Characterization and selection of Bacillus thuringiensis isolates effective against Sitophilus oryzae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos de herbicidas nos teores de clorofilas de Spirodela punctata

Com o objetivo de verificar na planta aquática Spirodela punctata o efeito de diferentes concentrações (0,005; 0,05; 0,5 e 5 mgL-1 de ingrediente ativo) dos herbicidas butachlor, glyphosate e propanil sobre os teores de clorofila a, b e na razão clorofila a/b, foram conduzidos, por sete dias completos, bioensaios em sala climatizada. Determinou-se os teores de clorofilas através de leituras espectrofotométricas nas absorbâncias A645 e A663 nm. Os resultados mostraram que o butachlor diminuiu o teor de clorofila b, o glyphosate o teor de clorofila a, enquanto o propanil diminuiu os teores de clorofila a, b e a razão a/b, provocando a maior redução de pigmentos na espécie.

Avaliação in vitro da eficácia de produtos homeopáticos contendo momordica charantia através de bioensaios

Homeopathic medicines have been used for over two hundred years without the examination of their effects on in vivo and in vitro assays, due to the peculiarity of homeopathic preparations, the high dilution, which creates a challenge for the use of usual analytical techniques of quality control of medicine.Although there is scarcity of literature and variety of experiments, recently there have been some studies with few in vitro assays which have shown positive responses when evaluating the mechanism of action of homeopathic medicines which are able to act on a specific system.The present study aims to evaluate the efficacy of homeopathic products containing Momordica charantia through bioassays.Homeopathic products were tested by the MTT to assess cytotoxicity in RAW 264.7 (macrophage-like cells) and in tumor cells HeLa (human cervical adenocarcinoma cells), CHO K1 (Chinese hamster ovary cells), PANC-1 (human pancreas cancer cells) and PC-3 (human prostate cancer cells), dosage of inflammatory mediators NO, TNF-α and IL-6 released by RAW 264.7 cells, analysis of the death process and cell cycle changes of PC-3 by flow cytometry. The data demonstrate that homeopathic products of Momordica charantia did not show cytotoxicity to RAW 264.7, increased the production of inflammatory mediators by RAW 264.7 synergistically with LPS, showed cytotoxicity to PC-3 with change in its cell cycle inhibiting its proliferation, being the 30CH the most potent sample. Correlation studies were conducted in order to evaluate the possible in vitro applicable models to the quality control of homeopathic products with Momordica charantia. The data showed that the best applicable models in assessing the quality are the MTT to assess cytotoxicity in RAW 264.7 and PC-3 in 24 hours for Momordica charantia fruit products and dosage of NO production by RAW 264.7 with and without LPS

Aspectos germinativos de capim-camalote (Rottboellia cochinchinensis)

O capim-camalote (Rottboellia cochinchinensis), originário da Índia, é encontrado em várias regiões do mundo, sendo uma espécie temida pelos agricultores devido ao seu difícil controle e avanço crescente. Objetivou-se com este trabalho estudar os fatores que afetam a germinação das sementes dessa espécie. Foram avaliados os métodos de superação de dormência: 1 - escarificação mecânica, 2 - escarificação química, 3 - tratamento pré-semeadura, 4 - tratamento químico, 5 - tratamento hormonal e 6 - sementes não tratadas. Foram estudados os efeitos da temperatura (5, 10, 15, 20, 25, 30, 35 e 40 ºC), do fotoperíodo (6, 8, 10, 12, 14 e 16 horas de luz), da qualidade da luz incidente (branca, vermelha, vermelha distante, amarela, verde, azul e ausência de luz), da disponibilidade de água (0,0, -0,2, -0,3, -0,4, -0,5, -0,6, -1,2 e -2,4 MPa) e da viabilidade de suas sementes quando armazenadas sob condições de câmara fria e seca e sob condições naturais. Todos os ensaios foram conduzidos por 30 dias em câmara de germinação, com os tratamentos arranjados em delineamento experimental inteiramente casualizado, em quatro repetições. Verificou-se que as sementes apresentaram elevado índice de germinação, praticamente não apresentaram dormência e não se mostraram fotoblásticas. A disponibilidade de água foi indispensável para a germinação, com temperatura ideal de 25 °C. As sementes recém-coletadas são inviáveis para o estudo da germinação, apresentando redução na germinação quando armazenadas sob condição de câmara fria e seca.

Atividade tóxica de isolados de Bacillus Thuringiensis a larvas de Aedes Aegypti (l.) (diptera: culicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identificação de genes cry de Bacillus thuringiensis no controle de Sphenophorus levis, o bicudo da cana-de-açúcar

A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis, por meio da caracterização morfológica e molecular, identificando as diferentes subclasses dos genes cry3 e cry35 e determinar a patogenicidade contra Sphenophorus levis, uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1163 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis. O material genético foi purificado pela matriz de troca iônica Instagene Matrix e submetido a PCR com iniciadores gerais cry3 e cry35 identificando-se 30 isolados contendo genes com potencial para o controle de coleópteros, os quais juntamente com as linhagens-padrão de B. thuringiensis var. tenebrionis, B. thuringiensis var. morrissone e B. thuringiensis var. tolworthi foram utilizados para a realização do bioensaio. Através de análise discriminante alocaram-se os isolados em quatro grupos quanto à toxicidade de B. thuringiensis. Os grupos ficaram assim definidos: um grupo que promovem até 10% de mortalidade contendo as testemunhas e duas linhagens;um grupo que causou 39% de mortalidade contendo três linhagens padrão e dez isolados; um grupo com 52% de mortalidade contendo treze isolados e um grupo com 70% de mortalidade contendo cinco isolados, os quais devem ser considerados promissores no controle biológico de S. levis.

Efeitos do enriquecimento com nutrientes (N e P) em diferentes condições de luz sobre o crescimento do fitoplâncton em um reservatório eutrófico no semi-árido brasileiro

The increasing of pollution in aquatic ecosystems in the last decades has caused an expansion of eutrophication and loss of water quality for human consumption. The increase of frequency and intensity of cyanobacteria blooms have been recognized as a major problem connected to water quality and eutrophication. The knowledge of environmental factors controlling these blooms is a key step towards the management for recovering aquatic ecosystems from eutrophic conditions. Primary productivity in aquatic ecosystems is dependent on light and nutrients availability. In the present work we evaluated the relative importance of the concentration of major nutrients, such as phosphorus and nitrogen, and light for phytoplankton growth in the main water reservoir of Rio Grande do Norte State, named Engenheiro Armando Ribeiro Gonçalves (EARG), which is an eutrophic system, dominated by potentially toxic cyanobacteria populations. Limitation of phytoplankton growth was evaluated through bioassays using differential enrichment of nutrients (N and/or P) under two light conditions (low light and high light) and monthly monitoring of chlorophyll-a and nutrients (total nitrogen and phosphorus) concentrations, and water transparency (Secchi depth) at the pelagic region. Our results confirm that EARG reservoir is an eutrophic system with a low water quality. Results of bioassays on the growth of phytoplankton limitation (N or P) were conflicting with the results predicted by the TN:TP ratios, which indicates that these ratios were not a good indicator of algal growth limitation. Nitrogen was the limiting nutrient, considering both frequency and magnitude. Light and hidrology affected phytoplankton response to nutrient enrichment. The extreme eutrophic conditions of this reservoir, dominated by cyanobacteria blooms, demand urgent managing strategies in order to guarantee the multiple uses for this system, including water supply for human population. Although nitrogen is the limiting nutrient, an effective management program must focus on the reduction of both phosphorus and nitrogen input

Efeitos de florações de cianobactérias sobre os cladóceros ceriodaphnia dubia e daphnia gessneri

Cyanobacterial blooms are common in eutrophic reservoirs in Brazilian northeastern semi-arid. Given this reality, the present study aimed to analyze the effect of potentially toxic cyanobacterial blooms in Gargalheiras reservoir (semi-arid) on the cladocerans Ceriodaphnia dubia and Daphnia gessneri. In vitro chronic bioassays were performed with reservoir water dilutions from August/2011 to May/2012 and the following effects were evaluated on: intrinsic rate of population growth (r), reproductive parameters (age of first reproduction and fecundity per capita) and cladocerans movements. Phytoplankton was dominated by Cylindrospermopsis raciborskii and Planktothrix agardhii and saxitoxin and microcystin were detected in reservoir water. In most months C. dubia showed differences in r between control (absence of cyanobacteria) and treatments, and has shown negative effects on reproductive parameters. In all months paralysis of swimming movements was observed in C. dubia when both C. raciborskii and saxitoxin (cyanotoxin neurotoxic) were present in water. While C. dubia was sensitive to the reservoir water containing cyanobacteria, D. gessneri showed less intense negative effects in r and reproductive parameters. Furthermore, D. gessneri showed no paralysis of swimming movements. These results support the hypothesis in the literature that D. gessneri is resistant to the Cylindrospermopsis effects. The clone‟s life history may be a key to understand the results. The C. dubia clone, isolated from eutrophicated environment, is in the lab for ten years and it is an exotic species in Brazil. D. gessneri is a common species in the country and this clone was isolated from the Gargalheiras reservoir (where there are constant blooms of potentially toxic cyanobacteria) a year ago. Perhaps the recent contact with cyanobacteria explain the higher resistance presented by this D. gessneri clone. In conclusion, the cladocerans studied have different levels of sensitivity to cyanobacteria, characterizing species-specific responses

Virulência de Aphanocladium album (Preuss) Gams e Verticillium lecanii (Zimm.) Viégas (Deuteromycotina: Hyphomycetes) para o Percevejo-de-renda da seringueira, Leptopharsa heveae (Drake & Poor) (Hemiptera: Tingidae)

The lace bug, Leptopharsa heveae is an insect that causes serious damage on rubber trees. In laboratory condition, the strains of Verticillium lecanii (ARSEF 6430, 6431 e 6432) and Aphanocladium album (ARSEF 6433) were tested on third-and fifth-instar nymphs and adults of L. heveae to evaluate their virulence using 2.4 x 10(5) and 2.4 x 10(7) conidia/mL. The bioassays were carried out using Petri dishes whose inner bottoms were covered with damp filter papers. Each Petri dish contained five insects and one rubber tree leaflet. The plates were covered with PVC film to provide high relative humidity, maintained at 26 +/- 0.5 degrees C and a photophase of 14 hours. The Probit analysis was calculated from mortality date of insects killed by fungi. In the highest concentrations, ARSEF 6430 was more virulent for third instar nymphs, and the LT50 was 1.9 days. For the fifth instar, the strains ARSEF 6430, 6433 and 6432 showed similar virulence with LT50 of 2.6, 2.6 and 3.2 days, respectively. For adults, ARSEF 6431 was the most virulent strain with the LT50 recorded at 2.0 days. The smallest concentration did not always cause more than 50% mortality.

Efeito de beauveria bassiana (bals.) Vuillemin eMetarhizium anisopliae (metsch.) sorokin nos parâmetros biológicos de trichogramma atopovirilia oatman & platner, 1983 (hymenoptera: trichogrammatidae)

Two bioassays were performed to evaluate the effect of two biopesticides based on Beauveria bassiana (Bals.) Vuillemin and Metarhizium anisopliae (Metsch.) Sorokin on the biological parameters of Trichogramma atopovirilia Oatman & Platner, 1983. In the first one, displays with S. frugiperda, 1797 eggs were dropped into the biopesticide suspension and offered to the parasitoid females for 24 hours. In the second one, parasitoid females were fed with a suspension containing honey and biopesticide suspension. In both cases, after the parasitoid death they were mantained into a humid chamber to observe fungus sporulation. The experiments were maintained in a climatized chamber at 25 +/- 1 degrees C, RH 70 +/- 10%, and photophase of 14 hours. The longevity and adult mortality, total parasitism, progeny emergency, number of individuals per egg and sexual ratio were analyzed. The biopesticides did not affect the evaluated parameters and no fungus sporulation was observed in dead females. It is possible to assert that the entomopathogenic fungi B. bassiana and M. anisopliae can be used with T. atopovirilia in IPM S. frugiperda systems.

Ação de inseticidas sobre os ovos e lagartas da broca-pequena-do-fruto do tomate, em bioensaio de laboratório

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicidade diferencial de produtos à base de abamectina ao ácaro Brevipalpus phoenicis (Geijskes, 1939) (Acari: Tenuipalpidae) em citros

O ácaro Brevipalpus phoenicis é uma das principais pragas dos citros por ser vetor do Citrus Leprosis Virus (CiLV), agente causal da leprose, uma das mais graves doenças da citricultura. Objetivou-se avaliar o efeito tóxico de produtos à base de abamectina sobre o ácaro B. phoenicis. Foram realizados um experimento de ação direta e três de ação residual no Laboratório de Acarologia do Departamento de Proteção de Plantas (Fitossanidade) da FCAV - UNESP, Jaboticabal-SP. O delineamento adotado nos bioensaios foi o inteiramente casualizado, onde 10 tratamentos foram repetidos 7 vezes, sendo cada repetição composta por um fruto de laranja. Os tratamentos estudados (mL p.c./100 L de água) foram: Acaramik a 20; 30; 40 e 50 mL; Vertimec a 30 e 40 mL; Abamectin Nortox a 30 e 40 mL; Tricofol a 77 mL e uma testemunha sem aplicação. Utilizaram-se frutos com presença de verrugose, que foram lavados e parcialmente parafinados, deixando-se uma área sem parafina, que foi circundada com cola entomológica para contenção dos ácaros. Transferiram-se 20 ácaros adultos B. phoenicis para cada fruto. No bioensaio de ação direta, a transferência foi realizada antes das aplicações e, nos bioensaios de ação residual, aos 5; 10 e 15 dias após a aplicação dos produtos. A aplicação dos produtos sobre os frutos foi realizada em Torre de Potter. Os resultados obtidos nos bioensaios evidenciaram que os melhores tratamentos foram: Tricofol a 77 mL, Acaramik a 40 e 50 mL e Vertimec a 40 mL. de forma geral, os produtos testados podem ser utilizados no controle do ácaro B. phoenicis.

Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines

Twelve pregnant female canines, naturally infected with Toxoplasma gondii, were reinfected with T. gondii: three (GI) received tachyzoites subcutaneously (1.0 x 107), three (GII) were orally inoculated with oocysts (1.5 x 104), and six (GIII) were kept as a nonreinfected control group. All the reinfected female canines (GI and GII) miscarried or presented fetal death, while only one GIII female presented a stillborn in a litter of four pups (P < 0.01). Fever, lymphoadenopathy, miscarriage, and fetal death were the main clinical alterations observed. The highest serological titers detected through the indirect fluorescence antibody test (IFAT) were 1,024 (GI) and 4,096 (GII). In group III, the titers ranged between 64 and 256. By bioassays in mice, T. gondii was isolated in 17 organs of the reinfected adult canines, in 11 of the control group, and in 20 of the neonates. Positive immunostaining of cysts and/or tachyzoites were observed in 26 canine tissues (14 from GI and GII and ten from GIII). The agent was detected by immunohistochemistry in the encephalon of a neonate and in the spinal cord of a stillborn, thus, confirming that T. gondii infected canine fetuses, provoking miscarriages, even in bitches that presented primoinfection.

Detection of Toxoplasma gondii oocysts in environmental samples from public schools

The number of Toxoplasma gondii oocysts that can be found in random environmental samples is probably low; in addition, these cysts may be confused with Hammondia spp. and Neospora spp. oocysts. The aim of the present work was to evaluate the presence of T. gondii oocysts in the soil of public elementary schools in the northwest area of the state of São Paulo, Brazil using mouse bioassays. A comparison was made between the different available bioassay techniques, such as squash, histopathology, immunohistochemistry and indirect fluorescent antibody test (IFAT). T. gondii was isolated by bioassay in mice (squash brain samples) from 22.58%(7/31) of the school playgrounds. Immunohistochemistry and IFAT showed positive results in 32.26% (10/31) and 25.80% (8/31) of samples, respectively. The sensitivity and specificity of the immunohistochemistry method were 85.71% and 83.33%, respectively. The IFAT results showed 100% sensitivity and 95.83% specificity. The presence of T. gondii was not detected in histopathological examinations. The results of the present study strongly suggest that T. gondii oocysts are widely distributed in elementary public schools in the region that was evaluated, likely constituting the main contamination source for these children. Educational programs directed at reducing environmental contamination with T. gondii would eventually lower the cost of treating humans for clinical toxoplasmosis. It is also possible to conclude that the use of IFAT in mouse bioassays can be recommended without the need for brain cysts research, which is extremely difficult and laborious. (C) 2010 Elsevier B.V. All rights reserved.

Toxoplasma gondii: Evidence for the transmission by semen in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)