Efficacy of focal applications of a mycoinsecticide to control Aedes aegypti in Central Brazil.

Entomopathogenic fungi can achieve important innovative outcomes for integrated mosquito control especially of Aedes aegypti, the key vector of arboviruses to humans in the tropics and subtropics. This study sought to design and to develop a simple dissemination device to attract and to infect gravid A. aegypti adults with a granular formulation of the ascomycete Metarhizium humberi IP 46, and to validate this device in the laboratory as well as in semi-field and field conditions. Hydrogel (polyacrylamide potassium polyacrylate) was confirmed to be a suitable substitute for water used in the device that attracted gravid females under field conditions. Females laid eggs on black polyethylene terephthalate carpet fixed in the device that also proved to be a suitable substrate for a granular formulation of fungal microsclerotia and/or conidia. The plastic device (29.5 cm high) was divided into a lower closed compartment with a water reservoir and an upper, laterally open but covered compartment with continuously hydrated gel and the fungal formulation attached to the carpet. The uppermost compartment permitted free circulation of mosquito adults. The device attracted both male and female A. aegypti. The fungal formulations of IP 46 propagules tested in the device were effective against adults in laboratory, semi-field, and field settings. Findings in the laboratory, semi-field, and especially in field conditions strengthen the value and utility of this innovative device for focal applications of a mycoinsecticide against this important mosquito vector.Key points• Low-cost and simple disseminating device for focal control of Aedes aegypti.• Granulized Metarhizium humberi IP 46 and hydrogel yield extended control.• Findings in field tests strengthen benefit of the device for focal application.

Lethal and sub-lethal activity of Brevibacillus laterosporus on the mosquito Aedes albopictus and side effects on non-target water-dwelling invertebrates.

The biocidal potential of Brevibacillus laterosporus against mosquitoes of major medical importance has been widely documented, but its effects on non-target invertebrates are still poorly known. In this study, we determined the lethal and sub-lethal effects of B. laterosporus strain UNISS 18, an entomopathogenic bacterium known for its effectiveness against synanthropic Diptera, on the larvae of the Asian tiger mosquito Aedes albopictus, a vector of several pathogens to humans. Moreover, we compared the larvicidal activity with the lethal action on the invasive snail Physella acuta and on two non-target water-dwelling species: the mayfly Cloeon dipterum, and the harlequin fly Chironomus riparius. B. laterosporus exhibited significant lethal effects on all the tested species with a concentration-dependent activity. However, the susceptibility varied among species, with a higher susceptibility of Ae. albopictus (LC50 = 0.16 × 107 spores mL-1) than the other species (LC50 = 0.31, 0.33, and 0.30 × 107 spores mL-1 for C. dipterum, C. riparius, and P. acuta, respectively). While 1st instar mosquito larvae were very susceptible to the bacterial infection, no effects on preimaginal development stages and adult emergence were observed at sub-lethal spores' concentrations. Even if the efficacy of B. laterosporus against Ae. albopictus and the invasive freshwater snail P. acuta is promising for their control, the susceptibility of non-target beneficial aquatic insects, highlights the need of accurate evaluations before applying B. laterosporus for pest management in water environments.

Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

UV-B tolerances of conidia, blastospores, and microsclerotia of Metarhizium spp. entomopathogenic fungi.

The aim of the present study was to analyze ten native Metarhizium spp. isolates as to their UV-B tolerances. Comparisons included: different fungal propagules (conidia, blastospores, or microsclerotia [MS]); conidia in aqueous suspensions or in 10% mineral oil-in-water emulsions; and conidia mixed with different types of soil. The UV-B effect was expressed as the germination of conidia or culturability of blastospores and MS relative to nongerminated propagules. Metarhizium anisopliae LCM S05 exhibited high tolerance as blastospores and/or MS, but not as conidia; LCM S10 and LCM S08 had positive results with MS or conidia but not blastospores. The formulations with 10% mineral oil did not always protect Metarhizium conidia against UV-B. Conidia of LCM S07, LCM S08, and LCM S10 exhibited the best results when in aqueous suspensions, 24 h after UV-B exposure. In general, conidia mixed with soil and exposed to UV-B yielded similar number of colony forming units as conidia from unexposed soil, regardless the soil type. It was not possible to predict which type of propagule would be the most UV-B tolerant for each fungal isolate; in conclusion, many formulations and propagule types should be investigated early in the development of new fungal biocontrol products.

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae).

Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL-1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.

Heat-stressed Metarhizium anisopliae: viability (in vitro) and virulence (in vivo) assessments against the tick Rhipicephalus sanguineus.

The current study investigated the thermotolerance of Metarhizium anisopliae s.l. conidia from the commercial products Metarril® SP Organic and Metarril® WP. The efficacy of these M. anisopliae formulations against the tick Rhipicephalus sanguineus s.l. was studied in laboratory under optimum or heat-stress conditions. The products were prepared in water [Tween® 80, 0.01 % (v/v)] or pure mineral oil. Conidia from Metarril® SP Organic suspended in water presented markedly delayed germination after heating to constant 40 °C (for 2, 4, or 6 h) compared to conidia suspended in mineral oil. Metarril® SP Organic suspended in oil and exposed to daily cycles of heat-stress (40 °C for 4 h and 25 °C for 19 h for 5 consecutive days) presented relative germination of conidia ranging from 92.8 to 87.2 % from day 1 to day 5, respectively. Conversely, germination of conidia prepared in water ranged from 79.3 to 39.1 % from day 1 to day 5, respectively. Culturability of Metarril® WP decreased from 96 % when conidia were cultured for 30 min prior to heat exposure (40 °C for 4 h) to 9 % when conidia were cultured for 8 h. Tick percent control was distinctly higher when engorged females were treated with oil suspensions rather than water suspensions, even when treated ticks were exposed to heat-stress regimen. Oil-based applications protected fungal conidia against heat-stress. Although Metarril® is not registered for tick control, it may be useful for controlling R. sanguineus, especially if it is prepared in mineral oil.

Effect of heat stress and oil formulation on conidial germination of Metarhizium anisopliae s.s. on tick cuticle and artificial medium.

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.

Conidiobolus macrosporus (Entomophthorales), a mosquito pathogen in Central Brazil.

A new fungal pathogen of Culicinae (Diptera: Culicidae) adults, Conidiobolus macrosporus (Entomophthorales: Ancylistaceae), was detected and isolated during a survey of mosquito pathogens close to the city of Aruanã, Goiás State, in December 2014. The morphological characteristics of C. macrosporus are presented, and reasons for some uncertainty about this identification are discussed. The pathogenicity and high virulence of this fungus for Aedes aegypti were confirmed in laboratory conditions. Mortality of adults exposed to conidia was observed within 24h of exposure to the pathogen, and increased to 100% as quickly as 3days after inoculation (with the highest conidial concentration tested, 8.3×10(4)conidia/cm(2)). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.

First report of Leptolegnia chapmanii (Peronosporomycetes: Saprolegniales) affecting mosquitoes in central Brazil.

Numerous isolates of an oomycete 'fungus', Leptolegnia chapmanii, are reported from Brazil for the first time. This aquatic pathogen was baited with Aedes aegypti sentinel larvae from stagnant, temporary bodies of water in selected locations under secondary tropical forest in and near the central Brazilian city of Goiânia and from more distant sites in the western and northern regions of the state of Goiás. Isolates were identified based on their morphological and developmental characters, comparative sequence data for the ITS and TEF loci, as well as their rapid activity against A. aegypti larvae. Taxonomic issues affecting the application of the name L. chapmanii and its typification are rectified. This study contributes to a better understanding of the presence and distribution of this oomycete in Brazil, its sequence-based identification, and of its potential as a biological agent against mosquito vectors.

Molecular and physiological effects of environmental UV radiation on fungal conidia.

Conidia are specialized structures produced at the end of the asexual life cycle of most filamentous fungi. They are responsible for fungal dispersal and environmental persistence. In pathogenic species, they are also involved in host recognition and infection. Conidial production, survival, dispersal, germination, pathogenicity and virulence can be strongly influenced by exposure to solar radiation, although its effects are diverse and often species dependent. UV radiation is the most harmful and mutagenic waveband of the solar spectrum. Direct exposure to solar radiation for a few hours can kill conidia of most fungal species. Conidia are killed both by solar UV-A and UV-B radiation. In addition to killing conidia, which limits the size of the fungal population and its dispersion, exposures to sublethal doses of UV radiation can reduce conidial germination speed and virulence. The focus of this review is to provide an overview of the effects of solar radiation on conidia and on the major systems involved in protection from and repair of damage induced by solar UV radiation. The efforts that have been made to obtain strains of fungi of interest such as entomopathogens more tolerant to solar radiation will also be reviewed.

Tolerance of entomopathogenic fungi to ultraviolet radiation: a review on screening of strains and their formulation.

Ultraviolet radiation from sunlight is probably the most detrimental environmental factor affecting the viability of entomopathogenic fungi applied to solar-exposed sites (e.g., leaves) for pest control. Most entomopathogenic fungi are sensitive to UV radiation, but there is great inter- and intraspecies variability in susceptibility to UV. This variability may reflect natural adaptations of isolates to their different environmental conditions. Selecting strains with outstanding natural tolerance to UV is considered as an important step to identify promising biological control agents. However, reports on tolerance among the isolates used to date must be analyzed carefully due to considerable variations in the methods used to garner the data. The current review presents tables listing many studies in which different methods were applied to check natural and enhanced tolerance to UV stress of numerous entomopathogenic fungi, including several well-known isolates of these fungi. The assessment of UV tolerance is usually conducted with conidia using dose-response methods, wherein the UV dose is calculated simply by multiplying the total irradiance by the period (time) of exposure. Although irradiation from lamps seldom presents an environmentally realistic spectral distribution, laboratory tests circumvent the uncontrollable circumstances associated with field assays. Most attempts to increase field persistence of microbial agents have included formulating conidia with UV protectants; however, in many cases, field efficacy of formulated fungi is still not fully adequate for dependable pest control.

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation.

The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.

Assessing gene expression during pathogenesis: Use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans.

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level.

New insights into the amphibious life of Biomphalaria glabrata and susceptibility of its egg masses to fungal infection.

The air-breathing snail Biomphalaria glabrata proliferates in stagnant freshwater, and nothing is known about the survival of eggs in intermittently (rather than perpetually) wet habitats. In the present study their egg masses matured, and juveniles subsequently eclosed and were mobile in a stable water film of transitory habitats simulated by two different simple test devices described here. The viability of eggs maintained in an unstable film however, was diminished. The maturation of egg masses in a water film or in water was significantly prevented by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. The efficiency depended on the fungal propagule and test environment. Hyphal bodies were more effective against egg masses than conidia. This appears to be a first report of activity of either entomopathogen against a mollusc. Both devices offer accurate and reproducible conditions to test both biological questions and the effects of substances or pathogens against B. glabrata egg masses in water films.

Effect of ultraviolet-A radiation on the production of Leptolegnia chapmanii (Saprolegniales: Saprolegniaceae) zoospores on dead Aedes aegypti (Diptera: Culicidae) larvae and their larvicidal activity.

Impact of UV-radiation in entomopathogens in aquatic environments remains little investigated. The present study reports on the effect of UV-A on the larvicidal activity of Leptolegnia chapmanii zoospores in Aedes aegypti; on the production of zoospores in larvae killed by the pathogen and then exposed to UV-A; and on the activity of these zoospores against healthy larvae. Whereas the virulence of free zoospores in A. aegypti larvae was affected by a UV-A exposure time longer than 10min, production of zoospores in larvae and their virulence were not hampered at a maximal 8h exposure of dead larvae to UV-A. Findings suggest that dead larvae and zoosporangia provide a certain protection to zoospores against UV-A and emphasize the susceptibility of free encysted zoospores to such radiation.

The influence of conidial Pr1 protease on pathogenicity potential of Metarhizium anisopliae senso latu to ticks.

Pr1 is a subtilisin-like protease produced by Metarhizium spp. entomopathogenic fungi, and it is recognized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, correlation was sought between mortality of tick larvae and conidial Pr1 levels of one Metarhizium anisopliae senso latu (s.l.) isolate (CG 148). Conidia with different levels of pr1 gene expression and enzymatic activity were obtained by producing them on either artificial medium (to yield low Pr1 activity) or on Rhipicephalus microplus cadavers (to yield high Pr1 activity). Conidial proteolytic activity was assessed using N-suc-ala-ala-pro-phe-ρNA as the chromogenic substrate, and pr1 expression was profiled by qPCR using three genes (gpd, try, and tef) as reference genes. Pr1 enzymatic (proteolytic) activity on conidia obtained from tick cadavers was 36 U mg(-1) in comparison to 4 U mg(-1) on conidia from PDA medium. Also, pr1 gene expression level was ten times higher in conidia from tick cadavers compared to PDA medium. Bioassays of M. anisopliae s.l. CG 148 spores with elevated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant change percent mortality of tick larvae). The minimal levels of Pr1 on conidia produced on artificial medium was adequate to afford high levels of virulence, and the elevated amounts of the enzyme on tick-cadaver-produced conidia did not induce elevated larval mortality. As long as some Pr1 activity was present, fungal virulence of isolate CG 148 against tick larvae was not elevated by increased levels of conidial Pr1.

Heat-induced post-stress growth delay: a biological trait of many Metarhizium isolates reducing biocontrol efficacy?

The habitats of many pest insects have fluctuating climatic conditions. To function effectively, the pathogens of these pests must be capable of infecting and developing disease at a wide range of temperatures. The current study examines ten Metarhizium spp. isolates as to their ability to recover normal metabolic activity after exposure to high temperature for several hours daily; and whether such recovery, with at least some isolates, requires a temporary repair ("retooling") period. Fungal colonies were exposed to 40°C for 4h or 8h followed by 20h or 16h at 28°C, respectively, for three consecutive days. Growth rates during treatments were compared to control plates (constant 28°C) and to plates with growth stoppage by cold treatment (4h or 8h at 5°C per day). All ten isolates survived 3days of cycled heat treatment and resumed normal growth afterward; some isolates however, were considerably more negatively affected by heat-cycling than others. In fact, some isolates underwent greatly reduced growth not only during 8h heating, but also some hours after cessation of heat treatment. This phenomenon is labeled in the current study as "post-stress growth delay" (PSGD). In contrast, all isolates stopped growing during 8h cold treatments, but immediately recommenced growing on return to 28°C. The delay in recommencing growth of some isolates after heat treatment amplifies the effect of this stress. In addition to the studies on the effects of heat cycling on fungal cultures, the effects of imposing such temperature cycling on fungal infection of insects was documented in the laboratory. Three Metarhizium isolates were bioassayed using Galleria mellonella larvae. Treated insects were placed at daily temperature regimes matching those used for the in vitro fungus rate-of-growth study, and insect mortality recorded daily. For all three isolates the levels of insect mortality at the highest-heat dose (40°C at 8h daily) significantly reduced infection. Fluctuating temperatures are likely to be a factor in most pest-insect habitats; therefore, the presence and level of PSGD of each isolate should be a primary consideration in selecting field-appropriate fungal isolates.

Impact of humidity on clustered tick eggs.

Eclosion of larvae from clustered tick eggs (1, 5, 10, 15, 20, 25, 50, and ±1,000 eggs) of Rhipicephalus sanguineus (Rs), Rhipicephalus microplus (Rm), Amblyomma cajennense (Ac), and Dermacentor nitens (Dn) was investigated at 43, 75, and > 98% relative humidity (RH) and 25 °C. Susceptibility of eggs to dehydration varied among tick species (Rs < Rm < Ac and Dn) and the number of clustered eggs but even singularized eggs of all tested species matured, and larvae eclosed at RH > 98%. A minimum of 25 clustered eggs and relative humidity close to saturation is suggested for quantitative tests on ovicidal activity of acaricides for related tick species.

Multifaceted Beauveria bassiana and Other Insect-Related Fungi.

Since Agostino Bassi first isolated the fungal pathogenic agent of the white muscardine in insects (later named Beauveria bassiana in his honor), and Ilya Mechnikov cultivated Metarhizium anisopliae as a first approach to use fungi as pest control agents, many other entomopathogenic fungi have been studied over the last two centuries [...].

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.