metarhizium anisopliae + Trichoderma harzianum presented some assays without control or with control near to or lower than 40%. For this, it is inferred that some stress occurred, due to competition for nutrients, imbalance of nutrients, or another kind of relationship, which may have hindered the production of spores and metabolites in some culture conditions. According to the ANOVA results (Supplementary Materials), the variable HAP was the most critical one in these double co-cultures. Considering the fungicide effects, the results of statistical analyses (Table 2) confirmed the inferences generated in the analysis of Figure 4. The control of Macrophomina phaseolina by Metarhizium anisopliae has few cases reported in the scientific literature, making this discovery promising. When studying Fragaria ananassa, Dara [23] observed that plants treated with Metarhizium anisopliae one week before Macrophomina phaseolina inoculation showed higher resistance compared to plants inoculated only with Macrophomina phaseolina. This scientific result is a confirmation parameter of the fungicidal action of Metarhizium anisopliae on the pathogenic fungus Macrophomina phaseolina.

In the statistical analysis, Sclerotinia sclerotiorum showed the highest inhibitions by the double and triple F_control of Beauveria bassiana, Trichoderma harzianum and Metarhizium anisopliae, and individual F_control of Trichoderma harzianum and Metarhizium anisopliae. Only the individual fungus Beauveria bassiana did not show a significant effect. These findings agree with the scientific literature, as Souza [24] concluded that strains of Trichoderma used in paired cultivation showed promising inhibition potential, ranging from 84 to 100% against the pathogen, pointing out the Trichoderma as a promising antagonist.

For Colletotrichum gloesporioides, the statistical analysis showed higher inhibition by the co-cultivation BVMT, followed by BV, BVMTTC, MT, BVTC, and MTTC (Table 2). For individual fungi, Trichoderma harzianum showed different inhibition from the others and with a lower value (approximately 10%). However, Sharma et al. [25] reported that the combined and individual applications of Trichoderma harzianum, Bacillus subtilis, and Pichia anomala biocontrol agents, by foliar spraying or fruit immersion, showed the potential to control anthracnose. Cruz-Quiroz et al. [26] performed in vitro tests with double culture and pointed out the use of different strains of Trichoderma sp. as an excellent biological agent in the control of fungal diseases caused by Colletotrichum gloeosporioides and Phytophthora capsici. Thus, this justifies the need for further studies to evaluate the fungicidal effect of Trichoderma harzianum.

Biocontrol activities were reported in in vitro assays involving another species of Trichoderma (Trichoderma viridae) and Beauveria bassiana against Colletotrichum gloeosporioides [27]. The support of the scientific literature demonstrates the effective control of Colletotrichum gloesporioides using combinations of entomopathogenic fungi, making effective the contribution of this research for the biological control of this pathogen.

The F_control did not differentiate the growth inhibition of the F_pathogen of Colletotrichum truncatum, remaining in the range of 30 to 50% (Table 2). These results corroborate the control efficacy demonstrated in the literature for other species of the genus Colletotrichum, such as for Colletotrichum gloesporioides. This statement is reinforced by Chagas et al. [28]. The authors used colony pairing and detected 46 strains of Trichoderma as efficient antagonists for the phytopathogens Colletotrichum cliviae and Colletotrichum truncatum. Regarding the inhibition of pathogen growth, 22 isolates of Trichoderma sp. were efficient antagonists, with more than 75% inhibition of Colletotrichum cliviae and three with more than 90% of Colletotrichum truncatum.

The threat of anthracnose caused by Colletotrichum truncatum in lima bean (Phaseolus lunatus L.) was also evaluated elsewhere [29]. The authors carried out a direct challenge test of pathogens in vitro with strains of Trichoderma that grew on the pathogen colony, occupying the entire Petri dish. It was observed that antagonistic fungi have potential against Colletotrichum truncatum in the antibiosis test, with inhibition of mycelial growth and sporulation ranging from 74.7 to 82.0% and 78.7 to 89.3%, respectively. In this research, promising results can be seen in the study of the fungicidal potential of entomopathogenic fungi, demonstrating good alternatives for the biological control of the pathogens discussed in this research.

In the context of economic issues, the use of biological products such as the presented ones emerges as a strategy to make agriculture more sustainable and compatible with the principles of the circular economy, which seeks to maximize resource efficiency and minimize waste. The development of biological products has enabled the creation of more effective and specific solutions. The fungi studied in this research can be easily replicated to have high biomass content, and the renewable nutrients can be easily found at low cost, since part of them are obtained from wastes. Therefore, the production can be affordable, while further studies dedicated to specific economic analysis are extremely welcome. Compared to traditional pesticides, biopesticides reduce the dependence on importing chemical products for several countries that do not produce sufficient pesticides. Also, as biological products are increasing, there is a crescent opportunity for industries to market novel and efficient bioproducts, thus contributing to the circular economy.

5. Conclusions

Most of the assays of cultivations and co-cultivations showed promising results for the biological control of Euschistus heros. The cultivations of Metarhizium anisopliae and Trichoderma harzianum, and the co-cultivations of Beauveria bassiana + Trichoderma harzianum and Beauveria bassiana + Metarhizium anisopliae are highlighted, which provided high percentages of insect mortality in most of the other assays as well. The fungicidal potential of entomopathogenic fungi is also promising. The duo Beauveria bassiana + Metarhizium anisopliae presented good inhibition against the phytopathogens Sclerotinia sclerotiorum, Macrophomina phaseolina, Colletotrichum gloesporioides, and Colletotrichum truncatum. The results found in this research are promising, as they point out effective and sustainable alternatives to complement the chemical control of the studied pathogens. In an integrated analysis and as a way forward, considering both results with insecticidal and fungicidal effects evaluated in this study, it is concluded that the submerged co-cultivation using Beauveria bassiana + Metarhizium anisopliae with glucose (30 g L⁻¹), sucrose (5 g L⁻¹), hydrolyzed animal protein (10 g L⁻¹), and soybean meal hydrolysate plus organic phosphorus (5 g L⁻¹) is the most suitable treatment to obtain the highest integrated control effects.