Made available in DSpace on 2016-04-28T20:15:25Z (GMT). No. of bitstreams: 1 2007-Ana Paula Rodrigues Moraes.pdf: 1193160 bytes, checksum: 61d4adc426c0fdbeab75966756651ece (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Stomoxys calcitrans is a hematophagous fly that is related to reduction in animal production due to the stress and dissemination of pathogens. The knowledge in plague control, using entomopathogenic fungi, has been growing year by year, but few works have been done to control S. calcitrans. The aims of this study were rearing a colony of S. calcitrans to make a bioassay of microbial control, verify the pathogenic potential of Metarhizium anisopliae in eggs, larvae and pupae and access three methods of egg s exposition to the studied fungi. After methodologies adaptation of rearing S. calcitrans that are described in literature, the colony was maintained in the Laborat?rio de D?pteros Hemat?fagos - UFRRJ. The bioassays were done in the Laborat?rio de Controle Microbiano de Atr?podes de Import?ncia Veterin?ria - UFRRJ, using the strain 959 of M. anisopliae in a water suspension. In the first method, eggs were immersed in the suspension (2.2 ? 108 conidia /ml and dilutions) and transferred to Petri plates with hydrophilic cotton. In the second methods (2.3 ? 108 conidia/ml and dilutions), eggs were immersed and transferred to the larval rearding medium. In the third method (2.1 ? 108 conidia/ml and dilutions), eggs were put in the larval rearding medium surface and one aliquot of fungus suspension were added. Larvae with nine days of development were immersed in suspension (2.1 ? 108 conidia/ml and dilutions) and after that were transferred to the larval rearding medium while pupae with 16 days of development were immersed in suspension (2.3 ? 108 conidia/ml and dilutions) and were held in Petri plates. Experiments were carried out with 107, 106 and 105 concentrations of conidia/ml and positive and negative controls. The mortality was assessed on the 10th day after fungus exposure. It was verified high mortality in all treated and control groups in the first method of eggs exposure. In the other two methods the eggs exposed to 108 conidia/ml concentration were 100% unviable. In the second method the eggs was 92.5% unviable and the third method was 53.33% both in 107 conidia/ml concentration. The subsequent concentrations did not show significant difference when compared to controls, neither between two methods of fungi exposure. Larvae and pupae exposure did not show statistic difference between treated and controls groups. Metarhizium anisopliae suspensions in high concentrations were capable to make unfeasible eggs of S. calcitrans; however, it did not happen in larvae and pupae experiments, in which fungi did not prevent their development. The second method was the best one to control immature forms of S. calcitrans but the third one resembles the nature conditions, in which has significant result only in the highest fungi concentration. Experimental methodologies that simulate nature conditions may optimize the utilization of fungi in arthropods control, in a way that it does not modify its action when applied in the field. / Stomoxys calcitrans ? uma mosca hemat?faga associada ? redu??o da produtividade, devido ao estresse causado aos animais e a dissemina??o de pat?genos. O conhecimento do controle de pragas, utilizando fungos entomopatog?nicos, vem crescendo no decorrer dos anos, por?m poucos trabalhos t?m sido realizados visando o controle de S. calcitrans. O objetivo deste estudo foi estabelecer uma col?nia de S. calcitrans direcionada ao bioensaio de controle microbiano, verificar o potencial patog?nico de Metarhizium anisopliae sobre ovos, larvas e pupas, e ainda, avaliar tr?s m?todos de exposi??o dos ovos ao fungo estudado. Ap?s adapta??o das metodologias de cria??o de S. calcitrans descritas na literatura, a col?nia foi mantida no Laborat?rio de D?pteros Hemat?fagos UFRRJ. Os bioensaios foram realizados no Laborat?rio de Controle Microbiano de Artr?podes de Import?ncia Veterin?ria UFRRJ, onde foi utilizada a cepa 959 de M. anisopliae para elabora??o das suspens?es aquosas. No primeiro m?todo, os ovos foram imersos na suspens?o (2,2 x 108 con?dios/ml e dilui??es) e transferidos para placa de Petri contendo algod?o hidr?filo. No segundo m?todo (2,3 x 108 con?dios/ml e dilui??es) os ovos foram imersos e transferidos para o meio de desenvolvimento larval. No terceiro m?todo (2,1 x 108 con?dios/ml e dilui??es), os ovos foram depositados sobre a superf?cie do meio de desenvolvimento larval e uma al?quota da suspens?o f?ngica foi adicionada. As larvas com nove dias de desenvolvimento foram imersas na suspens?o (2,1 x 108 con?dios/ml e dilui??es) e transferidas para o meio de desenvolvimento larval, enquanto que as pupas de 16 dias de desenvolvimento foram imersas na suspens?o (2,3 x 108 con?dios/ml e dilui??es) e depositadas em placas de Petri. Foram utilizadas dilui??es na concentra??o de 107, 106 e 105 con?dios/ml e controle positivo e negativo. A mortalidade foi avaliada no d?cimo dia ap?s exposi??o, onde foi observado se ocorria mudan?a de est?gio. Foi verificada alta mortalidade em todos os grupos (tratados e controles) no primeiro m?todo de exposi??o de ovos. Nos outros dois m?todos, foram obtidos 100% de inviabilidade dos ovos expostos ? concentra??o de 108 con?dios/ml. No segundo m?todo, houve 92,5% de inviabilidade dos ovos, enquanto que no terceiro m?todo, a inviabilidade foi de 53,33%, ambos na concentra??o de 107 con?dios/ml. As concentra??es subseq?entes n?o apresentaram diferen?a significativa frente aos controles e nem entre os dois m?todos de exposi??o f?ngica. Nas exposi??es de larvas e pupas n?o ocorreu diferen?a estat?stica entre os tratamentos e os controles. Suspens?es de M. anisopliae em altas concentra??es s?o capazes de inviabilizar ovos de S. calcitrans, entretanto, isto n?o ocorreu em larvas e pupas, pois o fungo n?o impediu o desenvolvimento destes. O m?todo mais efetivo para o controle das formas imaturas de S.calcitrans foi o segundo m?todo, entretanto, o que mais se assemelha ?s condi??es naturais seria o terceiro, onde foi obtido resultado significativo apenas na maior concentra??o f?ngica. A ado??o de metodologias experimentais que simulem condi??es naturais pode otimizar a utiliza??o de fungos no controle de artr?podes, de forma que sua a??o n?o seja comprometida, quando aplicados no campo.

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Made available in DSpace on 2014-06-12T15:55:40Z (GMT). No. of bitstreams: 2 arquivo9374_1.pdf: 2655923 bytes, checksum: 4a8cd8dfda72129aa8308dbf088c4d10 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Beauveria bassiana, Metarhizium anisopliae var. anisopliae e Metarhizium flavoviride var. flavoviride são fungos entomopatogênicos com ação comprovada contra várias espécies, embora não tenham sido testados em Chrysomya albiceps, um díptero de importância na saúde pública. Assim, objetivou-se avaliar a ação de B. bassiana, M. anisopliae var. anisopliae e M. flavoviride var. flavoviride em ovos, larvas e adultos de C. albiceps, utilizando as concentrações 104, 105, 106, 107, 108 conídios.mL-1, considerando o percentual de eclosão de larvas, período de pré-pupa, pupa, percentual de emergência de adultos, ritmo de emergência, morte acumulativa, longevidade, período de postura e percentual de eclosão a partir de fêmeas infectadas. O comportamento dos fungos reisolados também foi avaliado através dos parâmetros biológicos: percentual de germinação, número de conídios, número e diâmetro de colônias, bem como a citologia no que se refere à descrição das estruturas vegetativas e reprodutivas. De acordo com a metodologia empregada, verificou-se que os três fungos apresentaram ação contra ovos, larvas e adultos de C. albiceps. Já em relação ao comportamento, foi observado que os fungos reisolados de larva apresentaram o melhor desempenho em relação ao controle e os aspectos citológicos não diferiram quando comparados ao controle. Esses resultados sugerem a possibilidade do emprego desses fungos no controle de C. albiceps.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-07-27Bitstream added on 2014-06-13T20:58:37Z : No. of bitstreams: 1 cintra_err_me_botfca.pdf: 215151 bytes, checksum: 7ae1761f27a1919df5207f8df40e958d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) / Neste trabalho estudou-se a patogenicidade de alguns isolados de Metarhizium anisopliae à cigarra do cafeeiro Fidicinoides pronoe. Além disso, utilizou-se o isolado IBCB 348 para determinação da CL90 e avaliação da compatibilidade do fungo com produtos fitossanitários empregados na cultura do café. Todos os isolados de M. anisopliae testados em cigarras do café foram patogênicos, causando mortalidades confirmadas de 50 %, com exceção dos isolados IBCB 410 e IBCB 348, que atingiram mortalidade de 60 %. Observou-se que a mortalidade foi maior nos 5 primeiros dias, havendo diminuição da mesma a partir do 6o dia. No teste de compatibilidade com produtos fitossanitários verificou-se que o fungicida oxicloreto de cobre, nas duas doses avaliadas (5.000g/ha e 2.000g/ha), resultou em maior produção de conídios e redução do crescimento vegetativo. O thiabendazole (35g/ha concentração mínima) e o hidróxido de cobre (5.000g/ha concentração máxima) não diferiram da testemunha quanto à produção de conídios. Já os produtos thiabendazole (1.500g/ha concentração máxima), benomyl (1.000g/ha), tebuconazole (100mL/ha), cyproconazole (200mL/ha) e mancozeb (5.000g/ha e 3.500g/ha) foram estatisticamente iguais entre si, mas apresentaram menor produção de conídios do que a testemunha. Através da fórmula de T, que avalia a compatibilidade dos produtos e sua toxicidade, o oxicloreto de cobre (2.000g/ha) e o thiabendazole (35g/ha) foram considerados compatíveis com M. anisopliae. Os demais fungicidas avaliados foram classificados como muito tóxicos e não apresentaram produção de conídios e crescimento vegetativo, com exceção de thiabendazole (1.500g/ha), que apresentou crescimento vegetativo. Quanto aos herbicidas avaliados, todos os produtos foram classificados como muito tóxicos nas concentrações testadas, afetando o crescimento vegetativo e a produção de conídios... / This paper deals with the pathogenicity of some isolates of Metarhizium anisopliae to the coffee cicada, Fidicinoides pronoe. Also, the isolate IBCB 348 were used to determine the LC90 and to evaluate the compatibility of this fungus with the pesticides used on the coffee crop. All M. anisopliae isolates were pathogenic to the coffee cicada, causing at least 50 % of confirmed mortality. The virulence of the fungus was higher in the first five days, decreasing after the sixth day. The fungicide copper oxicloreto, in both concentrations, resulted in higher production of conidia and reduction of vegetative growth. Thiabendazole (minimum concentration) and copper hydroxide (maximum concentration) led to conidia production similar to the control. Thiabendazole (maximum concentration), benomyl, tebuconazole, cyproconazole and mancozeb (both concentrations) showed lower production of conidia than the control. By using the T formula, which evaluate the compatibility of the pesticides and their toxicity, copper oxicloreto and thiabendazole were considered compatibles with M. anisopliae. The remaining fungicides were classified as very toxic and showed neither production of conidia nor vegetative growth, excepting thiabendazole (maximum concentration), which resulted in vegetative growth. All herbicides were very toxic to the fungus. Among the insecticides, thiamethoxam were considered compatible to M. anisopliae; the other products resulted in lower production of conidia and were considered from moderately to very toxic.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-29Bitstream added on 2014-06-13T20:44:14Z : No. of bitstreams: 1 mochi_da_dr_jabo.pdf: 1918684 bytes, checksum: f29aa98fefb34604c991782bab720e3b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A mosca-dos-chifres Haematobia irritans é um dos principais ectoparasitos associados a bovinos em pastagens. O presente trabalho objetivou analisar a ação patogênica dos isolados E9, IBCB425 e IBCB159 do fungo Metarhizium anisopliae, JAB06, JAB07 e AM09 de Beauveria bassiana, IBCB133 e CB75 de Isaria fumosorosea (=Paecilomyces fumosoroseus) e CG189 e CG195 de Isaria farinosa (=Paecilomyces farinosus) para a mosca-dos-chifres H. irritans em laboratório e campo. Avaliou-se em laboratório, a ação de todos os isolados dos fungos sobre ovos, larvas, pupas e adultos. Grupos de 30 ovos foram inoculados com suspensões fúngicas, e colocados sobre papel de filtro esterilizado, em 5 g de fezes bovinas frescas, acondicionadas em tubos de plástico. No ensaio com larvas, pedaços de papel filtro esterilizado contendo na superfície grupos de 30 ovos foram colocados sobre 5 g de fezes bovinas frescas, contidas em tubos de plástico, às quais incorporaram-se diversas concentrações de conídios mg fezes-1. No ensaio com ovos e larvas, o término do experimento ocorreu com a emergência dos adultos. Grupos de 20 pupas foram imersas por 60 segundos em suspensão do fungo, e colocadas em placas de Petri. Após a emergência, os adultos foram transferidos para gaiolas de isopor pequenas. Para o ensaio com adultos, grupos de 30 adultos foram separados e pulverizados com 0,3 mL das suspensões de conídios. Doze horas após, as moscas foram transferidas para gaiolas de isopor e avaliadas diariamente até o 15o dia. Para todos os ensaios, foram utilizadas suspensões contendo 106, 107 e 108 conídios mL-1. O isolado que promoveu os melhores resultados nos ensaios anteriores, foi usado em um experimento de campo para avaliar a ação fúngica em larvas e adultos de H. irritans em bovinos naturalmente infestados. Para o controle de larvas, foi oferecido aos bovinos conídios de M. anisopliae isolado... / Horn fly Haematobia irritans is one of the main ectoparasites associated to cattle on pastures. The present work had the objective of analyzing the pathogenic action of the isolates E9, IBCB425 and IBCB159 of the fungus Metarhizium anisopliae, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) for horn fly H. irritans, in laboratory and field conditions. The action of all the fungi isolates over the eggs, larvae, pupae and adults were evaluated in laboratory. Groups of 30 eggs were inoculated with fungal suspensions and placed over sterilized filter paper on top of 5 g of fresh bovine feces, put in plastic tubes. In the larvae assay, peaces of sterilized filter paper, containing groups of 30 eggs on the surface, were placed over 5 g of fresh bovine feces, contained in plastic tubes, in which different concentrations of conidia mg feces-1 were incorporated. In the eggs and larvae assay, the end of the experiment matched the emergence of the adults. Groups of 20 pupae were immersed for 60 seconds in the fungus suspension and placed on Petri dishes. After the emergence, the adults were transferred to small Styrofoam cages. In the adults assay, groups of 30 adults were separated and pulverized with 0,3 ml of the conidia suspensions. After twelve hours, the adults were transferred to Styrofoam cages and the evaluations were done daily until the 15th day. Suspensions containing 106, 107 and 108 conidia ml-1 were used for all the assays. The isolate that promoted best results on the previous assays was used in a field experiment, where the fungal action on larvae and adults of H. irritans, on naturally infested bovines, was evaluated. For the larvae control conidia of the E9 isolate of M. anisopliae, microencapsulated... (Complete abstract click electronic access below)

Made available in DSpace on 2015-03-03T11:52:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-22Bitstream added on 2015-03-03T12:07:10Z : No. of bitstreams: 1 000807109.pdf: 457835 bytes, checksum: c8b102f16b86a5f3ecea8c8ee3c9df40 (MD5) / A pesquisa objetivou analisar a suscetibilidade de duas populações distintas de Helicoverpa armigera a produtos comerciais à base de Bacillus thuringiensis (Bt) e isolados de Beauveria bassiana, Metarhizium anisopliae e Metarhizium rileyi. As populações foram coletadas em lavouras de soja nos municípios de Rio Verde (GO) e Luís Eduardo Magalhães (BA) e criadas em laboratório até a décima geração antes do início dos bioensaios. Os formulados utilizados foram Dipel® PM (B. thuringiensis var kurstaki), Xentari® WG (B. thuringiensis var. aizawai) e Agree® WG (B. thuringiensis var. aizawai GC91 transconjugado com B. thuringiensis var. kurstaki), com a aplicação de 50 ?L da suspensão do produto biológico, na concentração de 107 esporos viáveis/mL, sobre a superfície da dieta acondicionada em recipientes esféricos de plástico (2 cm de diâmetro x 3 cm de altura). Em cada recipiente foi inserida uma lagarta, de cada instar larval, em um total de 100 lagartas por tratamento distribuídas em 10 repetições. As avaliações referentes à mortalidade foram efetuadas a cada 24 horas, até o sétimo dia após a aplicação dos tratamentos. Os fungos B. bassiana e M. anisopliae foram obtidos a partir dos produtos comerciais biológicos Boveril® PM e Metarril® PM, respectivamente, enquanto que M. rileyi foi isolado de cadáveres de H. armigera, sendo padronizados na concentração de 108 conídios viáveis/mL. Como unidade experimental foram utilizados recipientes de plástico (2 cm de diâmetro x 3 cm de altura), com dieta artificial na qual uma alíquota de 50 ?L do fungo foi misturada ou aplicada na superfície. Em cada recipiente foi inserida uma lagarta de primeiro instar, com menos de 24 h de vida, totalizando 100 lagartas por tratamento distribuídas em 10 repetições. A mortalidade foi avaliada a cada 24 h, até o décimo dia após a aplicação dos tratamentos. Lagartas de primeiro instar de H. armigera, de ambas ... / The research aimed to assess the suscetibility of two distinct Brazilian populations of Helicoverpa armigera to commercial Bacillus thuringiensis insecticides and isolates of Beauveria bassiana, Metarhizium anisopliae and Metarhizium rileyi.The pest populations were collected in soybean fields located in Rio Verde (GO) and Luís Eduardo Magalhães (BA), taken to laboratory and reared until the tenth generation on artificial diet, to be used in the experiment.The bioassays were performed using spherical plastic receptacles (2-cm diameter x 3- cm height) containing artificial diet until complete the half volume. The formulated products used were Dipel® WP (B. thuringiensis var kurstaki), Xentari® WG (B. thuringiensis var. aizawai) and Agree® WG (B. thuringiensis var. aizawai GC91 with B. thuringiensis var. kurstaki), spraying 50 ?L of biological product solution at 107 viable spores/mL, on the surface of the diet. One caterpillar was inserted in each receptacle, related with larval instar, totaling 100 caterpillars per treatment and each group containing 10 caterpillars were considered a replication. Mortality were assessed each 24h, until the seventh day after the beginning of the bioassay. To perform the bioassays with entomopathogenic fungi, B. bassiana and M. anisopliae were obtained from the bio-insecticides Boveril® WP and Metarril® WP, respectively and M. rileyi was isolated from H. armigera cadavers at concentration of 108 viable conidia/mL. The bioassays were performed using spherical plastic receptacles (2-cmdiameter x 3-cm height) containing artificial diet until complete the half volume. After this step, a solution of 50 ?L containing the entomopathogenic fungi was sprayed on the diet surface. One caterpillar was inserted in each receptacle, with less than 24 h after hatch, totaling 100 caterpillars per treatment. Each group containing 10 caterpillars were considered a replication. Mortality were assessed each 24h, until ...

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A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.