Toxicity of Post-Emergent Herbicides on Entomopathogenic Fungi Used in the Management of Corn Leafhopper: In Vitro and In Vivo Assessments

Abstract

This is the first study to assess the physicochemical and biological compatibility of herbicides used in corn crops with entomopathogenic fungi used in the management of Dalbulus maidis in Brazil. The biological index was employed to ascertain the in vitro compatibility of the herbicides with pure spores (not formulated) of tested fungal isolates (Esalq-1296 of Cordyceps javanica and IBCB66 and Simbi BB15 of Beauveria bassiana). The results indicated a significant interaction between herbicides and fungal isolates when colony diameter and colony-forming units (CFU) were considered. Furthermore, changes in physicochemical characteristics were observed in some mixtures of herbicides and mycoinsecticides tested. The number of CFU was significantly reduced as the exposure time increased in the mixtures containing all the herbicides tested. In general, the Esalq-1296 isolate of C. javanica, formulated in a suspension concentrate (Octane^®), proved to be more sensitive to the herbicides studied. In vivo bioassays demonstrated that, despite the synergistic effect of the binary mixtures of herbicides and mycoinsecticides on D. maidis mortality, the presence of the herbicide in the mixtures prevented the extrusion of entomopathogens from cadavers; therefore, caution is recommended when combining mycoinsecticides and post-emergent herbicides in tank mixtures aiming to manage D. maidis.

1. Introduction

The productivity of maize [Zea mays L. (Poales: Poaceae)] crops can be negatively impacted by biotic and abiotic factors, including weed competition, which is particularly prevalent during the initial stage of crop development [1]. At this stage, competition between maize plants and weeds for essential environmental resources (i.e., water, light, space, and nutrients) can result in yield reductions of up to 70% [2,3], which can significantly impact the profitability and viability of the crop. In this context, synthetic herbicides have been employed as the primary post-emergence weed management strategy [4,5].

Besides weeds, the occurrence of arthropod pests in the early stages of maize development—such as the fall armyworm [Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)], the green-belly stink bug complex [Diceraeus spp. (Hemiptera: Pentatomidae)], and the corn leafhopper [Dalbulus maidis (DeLong and Wolcott) (Hemiptera: Cicadellidae)] [6,7,8]—have been identified as important threats to the yield of corn crops in Brazil. The critical period for weed incidence coincides with that of the key insect pests of the initial stages of the crop, and as a result, the use of herbicides in association with insecticides in the spray tank has become a common practice among Brazilian producers, with over 70% of them adopting this approach [9]. This practice has contributed to a reduction in the cost and time of the operations, as well as the transit of machinery through the fields, the use of fuel and water, and even the number of machines needed to carry out these operations [9]; however, the compatibility of these mixtures in the spray tank has been rather variable [10], which has often jeopardized the effectiveness of the herbicides and/or synthetic insecticides in controlling weeds/insect pests that occur in the crop [11].

In addition to synthetic pesticides, entomopathogenic fungi-based products (mycoinsecticides) have also been employed to manage arthropod pests in Brazil and elsewhere [12,13]. In recent years, the use of mycoinsecticides for the control of agricultural pests has increased considerably due to both the high epizootic capacity of these microorganisms against various species of arthropod pests of agricultural relevance and the commercial availability [14,15,16]. For example, the use of mycoinsecticides based on Beauveria bassiana (Bals.-Criv.) Vuill. and Cordyceps javanica (Wize) (both Hypocreales: Cordycipitaceae) exceeds 1.8 million hectares in corn crops in Brazil [17,18]. The corn leafhopper, considered one of the most important pests of corn crops in the Neotropical region [8], has been identified as a primary target for mycoinsecticides [19,20]. The corn leafhopper causes direct damage to maize plants by sucking sap and injecting toxins [21]. It also causes damage indirectly by acting as a vector for two mollicutes [corn stunt spiroplasma (CSS, Spiroplasma kunkelii) and maize bushy stunt phytoplasma (MBSP, Candidatus phytoplasma asteris)] and two viruses [maize rayado fino virus (MRFV, Marafivirus) and maize striate mosaic virus (MSMV, Geminiviridae)] responsible for the corn stunt complex (CSC) in maize plants [22,23,24].

Given the increasing damage caused by D. maidis and the lack of curative measures for CSC, the management of this pathosystem is complex and requires the integration of strategies in a regional context [25], including the removal of volunteer maize plants in the off-season, the use of tolerant cultivars, and population control of the insect vector in the post-emergence of the crop [19,20]. In Brazil, management of D. maidis populations has been conducted by applying synthetic insecticides alone or in a mixture with mycoinsecticides based on spores of B. bassiana and C. javanica [20,26]. However, mycoinsecticides are composed of fungal isolates with varying degrees of susceptibility to the agrochemicals utilized in spray tank mixtures. Furthermore, synthetic pesticides utilize diverse additives and adjuvants in their formulation, which can influence their compatibility with these microorganisms [27], modify the physicochemical characteristics of spray solutions in tank mixtures [28], and induce changes in the immune system of target pests [29].

Given the temporal coincidence of weeds and insect pests in corn production areas and the operational advantages of the associated spraying of phytosanitary products, we tested the primary hypothesis that there is the possibility of associating post-emergent herbicides from different chemical classes with isolates of entomopathogenic fungi used in commercial products (mycoinsecticides) in Brazil. As a secondary hypothesis, we predict that the levels of toxicity of post-emergent herbicides are variable according to fungal isolates available on the market for the control of D. maidis in cornfields. To this end, we evaluated the in vitro and in vivo compatibility of different post-emergent herbicides used in maize crops with the isolates IBCB 66 and Simbi BB15 of B. bassiana and Esalq-1296 of C. javanica. Furthermore, the physicochemical compatibility of the mycoinsecticide and herbicide formulations was evaluated, as well as the effect of two formulations [oil dispersion (OD) and suspension concentrate (SC)] on the compatibility of mycoinsecticides and herbicides available on the Brazilian market. The findings of this study will not only serve to verify the compatibility of mycoinsecticides with post-emergent herbicides but will also elucidate the effectiveness of these mixtures in suppressing D. maidis populations in maize crops within an integrated pest management (IPM) framework.

2. Materials and Methods

2.1. Herbicides and Mycoinsecticides

The commercial herbicides and mycoinsecticides utilized in the bioassays are detailed in

Table 1. Synthetic herbicides used in the laboratory bioassays of compatibility with entomopathogenic fungal isolates or mycoinsecticide formulations.

Pesticides (Concentration g L−1 or g kg−1)	Commercial Brands	Formulation 1	Tested Dose 2 (mL or g ha−1)	Manufacturer
Synthetic Herbicides
Glyphosate (577.0)	Roundup Original Mais®	SL	6000.0	Bayer S/A (São José dos Campos, São Paulo, Brazil)
Glyphosate (792.5)	Roundup WG®	WG	3500.0	Bayer S/A (São José dos Campos, São Paulo, Brazil)
Glufosinate–ammonium (200.0)	Finale®	SL	2000.0	Basf SE (Guaratinguetá, São Paulo, Brazil)
Atrazine (400.0)	Primóleo®	SC	6000.0	Syngenta Proteção de Cultivos Ltda. (Paulínia, São Paulo, Brazil)
Tembotrione (420.0)	Soberan®	SC	240.0	Bayer S/A (São José dos Campos, São Paulo, Brazil)
Atrazine + simazine (250.0 + 250.0)	Primatop SC®	SC	8000.0	Syngenta Proteção de Cultivos Ltda. (Paulínia, São Paulo, Brazil)
Atrazine + mesotrione (50.0 + 500.0)	Calaris®	SC	2400.0	Syngenta Proteção de Cultivos Ltda. (Paulínia, São Paulo, Brazil)
Mycoinsecticides
Beauveria bassiana (Simbi BB15 isolate)	FlyControl®	OD	500.0	Simbiose Ltda. (Cruz Alta, Rio Grande do Sul, Brazil)
Beauveria bassiana (IBCB 66 isolate)	Bovenat®	WP	500.0	Bionat Soluções Biológicas Ltda. (Olímpia, São Paulo, Brazil)
Cordyceps javanica (Esalq-1296 isolate)	Octane®	SC	500.0	Koppert do Brasil Ltda. (Charqueada, São Paulo, Brazil)

¹ Formulations: SL = Soluble concentrate; WG = dispersible granules; SC = Suspension concentrate; OD = Oil dispersion and WP = Wettable powder.

. The herbicides and mycoinsecticides were tested at the maximum doses recommended by the manufacturers for corn cultivation in Brazil [26]. The herbicides and mycoinsecticides were dissolved in distilled water in a total volume corresponding to 150 L ha⁻¹.

2.2. Tested Insects

For the in vivo tests, D. maidis adults were obtained from a laboratory colony established with specimens collected in cornfields located in the municipalities of Mogi Mirim, SP, Brazil (22°27′06.4″ S and 47°04′12.9″ W), and Chapecó, SC, Brazil (27°05′16.0″ S and 52°38′14.4″ W). For rearing, P3016VYHR hybrid maize plants (Corteva Agriscience do Brasil Ltd.a., Barueri, SP, Brazil) in the V3–V4 phenological stages [30] were used, which were cultivated in pots (400 cm⁻³) containing sterilized substrate based on the Pinus sp. bark, peat, and expanded vermiculite (Tropstrato^®; Vida Verde Indústria e Comércio de Insumos Orgânicos Ltd.a., Mogi Mirim, SP, Brazil). The plants were placed in cages (35 × 30 × 30 cm in height, length, and width, respectively) made from plastic frames and covered with anti-aphid mesh (200-millimeter mesh). The corn plants were replaced every two days throughout the insect’s life cycle. The rearing process was conducted in a climate-controlled room at 25 ± 1 °C, 60 ± 10% relative humidity (RH), and with a 14 L:10 D hour photoperiod.

2.3. Obtaining and Propagating Entomopathogenic Fungal Isolates

Pure plates of the fungal isolates IBCB66 and Simbi BB15 of B. bassiana and Esalq-1296 of C. javanica were obtained by isolating the commercial mycoinsecticides used in this study (Table 1). To achieve this, the fungal isolates were grown on potato dextrose agar-based culture medium [PDA Difco^® (Becton-Dickinson Company, Franklin Lakes, NJ, USA)] with 5 g L⁻¹ of streptomycin in sterile Petri dishes (9 cm in diameter) in a vertical airflow chamber. Subsequently, the purified plates were incubated in a biochemical oxygen demand (BOD) chamber at a temperature of 25 ± 1 °C, 75 ± 10% RH, with a photoperiod of 14 L:10 D hours.

2.4. In Vitro Bioassays

All in vitro bioassays were conducted in BOD chambers maintained at 25 ± 1 °C, 75 ± 10% RH, with a 14 L:10 D hour photoperiod. Physicochemical compatibility studies of the herbicides with the mycoinsecticides were conducted in a climate-controlled room (25 ± 1 °C and 65 ± 10% RH). This study was conducted during the years 2023 and 2024.

2.4.1. Effects of Herbicides on Colony-Forming Units (CFU) of Pure Isolates of Entomopathogenic Fungi

To assess the effect of the herbicides on the number of colony-forming units (CFU), fungal suspensions containing the pure isolates were quantified in a Neubauer chamber (Kasvi Importação e Distribuição de Produtos para Laboratórios Ltd.a., São Paulo, SP, Brazil). The concentration was standardized at 500 spores mL⁻¹ in a volume of 10 mL of solution using a Zeiss AX10 (400×) microscope (Zeiss AG Corp., Oberkochen, Germany). Subsequently, the adhesive spreader Tween 80^® at 0.1% (Sigma-Aldrich Corp., Sydney, Australia) was added, and the suspensions were added to the herbicides recommended for corn cultivation in Brazil (Table 1, [26]) following the procedure described by Ribeiro et al. [20].

The suspensions were homogenized in a vortex shaker and kept in BOD chambers at 25 ± 1 °C for periods of 1.5 and 3.0 h of exposure. Following this period, a 200 µL aliquot of each suspension (treatment) was transferred to sterile Petri dishes containing Difco^® PDA culture medium with 5 g L⁻¹ streptomycin and spread on the dish with a Drigalski loop. A negative control was established by using sterile distilled water (200 µL). Afterward, the plates were kept in a BOD chamber for four days to count the CFU. Five Petri dishes (replicates) were used for each treatment level. The bioassay was conducted using a completely randomized design in a 7 × 3 × 2 factorial scheme (herbicides × fungal isolates × exposure times).

2.4.2. Vegetative Growth of Fungal Isolates Exposed to Herbicide-Treated Culture Media

The impact of the herbicides on the vegetative growth of the fungal isolates was evaluated following the protocol established by the International Organization for Biological and Integrated Control (IOBC) for testing the adverse effects of pesticides on entomopathogenic fungi [31], with adaptations suggested by Ribeiro et al. [20]. Firstly, the Difco^® PDA culture medium was sterilized at 121 °C for 15 min, cooled to 45 °C, an aliquot (5 g L⁻¹) of streptomycin was added and, subsequently, the herbicide solutions at the pre-defined concentrations (Table 1) were incorporated into the culture medium using manual stirring. Afterward, the culture media treated with the different treatments (15 mL) were transferred to sterile Petri dishes, and conidia of the fungal isolates previously extracted from pure sporulated cultures were inoculated. The inoculum of each isolate was obtained by scraping the surface of the pure plates with sterile spatulas in 5 mL of sterile water and 0.1% Tween 80^®. Each suspension of fungal isolates was homogenized in a vortex and the concentration was adjusted to 1 × 10⁸ conidia mL⁻¹. After the culture medium had solidified in the treated plates, 10 µL of the solution of each fungal isolate was inoculated into the center of each Petri dish. For each treatment, 10 Petri dishes (replicates) were used. Sterile distilled water was incorporated in the same proportion into the culture medium and used as a negative control. After inoculation, the Petri dishes were placed in BOD chambers to assess the vegetative growth of the fungal isolates.

A completely randomized design was employed in a 7 × 3 factorial scheme (herbicides × fungal isolates) for this bioassay. The vegetative growth of the fungal isolates was assessed 12 days after incubation (when the fungi had almost completely colonized the culture medium of the control treatment) by measuring the diameter of the colonies. The diameter of the colonies was quantified based on the average of two orthogonal measurements obtained using a digital electronic caliper (Worker Ferramentas Ltd.a., São José dos Pinhais, PR, Brazil).

2.4.3. Conidial Production and Viability of Fungal Isolates Exposed to Herbicide-Treated Culture Media

After assessing vegetative growth, five representative colonies were randomly selected and used to estimate the production (conidiogenesis) and viability of conidia. To assess conidia production, colonies were excised with a sterile scalpel and transferred to test tubes containing 10 mL of sterile distilled water + 0.1% Tween 80^®, which were shaken in a vortex shaker at 2800 rpm for 5 min. Subsequently, the suspensions were serially diluted and the conidia of the samples were quantified in a Neubauer chamber under an optical microscope (400×).

To assess conidial viability, conidial suspensions obtained from the previously selected colonies were adjusted to a concentration of 1 × 10⁶ conidia mL⁻¹, and an aliquot (600 µL) of the suspension was removed and spread on Rodac Petri dishes (60 mm in diameter) with a Drigalski loop. The germination of conidia was assessed between 14 and 18 h after inoculation under an optical microscope (400×). The bioassays (conidiogenesis and conidia germination) were conducted under a completely randomized design in a 7 × 3 factorial scheme (herbicides × fungal isolates).

2.4.4. Determination of Compatibility Between Herbicides and Pure Entomopathogenic Fungi

The in vitro biological compatibility of the synthetic herbicides with pure fungal isolates was determined based on the estimation of the biological index (BI) using the formula $BI:\left[47\times \left(VG\right)+43\times \left(SPO\right)+10\times GERM\right]/100,$ as proposed by Rossi-Zalaf et al. [32], where VG is the percentage of vegetative growth compared to the control; SPO is the percentage of sporulation compared to the control; GERM is the percentage of conidial germination compared to the control. The effect of the synthetic herbicides on the fungi isolates was classified according to the following criteria: incompatible (0 ≤ BI ≤ 41), moderately compatible (41 < BI ≤ 66), and compatible (BI > 66).

2.5. Physicochemical Analysis

The physicochemical characteristics of the herbicide and mycoinsecticide spray solutions, used alone or in binary mixtures at the respective concentrations tested (Table 1), were evaluated according to the technical standards recommended by the Brazilian Association of Technical Standards (ABNT) and described in the Brazilian Standard (NBR) 13875:2014 [28,33]. For these experiments, standard water with a hardness of 20 mg kg⁻¹ CaCO₃ was utilized. This was performed using graduated glass beakers to add 150 mL of standard water plus herbicides/mycoinsecticides, which were then rotated (180°) every 2 s for 10 times. Subsequently, 100 mL of standard water was added to the mixture, which was then transferred to Becker-type glass containers and kept under agitation in an IKA^® RT-15 magnetic stirrer (IKA Brasil Equipamentos Laboratoriais, Analíticos e Processos Ltd.a., Campinas, SP, Brazil) for 2 h. Following this, the spray solutions were allowed to stand for 10 min and visually assessed for physical characteristics. These included the presence (P) or absence (A) of homogeneity, flocculation, sedimentation, phase separation, oil separation, crystal formation, cream, foam, lump formation, and the presence of lumps larger than 149 µm, as required by NBR NM ISO 3310-1:2010 [34].

The chemical characteristics of the spray solutions were evaluated through the measurement of hydrogenionic potential (pH) with a DM20 pH meter (Digimed Instrumentação Analítica Ltd.a., São Paulo, SP, Brazil) and the electrical conductivity (EC) with an Orion 5-Star conductivity meter (Thermo Fischer Scientific Corp., Waltham, MA, USA). The order in which the products were added to form the treatments was as follows. First, the products with solid formulations were added to the beaker in sequence, dispersible granules (WG) and wettable powders (WP), and then the liquid formulations were added in sequence, emulsion oil-in-water (EW), suspension concentrate (SC), and emulsifiable concentrate (EC), as described by Rakes et al. [28].

2.6. Effects of Herbicides on the CFU of Mycoinsecticide Commercial Formulations

To ascertain the differences in the sensibility of the pure spores of the tested fungal isolates and those contained in the commercial formulations [FlyControl^® (OD formulation) and Octane^® (SC formulation); Table 1] to selected post-emergent herbicides, a new bioassay was conducted. To this purpose, the maximum doses recommended by the manufacturers were utilized to assess the effect on the CFU number of each mycoinsecticide exposed to herbicides in a spray solution, simulating a tank mixture (Table 1). Thus, the mycoinsecticides were mixed with the herbicides and the resulting suspensions were homogenized in a vortex-type shaker. The suspensions were then placed in BOD-type chambers at 25 ± 1 °C for 1.5 or 3.0 h of exposure.

Following the before-mentioned independent sampling periods, the spray solutions (binary mixtures of mycoinsecticides and herbicides) were diluted 20 times (with 1 part of the suspension containing the binary mixture of mycoinsecticide and herbicide to 19 parts of sterile distilled water). This dilution was necessary to facilitate the counting of the colonies on the plates. Subsequently, 200 µL of each suspension (treatment) was transferred to sterile Petri dishes containing Difco^® PDA culture medium with 5 g L⁻¹ streptomycin and spread with a Drigalski loop. The number of CFU for each treatment was quantified 4 days after the plates were placed in a BOD chamber. The application of 200 µL of sterile distilled water was used as a negative control. A total of five Petri dishes (replicates) were used for each treatment level. The bioassay was conducted using a completely randomized design in a 7 × 3 × 2 factorial scheme (herbicides × fungal isolates × exposure times).

2.7. Comparison of the Reduction in the Number of CFU of Pure and Formulated Spores (OD and SC Formulations) When Mixed with Herbicides

To ascertain the impact of synthetic herbicides when mixed with the pure spores of Simbi BB15 from B. bassiana and Esalq-1296 from C. javanica in comparison to their respective commercial formulations [FlyControl^® (oil dispersion—OD) and Octane^® (suspension concentrate—SC)], a descriptive reduction analysis was conducted following the method established by the IOBC [35] and calculated using the following equation:

$$E=(1-T/C)\times 100$$

where E is the percentage of the reduction in the number of CFU; T is the mean CFU for the treatment; and C is the mean CFU for the control treatment.

2.8. In Vivo Bioassays

All in vivo bioassays were conducted in BOD chambers maintained at 25 ± 1 °C, 80 ± 10% RH, with a 14 L:10 D hour photoperiod. For this purpose, adults of D. maidis (5–7 days old) from the laboratory population were anesthetized with CO₂ and placed in a dorsal position on sterilized Petri dishes for the application of the treatments. The treatments were mixed for 1.5 h (Table 1) and applied to the insects using an airbrush (OVD Importadora e Distribuidora Ltd.a., Novo Hamburgo, RS, Brazil) equipped with a 0.3 mm spray tip, a working pressure of 20 PSI, and an application volume of 1 mL, which was equivalent to a spray volume of 150 L ha⁻¹. Following the administration of the treatments, the insects were placed in cylindrical cages (700 mL) made from polyethylene terephthalate with side openings lined with voile-type fabrics. A corn plant (V2 stage) was added to each cage to provide the insects with a feed source. Insect mortality was assessed daily for 9 days. Insects that did not display any movement when stimulated with a fine-tipped brush were considered dead.

The dead insects (insect cadavers) were removed from the cages, superficially sterilized with sodium hypochlorite (1%, v/v) for 20 s, and immediately immersed in sterile distilled water three times. Subsequently, each sterilized insect was transferred individually to a sterile 2 mL cryogenic tube. Sterile hydrophilic absorbent cotton moistened with sterile distilled water was placed inside the lid of each cryogenic tube to maintain the humidity inside the tubes. The cryogenic tubes were kept in a BOD chamber (25 ± 1 °C; 75 ± 10% RH, with a 12 L:12 D hour photoperiod) for 10 days.

Following this period, the number of extruded insects was quantified. The extrusions were identified using the morphological characters described by Barnett and Hunter [36]. Subsequently, the contents of the cryogenic tubes were washed with 1 mL of sterile distilled water + 0.1% Tween 80^®. Afterward, the contents were transferred to test tubes containing 9 mL of sterile distilled water + 0.1% Tween 80^® and shaken in a vortex at 2800 rpm for 5 min. The suspensions were then serially diluted and the conidia from the samples were quantified in a Neubauer chamber under an optical microscope (400×). A completely randomized design was employed, with five experimental units (replicates) utilized for each treatment and 10 insects per replicate (n = 50).

2.9. Data Analysis

For the in vitro bioassays, the obtained data were initially subjected to the Shapiro–Wilk test to verify the residue’s normality and Bartlett’s test [37] to check the variance homogeneity. The transformation based on the optimal power method of Box–Cox [38] was used when assumptions were not met. For data that met the assumptions, an analysis of variance (ANOVA) was conducted and the means were then compared using Tukey’s or the Kruskall–Wallis test (p < 0.05).

For the in vivo bioassays, a generalized linear model (GLM) [39] with quasi-binomial and quasi-Poisson distributions was employed to analyze the proportion of mortality and the number of conidia, respectively. The goodness-of-fit was assessed through half-normal plots with simulation envelopes using the “hnp” package [40]. In the case of significant differences between treatments, multiple comparisons were conducted using Tukey’s test (p < 0.05) with the “glht” function of the “multcomp” package and with adjusted p values [41]. Finally, the effect of treatments on D. maidis survival was determined based on survival curves with their respective median lethal times using Kaplan–Meier analysis, with comparisons of survival curves conducted using the log-rank test. All statistical analyses were performed using R software, version 3.6.2 [42].

3. Results

3.1. In Vitro Compatibility

3.1.1. Effects of Herbicides on Colony-Forming Units (CFU) of Entomopathogenic Fungi Pure Isolates

After 4 days of incubation, a significant interaction between herbicides × fungal isolates × exposure times (F = 98.18; Df = 14, 192; p < 0.0001) was observed when evaluating the number of colony-forming units (CFU) (

Table 2. Colony-forming units (CFU) of Esalq-1296 of Cordyceps javanica and isolates IBCB66 and Simbi BB15 of Beauveria bassiana exposed to different herbicides in tank mixtures for two exposure times (1.5 or 3.0 h).

Synthetic Herbicides	Esalq-1296		IBCB66		Simbi BB15
	1.5 h	3.0 h	1.5 h	3.0 h	1.5 h	3.0 h
Glyphosate (SL formulation)	115.00 ± 1.44 dBA	78.60 ± 2.16 dBB	305.80 ± 6.50 bAA	200.00 ± 3.16 dAB	32.40 ± 1.68 cCA	10.4 ± 1.15 eCB
Glyphosate (WG formulation)	159.80 ± 5.26 cAA	32.80 ± 0.95 eAB	107.20 ± 3.30 eBA	0.00 ± 0.00 gBB	0.40 ± 0.21 dCA	0.00 ± 0.00 gBA
Glufosinate–ammonium	69.80 ± 2.68 fBA	23.20 ± 1.28 fAB	115.80 ± 2.59 eAA	15.80 ± 1.27 fBB	34.00 ± 2.00 cCA	19.80 ± 1.65 dABB
Atrazine	198.60 ± 4.37 abAA	191.40 ± 5.47 bcAA	138.80 ± 3.13 dBA	103.80 ± 2.16 eBB	106.80 ± 1.58 aCA	53.40 ± 3.13 bCB
Tembotrione	197.80 ± 3.57 abBA	195.60 ± 4.64 abBA	300.20 ± 4.20 bcAA	254.80 ± 4.10 cAB	71.20 ± 1.30 bCA	40.20 ± 1.24 cCB
Atrazine + simazine	100.40 ± 2.32 eBA	84.20 ± 1.42 dBB	306.20 ± 3.21 bAA	285.60 ± 3.33 bAB	65.40 ± 3.37 bCA	5.00 ± 1.57 fCB
Atrazine + mesotrione	184.20 ± 3.05 bBA	176.80 ± 3.52 cBA	282.60 ± 5.27 cAA	242.00 ± 6.13 cAB	33.40 ± 1.90 cCA	15.80 ± 1.53 deCB
Negative control (distilled water)	207.40 ± 3.75 aBA	210.60 ± 3.57 aBA	348.20 ± 2.81 aAA	342.20 ± 2.87 aAA	109.00 ± 3.05 aCA	106.40 ± 2.12 aCA
Herbicides (F = 2129.22; Df = 7, 192; p < 0.0001)
Isolates (F = 9529.18; Df = 2, 192; p < 0.0001)
Exposure times (F = 2298.12; Df = 2, 192; p < 0.0001)
Interaction between herbicides × isolates (F = 491.94; Df = 14, 192; p < 0.0001)
Interaction between herbicides × exposure times (F = 158.04; Df = 7, 192; p < 0.0001)
Interaction between isolates × exposure times (F =74.25; Df = 2, 192; p < 0.0001)
Interaction between herbicides × isolates × exposure times (F = 98.18; Df = 14, 192; p < 0.0001)

Data (mean ± SE) followed by the same lowercase letters, to compare the different herbicides within each isolate and exposure time, do not differ significantly by Tukey’s test (p > 0.05); data (mean ± SE) followed by the same uppercase letters, to compare the different isolates within each herbicide and exposure time, do not differ significantly by Tukey’s test (p > 0.05); data (mean ± SE) followed by the same superscript letters in bold, to compare the different exposure times for each herbicide and isolate, do not differ significantly by the Tukey test (p > 0.05).

). For the Esalq-1296 isolate of C. javanica (1.5 h exposure time), only the herbicides formulated with atrazine and tembotrione did not reduce the number of CFU compared to the control. However, for the 3 h exposure time to the same isolate, all the herbicides (except tembotrione) reduced the number of CFU compared to the control (Table 2). For the B. bassiana isolate IBCB66, all the herbicides reduced the number of CFU at the two exposure times evaluated, indicating that this isolate is the most susceptible to the herbicides used in the spray tank mixture (Table 2). Moreover, for the Simbi BB15 isolate of B. bassiana, all the herbicides (except atrazine) reduced the number of CFU in the 1.5 h exposure time. However, after 3.0 h of exposure, all the herbicides tested reduced the number of CFU of this isolate (Table 2).

Following a 1.5 h exposure period to the spray solutions, the largest number of CFU was observed in isolate IBCB66 of B. bassiana in all the herbicides tested, except for the treatments consisting of glyphosate-based herbicides (WG formulation) and atrazine, in which the largest numbers of CFU were observed in isolate Esalq-1296 of C. javanica (Table 2). Similar results were obtained for the 3 h exposure period, in which the largest number of CFU was observed in isolate IBCB66 of B. bassiana, except for the treatments consisting of the herbicides glyphosate (WG formulation), glufosinate–ammonium, and atrazine (Table 2), in which the highest values were again observed in isolate Esalq-1296 of C. javanica. The smallest number of CFU was also observed in the Simbi BB15 isolate of B. bassiana, regardless of the treatment or exposure time.

In general, increasing the exposure time (from 1.5 to 3.0 h) resulted in a reduction in the number of CFU after exposure to herbicide solutions, except for treatments consisting of herbicides based on atrazine, tembotrione, and atrazine + mesotrione for the C. javanica isolate Esalq-1296 or the treatments consisting of the glyphosate-based herbicide (WG formulation) and the Simbi BB15 isolate of B. bassiana, in which no effect of exposure time to the solutions of these herbicides was observed (Table 2).

3.1.2. Vegetative Growth of Fungal Isolates Exposed to Herbicides-Treated Culture Media

After 12 days of incubation, a significant interaction between herbicides × fungal isolates (F = 74.23; Df = 7, 216; p < 0.0001) was observed when evaluating the diameter of colonies of different entomopathogenic fungal isolates developed in PDA culture media treated with the different herbicides (

Table 3. Colony diameter (mm) of isolate Esalq-1296 of Cordyceps javanica and isolates IBCB66 and Simbi BB15 of Beauveria bassiana exposed to culture media containing different synthetic herbicides after 12 days of incubation.

Synthetic Herbicides	Colony Diameter (mm) 1
	Esalq-1296	IBCB66	Simbi BB15
Glyphosate (SL formulation)	0.00 ± 0.00 eB	6.93 ± 0.14 dA	6.59 ± 0.19 eA
Glyphosate (WG formulation)	31.36 ± 0.44 bB	32.87 ± 0.50 bAB	33.66 ± 0.39 bA
Glufosinate–ammonium	0.00 ± 0.00 eA	0.00 ± 0.00 eA	0.00 ± 0.00 fA
Atrazine	16.74 ± 0.29 dA	0.00 ± 0.00 eC	13.59 ± 0.17 dB
Tembotrione	31.78 ± 1.17 bB	31.35 ± 0.49 bB	34.33 ± 0.68 bA
Atrazine + simazine	0.00 ± 0.00 eB	17.29 ± 0.28 cA	16.87 ± 0.30 cA
Atrazine + mesotrione	27.02 ± 0.71 cB	32.18 ± 0.22 bA	32.77 ± 0.52 bA
Negative control (distilled water)	35.79 ± 0.72 aC	47.25 ± 1.77 aA	45.05 ± 0.61 aB
Herbicides (F = 2043.30; Df = 7, 216; p < 0.0001)
Isolates (F = 139.97; Df = 2, 216; p < 0.0001)
Interaction between herbicides × isolates (F = 74.23; Df = 14, 216; p < 0.0001)

¹ Data (mean ± SE) followed by the same lowercase letters, to compare the different herbicides within each isolate, do not differ significantly by Tukey’s test (p > 0.05); data (mean ± SE) followed by the same uppercase letters, to compare the different isolates within each herbicide, do not differ significantly by Tukey’s test (p > 0.05).

; Figure 1). Regardless of the herbicide tested, a reduction in colony diameter was observed for the three fungal isolates tested (Table 3).

The vegetative growth of isolate Esalq-1296 was completely inhibited when exposed to the herbicides glyphosate (SL formulation) and atrazine + simazine (Table 3). In contrast, the mycelial growth of the B. bassiana isolate IBCB66 was completely inhibited when the herbicide atrazine was incorporated into the PDA medium (Table 3). Furthermore, regardless of the fungal isolate, there was no vegetative growth when the glufosinate–ammonium herbicide was incorporated into the culture medium (Table 3).

The analysis of the susceptibility levels of fungal isolates within each herbicide indicated that the Esalq-1296 isolate of C. javanica was the most susceptible to herbicides when mycelial growth was evaluated (Table 3). This isolate differed from the two B. bassiana isolates (Simbi BB15 and IBCB66) when exposed to most herbicides, except for atrazine, which showed greater mycelial growth, and for the treatments using herbicides based on tembotrione and glyphosate (WG formulation), where the Esalq-1296 isolate exhibited a mycelial growth similar to that of the B. bassiana isolate IBCB 66 (Table 3).

3.1.3. Conidial Production and Viability of Fungal Isolates Exposed to Herbicide-Treated Culture Media

Significant differences were observed in the estimation of the average number of conidia produced by the colonies of fungal isolates exposed to post-emergent herbicides (

Table 4. Number of conidia (×10⁷) produced by each colony of isolate Esalq-1296 of Cordyceps javanica and isolates IBCB66 and Simbi BB15 of Beauveria bassiana in a culture medium containing different synthetic herbicides after 12 days of incubation.

Synthetic Herbicides	Number of Conidia (×107) 1			(χ2; p-Value)
	Esalq-1296	IBCB66	Simbi BB15
Glyphosate (SL formulation)	-	0.02 ± 0.005 fA	0.01 ± 0.004 eA	χ2 = 3.03; Df = 1; p = 0.0816
Glyphosate (WG formulation)	70.30 ± 2.96 aA	9.48 ± 1.66 dB	2.68 ± 0.49 cC	χ2 = 12.50; Df = 2; p = 0.0019
Glufosinate–ammonium	-	-	-	-
Atrazine	20.30 ± 1.25 cA	-	2.21 ± 0.21 cB	χ2 = 6.81; Df = 1; p = 0.0090
Tembotrione	35.50 ± 4.26 bA	48.20 ± 3.30 cA	20.60 ± 1.94 aB	χ2 = 9.57; Df = 2; p = 0.0083
Atrazine + simazine	-	0.39 ± 0.03 eB	0.90 ± 0.07 dA	χ2 = 6.82; Df = 1; p = 0.0090
Atrazine + mesotrione	17.90 ± 3.61 cB	121.89 ± 16.00 bA	6.60 ± 0.97 bC	χ2 = 10.82; Df = 2; p = 0.0044
Negative control (distilled water)	81.20 ± 5.25 aB	319.50 ± 31.20 aA	23.00 ± 2.67 aC	χ2 = 12.50; Df = 2; p = 0.0019
χ2	19.63	28.24	32.00
Df	4	5	6
p-value	<0.0001	<0.0001	<0.0001

¹ Means followed by lowercase letters in the column and uppercase letters in the row do not differ by the Kruskal–Wallis test (p > 0.05).

). In comparison to the negative control, the number of conidia produced by the Esalq-1296 isolate of C. javanica was found to be lower when the herbicides atrazine + mesotrione, tembotrione, and atrazine were incorporated into the PDA medium (χ² = 19.63; Df = 4; p < 0.0001) (Table 4). Similarly, the number of conidia produced by the Simbi BB15 isolate of B. bassiana was also lower when herbicides were incorporated into the medium (χ² = 32.00; Df = 6; p < 0.0001), except for the treatment using the herbicide tembotrione (Table 4). In contrast, all herbicides studied demonstrated a reduction (χ² = 28.24; Df = 5; p < 0.0001) in the conidiogenesis of the B. bassiana isolate IBCB66 (Table 4). When comparing the different isolates within the negative control, the B. bassiana isolate IBCB66 produced the largest number of spores (Table 4).

Except for the herbicide glyphosate (WG formulation) for isolate IBCB66 of B. bassiana, all the herbicides that were tested affected the viability of conidia produced by the three fungal isolates tested (

Table 5. Conidial germination (%) of isolates Esalq-1296 of Cordyceps javanica and IBCB66 and Simbi BB15 of Beauveria bassiana in a culture medium containing different synthetic herbicides after 12 days of incubation.

Synthetic Herbicides	Conidial Germination (%) 1			(χ2; Df; p-Value)
	Esalq-1296	IBCB66	Simbi BB15
Glyphosate (SL formulation)	-	-	-	-
Glyphosate (WG formulation)	47.40 ± 0.84 cB	96.80 ± 0.72 aA	94.10 ± 0.99 bA	χ2 = 10.67; Df = 2; p = 0.0048
Glufosinate–ammonium	-	-	-	-
Atrazine	19.30 ± 1.00 dA	-	19.70 ± 1.52 eA	χ2 = 0.04; Df = 1; p = 0.8340
Tembotrione	67.50 ± 1.37 bC	76.10 ± 3.05 bB	86.30 ± 1.29 cA	χ2 = 9.74; Df = 2; p = 0.0076
Atrazine + simazine	-	3.50 ± 0.61 cB	26.30 ± 2.18 dA	χ2 = 6.86; Df = 1; p = 0.0088
Atrazine + mesotrione	64.60 ± 1.63 bC	84.50 ± 1.70 bB	89.30 ± 1.25 cA	χ2 = 10.85; Df = 2; p = 0.0044
Negative control (distilled water)	97.20 ± 0.46 aA	97.50 ± 0.50 aA	98.70 ± 0.48 aA	χ2 = 3.60; Df = 2; p = 0.1652
χ2	22.23	21.41	26.83
Df	4	4	5
p-value	<0.0001	<0.0001	<0.0001

¹ Means followed by lowercase letters in the column and uppercase letters in the row do not differ by the Kruskal–Wallis test (p > 0.05).

). Among the fungal isolates, Simbi BB15 of B. bassiana exhibited greater tolerance to herbicides when considering the conidial viability (Table 5).

3.1.4. Determination of Compatibility Between Herbicides and Pure Isolates of Entomopathogenic Fungi

The biological index (BI) indicated that the herbicides glyphosate (SL formulation), glufosinate–ammonium, atrazine, and atrazine + simazine were considered incompatible with the pure isolates of Esalq-1296 of C. javanica and IBCB66 and Simbi BB15 of B. bassiana, while the herbicide atrazine + mesotrione was considered moderately compatible with the three fungal isolates, and the herbicide tembotrione was compatible with the isolates Esalq-1296 of C. javanica and Simbi BB15 of B. bassiana (

Table 6. In vitro toxicity classification of synthetic herbicides to isolate Esalq-1296 of Cordyceps javanica and isolates IBCB66 and Simbi BB15 of Beauveria bassiana.

Synthetic Herbicides	Commercial Brands	Toxicity Classification
		Esalq-1296		IBCB 66		Simbi BB15
		BI 1	Classification 2	BI 1	Classification 2	BI 1	Classification 2
Glyphosate (SL formulation)	Roundup Original Mais®	0.00	Incompatible	6.87	Incompatible	6.77	Incompatible
Glyphosate (WG formulation)	Roundup WG®	82.82	Compatible	44.36	Moderately compatible	49.53	Moderately compatible
Glufosinate–ammonium	Finale®	0.00	Incompatible	0.00	Incompatible	0.00	Incompatible
Atrazine	Primóleo®	33.58	Incompatible	0.00	Incompatible	20.68	Incompatible
Tembotrione	Soberan®	68.11	Compatible	46.58	Moderately compatible	80.46	Compatible
Atrazine + simazine	Primatop SC®	0.00	Incompatible	17.77	Incompatible	22.28	Incompatible
Atrazine + mesotrione	Calaris®	50.09	Moderately compatible	57.78	Moderately compatible	54.83	Moderately compatible

¹ BI: biological index proposed by Rossi-Zalaf et al. (2008). ² Classification: incompatible (0 ≤ BI ≤ 41); moderately compatible (41 ≤ BI ≤ 66); and compatible (BI > 66).

).

3.2. Physicochemical Analysis

Synthetic herbicides and mycoinsecticides alone exhibited pH values ranging from 3.14 to 6.98 (

Table 7. Physical–chemical compatibility of mixtures of different synthetic herbicides registered for corn management and mycoinsecticides registered for Dalbulus maidis management in Brazil and tested according to criteria of dynamic assays proposed by the Brazilian Association of Technical Standards (ABNT NBR 13875:2014).

Commercial Brands	pH	EC (µS/cm)	ho	fl	sd	ps	pg	os	cf	cr	fo	pg *
Synthetic Herbicides
Glyphosate (SL formulation)	6.42	16.98	P	A	A	A	A	A	A	A	A	A
Glyphosate (WG formulation)	3.56	9.91	P	A	A	A	A	A	A	A	A	A
Glufosinate–ammonium	6.30	1824.00	P	A	A	A	A	A	A	A	A	A
Atrazine	6.98	96.00	P	A	A	A	A	A	A	A	A	A
Tembotrione	3.63	59.50	P	A	A	A	A	A	A	A	A	A
Atrazine + simazine	6.76	336.00	P	A	A	A	A	A	A	A	A	A
Atrazine + mesotrione	3.14	325.00	P	A	A	A	A	A	A	A	A	A
Mycoinsecticides
FlyControl®	5.39	6.58	P	A	A	A	A	A	A	A	A	A
Bovenat®	5.48	9.09	P	A	A	A	A	A	A	A	A	A
Octane®	5.50	8.09	P	A	A	A	A	A	A	A	A	A
Water	5.40	3.95	-	-	-	-	-	-	-	-	-	-
Synthetic herbicides + mycoinsecticides
Glyphosate (SL formulation) + FlyControl®	6.41	17.02	A	A	A	P	A	A	A	A	A	A
Glyphosate (WG formulation) + FlyControl®	3.59	9.87	A	A	A	P	A	A	A	A	A	A
Glufosinate–ammonium + FlyControl®	6.28	1819.00	A	A	A	P	A	A	A	A	P	A
Atrazine + FlyControl®	6.95	99.00	A	A	A	P	A	A	A	A	A	A
Tembotrione + FlyControl®	3.72	56.70	A	A	A	P	A	A	A	A	A	A
Atrazine + simazine + FlyControl®	6.80	328.00	A	A	A	P	A	A	A	A	A	A
Atrazine + mesotrione + FlyControl®	3.30	321.00	A	A	A	P	A	A	A	A	A	A
Glyphosate (formulation SL) + Octane®	6.42	16.73	P	A	A	A	A	A	A	A	A	A
Glyphosate (formulation WG) + Octane®	3.70	9.66	P	A	A	A	A	A	A	A	A	A
Glufosinate–ammonium + Octane®	6.43	1726.00	P	A	A	A	A	A	A	A	P	A
Atrazine + Octane®	7.03	101.00	P	A	A	A	A	A	A	A	A	A
Tembotrione + Octane®	3.69	58.80	P	A	A	A	A	A	A	A	A	A
Atrazine + simazine + Octane®	6.86	335.00	P	A	A	A	A	A	A	A	A	A
Atrazine + mesotrione + Octane®	3.20	327.00	P	A	A	A	A	A	A	A	A	A
Glyphosate (SL formulation) + Bovenat®	6.41	15.19	P	A	A	A	A	A	A	A	A	A
Glyphosate (WG formulation) + Bovenat®	3.66	10.14	P	A	A	A	A	A	A	A	A	A
Glufosinate–ammonium + Bovenat®	6.36	1752.00	P	A	A	A	A	A	A	A	P	A
Atrazine + Bovenat®	6.96	93.90	P	A	A	A	A	A	A	A	A	A
Tembotrione + Bovenat®	3.96	93.30	P	A	A	A	A	A	A	A	A	A
Atrazine + simazine + Bovenat®	6.71	283.40	P	A	A	A	A	A	A	A	A	A
Atrazine + mesotrione + Bovenat®	3.26	304.00	P	A	A	A	A	A	A	A	A	A

P, presence, or A, absence (from assessment criteria). pH after agitation; EC: electrical conductivity; ho: homogeneity; fl: flocculation; sd: sedimentation; ps: phase separation; pg: presence of groats; os: oil separation; cf: crystal formation; cr: cream; fo: foam; pg *: presence of groats (149 μm sieve).

). Additionally, the mycoinsecticide based on B. bassiana isolate Simbi BB15 demonstrated lower electrical conductivity (EC) values (6.58 µS cm⁻¹), while the herbicide glufosinate–ammonium (1824.00 µS cm⁻¹) showed the highest values. In general, binary mixtures containing herbicides and mycoinsecticides did not demonstrate significant changes in pH and EC values when compared to isolated products, except for the mixtures of glufosinate–ammonium + Octane^® and glufosinate–ammonium + Bovenat^®, which exhibited reductions in EC values more than 95 µS cm⁻¹ when compared to the herbicide evaluated alone (Table 7).

Physical compatibility assessments revealed a lack of homogeneity and phase separation following the shaking of herbicide mixtures with the FlyControl^® mycoinsecticide (Table 7). Additionally, all mixtures containing the herbicide glufosinate–ammonium exhibited the presence of foam during the evaluation period following the stirring period (Table 7); conversely, the other herbicides evaluated alone or in mixture with entomopathogenic fungi demonstrated homogeneous physical characteristics (Table 7).

3.3. Effects of Herbicides on CFU of Mycoinsecticide Commercial Formulations

After 4 days of incubation, a significant interaction between herbicides × mycoinsecticides × exposure times (F = 157.53; Df = 7, 128; p < 0.0001) was observed for the number of CFU (

Table 8. Colony-forming units (CFU) of the commercial mycoinsecticides Octane^® (isolate Esalq-1296 from Cordyceps javanica) and FlyControl^® (isolate Simbi BB15 from Beauveria bassiana) exposed to different pesticides in tank mixtures at two exposure times (1.5 and 3 h).

Synthetic Herbicides	Octane®		FlyControl®
	1.5 h	3 h	1.5 h	3 h
Glyphosate (SL formulation)	14.40 ± 0.67 eBA	0.00 ± 0.00 fBB	515.60 ± 8.77 bAA	299.40 ± 7.17 cAB
Glyphosate (WG formulation)	1.20 ± 0.52 fBA	0.00 ± 0.00 fAB	158.60 ± 6.48 fAA	0.00 ± 0.00 fAB
Glufosinate–ammonium	319.20 ± 2.96 cBA	269.80 ± 3.67 dBB	543.80 ± 9.84 bAA	292.40 ± 7.27 cAB
Atrazine	976.60 ± 5.79 aAA	836.80 ± 5.56 bAB	283.80 ± 5.50 dBA	278.40 ± 7.44 cBA
Tembotrione	57.40 ± 1.34 dBA	57.60 ± 1.97 eBA	338.40 ± 8.62 cAA	226.40 ± 5.89 dAB
Atrazine + simazine	669.40 ± 4.53 bAA	480.20 ± 3.70 cAB	367.20 ± 6.66 cBA	348.80 ± 7.37 bBA
Atrazine + mesotrione	62.00 ± 1.88 dBA	46.20 ± 1.63 eBB	197.00 ± 5.99 eAA	62.40 ± 4.08 eAB
Negative control (distilled water)	997.20 ± 5.98 aAA	995.60 ± 6.76 aAA	592.80 ± 12.75 aBA	592.60 ± 11.25 aBA
Herbicides (F = 7468.17; Df = 7, 128; p < 0.0001)
Mycoinsecticides (F = 690.44; Df = 1, 128; p < 0.0001)
Exposure times (F = 1937.33; Df = 1, 128; p < 0.0001)
Interaction between herbicides × mycoinsecticides (F = 2703.91; Df = 7, 128; p < 0.0001)
Interaction between herbicides × exposure times (F = 122.98; Df = 7, 128; p < 0.0001)
Interaction between mycoinsecticides × exposure times (F = 371.25; Df = 1, 128; p < 0.0001)
Interaction between herbicides × mycoinsecticides × exposure times (F = 157.53; Df = 7, 128; p < 0.0001)

Data (mean ± SE) followed by the same lowercase letters, to compare the different herbicides within each mycoinsecticide and exposure time, do not differ significantly by Tukey’s test (p > 0.05); data (mean ± SE) followed by the same uppercase letters, to compare the different mycoinsecticides within each herbicide and exposure time, do not differ significantly by Tukey’s test (p > 0.05); data (mean ± SE) followed by the same superscript letters in bold, to compare the different exposure times for each herbicide and mycoinsecticide, do not differ significantly by Tukey’s test (p > 0.05).

). Regardless of the mycoinsecticide and exposure time, all herbicides exhibited a reduction in the number of CFU compared to the negative control (Table 8), except for the herbicide atrazine for the mycoinsecticide Octane^® for the spray solution exposure time of 1.5 h (Table 8).

Except for the treatments using the herbicides atrazine and atrazine + simazine, the mycoinsecticide FlyControl^® exhibited the largest number of CFU after 1.5 h of exposure to the spray (Table 8). Similar results were observed for the 3 h exposure time (Table 8). Regardless of the mycoinsecticide and herbicide, an increase in exposure time in the spray solution resulted in a reduction in the number of CFU, except for the tembotrione-based herbicide for the mycoinsecticide Octane^® and the herbicides atrazine and atrazine + simazine for the mycoinsecticide FlyControl^® (Table 8).

3.4. Comparison of the Reduction in CFU Number of Fungi Isolates and Their Respective Commercial Formulations When Mixed with Synthetic Herbicides

A comparison of the percentage of CFU reduction in relation to the respective negative controls revealed a variation in the susceptibility of the fungal isolates when the mycoinsecticides were exposed to synthetic herbicides in the form of pure spores or in their respective commercial formulations (

Table 9. Reduction in the number of colonies forming units (CFU) of one pure isolate (Esalq-1296) and commercial mycoinsecticide Octane^® of Cordyceps javanica and one pure isolate (Simbi BB15) and commercial mycoinsecticide FlyControl^® of Beauveria bassiana exposed to different pesticides in tank mixtures for two (1.5 and 3 h) exposure times.

Synthetic Herbicides	Esalq-1296		Octane®		Simbi BB15		FlyControl®
	1.5 h	3 h	1.5 h	3 h	1.5 h	3 h	1.5 h	3 h
Glyphosate (SL formulation)	44.55	62.68	98.55	100.00	70.28	90.23	12.87	49.30
Glyphosate (WG formulation)	22.95	84.43	99.88	100.00	99.63	100.00	73.19	100.00
Glufosinate–ammonium	66.35	88.98	67.99	72.90	68.81	81.39	8.14	50.46
Atrazine	4.24	9.12	2.05	15.92	2.02	49.81	51.99	51.97
Tembotrione	4.63	7.12	94.24	94.21	34.68	62.22	42.69	61.80
Atrazine + simazine	51.59	60.02	32.87	51.76	40.00	95.30	37.87	40.96
Atrazine + mesotrione	11.19	16.05	93.78	95.35	69.36	85.15	66.68	89.46

E= (1 − T/C) × 100, where E is the percentage of CFU; T is the mean CFU for the treatment; and C is the mean CFU for the control treatment.

). In general, the suspension concentrate (SC) formulation present in the mycoinsecticide Octane^® was found to be more susceptible to herbicides, at both exposure times, due to the greater reductions in CFU, except for the herbicides glufosinate–ammonium and atrazine + simazine (Table 9). Conversely, the oil dispersion (OD) formulation, present in FlyControl^®, demonstrated a lower degree of susceptibility when exposed to the herbicides glyphosate (SL formulation), glufosinate–ammonium, atrazine + simazine, and atrazine + mesotrione after 1.5 and 3.0 h of exposure when compared to the pure isolate exposed to the same herbicides (Table 9). In contrast, the pure isolate of B. bassiana (Simbi BB15) demonstrated a lower susceptibility to the herbicides atrazine and tembotrione under a 1.5 h exposure time (Table 9).

3.5. In Vivo Bioassays

After 9 days of topical exposure of D. maidis adults to synthetic herbicides and mycoinsecticides applied alone or in binary mixtures, significant differences were observed in the percentage of insect mortality (F = 5.52; Df = 23,96; p < 0.0001) (

Table 10. Percentages of Dalbulus maidis mortality and proportion of extruded adults as well as the number of conidia produced in insect cadavers exposed to mixtures containing herbicides, mycoinsecticides, and a mixture of them.

Treatment	% Mortality 1	% Extrusion 3	Number of Conidia (×107) 2
Glyphosate (SL formulation)	32.00 ± 5.21 bcde	0.00	-
Glyphosate (WG formulation)	14.00 ± 3.57 efghi	0.00	-
Glufosinate–ammonium	16.00 ± 2.19 defghi	0.00	-
Atrazine	14.00 ± 6.69 efghi	0.00	-
Tembotrione	4.00 ± 2.19 hi	0.00	-
Atrazine + simazine	8.00 ± 1.78 ghi	0.00	-
Atrazine + mesotrione	4.00 ± 2.19 hi	0.00	-
Glyphosate (SL formulation) + Octane®	34.00 ± 6.69 bcd	0.00	-
Glyphosate (WG formulation) + Octane®	14.00 ± 6.06 efghi	0.00	-
Glufosinate–ammonium + Octane®	52.00 ± 10.73 ab	0.00	-
Atrazine + Octane®	28.00 ± 5.93 cdef	0.00	-
Tembotrione + Octane®	14.00 ± 3.57 efghi	0.00	-
Atrazine + simazine + Octane®	10.00 ± 5.65 fghi	0.00	-
Atrazine + mesotrione + Octane®	10.00 ± 2.82 fghi	0.00	-
Glyphosate (SL formulation) + FlyControl®	58.00 ± 10.73 a	0.00	-
Glyphosate (WG formulation) + FlyControl®	48.00 ± 5.21 abc	0.00	-
Glufosinate–ammonium + FlyControl®	52.00 ± 7.69 ab	0.00	-
Atrazine + FlyControl®	26.00 ± 6.69 cdef	0.00	-
Tembotrione + FlyControl®	14.00 ± 2.19 efghi	0.00	-
Atrazine + simazine + FlyControl®	28.00 ± 7.69 cdef	0.00	-
Atrazine + mesotrione + FlyControl®	20.00 ± 6.92 defgh	0.00	-
C. javanica Esalq-1296 (Octane®)	24.00 ± 6.69 defg	25.00	1.34 ± 0.07 a
B. bassiana Simbi BB 15 (FlyControl®)	18.00 ± 8.19 defghi	33.30	0.74 ± 0.05 b
Bifenthrin + carbosulfan	100.00 ± 0.00 *	0.00	-
Negative control (distilled water)	2.00 ± 1.78 i	0.00	-
F	5.52	-	33.75
Df	23, 96	-	1, 4
p	<0.0001	-	=0.0043

¹ Data (mean ± SE) followed by the same letter in a column do not differ significantly (GLM with quasi-binomial distribution, followed by Tukey’s post hoc test; p > 0.05); ² data (mean ± SE) followed by the same letter in a column do not differ significantly (GLM with quasi-poisson distribution, followed by Tukey’s post hoc test; p > 0.05); ³ percentage of extruded insects concerning the total number of deaths; * treatment not included in the statistical analysis (null variance).

). The highest mortality rate (58%) was observed in the treatment using the herbicide glyphosate (SL formulation) in combination with the fungal isolate Simbi BB15 of B. bassiana (FlyControl^®), which did not differ from the treatments using glufosinate–ammonium + Octane^®, glyphosate (WG formulation) + FlyControl^® and glufosinate–ammonium + FlyControl^®, which exhibited mortality rates above 45% (Table 10).

Significant differences were observed (Kaplan–Meier Log-rank: p < 0.0001) in the survival probability of D. maidis adults exposed to synthetic herbicides and mycoinsecticides applied alone (Figure 2A) or in binary mixtures using synthetic herbicides and the mycoinsecticides Octane^® (Figure 2B) and FlyControl^® (Figure 2C). In general, the application of isolated herbicides resulted in a reduction in the survival probability of D. maidis until the third day of evaluation. Furthermore, the herbicide glyphosate (SL formulation) was observed to reduce the survival of D. maidis by more than 25% (Figure 2A). Conversely, treatments utilizing the mycoinsecticides FlyControl^® and Octane^® applied alone resulted in reductions in the survival probability of D. maidis only after the fifth day of application of the products (Figure 2A). On the other hand, the positive control, which consisted of an insecticide based on bifenthrin + carbosulfan [Talisman^® (FMC Química do Brasil Ltd.a., Campinas, São Paulo State, Brazil)], resulted in 100% mortality 24 h after application (Figure 2A).

In general, when herbicides were mixed with mycoinsecticides, the probability of insect survival was reduced compared to the same treatments applied alone (Figure 2B,C). Furthermore, binary mixtures comprising glufosinate–ammonium + Octane^®, glufosinate–ammonium + FlyControl^®, glyphosate (WG formulation) + FlyControl^®, and glyphosate (SL formulation) + FlyControl^® resulted in reductions in insect survival higher than 45% at the bioassay’s endpoint (Figure 2B,C). The p-values of the pairwise comparisons of all treatments are presented in the Supplementary Materials (Table S1 in Supplementary Materials).

From the dead insects of D. maidis collected and maintained in conditions suitable for the development of entomopathogens, only those exposed to treatments with the mycoinsecticides FlyControl^® and Octane^® applied alone exhibited fungal extrusions in 33.3 and 25.0% of the insect cadavers, respectively (Table 10). Furthermore, a significant difference was observed in the number of conidia produced per dead insect (F = 33.75; Df = 1, 4; p = 0.0043), with the mycoinsecticide Octane^® producing a larger number of conidia per insect cadaver (Table 10).

4. Discussion

The results from our in vitro bioassays indicated that the toxicity of post-emergent herbicides varied depending on the fungal isolate, the biological variable assessed, and the time of exposure of the entomopathogens to tested herbicides. However, despite the lack of effects of herbicides on some biological variables of the fungal isolates observed in the in vitro bioassays and the indication of a possible synergistic effect on the mortality of D. maidis in the in vivo trials, no fungal extrusion was observed on the cadavers of leafhoppers exposed to binary mixtures of herbicides and mycoinsecticides. This compromises horizontal transmission and, consequently, the secondary cycles of epizootics in agroecosystems. Although the use of tank mixtures is a common practice among corn producers in Brazil, this is the first study devoted to evaluating the physical, chemical, and biological compatibility of post-emergent herbicides, through lethal and sublethal parameters, with mycoinsecticides based on B. bassiana and C. javanica, which are widely used in the management of corn leafhopper in corn crops in Brazil.

The germination of the reproductive structure of fungi (spores) and, consequently, the formation of a colony are key points for the initiation of the epizootic under field conditions [43,44,45]. Furthermore, the probability of in vitro occurrence of a biological incompatibility between pesticides and entomopathogenic fungi may be attributed to several factors, with exposure time being a significant one [20,43]. In the field, the biocontrol agent is typically exposed to pesticides for an average of up to 4 h in the spray tank, including the stages of preparing the mixture and spraying [43]. The results of our bioassays, which are considered the direct contact of conidia with herbicides simulating the condition of a tank mixture, demonstrated that the majority of herbicides tested reduced the number of CFU, depending on the fungal isolate exposed. Although a significant interaction between herbicides × isolates was observed, increasing the exposure time from 1.5 to 3 h generally resulted in a reduction in the number of CFU of the different fungal isolates tested. Similarly, Ribeiro et al. [20] also concluded that longer exposure times of mycoinsecticides to pesticides in the spray tank should be avoided.

In addition to changes in biological compatibility in spray tank mixtures, the physical and chemical characteristics of spray mixtures, including changes in pH and electrical conductivity, are important points to be considered in the associated use of phytosanitary products [28,46]. Concerning herbicides, the chemical characteristics of the mixture are even more important as they can affect the absorption and xylem and/or phloem translocation of the active ingredient in treated plants [47,48] as well as causing variations in the water solubility and hydrolysis rate of products [49,50]. Furthermore, the occurrence of physical incompatibility can result in the clogging of spray tips and filters, equipment wear, reduced spraying effectiveness, and increased crop production costs [9]. Our results indicated small reductions in pH values and electrical conductivity of the spray mixtures when mycoinsecticides were mixed with herbicides in the spray tank. Moreover, physical analyses have indicated that constant agitation of the spray solution is necessary when the mycoinsecticide formulated based on the Simbi BB15 isolate of B. bassiana (FlyControl^®) is mixed with herbicides due to the possibility of the occurrence of phase separation.

As recommended by the International Organization for Biological Control—IOBC [31], evaluation methods using the incorporation of synthetic pesticides into culture medium are widely used in studies of this nature [20,43,45,51,52,53,54]. This protocol recommends the evaluation of additional important parameters of the fungus, including vegetative growth and the number and viability of the conidia produced [44]. Todorova et al. [52] reported an inhibition of the mycelial growth of B. bassiana when glufosinate–ammonium (formulated in aqueous suspension) was incorporated into the culture medium. Our findings align with those of Silva et al. [43], who observed a reduction in sporulation and mycelial growth rate in the biocontrol agents Trichoderma harzianum Rifai and Trichoderma asperellum (Samuels, Lieckf, and Nirenberg) (both Hypocreales: Hypocreaceae) following exposure to glyphosate N-ammonium salt.

Regarding the toxicity of synthetic pesticides, the physicochemical nature of the active ingredient, the dose or concentration used, and the product formulation can affect the toxicity of pesticides on entomopathogens [55,56,57]. In this sense, our results demonstrated that the two glyphosate formulations exhibited differential toxicity levels on the three fungal isolates tested. In general, the SL formulation of glyphosate was found to be more toxic to the three fungal isolates. In addition to the active ingredients and doses, the toxicity of pesticides to entomopathogens is hypothetically related to the inert ingredients and adjuvants present in the formulation [54]; however, further studies should be conducted to evaluate this hypothesis.

The development of mycoinsecticide formulation technologies is of paramount importance for the commercial robustness and increased shelf life of products, as well as for the reduction in rates of degradation by solar radiation—particularly by UV-A and UV-B spectra—and low relative humidity [58,59]. However, the diverse formulations of mycoinsecticides utilized in the present study also resulted in alterations in the susceptibility levels of fungal isolates to herbicides. This phenomenon is likely due to the enhanced protection of the conidia. Lopes et al. [27] reported that the germination of B. bassiana conidia, formulated in oil, can protect against damage caused by triadimefon-based fungicide. However, the results demonstrated that the formulation present in the mycoinsecticide Octane^® (SC formulation) exhibited greater susceptibility to herbicides than the pure isolate (Esalq-1296 of C. javanica), as evidenced by a greater percentage of reduction in the number of CFU. Conversely, the OD formulation present in the FlyControl^® mycoinsecticide exhibited a partial degree of mitigation of the impact of herbicides on the number of CFU.

Regarding the in vivo effects of entomopathogenic fungi, the virulence of each species is contingent upon the susceptibility of the target pest [60]. However, when combined with other pesticides, there is the potential for enhanced mortality of different insect groups, as evidenced by previous studies [20,61,62,63]. These interactions are hypothetically caused initially by the action of pesticides, mycoinsecticides, and/or their mixtures on the epithelial tissue [62], which can cause the solubilization of cuticular wax [64,65,66], as well as by the greater adhesion of chemical compounds to the insect’s integument due to their lipophilic characteristics [67]. In light of the above-mentioned findings, our results also indicated a synergistic effect of binary mixtures on the mortality of D. maidis in the majority of mixtures in comparison to treatments utilizing isolated products. Despite the observed synergistic interaction on mortality rates (up to the third day), there were no individuals extruded in the treatments containing mixtures of herbicides and mycoinsecticides. This compromises the persistence and effectiveness of the entomopathogen in controlling the pest in crops.

Given the high biotic potential of the corn leafhopper [8], the high active and passive dispersion of the pest [68], and the long critical period of the crop for pest damage (VE—V8) [25], it is important to consider the horizontal transmission of entomopathogens and the occurrence of secondary cycles of epizootics within an IPM program of pathosystem D. maidis vs. CSC. Although the operational benefits of using tank mixtures are evident, the results of the in vitro and in vivo bioassays indicate that caution should be exercised when recommending mixtures of mycoinsecticides and post-emergent herbicides for the management of this pathosystem in corn crops.

5. Conclusions

In vitro, the toxicity of tested herbicides on the fungal isolates Simbi BB15 and IBCB66 of B. bassiana and Esalq-1296 of C. javanica varied according to the active ingredient, the variable analyzed, and the exposure time. The biological index (BI) indicates that herbicides based on atrazine + simazine, atrazine, glufosinate–ammonium, and glyphosate (SL formulation) are incompatible with the three fungal isolates tested. Conversely, physical–chemical compatibility assessments revealed alterations in the electrical conductivity of binary mixtures with the herbicide glufosinate–ammonium and the fungal isolates Esalq-1296 from C. javanica and IBCB66 from B. bassiana. Furthermore, physical incompatibilities were observed based on the lack of homogeneity as well as phase separation when mixtures of the B. bassiana mycoinsecticide Simbi BB15 were made with post-emergent herbicides. In addition, an in vitro tank-mix assay indicated that the suspension concentrate (SC) formulation present in the commercial mycoinsecticide Octane^® was more susceptible to herbicides for both exposure times tested (1.5 and 3.0 h). Additionally, a synergistic effect was observed in most mixtures of herbicides with mycoinsecticides on the mortality of D. maidis adults (in vivo bioassays); however, there was no fungal extrusion in any cadaver collected in treatments containing a mixture of herbicides and mycoinsecticides. Therefore, caution is recommended when using tank mixtures of mycoinsecticides and post-emergent herbicides for the management of D. maidis in cornfields.