Resumo:O objetivo deste trabalho foi selecionar e caracterizar molecularmente isolados de Bacillus thuringiensistóxicos a Spodoptera eridaniaeS. frugiperda. Trinta e quatro isolados foram submetidos ao bioensaio, dos quais três foram selecionados e usados para a estimativa da CL50. Os isolados selecionados não diferiram da linhagem padrão HD-1. Na caracterização molecular, identificou-se a presença dos genes cry1 e cry2, nos isolados BR37 e BR94, e dos genes cry4A, cry4B, cry10, cry11 e cyt1 no isolado BR58, o que confirmou o perfil proteico obtido de 130, 70 e 65 kDa. Foram identificados cristais bipiramidais e esféricos. O isolado BR58, apesar de não conter os genes relacionados à toxicidade a Lepidoptera, causa mortalidade em ambas as espécies

Abstract:The objective of this work was to select and molecularly characterize Bacillus thuringiensis isolates toxic to Spodoptera eridaniaand S. frugiperda. Thirty four isolates were subjected to the bioassay, of which three were selected and used to estimate the LC50. The selected isolates did not differ from the HD-1 standard line. In the molecular characterization, the presence of the cry1 and cry2 genes was identified in the BR37 and BR94 isolates, and of the cry4A, cry4B, cry10, cry11, and cyt1 genes in the BR58 isolate, which confirmed the obtained protein profile of 130, 70, and 65 kDa. Bipyramidal and spherical crystals were identified. The BR58 isolate, although not containing the genes related to toxicity to Lepidoptera, causes mortality in both species.

El objetivo del trabajo fue evaluar la eficiencia de los insecticidas metoxifenozide, teflubenzuron, piriproxifeno, clorantraniliprole y Azadirachta indica en el control del escarabajo de la cama Alphitobius diaperinus. Se realizaron dos bioensayos en el laboratorio del control microbiano de la Universidade Estadual Londrina (Brasil). En el primero, las larvas de A. diaperinus fueron alimentadas con pienso de maíz tratado con los insecticidas en siete dosis (i.e. dosis recomendada - DR y porcentajes del 50; 25; 12,5; 6,25; 3,12 y 1,65 de la misma). En el segundo bioensayo, lós diferentes tratamientos fueron aplicados en las larvas pero sólo en las tres más altas dosis. La cuantificación de la mortalidad de larvas se hizo diariamente hasta el día 30, cuando se evaluó el número de pupas y adultos. Las mayores mortalidades de larvas se presentaron en larvas alimentadas con raciones con teflubenzuron, clorantraniliprole y A. indica. Los tratamientos con metoxifenozide y piriproxifeno causaron menor mortalidad, sin embargo, con este último, las larvas no alcanzaron a formar pupas ni adultos. Teflubenzuron presentó la mayor toxicidad con menor TL50. Cuando los productos fueron aplicados directamente en las larvas, los tratamientos más eficientes fueron teflubenzuron, clorantraniliprole y A. indica, sin embargo, hubo reducción en los porcentajes de la mortalidad con teflubenzuron y clorantraniliprole. Para A. indica, el método de aplicación no interferió con los niveles de mortalidad. Piriproxifeno, que no había causado mortalidad en el consumo de ración, aumentó hasta el 30% cuando fue aplicado a la larva.

The purpose of this study was to evalúate the efficiency of the insecticides methoxyphenozide, teflubenzuron, pyriproxyfen, chlorantraniliprole and also Azadirachta indica in the control of Alphitobius diaperinus (Coleoptera: Tenebrionidae). The study was carried out in the Microbial Control laboratory of Universidade Estadual Londrina (Brazil) and consisted of two bioassays. In the first, A. diaperinus larvae were fed with corn feed treated with the insecticides in seven dosages. In the second bioassay, the different treatments were sprayed on the larvae only at the three highest dosages. Evaluations were performed daily, quantifying larval mortality, and on the 30th day, the number of pupae and adults were evaluated. Teflubenzuron, chlorantraniliprole and A. indica were responsible for the greatest percentage of mortalities in larvae fed with treated feed. The treatments with methoxyphenozide and pyriproxyfen caused the lowest mortalities; however, with pyriproxyfen the larvae were not transformed into pupae and adults up to the 30th day of assessment. The treatment with teflubenzuron showed the greatest toxicity, with the lowest survival time (ST50) When the products were applied directly on to the larvae, the most efficient treatments were teflubenzuron, chlorantraniliprole and A. indica; however, there was a reduction in the mortality percentages with teflubenzuron and chlorantraniliprole in relation to the feed bioassay. For A. indica, the application method did not affect the mortality levels. Pyriproxyfen, which had not led to mortality through feed ingestion, obtained an increase of up to 30% after being applied directly on to the larvae.

El objetivo de este trabajo fue evaluar el efecto de pasajes sucesivos de Beauveria bassiana por Alphitobius diaperinus en relación al crecimiento vegetativo, a la producción de conidios, a la virulencia, a la sensibilidad frente a la temperatura y a la radiación UV. Fueron utilizados tres aislados inicialmente multiplicados en medio de cultivo y pasados 15 veces por los insectos. Los conidios de cada aislado, provenientes de los insectos muertos, correspondientes al primer, quinto, décimo y décimo quinto pasaje fueron multiplicados en medio de cultivo y empleados en los experimentos. Los pasajes del hongo por el huésped afectaron la calidad de los conidios, lo que varió en los diferentes aislados. De manera general, hubo reducción del crecimiento vegetativo y de la producción de conidios para los aislados Unioeste 4 y Unioeste 40 a medida que el hongo fue pasando por el huésped. En contraste, el crecimiento vegetativo y la producción de conidios del aislado CG 152 no fueron afectados. Para los tres aislados, la producción de conidios en arroz y la virulencia aumentaron después de los pasajes por el huésped, principalmente en el décimo y décimo quinto pasaje. La sensibilidad a la radiación UV fue reducida con el aumento del número de pasajes en los tres aislados, mientras que la sensibilidad a la temperatura aumentó principalmente en los décimo y décimo quinto pasajes. La calidad de los conidios de B. bassiana en relación a la producción de conidios en arroz, virulencia y sensibilidad frente a la temperatura, fue favorecida por los pasajes del hongo por el huésped.

The vegetative growth, conidial production, conidial yield on rice, virulence, and heat and UV radiation tolerances of Beauveria bassiana after successive passages through Alphitobius diaperinus were investigated. Three strains of B. bassiana were cultivated in a culture medium, and passed them up to 15 times through the host insect. The conidia of each strain, derived from dead insects, were cultivated in a culture medium. We used conidia corresponding to the first, fifth, tenth, and fifteenth passages in our experiments. Our results showed that successive passages of the fungus through the host insect affected the quality of the conidia; moreover, the effect on conidial quality varied among the strains. For strains Unioeste 4 and Unioeste 40, successive passages through the host insect resulted in reduced vegetative growth and conidial production. In contrast, vegetative growth and conidial production of strain CG 152 were unaffected by successive passages through the host insect. For all 3 strains, successive passages through the host insect resulted in a higher conidial yield on rice and increased virulence, especially after the tenth and fifteenth passages. In addition, an increase in the number of passages through the host insect led to a decrease in the UV radiation tolerance, but an increase in the heat tolerance, especially after the tenth and fifteenth passages. Our results indicate that the conidial yield on rice, virulence, and heat tolerance of B. bassiana are favored by successive passages through A. diaperinus.

El efecto de subcultivos sucesivos in vitro de Beauveria bassiana a diferentes condiciones nutricionales fue evaluado mediante el crecimiento vegetativo, la producción conidial, la virulencia, tolerancia a la radiación UV y al calor. Se usaron los medios agar dextrosa papa (PDA) y un medio basado en adultos de Alphitobius diaperinus (MAD) para la producción conidial. El hongo se inoculó en los insectos, se aisló, y subcultivó 17 veces en medios diferentes. Después de subcultivados, los hongos se inocularon en los insectos, y se aislaron nuevamente a partir de ellos. Los subcultivos sucesivos y las condiciones nutricionales influenciaron la calidad de los hongos. El medio MAD mantuvo el crecimiento vegetativo y la producción conidial en cultivo y en arroz, después de un gran número de subcultivos en PDA. Así mismo, la reducción en la producción de conidias en MAD fue poco percibida comparado con la producción en PDA. El cultivo de conidias en MAD mantuvo la virulencia luego de 17 subcultivos sucesivos. Las conidias cultivados en PDA preservaron los niveles iniciales de termotolerancia. La viabilidade de los conídios disminuyó después de recibir radiación UV, pero esta disminución no afectó los subculivos sucesivos en PDA y MAD. La virulencia, la producción conidial y la tolerancia a alta temperatura, se redujeron después de los sucesivos subcultivos in vitro, pero restauradas con el paso de los hongos a través del hospedero.

The effect of successive subculturing of Beauveria bassiana under different in vitro nutritional conditions was evaluated on vegetative growth, conidial production, virulence, tolerance to UV radiation and heat tolerance. Potato dextrose agar (PDA) and a medium based on adult Alphitobius diaperinus (MAD) were used. The fungus was inoculated on the insects, isolated, and subcultured 17 times on the different media. After subculturing, the fungus was again inoculated on the insects, and it was then re-isolated. Successive subculturing and nutritional conditions influenced fungus quality. MAD favored maintenance of vegetative growth and conidial production on culture medium and on rice after a greater number of successive subcultures than PDA. Furthermore, a decrease in conidial production in MAD was less pronounced than in conidia from the PDA medium. Conidia cultured on MAD maintained virulence after 17 successive subcultures. Conidia cultured on PDA retained initial thermotolerance levels. Conidial viability decreased after UV irradiation, but this decrease was unaffected by successive subculturing on PDA and MAD. Virulence, conidial production, and temperature tolerance, which were reduced after successive in vitro subculture, were restored with fungus passage through the host.

O objetivo deste trabalho foi avaliar a eficiência de Beauveria bassiana (Bb), da terra diatomácea (TD) e dos pós de rocha Protesyl e Rocksil, isoladamente ou em associações, para o controle de Alphitobius diaperinus. Os agentes de controle foram aplicados na forma sólida misturados à cama de frango. A concentração de conídios de Bb foi de 0,08% e, para os pós inertes, as concentrações foram de 0,5; 1,0 e 1,5%. Testaram-se 20 tratamentos, com cinco repetições de 200 insetos, por 10 dias. Os tratamentos que apresentaram maior eficiência foram TD 1,5% e Bb 0,08% + TD 1,5%, com 60,2 e 57,8% de mortalidade, respectivamente. Protesyl e Rocksil, nas três concentrações, e TD 0,5% não diferiram da testemunha. Na interação entre os agentes de controle, ocorreu efeito aditivo para Bb + TD e Bb + Rocksil, nas três concentrações, e para Bb + Protesyl a 1,5%. A eficiência do fungo foi reduzida na associação com Protesyl, com efeito não aditivo antagônico nas concentrações de 0,5 e 1,0%. A terra diatomácea, isolada ou associada à B. bassiana, apresenta-se como boa alternativa para controle de A. diaperinus. Já os pós de rocha Protesyl e Rocksil não foram eficientes para o controle da praga.

The objective of this study was to evaluate the efficacy of Beauveria bassiana (Bb) of diatomaceous earth (TD) and the dust of rock Protesyl and Rocksil, alone or in combinations, for Alphitobius diaperinus to control. The control agents were applied in dried by mixing with the poultry liter. The concentration of Bb conidia was 0.08% on the weight of the substrate, and for the dust inert were 0.5, 1.0 and 1.5%. Twenty treatments with five repetitions of 200 insects were tested for 10 days. The treatments with highest efficiency were TD 1.5% and Bb 0.08% + TD 1.5%, with 60.2 and 57.8%, respectively. Protesyl and Rocksil in the three concentrations and TD 0.5% did not differ from control. For the interaction between control agents no advantage was observed for the association. Additive effect occurred for Bb + TD and Bb + Rocksil at the three concentrations and for Bb + Protesyl at 1.5%. The action of the fungus was reduced in association with Protesyl with antagonistic non-additive effect in 0.5 and 1.0%. Diatomaceous earth, isolated our in association with B. bassiana its a good alternative for A. diaperinus control. Just rock dust Protesyl and Rochsil were not efficient for insect control.

O objetivo deste trabalho foi avaliar a eficiência de Beauveria bassiana (Bb), terra diatomácea (TD) e microrganismos eficazes (EM-4), associados ou não, no controle de Alphitobius diaperinus, e o efeito de EM-4 no desenvolvimento de B. bassiana. Os agentes de controle (Bb, TD e EM-4), em diferentes concentrações e combinações, foram aplicados em uma mistura de cama-de-frango e ração, em que os insetos foram acondicionados por dez dias. Avaliaram-se, in vitro: a germinação, as unidades formadoras de colônia (UFC), o crescimento vegetativo e a produtividade de conídios de Bb em contato com suspensão aquosa de EM-4 (1%) não filtrada e filtrada. Os maiores índices de mortalidade foram observados nos tratamentos TD + Bb + EM-4 e TD + Bb, nas maiores concentrações. Verificou-se efeito não-aditivo sinérgico para TD + Bb nas três concentrações. Nos testes com EM-4 filtrado, não houve diferença em relação à testemunha quanto à germinação e às UFC, entretanto, o crescimento vegetativo e a produtividade de conídios foram negativamente afetados. O uso conjunto de B. bassiana e terra diatomácea, para o manejo de populações de A. diaperinus, pode reduzir o uso de produtos químicos.

The objective of this work was to evaluate Beauveria bassiana (Bb), diatomaceous earth (TD) and effective microorganisms (EM-4), associated or not, to Alphitobius diaperinus control, and the EM-4 effect on B. bassiana development. The control agents (Bb, TD and EM-4), in different concentrations and combinations, were applied in a mixture of litter and chicken food, where insects had been left per ten days. Conidia germination, colony-forming units (CFU), vegetative growth and the productivity of Bb conidia were evaluated when in contact with a filtered and non filtered watery EM-4 suspension (1%). The highest indices of mortality were observed in the treatments TD + Bb + EM and TD + Bb in the highest concentrations. A synergic non additive effect was observed for TD + Bb in the three concentrations. In filtered EM-4 tests, no difference was observed related to the control of germination and CFU, however, vegetative growth and conidia productivity were negatively affected. The joint use of B. bassiana and diatomaceous earth for A. diaperinus populations management can minimize the use of chemical products.