Biological control potential of two Beauveria bassiana isolates against the stink bugs Nezara viridula L. and Piezodorus guildinii Westwood (Hemiptera: Pentatomidae) in common bean

Background The stink bugs, Nezara viridula L. and Piezodorus guildinii Westwood (Hemiptera: Pentatomidae) are the most important and widespread species of polyphagous stink bugs in the tropical and subtropical regions of Latin America, which affect the quality and yield of the common bean ( Phaseolus vulgaris L.). The use of synthetic chemical insecticides is the major control strategy to manage stink bugs in common beans and alternatives are needed. In this study, mortality and median Lethal Time (LT 50 ) of two Cuban isolates of the entomopathogenic fungus Beauveria bassiana (18 S-O-R and 96 P-O-E), as well as one commercial Cuban isolate (Bb-18), at a concentration of 1 × 10 8 conidia/ml were evaluated. These evaluations were conducted against both stink bug species using Petri dish bioassays and a semi-field experiment in common beans. Results In Petri dish bioassays, the isolates 18 S-O-R and 96 P-O-E caused 100% mortality of both N. viridula and P. guildinii . This was significantly higher than for isolate Bb-18, which caused 86.3% N. viridula and 81.3% P. guildinii mortality. In the semi-field experiment, when pooling both stink bug species, total mortality after 14 days was 91.3% for 18 S-O-R, 80.0% for 96 P-O-E and 73.8% for Bb-18 isolates. LT 50 value for isolate 18 S-O-R tested under laboratory conditions was 6.04 ± 0.18 days for N. viridula and 5.32 ± 0.14 days for P. guildinii at the same concentration of 1 × 10 8 conidia/ml . LT 50 value for isolate 18 S-O-R in semi field was 6.79 ± 0.37 days for N. viridula and 7.71 ± 0.32 days for P. guildinii at 1 × 10 8 conidia/ml. Conclusion The study highlights the potential of B. bassiana 18 S-O-R as a promising candidate for control of stink bugs in common bean under tropical conditions as an alternative to conventional chemical insecticides in integrated pest management (IPM) programs. Moving forward, further research should focus on validating the efficacy under diverse field conditions and integrating application methods into practical IPM approaches. Future use of B. bassiana will enhance sustainability and reduce environmental impacts associated with pesticide use.


Background
Common bean (Phaseolus vulgaris L.) is the most important edible legume for direct consumption in the world (Suárez-Martínez et al. 2015) and it is part of the daily diet of many Cubans (Morales-Soto et al. 2022) and elsewhere in Latin America (Lopes et al. 2015a).Attack by stink bugs (Hemiptera: Pentatomidae) during the pod stage affects the quality and quantity of the common bean crop (Ramos et al. 2017a).The most important and widespread species of polyphagous stink bugs in the tropical and subtropical regions of Latin America are the red banded stink bug, Piezodorus guildinii Westwood and the southern green stink bug Nezara viridula L. (Souza et al. 2015).The two pests introduce their piercingsucking mouthparts through the pods and inject salivary secretions to feed on the seed nutrient contents (Depieri and Panizzi 2011).This feeding activity can cause seed abortion or deformation and the feeding injury facilitates plant pathogen infection (Zerbino and Panizzi 2019).
Use of synthetic chemical insecticides is the major control strategy to manage stink bugs in soybeans (Marques et al. 2019) and insecticide resistance in stink bug populations is now a big challenge (Ademokoya et al. 2022).When pesticide efficacy is reduced, application rates often increase.World-wide there is, however, an increasing focus on sustainable use of chemical pesticides to reduce the risks and impacts on human health and the environment.This has led to the promotion of Integrated Pest Management (IPM) and of alternative approaches or techniques that may be used as alternatives for chemical pesticides.Many countries have established guidelines for this e.g. the EU Directive 2009/128/EC.Improved IPM strategies are continuously being developed and several have proved to be successful also against sink bugs e.g.use of synthetic insecticides when needed (Brown et al. 2012), use of botanical insecticides (Werdin et al. 2013), use of beneficial insects such as predators and parasitoids (Cingolani et al. 2013) as well as entomopathogenic fungi (EPF) (Oliveira et al. 2016).
Beauveria spp.(Ascomycota: Hypocreales) are among the most widely used entomopathogenic fungal taxa for microbial control (Mascarin and Jaronski 2016).The effect of B. bassiana sensu lato isolates against different species of stink bugs under controlled conditions have been the subject of various studies (Silva-Santana et al. 2022).However, the potential of B. bassiana isolates as a microbial control agent against N. viridula and P. guildinii in common beans in the tropics and the effect against stink bugs in semi-field conditions has not been evaluated.B. bassiana was widespread in common bean fields in Cuba and isolates could be obtained from soil, as endophytes of common bean plants and as natural infections in stink bugs (Ramos et al. 2017b).However, the potential of these isolates for inundation or inoculation biological control of stink bugs has not been evaluated.
The aim of this study was to evaluate the biocontrol potential of three Cuban B. bassiana isolates, Bb-18 (formulated and sold commercially in Cuba), and two naturally occurring isolates (18 S-O-R and 96 P-O-E) against N. viridula and P. guildinii compared to the control potential of the synthetic insecticide Methamidophos under laboratory and semi-field conditions.

Cultivation of common bean plants
Seeds of the common bean cultivar 'Chévere' were sown separately inside polyethylene bags containing 500 g of an Inceptisol soil (Soil taxonomy; USDA) collected at the Experimental Station "Álvaro Barba Machado" belonging to Universidad Central "Marta Abreu" de Las Villas, Villa Clara, Cuba.The soil was sterilized by heating it to 121 °C three times before being used in the experiment.Bags with soil and seed were then placed in a climatic chamber at 20 ± 1 °C, 50% relative humidity (RH), 14-h of light and 10-h of darkness (L14:D10) and fluorescent light of 40 W/m 2 (Dobben et al. 1981) and watered daily.After six days, when seedlings had unfolded leaves, they were placed in a greenhouse with day temperatures of 30 °C and night temperatures of 20 °C.Plants were irrigated twice per day, at 9.00 am and 5.00 pm.

Rearing of N. viridula and P. guildinii
Nezara viridula and P. guildinii were collected from a common bean field (cv.'Chévere' in 2016) located in the Sandino orchard (22°57′56"N, 79°28′34''W), Remedios, Cuba.Sixty adults N. viridula and sixty adults P. guildinii (sex ratio 1:1) were distributed in three glass containers (30 × 18 cm, covered with lids perforated with 20 holes for aeration), separately for each species (20 stink bugs per container), and reared in a climatic chamber (Memmert, Germany) at 26 ± 2 °C, 65 ± 10% RH, 14L:10D as described by Silva et al. (2013).The insects were fed on a diet of fresh pods of common bean and soybean (Glycine max L.) obtained from an indoor plant culture.Food was replaced every two days.As described by Bentivenha et al. (2018), cotton pieces (3 × 3 cm) moistened with distilled water were placed in the containers to obtain appropriate humidity.Cotton pieces were replaced every two days.To facilitate oviposition, paper towels were placed inside each container.Egg masses were collected daily and transferred to plastic cages with moistened filter paper for hatching, and emerging nymphs were transferred to glass containers (30 × 18 cm) with pods of bean and soybean and covered with a mesh (1.35 µm).

Fungal isolates and synthetic insecticide
Two B. bassiana isolates, 18 S-O-R (isolated from soil in a common bean field in Remedios, Cuba at 22° 57′ 56″ N, 79° 28′ 34″ W) and 96 P-O-E (isolated from common bean plant tissue in a common bean field in Encrucijada, Cuba at 22° 37′ 01″ N, 79° 51′ 58″ W) (Ramos et al. 2017b), were used in the experiments.The isolates were stored at 4 °C in 40 ml test tubes with Sabouraud Dextrose Agar (SDA) (BioCen, Cuba) at the Universidad Central "Marta Abreu" de Las Villas collection of EPF, Santa Clara, Cuba.In addition, the B. bassiana isolate Bb-18 was supplied by the Plant Health Laboratory in Santa Clara belonging to the Cuban Ministry of Agriculture.This commercial strain (commercialized in Cuba) was originally isolated from Hypothenemus hampei Ferrari (Coleoptera: Curculionidae) in Manicaragua, Cuba and it is the most used mycoinsecticide in Cuba (Baldiviezo et al. 2020).All three isolates used were confirmed by molecular methods in earlier studies to be B. bassiana sensu stricto (Ramos et al. 2017b).Mycelia from each of the three B. bassiana isolates were obtained by inoculation of 100 ml Sabouraud Dextrose Broth (SDB) (BioCen, Cuba) with 5 mm plugs of 14-days-old fungal cultures grown on SDA at 25 °C.The fungal cultures were grown on an orbital shaker (120 rpm) for five days at 25 °C and in the dark as described in Ramos et al. (2017b).Sixty milliliters of mycelium were then harvested from the SDB fungal culture and used to inoculate 300 g autoclaved (30 min at 120 °C) rice (Oryza sativa L.) in polypropylene bags (35 × 22 cm).Afterwards, bags were sealed and incubated at 25 °C, 75% RH in the dark for 14 days.Rice with fungi was then air-dried in a laminar flow hood to allow harvesting of dry conidia.Dry conidia were manually harvested by adding 100 g of rice to a 0.25 m 2 sieve with a 5 mm mesh and shake for 10 min.Finally, conidia from each B. bassiana isolate were suspended in sterile distilled water with 0.01% Tween 80 and adjusted to a concentration of 1 × 10 8 conidia/ml by using a Neubauer hemocytometer chamber (Brand, Germany).Before using this concentration in the experiment, it was checked for conidial viability.This was done by counting viable and non-viable conidia under a microscope (400 ×) 22 h after fungal inoculation on an SDA plate, following the procedure described by Oliveira et al. (2015).Conidia were considered viable when germ tubes were longer than the conidial diameter; all suspensions used showed germination rates ≥ 95%.
The chemical insecticide Tamaron 60 Suspension Concentrate (SC) with the active ingredient Methamidophos (1.5 g/l a.i.) was used at the concentration recommended on the label by the supplier (Bayer Crop Science, Germany).Tamaron 60 SC is an organophosphate used against piercing-sucking insects and it is reported to be effective against stink bugs (Sosa-Gómez et al. 2001).Sterile distilled water was used as the control treatment in all experiments.

Experimental set up: bioassays
Twenty adults (34-days-old) N. viridula 10 females/10 males and 20 adults (31-days-old) P. guildinii 10 females/10 males were individually dipped for 15 s in 10 ml of each of the five treatments shown in Table 1, using the method described by Raafat et al. (2015).This procedure was repeated at separate occasions, using new conidial suspensions.Each stink bug was then placed separately in a sterile Petri dish (12.5 cm) with a moistened filter paper and a fully-grown common bean pod of the cultivar 'Chévere' collected from plants in growth stage BBCH 75 (Meier 2001) from the indoor plant culture described above.Common bean pods were replaced every two days.Each pod was superficially sterilized before use by dipping it for 3 min in 1.5% sodium hypochlorite, 2 min in 70% ethanol and then rinsed three times in sterile distilled water (Parsa et al. 2013).Petri dishes with stink bugs and common bean pods were then placed at 25 ± 1 °C, 85% RH, 14L:10D and observed daily for 11 days to evaluate stink bug mortality.Dead stink bugs from the B. bassiana treatments were surface sterilized as described before.Disinfected dead stink bugs were placed individually in a sterile plastic cup (40 ml) containing 1.5% water agar (BioCen, Cuba) and a moistened filter paper piece (2 × 2 cm) and sealed with a lid.These were incubated at 25 ± 1 °C, 85% RH in the dark for five days to observe for fungal growth.Presence of Beauveria was confirmed by morphological identification of fungal structure under microscope, according to Humber (2012).The experiments were repeated four times over time.

Experimental set up: semi-field experiment
Bean plants from the commercial common bean cv.'Chévere' from the indoor plant culture described above were used.When half of the plants had reached growth stage BBCH 75 and its foliage had 15 cm in diameter, the five different treatments (Table 1) were applied to each of five bean plants at a total rate of 10 ml per plant according to the recommended concentration and as described by Afandhi et al. (2019).Each plant was sprayed independently by the use of a backpack sprayer (Hudson, USA; 10 l capacity).The backpack sprayer was disinfected with tap water, ethanol 70%, acetone 99% and washed three times with distilled water between treatments to avoid contamination among treatments (Castro et al. 2015).
After spraying, plants were allowed to air dry before they were placed into a cage (30 × 30 × 30 cm) covered with a mesh (3350 BT Bionete 50 mesh; Technical Protection Textiles, Arrigoni 1936 ® , Italy).Four replicate cages per treatment with five plants in each were prepared for each stink bug species, placed outside and hence exposed to ambient weather conditions.Twenty adults (10 females/10 males) of either N. viridula (34-days-old) or P. guildinii (31-days-old) from the insect rearing were released into each of the four separate cages for each of the five treatments.Bean plants were irrigated twice a week.Stink bugs were checked on the plants every day for 14 days for mortality.Dead stink bugs from the B. bassiana treatments were surface sterilized, placed into a moist chamber as described above and observed for fungal growth for five days.Presence of Beauveria was confirmed by morphological identification of fungal structure under microscope, according to Humber (2012).The experiments were repeated at four separate occasions using different generations of reared stink bugs and new suspensions of B. bassiana.

Data analysis
Survival data for stink bugs in Petri dish bioassays and semi-field experiments were analyzed separately for each species with the PROC LIFETEST procedure in SAS 9.3, SAS Institute 2009 (Littell et al. 1996) using the log-rank test to evaluate equality among treatments with post-hoc comparisons by the Sidak adjustment.Proportions of dead stink bugs with fungal outgrowth identified to Beauveria as described above were analyzed by logistic regression using the GLIMMIX procedure in SAS including fungal isolate as fixed effect and experimental repetition as random effect.If significant effects were found (P < 0.05), means were separated by LS-means using the Tukey adjustment.

Bioassays
In the Petri dish bioassays, Tamaron 60 SC (a.i.Methamidophos) caused 100% mortality for both stink bug species the first day after exposure and data of this treatment were therefore not included in survivorship analyses.Survival curves of N. viridula adults over time after exposure to the three B. bassiana isolates and the control were significantly different (Log-rank test: χ 2 = 258.65,df = 3, P < 0.0001) (Fig. 1a).N. viridula (n = 80) had LT 50 values of 6.04 ± 0.18 (SE) days when exposed to B. bassiana isolate 18 S-O-R, 6.75 ± 0.22 (SE) days when exposed to 96 P-O-E and 8.33 ± 0.23 (SE) days when exposed to the commercial isolate Bb-18 at the same concentration of 1 × 10 8 conidia/ml.All (100%) N. viridula were dead within 8 days (isolate 18 S-O-R) and 10 days (isolate 96 P-O-E) after exposure.This was significantly higher than for the commercial isolate Bb-18 that gave 86.3% N. viridula mortality at day 11 (Fig. 1a).No mortality was observed among control treated stink bugs after 11 days (Fig. 1a).All B. bassiana isolates led to fungal growth and sporulation of cadavers, and the highest proportion of cadavers developing mycosis was observed for isolate 18 S-O-R (Table 2).Survivorship curves of P. guildinii stink bugs were also significantly different among treatments when excluding Tamaron 60 SC (a.i.Methamidophos) (Log-rank test: χ 2 = 252.05,df = 3, P < 0.0001) (Fig. 1b).P. guildinii had LT 50 values of 5.32 ± 0.14 (SE) days when exposed to 18 S-O-R, 5.63 ± 0.12 (SE) days when exposed to 96 P-O-E and 7.11 ± 0.21 (SE) days when exposed to the commercial isolate Bb-18 at the same concentration of 1 × 10 8 conidia/ml.All (100%) P. guildinii were dead after 7 days when exposed to 18 S-O-R and 96 P-O-E while the commercial isolate Bb-18 resulted in a maximum mortality of 81.3% at day 9.No control mortality was detected among P. guildinii (Fig. 1b).The proportion of cadavers developing mycosis was not significantly different between B. bassiana isolates (Table 2).

Semi-field experiment
In the semi-field experiment, Tamaron 60 SC (a.i.Methamidophos) caused 100% mortality one day after application for both stink bug species and the survival data of this treatment were therefore not included in further analyses.Pooling both stink bug species, total mortality after 14 days, when observations ended, was 91.3% for 18 S-O-R, 80.0% for 96 P-O-E and 73.8% for For the 18 S-O-R isolate sprayed on plants, 91% of N. viridula adults were dead after 12 days when observations ended and had LT 50 value of 6.79 ± 0.37 (SE) days at concentration of 1 × 10 8 conidia/ml.For the 96 P-O-E isolate sprayed on plants, 80% of N. viridula adults were dead after 13 days when observations ended and had LT 50 value of 8.28 ± 0.37 (SE) days, and for the commercial isolate Bb-18 sprayed on plants, 68% of N. viridula adults were dead after 14 days when observations ended and had LT 50 of 10.05 ± 0.39 (SE) days at 1 × 10 8 conidia/ml.No mortality was observed in the control treatment.Proportions of dead N. viridula with mycosis were not significantly different between the three fungal isolates (Table 3).

Days post inoculation
Piezodorus guildinii survival curves were significantly different among the three B. bassiana isolates and control treatments (Log-rank test: χ 2 = 207.28,df = 3, P < 0.0001) with no mortality observed in the control (Fig. 2b).The B. bassiana isolates 18 S-O-R and 96 P-O-E induced mortalities in adults of P. guildinii from day 4 after inoculation while Bb-18 caused mortality from day 5 after inoculation.All (100%) P. guildinii on plants sprayed with 18 S-O-R were dead 12 days after inoculation and had LT 50 of 7.71 ± 0.32 (SE days) at 1 × 10 8 conidia/ml.For the 96 P-O-E isolate, 92.5% of P. guildinii adults were dead after 14 days when observations ended and had LT 50 of 9.28 ± 0.31 (SE) days at concentration of 1 × 10 8 conidia/ml.For the commercial isolate Bb-18 isolate, 53% of P. guildinii adults were dead after 14 days when observations ended and had LT 50 of 10.03 ± 0.27 (SE) days at 1 × 10 8 conidia/ml (Fig. 2b).Proportions of dead P. guildinii with mycosis were non-significantly different among the three fungal isolates (Table 3).

Discussion
The study results revealed that the stink bug species N. viridula and P. guildinii were susceptible to all three B. bassiana isolates tested in Petri-dish bioassays and semifield experiments.These findings are consistent with previous reports that B. bassiana isolates can infect stink bugs.Lopes et al. (2015b) demonstrated in a laboratory study that the stink bug species Diceraeus melacanthus Dallas (Heteroptera: Pentatomidae), Euschistus heros Fabricius (Heteroptera: Pentatomidae) and Chinavia ubica Rolston (Heteroptera: Pentatomidae) were susceptible to the Brazilian B. bassiana isolate CG1105.Further, an Egyptian B. bassiana strain isolated from N. viridula and the strain B. bassiana ARSEF2011 were reported to have damaging effects on the epicuticle and procuticle of N. viridula 72 h after infection (Raafat et al. 2015).Also, Nora et al. (2021) reported that B. bassiana isolates (UFSM-1 and UFSM-2 from the stink bug E. heros in soybean fields in Brazil) caused a high mortality of stink bugs over 15 days at a concentration of 1 × 10 8 conidia/ ml, under laboratory conditions.However, in the present study, it was further confirmed in a series of experiments, from Petri-dish bioassays and semi-field experiments, that B. bassiana isolates resulted in relatively high stink bug mortality, indicating potential for use in biological control of these pests in common bean.
In the laboratory study (at 25 ± 1 °C, 85% RH, 14L:10D), the B. bassiana isolate 18 S-O-R at a concentration of 1 × 10 8 conidia/ml caused N. viridula and P. guildinii mortality of 100% after 8 and 7 days, respectively.Silva-Santana et al. (2022) demonstrated in a laboratory study (at 26 ± 2 °C, 60% RH and 14L:10 D) that the isolate of B. bassiana Unioeste 76 at a concentration of 1 × 10 8 conidia/ml caused 96% mortality of E. heros 14 days after treatment.Parys and Portilla (2020) showed through a laboratory assay at 27 °C that the strains of B. bassiana BotaniGard ® and NI8 at 5 × 10 7 conidia/ml caused 87 and 88% mortality of P. guildinii, respectively.This suggested that the Cuban B. bassiana isolate 18 S-O-R used against stink bug species (N.viridula and P. guildinii) in common Further, our semi field experiment showed that the most promising B. bassiana isolate 18 S-O-R (at 1 × 10 8 conidia/ml) resulted in 91.3% mortality (both stink bug species pooled) and was comparable to the chemical viridula at concentration of 1 × 10 8 conidia/ml) in present study.
Our results are consistent with those obtained by Silva-Santana et al. (2022) in a semi-field experiment on soybeans, where the isolate B. bassiana Unioeste 76, at concentrations of 1 × 10 8 conidia/ml and 1 × 10 9 conidia/ ml, resulted in mortality rates of 80 and 90% on E. heros, respectively.Patel et al. (2006) demonstrated that the soil-derived B. bassiana LRC28 isolate applied at a concentration of 4 × 10 7 conidia/ml exhibited higher virulence towards adult rice stink bugs (Oebalus pugnax Fabricius, Heteroptera: Pentatomidae) compared to B. bassiana isolated from O. pugnax applied at a concentration of 4 × 10 7 conidia/ml.The authors reported that a single application of B. bassiana LRC28 reduced 44% of the rice stink bug populations on six out of the nine sampling dates.Sosa-Gómez and Moscardi (1998) also demonstrated the susceptibility of N. viridula and P. guildinii to B. bassiana (CNPSo-Bb56) applied at a concentration of 1.5 × 10 13 conidia/ml in field cage experiments with soybean, resulted in mortalities of 48% and 41%, respectively.

Conclusion
Obtained results indicated that the B. bassiana 18 S-O-R isolate had a potential as a biological control agent against stink bugs in common bean and suggested that it could be used as an alternative tool that may substitute traditional chemical insecticides in IPM programs.However, B. bassiana 18 S-O-R killed slower (higher LT 50 values) than the chemical insecticide Tamaron 60 SC (a.i.Methamidophos) and therefore will not provide an immediate effect.This aspect needs to be emphasized when guiding farmers in effective application of this fungus as a microbial control agent.Further field studies are still needed to confirm the use of B. bassiana against the sting bugs in IPM programs.

Fig. 1
Fig. 1 Survivorship curves over 11 days for Nezara viridula (a) and Piezodorus guildinii (b) exposed to three different Beauveria bassiana isolates (Bb-18, 96 P-O-E, 18 S-O-R) at a concentration of 10 8 conidia/ml or a control in Petri dish bioassays (n = 80 stink bugs per treatment).Curves with different letters are significant different

Fig. 2
Fig. 2 Survivorship curves over 14 days for Nezara viridula (a) and Piezodorus guildinii (b) exposed to three different Beauveria bassiana isolates (Bb-18, 96 P-O-E, 18 S-O-R) at a concentration of 10 8 conidia/ml or a control in semi-field experiments with whole common bean plants (n = 20 per treatment and per stink bug specie).Curves with different letters are significant different

Table 1
List of treatments, origin of Beauveria bassiana isolates and concentration rates used in Petri dish bioassays and semi-field experiments with Nezara viridula and Piezodorus guildinii SC Soluble concentrate, a.i.Active ingredient *The synthetic insecticide Tamaron 60 SC was used in both Petri dish bioassays and semi field experiments

Table 2
Mean (± SE) proportion of cadavers developing mycosis in the two species of stink bugs exposed to three different Beauveria bassiana isolates in Petri dish bioassays Different letters within a row indicate significant different proportions.It was applied if tests showed significant differences between treatments on sporulation proportion beans was comparable to BotaniGard ® and maybe better than the B. bassiana isolate NI8 tested against and P. guildinii, in laboratory studies.

Table 3
Mean (± SE) proportion of cadavers developing mycosis in the two species of stink bugs exposed to three different Beauveria bassiana isolates in semi-field experiments