Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification / Toxicidade e atividade larvicida sobre Aedes aegypti de óleo essencial de citronella submetido à esterificação enzimática

The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 μg mL-¹ were obtained for the essential oil and 4.36 μg mL-¹ for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 μg mL-¹ for the essential oil of citronella and 86.30 μg mL-¹ for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 μg mL-¹ para o óleo essencial e 4,36 μg mL-¹ para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 μg mL-¹ para o óleo essencial de citronela e 86,30 μg mL-¹ para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification

Abstract The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 g mL-1 were obtained for the essential oil and 4.36 g mL-1 for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 g mL-1 for the essential oil of citronella and 86.30 g mL-1 for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

Resumo O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 g mL-1 para o óleo essencial e 4,36 g mL-1 para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 g mL-1 para o óleo essencial de citronela e 86,30 g mL-1 para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Toxicity and larvicidal activity on Aedes aegypti of citronella essential oil submitted to enzymatic esterification / Toxicidade e atividade larvicida sobre Aedes aegypti de óleo essencial de citronella submetido à esterificação enzimática

Abstract The essential oil of citronella (Cymbopogon winterianus) has several biological activities, among them the insect repellent action. Some studies showed that cinnamic acid esters can be applied as natural pesticides, insecticides and fungicides. In this context, the objective of the present work was to evaluate the production of esters from citronella essential oil with cinnamic acid via enzymatic esterification. Besides, the essential oil toxicity before and after esterification against Artemia salina and larvicidal action on Aedes aegypti was investigated. Esters were produced using cinnamic acid as the acylating agent and citronella essential oil (3:1) in heptane and 15 wt% NS 88011 enzyme as biocatalysts, at 70 °C and 150 rpm. Conversion rates of citronellyl and geranyl cinnamates were 58.7 and 69.0% for NS 88011, respectively. For the toxicity to Artemia salina LC50 results of 5.29 μg mL-1 were obtained for the essential oil and 4.36 μg mL-1 for the esterified oils obtained with NS 88011. In the insecticidal activity against Aedes aegypti larvae, was obtained LC50 of 111.84 μg mL-1 for the essential oil of citronella and 86.30 μg mL-1 for the esterified oils obtained with the enzyme NS 88011, indicating high toxicity of the esters. The results demonstrated that the evaluated samples present potential of application as bioinsecticide.

Resumo O óleo essencial de citronela (Cymbopogon winterianus) possui diversas atividades biológicas, entre elas a ação repelente a insetos. Alguns estudos mostraram que os ésteres do ácido cinâmico podem ser aplicados como pesticidas naturais, inseticidas e fungicidas. Nesse contexto, o objetivo do presente trabalho foi avaliar a produção de ésteres a partir do óleo essencial de citronela com ácido cinâmico via esterificação enzimática. Além disso, foi investigada a toxicidade do óleo essencial antes e após a esterificação contra Artemia salina e a ação larvicida sobre Aedes aegypti. Os ésteres foram produzidos utilizando ácido cinâmico como agente acilante e óleo essencial de citronela (3: 1) em heptano e 15% em peso da enzima NS 88011 como biocatalisadores, a 70 ° C e 150 rpm. As taxas de conversão de cinamatos de citronelil e geranil foram 58,7 e 69,0% para NS 88011, respectivamente. Para a toxicidade sobre Artemia salina foram obtidos CL50 de 5,29 μg mL-1 para o óleo essencial e 4,36 μg mL-1 para os óleos esterificados com NS 88011. Na atividade inseticida contra larvas de Aedes aegypti, obteve-se CL50 de 111,84 μg mL-1 para o óleo essencial de citronela e 86,30 μg mL-1 para os óleos esterificados com a enzima NS 88011, indicando alta toxicidade dos ésteres. Os resultados demonstraram que as amostras avaliadas apresentam potencial de aplicação como bioinseticida.

Spores of Beauveria bassiana and Trichoderma lignorum as a bioinsecticide for the control of Atta cephalotes

Background: The leafcutter ant (Atta cephalotes) is associated with losses in the agricultural sector, due to its defoliating activity; for its control, biological, mechanical and chemical methods have been developed, the latter associated with adverse effects on human and environmental health. This research validated in the field for the control of the leafcutter ant (A. cephalotes) using a mixture of Beauveria bassiana and Trichoderma lignorum spores. Methods: The effectiveness from the combination of spores of B. bassiana and T lignorum with an initial concentration of 2 × 109 spores/ml, in the following proportions of B. bassiana and T. lignorum, A (1:1), of each fungus. It was evaluated within the university campus, comparing it with two commercial formulations, Mycotrol (B. bassiana) and Mycobac (T. lignorum). Additionally, this formulation was evaluated in 49 nests distributed 16 in 14 locations in Colombia. The formulation application was carried out by direct application, using a pump at a speed of 10 ml/m2. The effectiveness was estimated from the reduction of the flow of ants, evaluating the statistically significant differences using the ANOVA and Tukey-test. Results: Effective control of 90% of the nests was observed in the field phase in 60 days, except in nests with areas > 50 m2 that were located in regions with high rainfall (annual average precipitation above 7000 mm), such as Buenaventura. Conclusions: In this work, it was demonstrated that the combination of B. bassiana and T. lignorum spores represent a viable alternative for the control of the leafcutter ant, in which the effectiveness is related to several factors, including the size of the nest and the rainfall in the area.

Astrodaucus persicus as a new source of bioinsectisides against malaria vector, Anopheles stephensi

OBJECTIVE@#To determine the larvicidal activities of petroleum ether, chloroform, ethyl acetate and methanol fractions of roots and fruits extracts of Astrodaucus persicus from Apiaceae family against malaria vector, Anopheles stephensi (An. stephensi).@*METHODS@#Twenty five third instar larvae of An. stephensi were exposed to various concentrations (10-160 g/L) of fractions and were assayed according to World Health Organization protocol. The larval mortality was calculated after 24 h treatment.@*RESULTS@#Among tested fractions, the highest larvicidal efficacy was observed from ethyl acetate fraction of fruits extract with 50% and 90% mortality values (LC and LC) of 34.49 g/L and 108.61 g/L, respectively. Chloroform fraction of fruits extract was the second larvicidal sample with LC of 45.11 g/L and LC of 139.36 g/L. Petroleum ether fractions of fruits and roots and methanol fraction of fruits showed moderate toxicity against An. stephensi.@*CONCLUSIONS@#Astrodaucus persicus is a potential source of valuable and natural larvicidal compounds against malaria vector, An. stephensi and can be used in mosquitoes control programs as an alternative to synthetic insecticides.

Astrodaucus persicus as a new source of bioinsectisides against malaria vector, Anopheles stephensi

Objective To determine the larvicidal activities of petroleum ether, chloroform, ethyl acetate and methanol fractions of roots and fruits extracts of Astrodaucus persicus from Apiaceae family against malaria vector, Anopheles stephensi (An. stephensi). Methods Twenty five third instar larvae of An. stephensi were exposed to various concentrations (10–160 g/L) of fractions and were assayed according to World Health Organization protocol. The larval mortality was calculated after 24 h treatment. Results Among tested fractions, the highest larvicidal efficacy was observed from ethyl acetate fraction of fruits extract with 50% and 90% mortality values (LC

Assessment of the Toxicity of Sub-chronic Low and High Doses of the Bio-insecticide Spinosad on the Liver, Kidney and the Cerebellum in Male Albino Mice

ABSTRACT Spinosad (SPD) is a highly selective insect control product. However, it was reported that SPD has toxicity toward other non-target organisms. This study was conducted to address the toxic effect of two sub-chronic low and high doses; 35 and 350 mg/kg SPD on some biochemical, histological and immunohistochemical parameters of the liver, kidney and cerebellum. Thirty-six male Swiss mice were divided into three groups of 12 mice each; first group (G1) served as a control, second group (G2) received a low sub-chronic dose of SPD that is equal to 35 mg/kg, and third group (G3) received a high sub-chronic dose of SPD that is equal to 350 mg/kg. The results showed that mice which were received 350 mg/kg SPD showed a significant decrease in the body weight and a significant increase in their relative kidney and spleen weights. They also showed a significant increase in alanine aminotransferase (ALT), triglycerides and urea levels. Histopathological examination showed cytoplasmic degeneration and cell necrosis in the liver and kidney. Immunohistochemical examination showed that cerebellum illustrated several neurodegenerative changes and a down-regulation of synaptophysin-Syp. In conclusion, exposure to a high dose of SPD that is equal to 350 mg/kg could cause a marked toxicity on the liver, kidney and cerebellum in male albino mice.

Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae)

We investigated the use of Bacillus thuringiensis isolated in the state of Amazonas, in Brazil, for the biological control of the dengue vector Aedes aegypti. From 25 soil samples collected in nine municipalities, 484 bacterial colonies were obtained, 57 (11.78%) of which were identified as B. thuringiensis. Six isolates, IBt-03, IBt-06, IBt-07, IBt-28, IBt-30, and BtAM-27 showed insecticidal activity, and only BtAM-27 presents the five genes investigated cry4Aa, cry4Ba, cry10Aa, cry11Aa, and cry11Ba. The IBt-07 and IBt- 28, with lower LC50 values, showed equal toxicity compared to the standards. The isolates of B. thuringiensis from Amazonas constitute potential new means of biological control for A. aegypti, because of their larvicidal activity and the possibility that they may also contain new combinations of toxins.

Propiedades insecticidas del polvo de Laurelia sempervirens L. para el control de Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) / Insecticidal properties of Laurelia sempervirens powder to Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) control

Sitophilus zeamais is a key pest of stored cereals. The insecticidal properties of Laurelia sempervirens powder against S. zeamais were assessed in laboratory. The variable evaluated were mortality of adult insects by contact and fumigant toxicity, mortality of immature insects by contact toxicity, residual effect of powder stored under environmental or refrigerated conditions, adult insect emergence (F1), repellency and weight reduction and germination of maize. The concentrations evaluated were 0, 0.25, 0.5, 1.0, 2.0 and 4.0 percent and the experimental design was completely randomly. In contact toxicity bioassay all treatments showed mortality over 90 percent while fumigant activity did not exceed 40 percent. The F1 only was registered in treatment of 0.25 percent and control showing all other treatments 0 percent. In immature control bioassay all treatments exhibited statistical differences with control. The storage of powder under refrigerated conditions not improved the residual effect. All concentrations of powder assessed showed repellence. Germination and grain weight loss of maize seeds not showed significant differences with control. We concluded that L. sempervirens has promissory perspectives to stored grain pests control...

Sitophilus zeamais es plaga primaria de los cereales almacenados. Se evaluaron, en laboratorio, las propiedades insecticidas del polvo de follaje de Laurelia sempervirens para el control de S. zeamais. Las variables evaluadas fueron mortalidad por contacto y fumigante de insectos adultos, toxicidad por contacto sobre estados inmaduros, efecto residual del polvo almacenado en condiciones ambientales y de refrigeración, emergencia de insectos adultos (F1), repelencia y pérdida de peso y germinación del maíz. Las concentraciones evaluadas fueron 0; 0,25; 0,5; 1,0; 2,0 y 4,0 por ciento y el diseño experimental fue completamente al azar. En el bioensayo de toxicidad por contacto todos los tratamientos mostraron una mortalidad sobre el 90 por ciento, mientras que la actividad como fumigante no superó el 40 por ciento. La F1 sólo se registró en el tratamiento de 0,25 por ciento y en el testigo mostrando todos los otros tratamientos 0 por ciento. En el bioensayo de control de estados inmaduros todos los tratamientos exhibieron diferencias significativas con el testigo. El almacenamiento del polvo en condiciones de refrigeración no mejoró el efecto residual. Todas las concentraciones de polvo evaluadas registraron efecto repelente. La germinación y pérdida de peso de las semillas de maíz no mostraron diferencias significativas con el testigo. Se concluye que L. sempervirens tiene perspectivas promisorias para el control de plagas de los granos almacenados...

Using ichthyotoxic plants as bioinsecticide: A literature review / Uso de plantas ictiotóxicas como bioinseticida: revisão de literatura

ABSTRACTSome ichthyotoxic plants are study object aiming to discover promising substances in the field of Biotechnology, in search of plant extracts which can be used or even transformed into natural insecticides. This paper presents a bibliographical survey in order to check the traditional use of ichthyotoxic plants as bioinsecticide. Among the plants identified as ichthyotoxic, the most cited in traditional use are those from the genera Derris, Serjania, Lonchocarpus, Magonia, and Tephrosia. The survey suggests that ichthyotoxic plant extracts can contain classes of chemical compounds such as isoflavonoids and tannins with a bioinsecticidal effect and, thus, they can be used in Biotechnology, contributing to reduce the use of synthetic insecticides that present a high toxicity level.

RESUMOUso de plantas ictiotóxicas como bioinseticida: revisão de literatura. Algumas plantas ictiotóxicas são objeto de estudos com a finalidade de descobrir substâncias promissoras no campo da Biotecnologia, na busca de extratos vegetais que possam ser usados ou mesmo transformados em inseticidas naturais. Esse artigo apresenta uma pesquisa bibliográfica sobre o uso tradicional de plantas ictiotóxicas como bioinseticida. Entre as plantas identificadas como ictiotóxicas, as mais citadas no uso tradicional são as dos gêneros Derris, Serjania, Lonchocarpus, Magonia e Tephrosia. A pesquisa sugere que extratos de plantas ictiotóxicas podem conter classes de compostos químicos, como isoflavonoides e taninos, com efeito bioinseticida e, assim, podem ser usados na Biotecnologia, contribuindo na redução do uso de inseticidas sintéticos que possuem alto nível de toxicidade.

Testes ecotoxicológicos de diferentes formulações do bioinseticida produzido na UNIVILLE submetidas ao teste de prateleira / Ecotoxicological testing of different formulations of bioinseticide produced at UNIVILLE subjected to a shelf test

O larvicida à base de Bacillus thuringiensis var. israelensis é utilizado para combater mosquitos e simulídeos atingindo o sistema digestivo de larvas do mosquito levando-as à morte. É importante conhecer a dose letal dos produtos (DL50) para os insetos alvo, bem como seu grau de ecotoxicidade (CEC) ao ambiente durante cada fase de caracterização dos produtos para estabelecer valores que não venham a ocasionar a morte ou intoxicação de outros seres vivos. O presente trabalho objetivou a determinação da DL50 através de bioensaios com larvas de Aedes albupictus, bem como o CEC com a utilização de testes agudos com o microcrustáceo Daphnia similis para o larvicida com diferentes formulações expostas ao teste de prateleira. A amostra que apresentou melhor valor para CEC foi B1 (sem aditivos) e para DL50 foi B2 (com cafeína na composição), sendo esses valores de 3526,000 e 16,815 mg.L−1, respectivamente.

The larvicide based on Bacillus thuringiensis var. israelensis is used to eliminate mosquitoes and simullidaes, acting on the larvae digestive system causing death. It is important to know the lethal dose (LD50) for the target insects and the environment ecotoxicity degree (CEC) during each characterization phase of the products in order to establish values that do not cause death or intoxication for other living beings. This article aimed to find the DL50 by bioassays with larvaes of Aedes albupictus, as well as the CEC using acute tests with the microcrustacean Daphnia similis for the product with different formulations exposed to a shelf test. The sample that showed the best CEC value was the B1 (without additives) and for LD50 it was the B2 (with caffeine in composition) that showed the following values: 3526.000 and 16.815 mg.L−1, respectively.

Toxicity and residual control of Plutella xylostella L. (Lepidoptera: Plutellidae) with Bacillus thuringiensis Berliner and insecticides / Toxicidade e controle residual de Plutella xylostella L. (Lepidoptera: Plutellidae) com Bacillus thuringiensis Berliner e inseticidas

Plutella xylostella L. is the most important worldwide pest of cruciferous plants and indiscriminate use of insecticides has led to the resistance of the species to different groups. This research was conducted to compare the toxicity and persistence of two strains of Bacillus thuringiensis to P. xylostella larvae. Concentrations between 125 and 500g 100L-1 of water of the commercial products were evaluated and compared to the insect growth inhibitor diflubenzuron and to the neurotoxic pyrethroid deltamethrin. The efficacy of the insecticides was compared between treated plants kept indoor greenhouse and outdoor. Third instar larvae were more susceptible to B. thuringiensis than first instar ones. Agree and Dipel showed similar control rates of third instars until 10 days after treatment, but on the 15th day, Agree was significantly more efficient than Dipel. Both B. thuringiensis formulations showed a reduction in mortality after 10 days when the treated plants were exposed to natural weather conditions in comparison to the same treatments kept inside greenhouse. Dimilin (100g 100L-1 of water) and deltamethrin (30ml of commercial product 100L-1 of water) were not efficient to control third instar larvae of P. xylostella. This inefficiency cannot be attributed to a resistant population of P. xylostella since the larval population used in the experiments was not subjected to insecticide pressure, once the crop was organically cultivated all year round. The results showed that both formulations of B. thuringiensis are sound alternatives for the control of the diamondback moth in organically conducted cruciferous crops, considering the high residual control provided under subtropical weather conditions.

Larvas de Plutella xylostella L. são as principais pragas de crucíferas cultivadas e o uso excessivo e indiscriminado de inseticidas tem levado a resistência da espécie para diferentes grupos de inseticidas. Este trabalho foi conduzido para comparar a toxicidade de duas formulações de Bacillus thuringiensis para larvas de primeiro e terceiro ínstar de P. xylostella. Concentrações entre 125 e 500g 100L-1 de água do produto comercial foram avaliadas e comparadas com o inibidor do crescimento diflubenzuron e com o piretroide deltametrina. A eficiência dos inseticidas foi comparada em plantas tratadas e mantidas dentro e fora da casa de vegetação. Larvas de terceiro ínstar foram mais suscetíveis a B. thuringiensis do que larvas de primeiro ínstar. Agree e Dipel apresentaram taxas de controle semelhantes até 10 dias após a aplicação, porém após 15 dias, Agree foi significativamente mais eficiente do que Dipel. Ambas as formulações apresentaram uma redução na mortalidade larval quando as plantas foram expostas às condições climáticas em comparação aos mesmos tratamentos mantidos em casa de vegetação. Dimilin (100g 100L-1 de água) e deltametrina (30ml de produto comercial 100L-1 de água) não foram eficientes para o controle de lagartas de terceiro ínstar de P. xylostella. Tal ineficiência não pode ser atribuída a um possível efeito de resistência da população de P. xylostella a esses produtos, uma vez que a colônia da espécie utilizada nos experimentos não estava sujeita à pressão de seleção, por se tratar de uma população coletada em área conduzida organicamente durante todo o transcorrer do ano. Os resultados mostram que as duas formulações de B. thuringiensis são alternativas viáveis para o controle de P. xylostella em cultivo orgânico de crucíferas, tendo em vista o expressivo controle residual observado para as condições climáticas dos subtrópicos.

Production of bio-inseticide Bacillus thuringiensis var. israelensis in semicontinuous processes combined with batch processes for sporulation

The aim of this work was to study the production of the bioinsecticide using the aerobic cultivation. Two-stage cultivation process was used: aerobic vegetative growth and end-phase of sporulation (with aeration and without aeration). The cuts of working volume evaluated were 20, 40, 60 and 80 percent. The highest value of spore concentration (2.17x10(9) CFU/mL) and spore productivity (7.2x10(7) CFU/mL.h) were observed using SP60. The lethal concentration (CL50) obtained through the aeration process and non-aeration process were 0.21 and 0.05 mg/L, respectively. The specific potential of the bioinsecticide produced without aeration (0.025 mg/10-12 CFU) was two times higher than that obtained with aeration (0.05 mg/10-12 CFU).

Produção semissólida de Metarhizium anisopliae e Beauveria bassiana em diferentes substratos e efeito da radiação ultravioleta e da temperatura sobre propágulos desses entomopatógenos / Semi-solid production of Metarhizium anisopliae (Metsch) Sorok and Beauveria bassiana (Bals) Vuill on different media and the effects of ultraviolet radiation and temperature on the infective propagules of these fungi

O trabalho teve os seguintes objetivos: 1) avaliar diferentes substratos para a produção semissólida dos fungos Metarhizium anisopliae e Beauveria bassiana; 2) verificar a tolerância dos conídios produzidos ao efeito da radiação ultravioleta e da temperatura; e 3) avaliar a patogenicidade dos conídios a Diatraea saccharalis. Para ambos os fungos, foram utilizadas 6 repetições para cada tratamento: amido de milho, arroz integral, arroz parboilizado tipo1, arroz tipo 1 e 2, aveia em flocos, canjiquinha, farelo de trigo, farinhas de mandioca crua, de milho amarela e de trigo especial, fubá, milho em grãos, polvilho azedo, soja em grãos, trigo moído e turfa. Os conídios foram quantificados em câmara de Neubauer e a determinação da viabilidade foi realizada através da observação em microscópio, dos conídios germinados e não germinados, após espalhamento da suspensão fúngica em placas de Petri contendo BDA. No ensaio com radiação os fungos foram expostos à radiação por 25 e 50 segundos e foi considerado um tratamento sem exposição; para temperatura os fungos foram expostos a 20, 25, 30 e 35o C. Utilizando a torre de Potter, 2 mL de cada suspensão fúngica dos tratamentos foram pulverizados em lagartas de D. saccharalis. As maiores concentrações de conídios de M. anisopliae e B. bassiana foram encontradas nos tratamentos com arroz parboilizado tipo 1, arroz tipo 1 e 2, farinha de milho amarela, fubá e trigo moído e a viabilidade dos conídios produzidos superou os 94%. Quanto maior o tempo de exposição à radiação menor foi o número de conídios viáveis; também ocorreu perda significativa da viabilidade dos conídios quando expostos à temperatura de 35o C. Os fungos foram patogênicos para D. saccharalis.

The present study was aimed to evaluate different (semi-solid) media for the production of Metarhizium anisopliae and Beauveria bassiana propagules, and to evaluate the tolerance of these propagules to ultraviolet radiation and temperature. The experiments were performed at the Biological Control Laboratory of the Instituto Biológico at Campinas, São Paulo, Brazil. For both fungi, 6 repetitions were performed for each of the 17 treatments: corn starch, full rice, parboiled rice, type-1 rice, type-2 rice, oat flakes, canjiquinha [grits], wheat flour, raw cassava flour, yellow corn flour, special wheat flour, corn flour, corn in grains, cassava starch, soy in grains, crushed wheat, and turf. The viability analysis was done in plastic plates containing BDA. For the bioassays involving exposure to ultraviolet light and temperature, BDA was also used for viability analysis, and each treatment was exposed to the UV radiation for 0, 25 and 50 seconds, the temperature exposure being at 20, 25, 30 and 35º C. Using a Potter tower, 2 mL of fungus suspension from each treatment was inoculated to the Diatraea saccharalis caterpillars. Regarding the sporulation, the largest concentrations of M. anisopliae and B. bassiana were found for the treatments with parboiled rice, type-1 rice, type-2 rice, yellow corn flour, corn flour and crushed wheat. The viability of all treatments was superior to 94.00%. Also, the longer the duration of the exposition to the UV, the 2Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, Botucatu, SP, Brasil.

Bioatividade de formulações de NIM (Azadirachta indica A. Juss, 1797) e de Bacillus thuringiensis subsp. aizawai em lagartas de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) / Bioactivity of neem (Azadirachta indica A. Juss, 1797) and Bacillus thuringiensis subsp. aizawai formulations in larvae of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

A utilização de inseticidas botânicos e bioinseticidas constitui uma alternativa promissora para o manejo de Spodoptera frugiperda (J.E. Smith, 1797), na cultura do milho. No presente trabalho, objetivou-se avaliar o efeito de formulações comerciais de nim e de Bacillus thuringiensis subsp. aizawai - Bta sobre esta praga em laboratório. Lagartas de S. frugiperda com 10 dias de idade foram alimentadas com folhas de milho submersas na calda dos inseticidas Neemseto®, Natuneem® e Xentari® (B. thuringiensis subsp. aizawai Bta) nas concentrações 2,5; 5,0; 7,5; 10 mL ou g/L e testemunha (água). Os efeitos dos inseticidas dependeram da concentração utilizada, pois provocaram mortalidade crescente das lagartas, alongaram o período larval e reduziram o peso larval. Em alguns casos, também, reduziram o peso das pupas, a viabilidade pupal e a longevidade de adultos. Lagartas com 0-24 h de idade foram mais susceptíveis às concentrações de Neemseto® e Xentari® a 5 e 10 mL ou g/L, em relação às com 10 dias de idade. Com o aumento da concentração, os efeitos sobre o peso das pupas e viabilidade pupal foram, também, maiores em lagartas com 0-24 h de idade.

The use of botanical and biological insecticides constitutes a promising alternative to control Spodoptera frugiperda on corn crop. Thus, the present work evaluated the effect of commercial formulations of neem (Natuneem® and Neemseto®) and Bacillus thuringiensis subsp. aizawai - Bta (Xentari®) on this pest under laboratory conditions. Newly hatched and 10 days old fall armyworm larvae were fed with corn leaves treated with the insecticides at concentration of 2.5, 5.0, 7.5, and 10 mL or g/mL and control (water). An increased effect of the insecticides was found as function of increasing concentration, causing higher larval mortality, longer larval period and lower larval weight. In some cases, reduction of pupa weight, pupa viability and longevity of adults were also verified. Newly hatched larvae (ca. 0-24 h old) were more susceptible to 5 and 10 mL or g/L of Neemseto® and Xentari® as compared with 10 days old larvae. With the increase of insecticide concentration, the effects on the pupa weight and pupa viability were also greater on larvae of 0-24h old.

Manejo da lagarta-do-cartucho em milho com formulações de nim e Bacillus thuringiensis subsp. aizawai / Management of fall armyworm in corn with commercial neem and Bacillus thuringiensis subsp. aizawai

A lagarta-do-cartucho, Spodoptera frugiperda, principal praga do milho, é comumente controlada com inseticidas sintéticos. Assim, na busca de alternativas eficientes e de baixo impacto ambiental, testou-se em campo as formulações comerciais de nim Natuneem® (10mL L-1 em 2005 e 15mL L-1 em 2006) e Neemseto® (5 e 10mL L-1 em 2005, e 10 e 15mL L-1 em 2006) e do inseticida biológico Bta (Xentari® 10g L-1 em 2005 e 2006), aplicados aos 15, 30 e 45 e aos 15, 25 e 35 dias, após a emergência das plantas, em 2005 e 2006, respectivamente. Antes de cada pulverização foram atribuídas notas de 0 a 5 para as injúrias foliares. Em 2005, somente Neemseto® 10mL L-1 foi eficiente aos 30 dias. Em 2006, Neemseto® 15mL L-1 e Bta 10g L-1, com notas médias de 0,93 e 0,98, diferiram da testemunha, com nota 2,38, na avaliação realizada aos 35 dias. Em casa-de-vegetação, plantas de milho foram pulverizadas com os inseticidas Natuneem®, Neemseto® e Xentari® e posteriormente infestadas, artificialmente, com lagartas recém-eclodidas. Após 10 dias, verificou-se que os tratamentos foram igualmente eficientes, diferindo da testemunha, tanto em relação às notas, quando ao número de lagartas sobreviventes.

The fall armyworm (Spodoptera frugiperda) is the main pest of corn in Brazil and the use of synthetic insecticides is the most commonly applied method to its control. Thus, searching for efficient and low environment impact alternatives, commercial formulations of the neem Natuneem® (10mL L-1 in 2005 and 15mL L-1 in 2006) and Neemseto® (5 and 10mL L-1 in 2005, and 10 and 15mL L-1 in 2006) and biological insecticide Bta Xentari® (10g L-1 in 2005 and 2006) applied at 15, 30 and 45 and at 15, 25 and 35 days after plant emerge in 2005 and 2006, respectively, were tested. Evaluations consisted of using a scale of foliar injury from 0 to 5 r before each spray. In 2005, only Neemseto® 10mL L-1 was efficient at 30 days. In 2006, Neemseto® 15mL L-1 and Bta Xentari® with average score of 0.93 and 0.98, respectively, were different from control with score of 2.38 at 35 days. At greenhouse corn plants were treated with Natuneem®, Neemseto® and Xentari® insecticides followed by artificial infestation with newly-hatched larvae. After 10 days, it was verified that treatment were equally efficient, differing from the control, regarding to the injury score, as well as, the number of surviving worms.

Heterologous expression of an insecticidal gene from the armed spider (Phoneutria nigriventer)

Insect-pests are global problems that cause severe damage to crop plants, and their control is commonly based on chemical insecticides. However, negative effects of pesticides on the environment and human health emphasize the necessity to develop alternative methods for insect-pest control. In the present study, a gene coding for the insecticidal peptide TX4(6-1) of the Brazilian armed spider (Phoneutria nigriventer) was cloned in fusion with maltose binding protein (MBP) and expressed in Escherichia coli. The affinity purified protein MBP-GlyTX4 was cleaved with the Xa factor and used for a bioassay against Spodoptera frugiperda and rabbit immunization. Five micrograms GlyTX4 protein injected into the hemocoel of larvae and abdominal cavity of adults produced trembling and uncoordinated movements immediately after injection and all adult insects died after 12h. After two days, larvae became paralyzed and the epidermal color changed to dark brown. Furthermore, the development stage was prolonged for two weeks. Alternatively, slices of maize leaves were imbibed with 15 micrograms of the recombinant protein cleaved with the Xa factor and used as diet for larvae. In this experiment, all larvae died in about 30 minutes. Polyclonal antibodies anti-MBP-GlyTX4 were effective for recognizing MBP and GlyTX4 in whole cell extract from E. coli expressing the recombinant protein.