Horizontal transmission of the entomopathogenic fungus <i>Beauveria bassiana</i> (Unioeste 76 strain) among adults of <i>Euschistus heros</i> (Fabricius, 1798) (Hemiptera: Pentatomidae) (2024)

The horizontal transmission of the entomopathogenic fungus Beauveria bassiana Unioeste 76 strain among adults of Euschistus heros was evaluated in the laboratory and greenhouse using sporulated cadavers and live insects contaminated as a source of inoculate. Adult insects in the laboratory bioassay either received treatments with dry conidia or were sprayed with an oily dispersion formulation, and the treatments were comprised of 10, 30, or 50% of contaminated insects. For the greenhouse bioassay, nylon cages were prepared, with soybean plants grown in pots. A total of 20 healthy brown stink bugs were released along with one or three sporulated cadavers of brown stink bugs, attached to the plant stem. In the bioassay with live insects, 20 stink bugs were released, and the treatments were comprised of 10, 30, or 50% contaminated insects (2:18, 6:14, and 10:10, fungus-treated insects : healthy insects). In all experiments, the control group was comprised only of healthy insects. Dead stink bugs were daily disinfested and transferred to a wet chamber for confirmation of death by fungus. Confirmed mortality was observed, which shows the occurrence of horizontal transmission. Mortality was higher when more contaminated insects were used in the population, in most treatments. The occurrence of transmission in the laboratory and the greenhouse emphasizes the potential of the Unioeste 76 strain as mortality agent and as an agent for maintaining the inoculum potential for the brown stink bug.

Keywords
auto-dissemination; biological control; microbial control; entomopathogen; soybeans

Soybean, Glycine max (L.) Merrill, is an oilseed with high protein content used in human diet and in the diet of animals, especially in the production of animal feed, bran, and soy oil (Gazzoni; Dall’agnol, 2018GAZZONI, D.L.; DALL’AGNOL, A. A saga da soja: de 1050 a.C. a 2050 d.C. Brasília: Embrapa, 2018.). Brazil is currently the largest world producer of soybeans, with a production of approximately 120 million tons (CONAB, 2022COMPANHIA NACIONAL DE ABASTECIMENTO (CONAB). Acompanhamento da safra brasileira de grãos: safra 2021/2022. Décimo primeiro levantamento. Brazil: CONAB, 2022. v. 9, n. 11. Available at: https://www.conab.gov.br/info-agro/safras/graos. Accessed on: Aug 11, 2022.
https://www.conab.gov.br/info-agro/safra... ).

The brown stink bug, Euschistus heros (Fabricius, 1798) (Hemiptera: Pentatomidae), is currently considered the most important soybean pest in Brazil, due to the damages it causes and because it is the species with the highest frequency of occurrence and with the highest abundance in the country (Zerbino; Panizzi, 2019ZERBINO, M.S.; PANIZZI, A.R. The underestimated role of pest pentatomid parasitoids in Southern South America. Arthropod-Plant Interactions, v.13, n.5, p.703-718, 2019. https://doi.org/10.1007/s11829-019-09703-1
https://doi.org/10.1007/s11829-019-09703... ; Sosa-Gómez et al., 2020SOSA‐GÓMEZ, D.R.; CORRÊA‐FERREIRA, B.S.; KRAEMER, B.; PASINI, A.; HUSCH, P.E.; DELFINO VIEIRA, C.E.; REIS MARTINEZ, C.B.; NEGRÃO LOPES, I.O. Prevalence, damage, management, and insecticide resistance of stink bug populations (Hemiptera: Pentatomidae) in commodity crops. Agricultural and Forest Entomology, v.22, n.2, p.99-118, 2020. https://doi.org/10.1111/afe.12366
https://doi.org/10.1111/afe.12366... ).

Chemical control is the most frequently used method to reduce the brown stink bug population (Bueno et al., 2013BUENO, A.F.; PAULA-MORAES, S.V.; GAZZONI, D.L.; POMARI, A.F. Economic thresholds in soybean-integrated pest management: old concepts, current adoption, and adequacy. Neotropical Entomology, v.42, n.5, p.439-447, 2013. https://doi.org/10.1007/s13744-013-0167-8
https://doi.org/10.1007/s13744-013-0167-... ; Tuelher et al., 2018TUELHER, E.S.; SILVA, É.H.; RODRIGUES, H.S.; HIROSE, E.; GUEDES, R.N.C.; OLIVEIRA, E.E. Area-wide spatial survey of the likelihood of insecticide control failure in the neotropical brown stink bug Euschistus heros. Journal of Pest Science, v.91, n.2, p.849-859, 2018. https://doi.org/10.1007/s10340-017-0949-6
https://doi.org/10.1007/s10340-017-0949-... ). However, the occurrence of populations resistant to chemical insecticides has become increasingly more frequent, and chemical control has become increasingly ineffective (Sosa-Gómez et al., 2020SOSA‐GÓMEZ, D.R.; CORRÊA‐FERREIRA, B.S.; KRAEMER, B.; PASINI, A.; HUSCH, P.E.; DELFINO VIEIRA, C.E.; REIS MARTINEZ, C.B.; NEGRÃO LOPES, I.O. Prevalence, damage, management, and insecticide resistance of stink bug populations (Hemiptera: Pentatomidae) in commodity crops. Agricultural and Forest Entomology, v.22, n.2, p.99-118, 2020. https://doi.org/10.1111/afe.12366
https://doi.org/10.1111/afe.12366... ). Entomopathogenic fungi are known to be an alternative for the control of the brown stink bug. Recent studies proved the virulence of some Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) strains with capacity for mass production in the laboratory (Dall Nora et al., 2021DALLA NORA, D.; PIOVESAN, B.C.; BELLÉ, C.; STACKE, R.S.; BALARDIN, R.R.; GUEDES, J.V.C.; MICHAUD, J.P.; JACCQUES, R.J.S. Isolation and evaluation of entomopathogenic fungi against the neotropical brown stink bug Euschistus heros (F.) (Hemiptera: Pentatomidae) under laboratory conditions. Biocontrol Science and Technology, v.31, n.1, p.22-34, 2021. https://doi.org/10.1080/09583157.2020.1826904
https://doi.org/10.1080/09583157.2020.18... ; Silva-Santana et al., 2022SILVA-SANTANA, M.F.; ALVES, L.F.; FERREIRA, T.T.; BONINI, A.K. Selection and characterisation of Beauveria bassiana fungus and their potential to control the brown stink bug. Biocontrol Science and Technology, v.32, n.1, p.90-102, 2022. https://doi.org/10.1080/09583157.2021.1970716
https://doi.org/10.1080/09583157.2021.19... ). In addition, there are several fungus-based products in Brazil for the control of the brown stink bug, although they are made from the same isolates (AGROFIT, 2022AGROFIT. Sistemas de agrotóxicos fitossanitários. AGROFIT. Available at: https://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons. Accessed on: Oct 8, 2022.
https://agrofit.agricultura.gov.br/agrof... ).

Fungi may also act by being transmitted inside a population, through the contact of a healthy insect with another insect, that is infected, or with sporulated cadavers (Hesketh et al., 2009HESKETH, H.; ROY, H.E.; EILENBERG, J.; PELL, J.K.; HAILS, R.S. Challenges in modelling complexity of fungal entomopathogens in semi-natural populations of insects. BioControl, v.55, n.1, p.55-73, 2009. https://doi.org/10.1007%2F978-90-481-3966-8_5
https://doi.org/10.1007%2F978-90-481-396... ). The capacity for horizontal transmission is a factor of great importance for its use as an agent for population control since infected individuals might be good vectors of the fungus in spreading the infection to healthy individuals and leading to the outbreak of epizootics in the field (Hajek; St. Leger, 1994HAJEK, A.E.; ST. LEGER, R.J. Interactions between fungal pathogens and insect hosts. Annual Review of Entomology, v.39, p.293-322, 1994. https://doi.org/10.1146/annurev.en.39.010194.001453
https://doi.org/10.1146/annurev.en.39.01... ; Furlong; Pell, 2001FURLONG, M.J.; PELL, J.K. Horizontal transmission of entomopathogenic fungi by the diamondback moth. Biological Control, v.22, n.3, p.288-299, 2001. https://doi.org/10.1006/bcon.2001.0981
https://doi.org/10.1006/bcon.2001.0981... ).

Even though the occurrence of horizontal transmission of B. bassiana has been proven between insects and mites (Cárcamo et al., 2015CÁRCAMO, M.C.; FELCHICHER, F.; DUARTE, J.P.; BERNARDI, E.; RIBEIRO, P.B. Horizontal Transmission of Beauveria bassiana (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in Musca domestica (Diptera: Muscidae). Journal of Economic Entomology, v.108, n.4, p.1579-1586, 2015. https://doi.org/10.1093/jee/tov163
https://doi.org/10.1093/jee/tov163... ; Forlani et al., 2015FORLANI, L.; PEDRINI, N.; GIROTTI, J.R.; MIJAILOVSKY, S.J.; CARDOZO, R.M.; GENTILE, A.G.; HERNÁNDEZ-SUÁREZ, C.M.; RABINOVICH, J.E.; JUÁREZ, M.P. Biological control of the Chagas disease vector Triatoma infestans with the entomopathogenic fungus Beauveria bassiana combined with an aggregation cue: field, laboratory and mathematical modeling assessment. PLoS Neglected Tropical Diseases, v.9, n.5, e0003778, 2015. https://doi.org/10.1371/journal.pntd.0003778
https://doi.org/10.1371/journal.pntd.000... ; Conceschi et al., 2016CONCESCHI, M.R.; D’ALESSANDRO, C.P.; MORAL, R.D.A.; DEMÉTRIO, C.G.B.; DELALIBERA JÚNIOR, I. Transmission potential of the entomopathogenic fungi Isaria fumosorosea and Beauveria bassiana from sporulated cadavers of Diaphorina citri and Toxoptera citricida to uninfected D. citri adults. BioControl, v.61, n.5, p.567-577, 2016. https://doi.org/10.1007/s10526-016-9733-4
https://doi.org/10.1007/s10526-016-9733-... ; Mannino et al., 2018MANNINO, M.C.; JUÁREZ, M.P.; PEDRINI, N. Tracing the coevolution between Triatoma infestans and its fungal pathogen Beauveria bassiana. Infection, Genetics and Evolution, v.66, p.319-324, 2018. https://doi.org/10.1016/j.meegid.2018.02.032
https://doi.org/10.1016/j.meegid.2018.02... ; Hassemer et al., 2020HASSEMER, M.J.; LOPES, R.B.; BORGES, M.; ALVES, L.F.A.; WITHALL, D.M.; PICKETT, J.A.; LAUMANN, R.A.; BIRKETT, M.A.; BLASSIOLI-MORAES, M.C. Development of an attract-and-infect device for biological control of lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae) in poultry houses. Biological Control, v.149, e104326, 2020. https://doi.org/10.1016/j.biocontrol.2020.104326
https://doi.org/10.1016/j.biocontrol.202... ; Nascimento et al., 2020NASCIMENTO, M.M.; ALVES, L.F.A.; OLIVEIRA, D.G.P.; LOPES, R.B.; GUIMARÃES, A.T.B. Laboratory and field evaluation of an autoinoculation device as a tool to manage poultry red mite, Dermanyssus gallinae, infestations with Beauveria bassiana. Experimental and Applied Acarology, v.80, n.2, p.151-165, 2020. https://doi.org/10.1007/s10493-020-00466-6
https://doi.org/10.1007/s10493-020-00466... ), there are no reports of its occurrence among pentanomids. Therefore, the present study aimed to evaluate the horizontal transmission of B. bassiana Unioeste 76 in adults of E. heros.

The experiments were conducted in two stages: in the laboratory and the greenhouse. All insects used in the experiment were reared in the laboratory, maintained in plastic containers, and fed with a natural diet, adapted from Oliveira et al. (2016)OLIVEIRA, D.G.P.; DUDCZAK, A.C.; ALVES, L.F.A.; SOSA-GOMEZ, D.R. Biological parameters of Euschistus heros (F.) (Heteroptera: Pentatomidae) and its susceptibility to entomopathogenic fungi when fed on different diets. Brazilian Archives of Biology and Technology, v.59, e16150141, 2016. https://doi.org/10.1590/1678-4324-2016150141
https://doi.org/10.1590/1678-4324-201615... .

Horizontal transmission was evaluated based on two different sources of conidia:

• Transmission of the fungus from infected cadavers (fungus-carrying cadavers) to healthy individuals;

• Transmission of the fungus from live infected stink bugs (fungus-treated insects) to healthy individuals with either dry conidia or oil dispersion formulation.

The B. bassiana Unioeste 76 strain was used, previously selected as the most virulent against the brown stink bug (Silva-Santana et al., 2022SILVA-SANTANA, M.F.; ALVES, L.F.; FERREIRA, T.T.; BONINI, A.K. Selection and characterisation of Beauveria bassiana fungus and their potential to control the brown stink bug. Biocontrol Science and Technology, v.32, n.1, p.90-102, 2022. https://doi.org/10.1080/09583157.2021.1970716
https://doi.org/10.1080/09583157.2021.19... ). The fungus was multiplied in pre-cooked parboiled rice and plastic bags (Leite et al., 2003LEITE, L.G.; BATISTA FILHO, A.; ALMEIDA, J.E.M.; ALVES, S.B. Produção de fungos entomopatogênicos. Ribeirão Preto: Alexandre de Sene Pinto, 2003.). Conidia were separated using a nylon sieve, and, after drying in a silica gel desiccator for seven days at 20 ± 1°C, they were separated using a metal 32-mesh sieve.

Preparing the fungus-treated insects

An oil dispersion formulation was prepared (a mixture of fungal conidia, emulsifier, and vegetable oil) (Faria; Wraight, 2007FARIA, M.R.; WRAIGHT, S.P. Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biological Control, v.43, n.3, p.237-256, 2007. https://doi.org/10.1016/j.biocontrol.2007.08.001
https://doi.org/10.1016/j.biocontrol.200... ; Alves et al., 2015ALVES, L.F.A.; OLIVEIRA, D.G.P.; LAMBKIN, T.; BONINI, A.K.; ALVES, V.; PINTO, F.G.S.; SCUR, M.C. Beauveria bassiana applied to broiler chicken houses as biocontrol of Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae), an avian pathogens vector. Brazilian Journal of Poultry Science, v.17, n.4, p.459-466, 2015. https://doi.org/10.1590/1516-635X1704459-466
https://doi.org/10.1590/1516-635X1704459... ). The formulation was diluted in distilled water (final concentration of 10⁹ conidia/mL). E. heros adults within 96 hours after emergence were dorsally identified in the pronotum with nontoxic yellow paint. The insects were submerged for 10 seconds in 4 mL of the formulation diluted in water and transferred to plastic containers. After drying, they were used in the laboratory and greenhouse experiments. Another group of identified insects was comprised of adult stink bugs and transferred to Petri dishes containing 0.5-g dry conidia for every 20 insects and slightly agitated for 60 seconds. The treated insects were called fungus-treated insects.

Preparing fungus-carrying cadavers

A suspension was prepared with dry conidia from the B. bassiana Unioeste 76 strain, in a 0.01% Tween 80 solution in distilled water (1×10⁹ conidia/mL). Euschistus heros adults were immersed in the suspension for 15 seconds, transferred to plastic containers, and maintained in an acclimatized room (26 ± 2°C, relative humidity 60 ± 10%, and 14 hours of light). Dead insects were daily removed and submerged in 70% alcohol, 2% sodium hypochlorite, and distilled water, and transferred to a moistened filter paper in Petri dishes. The dishes were placed in a plastic container with polyurethane foam saturated with distilled water (wet chamber) and incubated for seven days to stimulate conidiogenesis (26 ± 2°C, relative humidity 60 ± 10%, and 14 hours of light). Cadavers were selected according to the uniformity of conidiogenesis. They were called fungus-carrying cadavers, and destined for the experiment in the greenhouse.

Transmission experiments in the laboratory

Fungus-treated insects (contaminated with the formulated fungus or with dry conidia) and healthy insects were transferred to plastic containers (8-cm height × 10-cm diameter) with a screened cap, at 2:18, 6:14, and 10:10 (fungus-treated insects : healthy insects ratio) ratios. In the control group, only 20 healthy insects were used. The insects were maintained in an acclimatized room (25 ± 1°C; 12 hours of photophase; relative humidity 60 ± 10%). Four replicates were prepared for each treatment and control group.

The experiment was daily evaluated for 10 days. The dead insects were removed, disinfected, and incubated in a wet chamber as previously described. The experiment was replicated twice.

Experiments in greenhouse

Plastic pots (4.9 L) were prepared with organic substrate and soil, in which soybean plants of the Brasmax Lança variety were grown and daily irrigated. When the plants reached the R1 phase, they were individualized in cages with nylon screens and metal structure (80-cm height × 30-cm diameter) and maintained in a greenhouse with controlled temperature. The pots were placed on a wooden structured and protected with plastic plates with water to prevent predator ants from attacking the stink bugs.

An Onset Hobo UX100-003 data logger was installed inside the greenhouse to monitor temperature and humidity continuously during the experimentation period.

Bioassay in a greenhouse with fungus-treated insects

The insects released into the previously prepared cages were already identified and treated with the fungus in the oil dispersion formulation at 2:18, 6:14, and 10:10 (fungus-treated insects : healthy insects) ratios. In the control group, only healthy insects were released. For each treatment and the control, five replicates were prepared.

Bioassay in a greenhouse with fungus-carrying cadavers

Another group of cages with soybeans plants was used with the fungus-carrying cadavers attached to the stems of soybeans using safety pins at random heights, at 1:20 and 3:20 (fungus-carrying cadavers : healthy insects) ratios per cage. Afterward, 20 laboratory-reared brown stink bugs healthy adults (96 hours after emergence) were released. In the control group, only healthy insects were released. Seven replicates were prepared for each treatment and the control.

In the two experiments, evaluations were daily performed for 10 days. The dead insects were removed, disinfected, and incubated in a wet chamber as previously described.

Evaluations and statistical analysis.

Total mortality data and confirmed mortality by fungus were corrected according to the Schneider-Orelli formula (Püntener, 1981PÜNTENER, W. Manual for field trials in plant protection. 2. ed. Basle: Ciba-Geigy Limited, Agricultural Division, 1981.), and, after that, they were analyzed regarding normality (Shapiro-Wilk’s test, p < 0.05) and hom*ogeneity of variance (Levene’s test, p < 0.05). Means were compared using the HSD Tukey’s test (p < 0.05).

Transmission experiments in the laboratory

In the cages with live insects contaminated by dry conidia, mortality varied between 73.5 and 100% and was higher in the presence of 10 fungus-treated insects (F = 46.22; p < 0.05), equivalent to 50% of the contaminated population (Table 1). Confirmed mortality ranged from 77.8 to 98.7% and was higher in the presence of 10 fungus-treated insects in the population (F = 144.73; p < 0.05) (Table 1). Total mortality of insects contaminated with the formulation varied between 10.7 and 53.4% and confirmed mortality ranged from 7.6 to 50.7%, with a significant difference among the three treatments (F = 114.7; p < 0.05). Confirmed mortality was higher when more fungus-treated insects were used in both laboratory bioassays (Table 1).

Comparing confirmed mortality from two different sources of inoculum, it was noticeable that transmission was higher when fungus-treated insects were infected with dry conidia than with the oil dispersion formulation, with significant difference in all treatments (Table 1).

Bioassay in a greenhouse with fungus-treated insects

Total mortality of E. heros was observed in the treatments with two, six, or ten fungus-treated insects among the total of 20 insects, ranging from 36.2 to 53.9%, with no significant difference between treatments (Table 2).

Comparing confirmed mortality among treatments, the treatment with two fungus-treated insects in the population obtained 20% of insects killed by the fungus (10% fungus-treated and 10% healthy). In the treatment with six fungus-treated insects, 41% of the insects were killed by the fungus (29% fungus-treated and 12% healthy). Finally, with 10 fungus-treated insects in the population, confirmed mortality was 49% (31% fungus-treated and 18% healthy). In the treatments with six and ten fungus-treated insects in the population, confirmed mortality was significantly higher than in the treatment with only two fungus-treated insects (F = 4.91; p < 0.05).

Temperatures between 12.1 and 32.1°C were observed, with daily mean temperature of 21.7°C and daily mean relative air humidity of 83.4%, ranging from 75.8 to 98.4% during the evaluation period.

Bioassay in a greenhouse with fungus-carrying cadavers

Mortality was 26.7% in the presence of one fungus-carrying cadaver, and the treatment with three cadavers had 29.7% mortality. There was no significant difference between the treatments (Table 3). In the presence of three fungus-carrying cadavers in the environment and 20 healthy insects, confirmed mortality was 31.1%, significantly higher than the treatment with one cadaver, which transmitted the fungus to an average of 19.4% of healthy insects. During the experimental period, the mean daily temperature was 24.4°C, with minimum and maximum temperatures of 19.4 and 34.6°C, respectively, and air relative humidity ranged between 74.8 and 90.6%, with a mean daily value of 83.8%.

Preliminary bioassays in the laboratory had higher values of confirmed mortality compared to those obtained in the greenhouse. This difference is likely because there is more control of environmental variables in the laboratory and insects were confined in containers with smaller space than the cages with plants, which promoted more encounters, that are directly proportional to the horizontal transmission of entomopathogenic fungi (Baverstock et al., 2010BAVERSTOCK, J.; ROY, H.E.; PELL, J.K. Entomopathogenic fungi and insect behavior: from unsuspecting hosts to targeted vectors. In: ROY, H.E.; VEGA, F.E.; CHANDLER, D.; GOETTEL, M.S.; PELL, J.; WAJNBERG, E. (eds.). The ecology of fungal entomopathogens. Dordrecht: Springer, 2009. p.89-102.). Gregarious behavior and population density are factors that might have benefitted transmission (Forlani et al., 2015FORLANI, L.; PEDRINI, N.; GIROTTI, J.R.; MIJAILOVSKY, S.J.; CARDOZO, R.M.; GENTILE, A.G.; HERNÁNDEZ-SUÁREZ, C.M.; RABINOVICH, J.E.; JUÁREZ, M.P. Biological control of the Chagas disease vector Triatoma infestans with the entomopathogenic fungus Beauveria bassiana combined with an aggregation cue: field, laboratory and mathematical modeling assessment. PLoS Neglected Tropical Diseases, v.9, n.5, e0003778, 2015. https://doi.org/10.1371/journal.pntd.0003778
https://doi.org/10.1371/journal.pntd.000... ; Conceschi et al., 2016CONCESCHI, M.R.; D’ALESSANDRO, C.P.; MORAL, R.D.A.; DEMÉTRIO, C.G.B.; DELALIBERA JÚNIOR, I. Transmission potential of the entomopathogenic fungi Isaria fumosorosea and Beauveria bassiana from sporulated cadavers of Diaphorina citri and Toxoptera citricida to uninfected D. citri adults. BioControl, v.61, n.5, p.567-577, 2016. https://doi.org/10.1007/s10526-016-9733-4
https://doi.org/10.1007/s10526-016-9733-... ; Mannino et al., 2018MANNINO, M.C.; JUÁREZ, M.P.; PEDRINI, N. Tracing the coevolution between Triatoma infestans and its fungal pathogen Beauveria bassiana. Infection, Genetics and Evolution, v.66, p.319-324, 2018. https://doi.org/10.1016/j.meegid.2018.02.032
https://doi.org/10.1016/j.meegid.2018.02... ).

It was also noticeable that mortality (both total and confirmed) in the treatment with formulated fungus was significantly lower than that obtained with dry conidia. The formulation might have influenced the time of conidial germination, growth, infection, and conidiogenesis. This contrasts with what was observed with dry conidia, which is the “naturally” found form of the fungus. Chergui et al. (2020)CHERGUI, S.; BOUDJEMAA, K.; BENZEHRA, A.; KARACA, I. Pathogenicity of indigenous Beauveria bassiana (Balsamo) against Ceratitis capitata Wiedemann (Diptera: Tephritidae) under laboratory conditions. Egyptian Journal of Biological Pest Control, v.30, 128, 2020. https://doi.org/10.1186/s41938-020-00331-z
https://doi.org/10.1186/s41938-020-00331... also found that different forms of application/contact or even sources of inoculum might have affected horizontal transmission.

The results also proved the occurrence of horizontal transmission of B. bassiana Unioeste 76 strain from contaminated E. heros, both sporulated cadavers and live individuals in the greenhouse. As horizontal transmission is an important aspect in the applicability of a biological fungus-based insecticide, its occurrence contributes to and emphasizes the potential of the Unioeste 76 strain of B. bassiana as a control agent for this species.

A previous study proved there was no conidiogenesis of B. bassiana on dead insects incubated above 34°C, while relative air humidity between 75 and 90% was considered suitable for conidia formation (Sosa-Gómez; Alves, 2000SOSA-GÓMEZ, D.R.; ALVES, S.B. Temperature and relative humidity requirements for conidiogenesis of Beauveria bassiana (Deuteromycetes: Moniliaceae). Anais da Sociedade Entomológica do Brasil, v.29, n.3, p.515-521, 2000. https://doi.org/10.1590/S0301-80592000000300014
https://doi.org/10.1590/S0301-8059200000... ). Temperature and relative air humidity are therefore key factors in infecting insects. During the experimental period, the variation in the environmental conditions of the greenhouse might have interfered with the results. However, even under unstable conditions, the horizontal transmission of the fungus between E. heros adults emphasizes the susceptibility of the brown stink bug to the Unioeste 76 strain and this strain capacity to infect and colonize the host.

Despite relatively low values of confirmed mortality in the greenhouse, the occurrence of horizontal transmission might help the fungal environment dissemination. Also, it might cause an early outbreak of the epizootic (Membang et al., 2021MEMBANG, G.; AMBANG, Z.; MAHOT, H.C.; KUATE, A.F.; FIABOE, K.K.M.; HANNA, R. Thermal response and horizontal transmission of cameroonian isolates of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae – Candidates for microbial controls of the banana root borer Cosmopolites sordidus. Fungal Ecology, v.50, 101042, 2021. https://doi.org/10.1016/j.funeco.2021.101042
https://doi.org/10.1016/j.funeco.2021.10... ) through the contamination of a healthy insect in contact with either a sporulated cadaver or an infected individual. It is worth noticing that transmission might occasionally be favored by air or water currents, which must be taken into consideration when using insect attract-and-infect devices using fungi (Vega et al., 1995VEGA, F.E.; DOWD, P.F.; BARTELT, R.J. Dissemination of microbial agents using an autoinoculating device and several insect species as vectors. Biological Control, v.5, n.4, p.545-552, 1995. https://doi.org/10.1006/bcon.1995.1064
https://doi.org/10.1006/bcon.1995.1064... ). Moreover, individuals sprayed with fungi might be potentially transmitters of the pathogen to other individuals in the same area that had not been sprayed with enough conidia. Additionally, they might migrate to nearby areas, thus favoring fungal dissemination by area. These phenomena need to be assessed in future studies under field conditions.

There was a horizontal transmission of the fungus B. bassiana Unioeste 76 strain from contaminated insects, both live and dead E. heros adults. The transmission rate, in most treatments, was higher when there was a greater quantity of inoculum source in the population, with mortality being higher with longer exposure times to the fungus.

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Agreement 4500058970

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.