Laboratory Assessment of Plant Losses by Sphenarium purpurascens and Control with Entomopathogenic Fungi in Oil Emulsions

Abstract

This study addresses the agricultural impact of the grasshopper Sphenarium purpurascens and evaluates the efficacy of entomopathogenic fungi (EPF), Beauveria bassiana, and Metarhizium robertsii, formulated in vegetable oil emulsions as sustainable pest control agents. The losses caused by S. purpurascens at different developmental stages (N4, N5, and adult) were assessed in five economically significant crops (Medicago sativa, Zea mays, Helianthus sp., Cynodon dactylon, and Cucurbita pepo), revealing a marked preference for Helianthus sp. and C. pepo, with consumption rates reaching 0.92 g/48 h during N4 and N5 stages, while adults showed preference for M. sativa (1.18 g/48 h) and Z. mays (1.15 g/48 h). The viability of EPF in oil emulsions (20% and 40% concentrations) was evaluated, demonstrating that formulations with Azadirachta indica and Moringa oleifera maintained over 99% fungal viability compared to the control absolute with distilled water (DW). The effectiveness of EPF against S. purpurascens adults was tested, with EPF on M. robertsii combined with Persea americana achieving 100% mortality within 72 h. Finally, the pathogenicity and dispersion of EPF in oil emulsions were evaluated, demonstrating that, at 240 h, the B. bassiana + A. indica strain (with three inoculated insects) achieved 100% mortality. It was observed that the number of inoculated adults directly influenced the mortality of S. purpurascens. These findings highlight the potential of EPF as a sustainable pest management strategy, emphasizing the need for further field trials to optimize its application and mitigate agricultural losses caused by S. purpurascens.

1. Introduction

Alfalfa (Medicago sativa L., 1753) is one of the main forage crops in Mexico, covering an area of 583,561 ha, which represents 57.1% of the total forage crop area. It is considered the most important forage legume in livestock farming, while corn (Zea mays L., 1753), oats (Avena sativa L., 1753), and sorghum (Sorghum bicolor (L.) Moench, 1794) together account for the remaining 42.9% of cultivated forage [1,2,3,4]. Z. mays is another key agricultural crop in Mexico, with significant cultural, economic, social and ecological importance due to its widespread distribution and essential role in people’s diets [4,5]. However, various factors, particularly biotic ones, have negatively affected crop growth, development, and productivity, with insect pests causing losses of up to 30% in agricultural production [5,6]. Orthoptera of the Pyrgomorphidae family, commonly known as locusts and grasshoppers, are among the insect pests responsible for such damage. These insects have adapted to various terrestrial habitats, with 12 species known to cause severe losses by feeding on the foliage of crops such as common beans (Phaseolus vulgaris L., 1753), M. sativa, pumpkin (Cucurbita pepo L., 1753) and Z. mays [7,8,9]. The genus Sphenarium is a group of grasshoppers of both economic and cultural importance, not only as a food source but also as an agricultural pest [10,11]. The grasshopper (Sphenarium purpurascens Charpentier, 1842) (Orthoptera: Pyrgomorphidae) is a wingless insect known for its distinctive body coloration during the adult stage, which varies from dark brown to bright olive green with black spots [12]. It has a univoltine life cycle, with egg hatching occurring between May and June following the rainy season in central and northern Mexico [13]. Its feeding activity intensifies as it matures, a behavior associated with the development of its mandibular structures. In recent years, this grasshopper has become one of the most significant agricultural pests due to its high reproductive capacity and broad range of host plants [7,14,15]. While pest control is commonly achieved using chemical pesticides, their excessive use has led to pesticide resistance and poses risks to human health and the environment [16,17]. Therefore, alternative biological control methods using beneficial organisms such as entomopathogenic fungi (EPF) are being explored [18,19]. These fungi can penetrate the insect cuticle, causing immobility, loss of coordination, feeding cessation and eventually, death [20,21]. Several EPF species with these unique insecticidal properties and specificity include Beauveria bassiana (Bals-Criv) Vuill., 1912, Metarhizium anisopliae (Metschn.) Sorokín (1883), Metarhizium acridum (Driver & Milner, 1998) and Entomophaga grylli (Fresen., 1964) A. Batko, all of which are natural pathogens of grasshoppers [8,14,21,22,23]. The genus B. bassiana is a cosmopolitan EPF that reproduces asexually and is associated with a wide range of hosts, infecting up to 707 insect species across the orders Orthoptera, Coleoptera, Lepidoptera, and Diptera [24]. Additionally, it can persist in the soil as a saprotrophic organism after killing the host, which exhibits a characteristic white-to-yellowish coloration [25,26]. Meanwhile, Metarhizium robertsii (Metschn., 1883) Sorokín) previously known as M. anisopliae, can infect over 200 species of insect pests [27]. Infected insects initially display white mycelial growth, which later develops into a green coloration as the fungus sporulates [26,28]. Furthermore, ecological alternatives such as plant extracts, aqueous solutions, and essential oils have demonstrated insecticidal activity. One study reported successful control of S. purpurascens in amaranth (Amaranthus hypochondriacus L., 1753) crops using a mixture of castor oil plant extract and chili, combined with B. bassiana and alternated with soap [29]. Additionally, the combination of B. bassiana with Persea americana (Mill, 1768) and Prunus dulcis (Mill, 1967) D.A. Webb., achieved 100% effectiveness against Strategus aloeus (L., 1758) within 48 h [30], while Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson, 2011, resulted in over 80% mortality in Leptopharsa gibbicarina (Froeschner, 1976) [31]. In Conotrachelus dimidiatus (Champion, 1904), mixtures of B. bassiana and plant extracts achieved 90% mortality, demonstrating the potential of EPF and botanical extracts for pest management [32].

This research was undertaken to address the growing need for biological alternatives to chemical insecticides in agricultural pest control, with a particular focus on S. purpurascens. Therefore, the objective of this study was to determine, under laboratory conditions, the losses in economically important crops that were caused by S. purpurascens and to evaluate control methods based on EPF, specifically B. bassiana and M. robertsii, formulated in vegetable oil emulsions of Azadirachta indica (A. Juss), Moringa oleifera (Lam, 1783), P. americana and P. dulcis.

2. Materials and Methods

This study was conducted in September to November 2024 at the Biological Control Laboratory at CIIDIR- Oaxaca located at 1564 m above sea level. Under laboratory conditions, the relative humidity (RH) levels varied from 56% ± 8.36% and dropped to 45% ± 8.68%. Meanwhile, the temperature varied from 23 °C ± 2.16 °C to 21 °C ± 2 °C.

The grasshoppers used in the experiment were obtained through direct collection methods using entomological nets in Santa Cruz Xoxocotlán (17°00′11.4″ N, 96°45′02.2″ W). For evaluation, only the required nymphal and adult stages of S. purpurascens were considered. Subsequently, the collected individuals were confined in four-liter plastic containers with modified lids, featuring a circular cut covered with a 1 mm diameter mesh to allow gas exchange, thereby facilitating their transport under optimal conditions to the laboratory. Once in the laboratory, grasshoppers were quarantined for 120 h in containers to allow for acclimatization and observation of possible infections. They were then separated into groups and fed with the evaluated crop, which had been previously disinfected with a 5% sodium hypochlorite solution for 20 min and rinsed with distilled water. Excreta were removed daily to maintain hygienic conditions.

The strains of EPF used in the experiment were obtained from the strain collection of the Biological Control Laboratory at CIIDIR-Oaxaca, and had been previously characterized.

2.1. Losses Caused by S. purpurascens in the N4, N5 Stages and Adult Crops of M. sativa, Z. mays, Helianthus sp., C. dactylon and C. pepo

Experiments were conducted from September 13 to October 4, 2024, to determine the losses caused by S. purpurascens at different developmental stages (N4 and N5 nymphs and adults). The specimens were selected based on their morphological characteristics [33,34]. Experiments were conducted using transparent plastic containers with a capacity of 300 mL. Each container houses one insect, which was fed 3 g of the evaluated crops: M. sativa, Z. mays, Helianthus sp. (L.), Cynodon dactylon (L.) Pers., 1805 and C. pepo. The weight of the foliage was recorded using a digital analytical balance PA2202C (OHAUS, Parsippany, Morris County, NJ, USA), with an accuracy of 200 g × 0.1 g.

Foliage weight was recorded and replaced every 48 h to document the weight loss per crop (g/48 h) and by the S. purpurascens stage of development. Since measurements were taken every 48 h, three measurements were collected each week (days 2, 4, and 6). The last measurement of each week (day 6) was averaged with the measurement taken at the start of the next week to maintain consistency in the calculations. The same insects were used throughout the experiment, allowing the change in their consumption to be assessed as their development progressed. A total of 15 treatments, with 12 repetitions per treatment over a period of 21 days, were approved.

2.2. Entomopathogenic Fungi and Their Culturing

The identification of EPF (M. robertsii and B. bassiana) was carried out using taxonomic keys and specific morphological traits of each species during September 2024. The EPF were propagated on a solid Potato Dextrose Agar (PDA) substrate in Petri dishes (80 × 10 mm) and kept at a regulated temperature of 25 ± 2 °C for 10 days. Conidia were extracted manually using a microbiological inoculation loop, then suspended in sterile distilled water (DW) at a concentration of 1 × 10⁹ conidia/mL and preserved in a beaker before being formulated in vegetable + oil emulsions.

2.3. Preparation of Oil Emulsion-Based Conidia Suspensions

During September 2024, M. robertsii and B. bassiana fungi were suspended in oil emulsions prepared using the following vegetable oils: A. indica (Herbofilia, Tlaxcala, Mexico), M. oleifera (Plant Guro Inc., Plainfield, NJ, USA), P. americana (Monte Persea Walmart, Oaxaca, Mexico) and P. dulcis (Aromatica Fresly, Oaxaca, Mexico). Tween 80 was used as a dispersing agent at 0.1% to facilitate the separation and dispersion of mycelia in water. Subsequently, serial dilutions were carried out using distilled water with 0.1% of Tween until a concentration of 1 × 10⁹ conidia/mL was achieved. Vegetable oil emulsion concentrations of 20% and 40% (by volume) were added to each glass Petri dish (55 mm diameter) using a 10–100 μL micropipette, (Pipet-Lite XLS, Rainin, Oakland, CA, USA). Finally, a volume of 0.5 mL of the aqueous suspension containing 1 × 10⁹ conidia was added to each glass Petri dish, and mixed for three minutes using a magnetic stirrer. The obtained conidia were counted using a Neubauer chamber at a concentration of 1 × 10⁹ conidia/mL.

2.4. Viability of Entomopathogenic Fungi in Vegetable Oil Emulsions

An oil emulsion with conidia in suspension (1 mL) was prepared and maintained at 26 ± 1 °C to evaluate conidial viability. Germination was analyzed for 96 h after preparation (from October 18 to 22, 2024) [35]. For microscopic observation, 1 μL of the suspension was placed on a glass slide and covered with a coverslip. A total of 100 conidia were counted per slide, and those whose germ tube was equal to or greater than their diameter were considered viable. A total of 18 treatments were evaluated, including an absolute control with DW each with 12 replicates. The formulations were prepared at two concentrations: 20% (0.2 mL of vegetable oil + 0.8 mL DW) and 40% (0.4 mL of vegetable oil + 0.6 mL DW). In both cases, the emulsions contained a conidial suspension with a concentration of 1 × 10⁹ conidia/mL.

2.5. Effectiveness of Entomopathogenic Fungi in Oil Emulsions on S. purpurascens Under Laboratory Conditions

Effectiveness was determined from October 28 to November 1, 2024, and was defined as the mortality of S. purpurascens insects after inoculation with B. bassiana and M. robertsii fungi. These fungi were applied at a concentration of 1 × 10⁹ conidia/insect in 2 mL of oil emulsions (prepared from A. indica, M. oleifera, P. americana and P. dulcis) at a 40% concentration. For emulsion preparation, a 100 μL micropipette was used with the following proportions: 0.40 μL of vegetable oil + 0.60 μL EPF suspension. S. purpurascens adults were placed individually in 300 mL cylindrical plastic containers, each containing M. sativa (2 g) as food. The M. sativa was previously disinfected with 5% sodium hypochlorite for 20 min and rinsed thoroughly with distilled water to ensure the removal of any contaminants. A 1 mL dose of the oil emulsion was administered to both the insects and their food. Mortality was recorded every 24 h until 96 h with deceased individuals added to the cumulative count. A total of 13 treatments were tested, including an absolute control with distilled water, each treatment replicated 12 times. Insects exhibiting symptoms of EPF infection were positioned in humid chambers at >90% RH and observed with a stereoscopic microscope at 200× magnification to determine the presence or absence of mycelial growth on the cadavers.

2.6. Pathogenicity and Dispersion of B. bassiana and M. robertssi in Oil Emulsions in S. purpurascens Adults

The dispersion and pathogenicity of adult S. purpurascens were determined by inoculating them with B. bassiana + A. indica or M. oleifera, and M. robertsii + P. americana or P. dulcis, between November 5 and November 15, 2024. Inoculation was carried out at a concentration of 1 × 10⁹ conidia/insect using oils at 40% purity.

In the experiment, ten adult S. purpurascens were placed in 4 L containers and fed M. sativa foliage. The insects were acclimated for 48 h before inoculation. To evaluate the ability of the fungi to infect healthy grasshoppers, inoculated insects were introduced into containers containing ten healthy grasshoppers. The number of inoculated insects varied according to the treatment, with 5, 3, or 1 inoculated insect per container. This experimental design allowed the evaluation of the fungi’s dispersal capacity and its effect on the mortality of healthy insects. Twelve treatments and absolute control were evaluated, with each treatment repeated 12 times to ensure the reliability and reproducibility of the results.

Observations were made every 48 h over a period of 240 h with deceased individuals added to the cumulative count. Dead insects were placed in humid chambers to induce mycelial growth, allowing for the determination of mortality induced by the fungi. Simultaneously, Koch’s postulates were applied to confirm the causal relationship between the fungi and insect mortality [36].

2.7. Statistical Analysis

The viability, efficacy, and pathogenicity percentages of the fungi were calculated using the following formula:

$$\%\mathrm{E}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{s}={\displaystyle \frac{Pi-Pf}{Pi}}\times 100$$

(1)

where Pi = initial population, Pf = Final population. The obtained data were normalized using an arcsine transformation and analyzed through an analysis of variance (ANOVA) to establish Tukey’s means at an alpha level of 0.05%, SAS^® Statistical software, version 9.1 [37].

3. Results

3.1. Losses Caused by the Developmental Stages of S. purpurascens in Five Crops

Average losses over a 21-day period per insect were calculated, with a preference observed in two crops at the N4 development stage: T3-Helianthus sp., with 0.91 g/48 h and T5-C. pepo with 0.93 g/48 h. In the N5 stage, the same preference was observed in T8-Heliantus sp., with 0.90 g/48 h, and T10-C. pepo with 0.95 g/48 h. These treatments in the N4 and N5 stages were statistically different from T1, T2, T4, T6, T7, and T9 treatments. The highest consumption was observed in the adult stage across all evaluated crops, with the most significant consumption recorded in T11-M. sativa with 1.18 g/48 h and T12-Z. mays with 1.15 g/48 h. Notably, a difference of more than 0.20 g/48 h was observed in the consumption of C. dactylon, Helianthus sp., and C. pepo (

Table 1. Average consumption of S. purpurascens in five cultures in three weeks (g/48 h).

Treatment	$\overline{\mathbf{X}}$/Week 1	$\overline{\mathbf{X}}$/Week 2	$\overline{\mathbf{X}}$/Week 3	$\overline{\mathbf{X}}$ Total
T1 N4 + M. sativa	0.56 ± 0.10 cd	0.64 ± 0.12 cd	0.65 ± 0.12 cd	0.61 ± 0.13 cd
T2 N4 + Z. mays	0.52 ± 0.09 cd	0.63 ± 0.11 cd	0.66 ± 0.16 cd	0.60 ± 0.09 cd
T3 N4 + Helianthus sp.	0.87 ± 0.15 ab	0.89 ± 0.17 ab	0.98 ± 0.10 ab	0.91 ± 0.16 ab
T4 N4 + C. dactylon	0.67 ± 0.20 cd	0.69 ± 0.13 cd	0.72 ± 0.09 cd	0.69 ± 0.34 cd
T5 N4 + C. pepo	0.87 ± 0.17 ab	0.94 ± 0.09 ab	0.99 ± 0.11 ab	0.93 ± 0.15 ab
T6 N5 + M. sativa	0.66 ± 0.25 cd	0.78 ± 0.15 cd	0.95 ± 0.16 ab	0.79 ± 0.15 cd
T7 N5 + Z. mays	0.65 ± 0.07 cd	0.70 ± 0.09 cd	0.94 ± 0.11 ab	0.76 ± 0.15 cd
T8 N5 + Helianthus sp.	0.85 ± 0.26 ab	0.88 ± 0.24 bc	0.98 ± 0.27 ab	0.90 ± 0.11 ab
T9 N5 + C. dactylon	0.75 ± 0.16 bc	0.76 ± 0.15 cd	0.80 ± 0.13 cd	0.77 ± 0.18 cd
T10 N5 + C. pepo	0.91 ± 0.15 ab	0.95 ± 0.15 ab	0.99 ± 0.11 ab	0.95 ± 0.16 ab
T11 Adult + M. sativa	1.06 ± 0.11 a	1.15 ± 0.21 a	1.33 ± 0.11 a	1.18 ± 0.14 a
T12 Adult + Z. mays	1.01 ± 0.03 a	1.10 ± 0.22 a	1.34 ± 0.14 a	1.15 ± 0.23 a
T13 Adult + Helianthus sp.	0.90 ± 0.13 ab	0.93 ± 0.17 bc	0.99 ± 0.21 ab	0.94 ± 0.11 ab
T14 Adult + C. dactylon	0.78 ± 0.15 bc	0.80 ± 0.14 bc	0.90 ± 0.18 ab	0.82 ± 0.15 bc
T15 Adult + C. pepo	0.91 ± 0.16 ab	0.95 ± 0.16 ab	1.10 ± 0.14 a	0.98 ± 0.15 ab

Mean ± standard deviation. Means with different letters in each column are statistically different (Tukey, p ≤ 0.05).

).

An increase in consumption was observed from week 1 to week 3, possibly due to the higher energy demands of S. purpurascens at more advanced developmental stages. Additionally, some crops may have experienced changes in their nutritional content or texture over time, influencing the consumption rate.

3.2. Evaluation of Entomopathogenic Fungi Viability in Vegetable Oil Emulsions

After 96 h, it was found that the oils of A. indica and M. oleifera, at concentrations of 20% and 40%, had no effect on the viability of B. bassiana and M. robertsii, registering percentages greater than 99% in all evaluated treatments (T1, T2, T5, T6, T9, T10, T13, and T14). In contrast, the absolute controls (T17 and T18), treated with distilled water, showed 0% survival, indicating complete fungal dehydration (

Table 2. Viability percentage for B. bassiana and M. robertsii to two concentrations.

Treatment	EPF	Oil	Concentration (%)	Viability (96 h)
T1	B. bassiana	A. indica	20	100 ± 0.0 a
T2	B. bassiana	M. oleifera	20	100 ± 0.0 a
T3	B. bassiana	P. americana	20	92.4 ± 4.4 ab
T4	B. bassiana	P. dulcis	20	90.5 ± 6.8 ab
T5	B. bassiana	A. indica	40	100 ± 0.0 a
T6	B. bassiana	M. oleifera	40	100 ± 0.0 a
T7	B. bassiana	P. americana	40	92.71 ± 6.8 ab
T8	B. bassiana	P. dulcis	40	94 ± 1.14 ab
T9	M. robertsii	A. indica	20	99.0 ± 1.14 a
T10	M. robertsii	M. oleifera	20	100 ± 0.0 a
T11	M. robertsii	P. americana	20	91.1 ± 1.14 ab
T12	M. robertsii	P. dulcis	20	97.2 ± 1.14 ab
T13	M. robertsii	A. indica	40	99.4 ± 6.8 a
T14	M. robertsii	M. oleifera	40	99.2 ± 3.9 a
T15	M. robertsii	P. americana	40	97.4 ± 6.8 ab
T16	M. robertsii	P. dulcis	40	96.57 ± 4.6 ab
T17	B. bassiana	DW (absolute control)	0	0 d
T18	M. robertsii	DW (absolute control)	0	0 d

Mean ± standard deviation. Values with different letters indicate a significant difference (p ≤ 0.05), according to Tukey’s test. DW: distilled water. Tukey’s mean. Values with different letters indicate a significant difference (p ≤ 0.05).

).

Both B. bassiana and M. robertsii responded similarly in terms of viability when formulated with A. indica and M. oleifera. Overall, the oil concentration (20% vs. 40%) did not have a negative impact on EPF viability. However, the results highlight the importance of oil selection in formulations, with A. indica and M. oleifera standing out as the most promising options for ensuring fungal stability.

3.3. Effectiveness of Entomopathogenic Fungi in Vegetables Oil Emulsions on S. purpurascens Adults Under Laboratory Conditions

The effectiveness began to be observed 24 h after inoculation in treatments T10 and T11, with a 57.14% mortality rate in S. purpurascens. Applying the effectiveness formula, it was determined that after 72 h, T11 (M. robertsii + P. americana) achieved 100% mortality, which was statistically different from the rest of the treatments. This suggests better compatibility between this fungus and the oil in controlling S. purpurascens. Meanwhile, four treatments (T4, T7, T8, T10) achieved mortality rates of 85.7%. At 96 h, only two treatments (T2, T3) showed mortality rates below 60%, which were statistically different from the other treatments. No mortality was recorded throughout the experiment’s evaluations in the case of the absolute control (T13) (

Table 3. Efficacy of B. bassiana and M. robertsii in 40% oil emulsions against S. purpurascens adults.

Treatment	Concentration (%)	Time (h)
		24	48	72	96
T1 A. indica	40	28.5 ± 0.4 c	42.8 ± 0.5 c	57.1 ± 0.5 d	85.7 ± 0.3 b
T2 M. oleifera	40	28.5 ± 0.4 c	28.5 ± 0.4 d	42.8 ± 0.5 e	57.1 ± 0.5 c
T3 P. americana	40	28.5 ± 0.4 c	28.5 ± 0.4 d	57.1 ± 0.5 d	57.1 ± 0.5 c
T4 P. dulcis	40	42.8 ± 0.5 b	57.1 ± 0.5 b	85.7 ± 0.3 b	100 ± 0.0 a
T5 B. bassiana + A. indica	40	14.2 ± 0.3 d	28.5 ± 0.4 d	57.1 ± 0.5 d	100 ± 0.0 a
T6 B. bassiana + M. oleifera	40	28.5 ± 0.4 c	42.8 ± 0.5 c	71.4 ± 0.4 c	100 ± 0.0 a
T7 B. bassiana + P. americana	40	0 ± 0.0 e	42.8 ± 0.5 c	85.7 ± 0.3 b	100 ± 0.0 a
T8 B. bassiana + P. dulcis	40	14.2 ± 0.3 d	71.4 ± 7.3 a	85.7 ± 0.3 b	85.7 ± 0.3 b
T9 M. robertsii + A. indica	40	14.2 ± 0.3 d	42.8 ± 0.5 c	57.1 ± 0.5 d	85.7 ± 0.3 b
T10 M. robertsii + M. oleifera	40	57.1 ± 0.53 a	71.4 ± 0.4 a	85.7 ± 0.3 b	85.7 ± 0.3 b
T11 M. robertsii + P. americana	40	57.1 ± 0.53 a	57.1 ± 0.5 b	100 ± 0.0 a	100 ± 0.0 a
T12 M. robertsii + P. dulcis	40	42.8 ± 0.0 b	71.4 ± 0.4 a	71.4 ± 0.4 c	100 ± 0.0 a
T13 DW (absolute control)	100	0 ± 0.0 e	0 ± 0.0 e	0 ± 0.0 f	0 ± 0.0 d

Mean ± standard deviation. Means with different letters in each column are statistically different (Tukey, p ≤ 0.05). DW: distilled water.

).

The treatments without EPF (T1, T2, T3, and T4) showed lower effectiveness compared to those containing EPF, with a slower effect and a lower mortality rate by the end of the experiment. However, P. dulcis oil (T4) exhibited a certain insecticidal effect on its own, with 100% mortality achieved at 96 h.

The use of B. bassiana and M. robertsii in emulsions significantly improved both mortality and the speed of action against S. purpurascens. These results highlight the higher effectiveness of B. bassiana compared to M. robertsii in infecting S. purpurascens.

3.4. Pathogenicity and Dispersion of B. bassiana and M. robertsii in Oil Emulsions on S. purpurascens Adults

Insect mortality progressively increased over time in all treatments, reaching its peak at 240 h. The treatment T2 B. bassiana + A. indica (three inoculated insects) showed the highest effectiveness, reaching 100% mortality at the end of the experiment. Other highly effective treatments were T4 B. bassiana + M. oleifera (five inoculated insects) and T7 M. robertsii + P. americana (five inoculated insects), both reaching 80% mortality at 240 h (

Table 4. Percentage of pathogenicity of B. bassiana and M. robertsii in S. purpurascens oil emulsions.

Treatments	Inoculated Insects	Time (h)
		48	96	192	240
T1 B. bassiana + A. indica	5	20 ± 0.3 e	33.3 ± 0.4 e	53.3 ± 0.4 ef	66.6 ± 0.7 c
T2 B. bassiana + A. indica	3	23 ± 0.4 d	38.4 ± 0.5 c	84.6 ± 0.5 a	100 a
T3 B. bassiana + A. indica	1	27.2 ± 0.3 b	36.3 ± 0.4 d	54.5 ± 0.6 de	63.6 ± 0.7 c
T4 B. bassiana + M. oleifera	5	26.6 ± 0.4 c	26.6 ± 0.4 g	73.3 ± 0.4 b	80 ± 0.1 b
T5 B. bassiana + M. oleifera	3	15.3 ± 0.4 f	15.3 ± 0.4 i	46.1 ± 0.2 fg	61.5 ± 0.6 c
T6 B. bassiana + M. oleifera	1	0 ± 0 h	9 ± 0.1 j	36.3 ± 0.4 h	45.4 ± 0.5 d
T7 M. robertsii + P. americana	5	33.3 ± 0.4 a	66.6 ± 0.7 a	74 ± 0.1 b	80 ± 0.1 b
T8 M. robertsii + P. americana	3	15.3 ± 0.2 f	30.7 ± 0.8 f	38.4 ± 0.5 d	46.1 ± 0.2 d
T9 M. robertsii + P. americana	1	0 ± 0 h	18.1 ± 0.2 i	36.3 ± 0.4 d	36.3 ± 0.4 e
T10 M. robertsii + P. dulcis	5	33.3 ± 0.4 a	46.6 ± 0.7 b	66.6 ± 0.7 bc	66.6 ± 0.7 c
T11 M. robertsii + P. dulcis	3	23 ± 0.1 d	38.4 ± 0.5 c	61.5 ± 0.6 bc	61.5 ± 0.6 c
T12 M. robertsii + P. dulcis	1	9 ± 0.1 g	9 ± 0.1 j	18.1 ± 0.2 i	36.3 ± 0.4 e
T13 DW (absolute control)	0	0 ± 0 h	0 ± 0 h	0 ± 0 h	0 ± 0 h

Mean ± standard deviation. Means with different letters in each column are statistically different (Tukey, p ≤ 0.05). DW: distilled water.

).

It was also observed that the number of inoculated insects influenced mortality, as it was higher when five infected insects were introduced into the experimental containers (Figure 1a). In contrast, treatments T8, T9, and T12 with M. robertsii + P. dulcis and P. americana showed lower effectiveness, with a maximum mortality of 46.1% at 240 h. The absolute control (T13) showed 0% pathogenicity at all time intervals, confirming that the observed effects were due to the combinations of fungi and oils.

Our results suggest that oil emulsion formulations can enhance the dispersion and pathogenicity of these EPF (Figure 1b,c).

4. Discussion

This study, performed under laboratory conditions, has provided valuable information on the feeding behavior of S. purpurascens at its N4, N5, and adult developmental stages. This research specifically focuses on the insect’s preference for certain crops and its potential impact on agricultural productivity. Previous study reported an incidence of 25 insects/m², considered a severe infestation threshold requiring control measures due to the damage and losses caused to producers [8,38]. In contrast, our findings indicate that feeding behavior is influenced by crop characteristics. Similar trends have been observed in other species: Dociostaurus maroccanus (Thunberg, 1815) and Tettigonia viridissima (Linnaeus, 1758) showed a preference for potato leaves over other plants, while Calliptamus barbarus barbarus (Costa, 1836) did not exhibit a significant preference [39]. In our study, C. dactylon exhibited lower consumption rates across all stages (N4, N5, and adults), averaging 0.76 g/48 h. Despite being replaced every 48 h, its rapid dehydration reduced its water content, decreasing its palatability. The ability of S. purpurascens to select food sources that support its growth and development is crucial for understanding its ecology and potential agricultural impact.

Currently, there are no reports or references on losses caused by S. purpurascens at its different development stages (N4, N5 and adults) with regard to the crops evaluated. However, the results of this study suggest a clear preference for specific crops at each of the insect’s developmental stages. A study on Scotussa lemniscata (Stal, 1861) revealed significant variations in consumption rates by developmental stage and sex, with pre-reproductive adults consuming the most. Notably, adult females consumed 118.81 ± 9.41 mg/day, significantly more than males and fifth-instar females [40]. This trend aligns with our findings, in which S. purpurascens adults exhibited the highest intake, particularly in M. sativa and Z. mays.

In addition to the developmental stage, food selection plays a crucial role in feeding dynamics. Previous research on Dichroplus maculipennis (Blanchard, 1851) demonstrated significant variations in consumption rates when they were fed Glycine max (Willd, 1802), Z. mays, Triticum aestivum (L., 1753), and A. sativa [41], highlighting the influence of nutritional composition and structural characteristics on dietary preferences. Our findings suggest that crops with higher moisture content are more attractive to S. purpurascens, likely due to their role in maintaining hydration and facilitating digestion.

Field studies on D. maculipennis reported an average intake of 236.0 ± 0.02 mg/individual/day in Festuca arundinacea (Schreb) pastures [42], reinforcing the idea that body size and metabolic demands drive feeding rates. The increased consumption observed in adult S. purpurascens supports this hypothesis, as larger individuals require more energy to sustain their physiological processes.

Our results highlight the importance of considering developmental stage and crop susceptibility when designing integrated pest management (IPM) strategies. The significant consumption of M. sativa and Z. mays by adult S. purpurascens suggests that these crops are particularly vulnerable in later instars, making biological control approaches, such as EPF in oil emulsions, a promising alternative.

The high viability observed in B. bassiana and M. robertsii exposed to A. indica and M. oleifera oils suggests that these oils represent an ideal option for the development of fungus-based biopesticides. Their compatibility with EPF ensures that these fungi maintain their survival capacity and efficacy when used under field conditions. The use of the highest concentration (40%) did not significantly reduce viability, which is particularly noteworthy. This indicates that these oils can be applied at higher doses without compromising fungal efficacy, a key advantage for field applications where higher concentrations may be required to enhance persistence or coverage. This emphasizes the importance of developing formulations compatible with the strains used to ensure their effective application against the target pest. For example, the use of Tween 80 at 0.05% as a surfactant enhances the dispersion and viability of B. bassiana [43], facilitating spore adhesion to the insect cuticle [44] and protecting them from external factors, thereby increasing conidia viability. The moderate (yet still high) viability observed with P. americana and P. dulcis oils suggests that these are also suitable, though slightly less effective compared to A. indica and M. oleifera. These findings align with previous research highlighting the importance of selecting compatible adjuvants for EPF formulations. For instance, a biopreparation based on Zeolite + Diatomite + B. bassiana was evaluated, reporting a conidial viability of 89.1% [23]. It was suggested that selecting preparations with higher viability is more effective for field applications, as these are more resistant to biotic and abiotic factors. Finally, it was noted that spore growth and germination are directly associated with the virulence of EPF [45].

Regarding the effectiveness of EPF in the field, previous studies have shown that combining extracts from Argemone mexicana (Linnaeus, 1753) and Capsicum frutescens (L., 1753) (chili) with B. bassiana reduced the incidence of S. purpurascens in A. hypochondriacus crops from 48% to 18%. Furthermore, B. bassiana strains at concentrations of 1 × 10⁸ and 1 × 10⁹ spores/mL caused 100% mortality in second- and fourth-stage nymphs after 8 days and 76–81% mortality in adults 6 days after application [28]. Similar results were obtained in our study, where vegetable oil emulsions with M. robertsii and P. americana caused 100% mortality in S. purpurascens adults after 72 h. These differences in effectiveness may be attributed to factors beyond concentration, such as the formulation type, application method, or environmental conditions. Although higher concentrations generally enhance strain efficacy, other studies have primarily focused on strain variability rather than the effects of concentration [45,46,47]. This finding highlights the importance of considering the developmental stage of the insect when designing biological control strategies. Reports of two M. anisopliae var. anisopliae isolates, Driver and Milner (Alberta 11S-1, Alberta 6W-2, and Green Guard), for Melanoplus bivittatus (Say) control at a 5 × 10⁷ conidia concentration in sunflower oil caused 50% mortality within 5 to 6 days and more than 90% by day 7 [48].

This bioassay helped us to understand the importance of ineffective processes, where EPF associate with healthy organisms, in relation to the ecology of the insects (habits). Grasshoppers act as spore dispersal vectors when they come into contact with the fungus, promoting its spread during movement. During mating, the transfer of spores between males and females can affect their reproductive viability. This is relevant, as a female lays between six and eight oothecas, each containing 20 to 40 eggs [49].

On the other hand, studies have demonstrated that combining canola oil with wheat bran and B. bassiana (1 × 10⁸ conidia) resulted in undesirable effects for third-stage nymphs of D. maculipennis in both laboratory and field cage settings. Mortality was observed between 10 and 15 days after inoculation [35]. In contrast, the combination of B. bassiana + Tween 80 at 0.05% + DW + A. indica showed 100% mortality 10 days after application under laboratory conditions. These results highlight the variability in efficacy depending on the formulation and target species. Furthermore, oil emulsions containing B. bassiana and M. robertsii significantly increased pathogenicity against S. purpurascens. This enhanced efficacy may be attributed to the oils’ ability to improve spore adhesion and distribution on the insect cuticle, a critical step in the fungal infection process. Moreover, fungal spores can persist in the environment for several months or even years. Additionally, by reproducing from dead insects, they maintain their role as infectious agents, enabling them to continue their life cycle and propagate [50,51]. This persistence and reproductive capability make EPF a sustainable and effective option for biological control. Similar results confirmed the potential of FPf for the control of Schistocerca gregaria (Forskal, 1775), highlighting B. bassiana and M. anisopliae var. acridum as effective alternatives to chemical insecticides. Green Muscle (M. anisopliae var. acridum) demonstrated the highest efficacy in the field, achieving up to 81.7% mortality in greenhouse trials in 14 days. However, B. bassiana isolates 341, 231, and 334, formulated in liquid paraffin, exhibited superior pathogenicity under laboratory conditions, exceeding 99% mortality in adult locusts in the same period [52,53].

5. Conclusions

In conclusion, this study demonstrates the potential of oil emulsions combined with B. bassiana and M. robertsii as effective biological control agents against S. purpurascens under laboratory conditions. The results emphasize the need for field trials to validate these findings across diverse crops and environments.

Furthermore, this study highlights the significant impact of S. purpurascens at different developmental stages on agricultural productivity, underscoring the importance of timely interventions by farmers.