Uma das principais espécies de ácaro praga é o ácaro rajado, Tetranychus urticae. Um dos inimigos naturais mais importantes deste ácaro é o patógeno Neozygites floridana. Para viabilizar o uso deste patógeno através de liberações inoculativas, ainda são necessárias informações básicas para subsidiar o desenvolvimento de uma metodologia de produção em escala e para liberações do fungo em campo. Para tanto, foi determinado em laboratório, a exigência de umidade e temperatura para a esporulação do fungo, colocando-se ácaros mumificados em folhas de feijão-de-porco. A 80 e 85% de umidade, em nenhuma das temperaturas (13, 17, 21 e 25°C) ocorreu esporulação. O fungo esporulou pouco a 90% UR em todas as temperaturas. As melhores condições para formação de capiloconídios foram a 95% e 100% UR a 21°C e 25°C. Ácaros mumificados foram colocados para esporular em feijão-de-porco em telados em diferentes épocas do ano e a temperatura e umidade monitorada. Notou-se que acima de 13°C o fungo conseguiu produzir um número considerável de conídios primários (>130) mesmo a 90% UR. Entretanto, nesta UR somente a 25°C a produção de capiloconídios foi significativa (>389). Determinou-se o efeito da exposição de duas intensidades luminosas (2.960 Lux e 15.392 Lux), três fotoperíodos (24h de luz, 12:12h(L:E) e 24h de escuro) e duas temperaturas (18°C e 23°C), na produção de conídios primários e de capiloconídios de isolados da Noruega e do Brasil. Ácaros mumificados foram expostos aos diferentes tratamentos em BOD. Não foram encontradas diferenças entre os dois isolados nas duas temperaturas testadas. Os resultados para o fotoperíodo de 12:12h(L:E) foram similares aos encontrados para 24h de escuro. A inibição da luz na formação de capiloconídios foi maior no isolado Norueguês. Baixa intensidade de luz por 24h teve efeito moderado na esporulação dos dois isolados, mas inibiu fortemente a formação de capiloconídios no isolado Norueguês. Foram realizados estudos para avaliar o efeito de agrotóxicos utilizados na produção de morango no Brasil e na Noruega sobre a esporulação e produção de capiloconídios de N. floridana em laboratório. Os resultados revelaram que Tebuconazol, Fenpropatrina na concentração recomendada e Abamectina na metade da concentração recomendada, foram os menos agressivos a N. floridana. Foram também realizadas tentativas de produção em escala deste fungo. Através de inoculações experimentais em telado foi possível induzir epizootias em populações de T. urticae em plantas de feijão de porco em vasos nos telados. Liberações de quatro ácaros mumificados por vaso foi a forma mais prática de inoculação. Maiores quantidades de ácaros mumificados foram produzidos quando as condições climáticas foram caracterizadas por alta UR e 20°C, sendo que durante o inverno a produção não foi eficiente. O uso de lâmpadas e ventiladores durante o período noturno ao final da epizootia, foi eficiente em inibir a esporulação e garantir a qualidade dos ácaros mumificados para o armazenamento. Embora tenha sido possível produzir um grande número de ácaros mumificados, vários estudos ainda são necessários para desenvolver uma metodologia de produção em escala para que seja economicamente viável a sua utilização em campo. / One of the main pest mite species is the Twospotted Spider Mite, Tetranychus urticae. The most important natural enemy of this mite is the pathogen Neozygites floridana. To enabling the use of this pathogen through inoculative releases, basic information is still needed to support the large scale methodology development for fungus production and release in the field. It was determined in the laboratory, the humidity and temperature requirements for fungus sporulation, by placing mummified mites on Jack-bean leaves. There was no sporulation at any of the temperatures tested (13, 17, 21 and 25°C) at 80% and 85% RH. The fungus had low sporulation at 90% RH at all temperatures. The best conditions for the capilloconidia development were 95% and 100%RH at 21°C and 25°C. Mummified mites were placed to sporulate in Jack-bean plants inside screenhouses at different times of year and temperature and humidity were monitored. It was noted that above 13°C the fungus can produce a considerable number of primary conidia (> 130) even at 90%RH. However, only at 90%RH and 25°C the capilloconidia production is significant (> 389). It was determined the effect of exposure of two light intensities (2.960 Lux and 15.392 Lux), three photoperiods (24h of light, 12:12h(L:D) and 24h of darkness) and two temperatures (18°C and 23°C), on the production of primary conidia and capilloconidia of N. floridana isolates from Norway and Brazil. Fungus-killed cadavers by each of the isolates were exposed to different treatments in BOD. No differences were found between the two isolates in the two temperatures tested. The results for the 12:12h(L:D) were similar to those found for 24h of darkness. Light inhibition of capilloconidia formation was greater on Norwegian isolate. Low light intensity for 24h had a mild effect on sporulation of the two isolates, but strongly inhibited the formation of capilloconidia of the Norwegian isolate. Studies were carried out to evaluate the effect of pesticides used in production of strawberries in both countries on sporulation and capilloconidia production of N. floridana in the laboratory. The results showed that Tebuconazole and Fenpropatrin in the recommended concentration and Abamectin in half of the recommended concentration were the less aggressive to N. floridana. Attempts to scale production of this fungus were also made. Through experimental inoculations it was possible to induce epizootics of the fungus in T. urticae populations on Jack-beans, planted in pots in screenhouses. The most convenient way for inoculation of the fungus was to release four mummified mites per pot. Larger quantities of mummified mites by N. floridana were produced when climatic conditions were characterized by high RH and 20°C, during the winter production was inefficient. The use of light and fans at night during the end of the epizootic phase, was effective in inhibiting the sporulation of the fungus and ensure the quality of mummified mites for storage. Although it was possible to produce a large number of mummified mites, several studies are still needed to develop an economically viable methodology for large scale production for field use.

Os fungos entomopatogênicos do gênero Metarhizium e Beauveria compreendem um importante grupo de patógenos de artrópodes-praga. A seleção de isolados de fungos promissores é a primeira e uma das mais importantes etapas no desenvolvimento de um biopesticida, pois alguns isolados podem apresentar alta virulência e não necessariamente boa produção em substrato e vice-versa, sendo interessante a combinação desses dois parâmetros para a viabilidade comercial de um produto. A dificuldade de criação ou manutenção de algumas espécies de pragas em laboratório é um limitante para a condução de testes de virulência, tornando-se interessante a utilização de espécies modelo, de fácil criação, nas etapas preliminares de seleção. Nesse sentido, este estudo objetivou selecionar isolados com alta produção de conídios e virulência, comparando a eficiência de controle de M. anisopliae e B. bassiana às pragas alvo Mahanarva fimbriolata e Bemisia tabaci Biótipo B, respectivamente, com a mortalidade em Tenebrio molitor. Inicialmente, foram selecionados 50 isolados a partir de 100 isolados de cada gênero, baseado em avaliações visuais do crescimento e esporulação em meio de cultivo em placas de Petri. Estes isolados selecionados foram cultivados em arroz parboilizado para quantificação do rendimento produtivo de conídios. Os 25 isolados mais produtivos de cada espécie de fungo foram utilizados nos bioensaios com T. molitor. Posteriormente, os cinco isolados que causaram maior e menor mortalidade de cada gênero, foram utilizados nos bioensaios com as respectivas pragas-alvo. A variação no rendimento de conídios de Beauveria spp., foi de 0,3 a 7,7 x 109 conídios.grama de arroz-1 e de Metarhizium spp. foi de 0,1 a 2,5 x 109 conídios.grama de arroz-1. A mortalidade confirmada de larvas de T. molitor por Beauveria spp., variou de 5,5 a 96,4% e por M. anisopliae variou de 29,1 a 89,1%. Alguns isolados causaram mortalidade elevada tanto no inseto modelo quanto na praga-alvo, porém, não foi verificada uma relação entre a virulência para as duas espécies. Da mesma forma, não foi observada associação entre os parâmetros produção de conídios e virulência. O isolado ESALQ 4958 de B. bassiana se destacou nos dois bioensaios apresentando mortalidades elevadas de ninfas de B. tabaci Biótipo B. Nos bioensaios utilizando ninfas de M. fimbriolata, ESALQ 1641 foi o isolado que apresentou os maiores percentuais de mortalidade nos dois bioensaios. Analisando conjuntamente as variáveis produção de conídios e virulência a T. molitor e a espécie alvo, os isolados ESALQ 540 (B. bassiana) e ESALQ 1116 (M. anisopliae) se destacaram por apresentarem valores elevados para todas as variáveis de interesse. Os resultados reforçam a necessidade de uma análise conjunta destas variáveis com um peso diferenciado para cada variável na seleção de isolados para utilização em produtos microbianos para o controle de pragas. / The genus Metarhizium spp. and Beauveria spp. are important entomopathogenic fungi used to control arthropod pests. The selection of promising fungal isolates is the first and one of the most important steps on the development of a biopesticide, since some isolates may present high virulence and not necessarily good production in substrate and vice-versa, being the combination of these two parameters important for the commercial viability. Difficulties of rearing or maintaining some species of pests in laboratory are limitations for the conduction of virulence tests, justifying the use of easy to breed model species on the preliminary steps of selection. Therefore, this study aimed to select isolates with high conidia production and virulence, comparing the control efficiency of M. anisopliae and B. bassiana to the target pests, Mahanarva fimbriolata and Bemisia tabaci biotype B, respectively, with mortality in Tenebrio molitor. At first, 50 isolates were selected from 100 isolates of each genus, based on growth and sporulation in culture medium on Petri dishes. These isolates were grown in parboiled rice to quantify the yield of conidia. The 25 most productive isolates of each fungus species were used in the bioassays with T. molitor. After, the five isolates that caused higher and lower mortality of each genus were used in the bioassays with the respective target pests. Beauveria spp. conidia yield ranged from 0.3 to 7.7 x 109 conidia.grams of rice-1 and Metarhizium spp. from 0.1 to 2.5 x 109 conidia.gram of rice-1. The confirmed mortality of T. molitor larvae by Beauveria spp. varied from 5.5 to 96.4% and M. anisopliae varied from 29.1 to 89.1%. Some isolates caused high mortality in both, model insect and the target pest; however, no relationship between the virulence of both species was observed. Similarly, there was no association between the parameters conidia production and virulence. The B. bassiana isolate ESALQ 4958 in both bioassays presented high mortalities of B. tabaci Biotype B. In bioassays using M. fimbriolata nymphs, ESALQ 1641 was the isolate that presented the highest mortalities in both bioassays. Analyzing the variables, conidia production and virulence to T. molitor and the target species, the isolates ESALQ 540 (B. bassiana) and ESALQ 1116 (M. anisopliae) showed high values for all variables of interest. The results reinforce the necessity of a joint analysis of these variables with different weight for each one in the selection of isolates, aiming to use them in microbial products for pest control.

Avaliou-se a patogenicidade de 50 isolados de 8 espécies de fungos entomopatogênicos e o produto comercial Boverilâ, formulado com isolado ESALQ-447 de B. bassiana para ninfas de 2 e 3 ínstar de Orthezia praelonga. Em outro experimento avalio-se a produção do isolado selecionado em dois sistemas de produção. Nos ensaios de seleção foram usados tubetes de citros infestados pela cochonilha. O patógeno foi inoculado pulverizando-se 5mL de suspensão conidial por muda, na concentração de 1 x 108 conídios/mL, utilizando-se um pulverizador com pressão constante de 0,5 libras/segundo. As mudas foram mantidas em estufas B.O.D. (25±0,5°C, 70±10% UR e 12 horas de fotofase). Dos isolados testados, 30 foram patogênicos para cochonilha causando mortalidade corrigida que variaram de 1,0 % a 46,6%, sendo que os melhores resultados foram obtidos com os isolados ESALQ-972 e ESALQ-1300 de V. lecanii que proporcionaram 46,6 % e 40,5%, respectivamente, após 12 dias da aplicação. Para os testes de produção foram utilizados dois métodos. O método da bandeja no qual se utilizou o arroz pré-cozido, esterilizado e inoculado com fungo em bandejas de plástico, com ciclo de produção de 15 dias. No outro método, utilizou-se de uma caixa plástica, contendo uma lâmina d’água no fundo do recipiente e um sistema de aeração constante para fornecer pressão positiva e manter o ambiente com umidade acima de 90 %. O isolado testado foi o ESALQ-972, e os métodos da bandeja e da caixa proporcionaram produção de 1,8 x 109 e 1,0 x 109 conídios/grama de arroz, respectivamente. / The pathogenicity was evaluated for 50 isolates of 8 species of entomopathogenic fungi and the commercial product Boverilâ, composed by the isolate ESALQ-447 of B. bassiana for nymphs of 2 and 3 instar of Orthezia praelonga. In another experiment the production of the isolated was selected in two production systems. In the selection tests, seedlings of citrus infested by the scale were used. The pathogen was inoculated pulverizing 5 ml of conidial suspension by seedlings, with a concentration of 1 x 108 conidia/ml, using a pulverizer with constant pressure of 0.5 lbs/second. The seedlings were kept in B.O.D. (25±0,5°C, 70±10% UR and 12 hours of photophase). From the isolates tested, 30 were pathogenic to the scale causing corrected mortality that varied from 1.0% to 46.6%, while the best results were obtained with the isolates ESALQ-972 and ESALQ-1300 of V. lecanii which generated 46.6% e 40.5%, respectively, after 12 days of aplication. For the production tests two methods were utilized. The tray method, which used the pre-cocked rice, sterilized and inoculated with fungus in plastic trays, with a production cicle of 15 days. In the other method, a plastic box was used, containing a water blade in the bottom and a constant ventilation system to provide positive pressure and keep the environment with humidity above 90%. The isolate tested was the ESALQ-972, resulting that the tray and the plastic box methods produced 1.88 x 109 and 1.0 x 109 conidia/gram of rice, respectively.

Avaliou-se a patogenicidade de diversas espécies de fungos entomopatogênicos a ninfas de 2º a 4º ínstares do psilídeo Diaphorina citri. Assim foram feitos bioensaios com Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarum, L. longisporum, Paecilomyces fumosoroseus, P. farinosus, Syngliocladium sp. na concentração de 5x107 conídios/mL para cada patógeno, com exceção de Hirsutella thompsonii que foi aplicado na concentração de 2,8x107 conídios/mL. Utilizou-se mudas de murta, Murraya paniculata, infestadas com ninfas do inseto que foram pulverizadas com as suspensões conidiais. Os fungos B. bassiana, M. anisopliae, H. thompsonii, L. muscarum e P. fumosorosus foram patogênicos para as ninfas do psilídeo. O isolado mais promissor foi o Esalq-PL63, de B. bassiana, que causou mortalidade de aproximadamente 72% das ninfas, sete dias após a inoculação. Esse fungo também afetou o processo de metamorfose das ninfas. A concentração letal média (CL50) foi calculada em 2,3 x 107 conídios/mL. O ciclo de infecção de B. bassiana sobre as ninfas do psilídeo foi estudado pulverizando-se a suspensão de 3x108 conídios/mL do fungo e, em seguida, observado em microscopia de luz e microscopia eletrônica de varredura, nos intervalos de tempo de 0, 24, 48, 72 e 168 horas após a inoculação. Constatou-se que o referido patógeno não conseguiu completar o desenvolvimento no corpo do hospedeiro, uma vez que a fase de conidiogênese é inibida, provavelmente, pela presença no interior do inseto de bactérias antagônicas ao seu desenvolvimento. O isolado Esalq-PL63 é um promissor agente de controle microbiano de ninfas de D. citri por afetar sua fisiologia e causar em altos índices de mortalidade. / It was evalueted the patogenicity of several species of entomopathogenic fungi against 2nd to 4th instar of Diaphorina citri. For the bioassays with Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarum, L. longisporum, Paecilomyces fumosoroseus, P. farinosus, Syngliocladium sp. a concentration of 5x107 conidia.mL-1 was used. For the Hirsutella thompsonii strain the concentration used was 2.8x107 conidia.mL-1. Seedlings of orange jasmine, Murraya paniculata, infested with nymphs of the insect were sprayed with the conidia suspensions. The fungi B. bassiana, M. anisopliae, H. thompsonii, L. longisporum and P. fumosoroseus were pathogenic to nymphs. The strain of B. bassiana (Esalq-PL63) was the most pathogenic causing 72% mortality after seven days of inoculation. This fungus also affected the process of nymph molting. The letal concentration (LC50) calculated was 2.3x107 conidia.mL-1. To prove mortality the pathogen was reisolated in media culture (AN, MC and BDA) and besides, observed under microscope examination. The infection cycle of B. bassiana in nymphs was studied after inoculation of 3x108 conidia.mL-1. This process were evaluated with the use of a light microscope and an electron scan microscope, after 0, 24, 48, 72 and 168 hours of conidia sprayed. This strain did not complete the development in the host because the conidiogenesis was inhibited, probably because it was found antagonistic bacteria into the host. However, this isolate is a potencial microbial control agent of nymphs of D. citri, affecting its physiology and causing high mortality.

A cultura dos citros é considerada de muita importância para a economia brasileira. Atualmente, o inseto Diaphorina citri tem sido considerado como o principal problema dessa cultura, por ser vetor da bactéria Candidatus Liberibacter asiaticus (?-Proteobacteria) causadora da doença conhecida como Greening. Além de D. citri, a cultura dos citros apresenta inúmeras espécies de insetos e ácaros pragas que reduzem a qualidade e a produção dos frutos e requerem medidas de controle. O controle das espécies pragas tem sido realizado quase exclusivamente por aplicações frequentes de pesticidas químicos. Recentemente, isolados dos fungos entomopatogênicos Isaria fumosorosea e Beauveria bassiana foram selecionados para o controle de D. citri. Nesse estudo investigou-se a patogenicidade destes isolados associados a adjuvantes no controle de ninfas e adultos dos insetos Diaphorina citri, Toxoptera citricida, Aleurocanthus woglumi, Praelongorthezia praelonga e adultos dos ácaros Brevipalpus phoenicis e Panonychus citri em condições de laboratório. Notou-se que os fungos estudados foram patogênicos para quase todas as espécies de pragas, com a execção do fungo B. bassiana para ninfas e adultos de P. praelonga. As maiores mortalidade provocadas pelos fungos entomopatogênicos foram obtidas para ninfas e adultos de D. citri (>86% e 55,7%, respectivamente) e T. citricida (>90% e 90,8%, respectivamente), onde também foi encontrado o maior nível de esporulação por indivíduo. Essas duas espécies pragas foram selecionadas para os ensaios de transmissão horizontal, onde foi avaliada a transferência de conídios dos fungos entomopatogênicos entre indivíduos esporulados e não infectados. Nos experimentos de laboratório foi observado que I. fumosorosea e B. basssiana apresentaram transmissão horizontal entre cadáveres e adultos não infectados de D. citri (intra-específica) e adultos de T. citricida para D. citri (inter-específica). Os mesmos resultados foram obtidos nos experimentos em semi-campo com adultos de D. citri. Podemos concluir que os fungos entomopatogênicos I. fumosorosea e B. bassiana apresentam potencial como agentes de controle de D. citri, T. citricida, A. woglumi, B. phoenicis, P. citri. A transmissão horizontal dos fungos apartir de T. citricida e D. citri infectados para adultos sadios de D. citri pode contribuir para o controle desta praga em campo. / The citrus crops are considered of great importance to the Brazilian economy. Currently, the insect Diaphorina citri is considered as the major pest of this crop, as a vector of the bacterium Candidatus Liberibacter asiaticus (? - Proteobacteria) causing the disease known as Greening. Besides D. citri, the orange crop has numerous species of insect and mites pests that reduce the quality and yield of fruits and require control. The control of pest species is carried out almost exclusively by frequent applications of chemical pesticides. Recently, isolates of the entomopathogenic fungi Isaria fumosorosea and Beauveria bassiana were selected for the control of D. citri. This study investigated the pathogenicity of the entomopathogenic fungi Beauveria bassiana and Isaria fumosorosea associated adjuvants on nymphs and adults of the insects Diaphorina citri, Toxoptera citricida, Aleurocanthus woglumi, Praelongorthezia praelonga and adult mites and B. phoenicis, Panonychus citri in laboratory conditions. The fungi studied were pathogenic to almost all pests species, with exception of the fungus B. bassiana against nymphs and adults of P. praelonga. The highest mortality caused by the entomopathogenic fungi were obtained for nymphs and adults of D. citri (> 86% and 55.7%, respectively) and T. citricida (> 90% and 90.8%, respectively), where we also found the highest level of sporulation per individual. These two pest species were selected to test horizontal transmission, which evaluated the transmission of disease between sporulated and healthy individuals. In the laboratory experiments, we observed that I. fumosorosea and B. basssiana showed horizontal transmission between cadavers and healthy adults of D. citri (intraspecific) and adults of T. citricida to D. citri (interspecific). Similar results were obtained in experiments in semi-field conditions with adults of D. citri. The results show that the potential of the entomopathogenic fungi I. fumosorosea and B. bassiana to control D. citri, T. citricida, A. woglumi, B. phoenicis, P. citri. Horizontal transmission of the disease from infected T. citricida and D. citri to health adults of D. citri may improve the control of D. citri in fields.

Dentre 45 isolados de Hyphomycetes testados, oito de Beauveria bassiana e quatro de Metarhizium anisopliae causaram em Tetranychus urticae, mortalidades superiores a 80 e 90%, respectivamente, cinco dias após a inoculação na concentração de 5x10 7 conídios/mL. Hirsutella sp. atingiu 73% de mortalidade na concentração de 1,7x10 7 conídios/mL. Entre 80 a 100% dos cadáveres de ácaros colonizados pelos isolados de B. bassiana e M. anisopliae apresentavam, internamente, cristais de cálcio. Conídio aéreo, blastósporo e célula de levedura de cinco isolados B. bassiana foram patogênicos a esta praga. Diferenças significativas (P ³ 0,05) para CL50 e coeficiente angular entre os isolados e entre as estruturas infectivas foram observadas. Os valores da CL50 variaram de 4,95x10 6 a 8,21x10 7 estruturas infectivas/mL. Não houve diferença significativa entre as estruturas infectivas para os dois parâmetros avaliados, contudo, houve diferenças significativas para a CL50 entre as estruturas infectivas em um mesmo isolado de B. bassiana. Três formulações de fungicidas, 24 de inseticidas e/ou acaricidas foram compatíveis com B. bassiana, sendo formulados com as seguintes moléculas: propamocarb hidrocloreto, enxofre, abamectin, acefato, acetamiprid, betacyflutrin, bifentrina, ciromazina, deltametrina, diafentiuron, diflubenzuron, dimetoato, fenpropatrina, fenpyroximate, fenvalerate, imidacloprid, metamidofós, propargite, tebufenozide e triclorfon. Houve grande variação na toxicidade dos produtos dentro de cada grupo químico e produtos formulados com a mesma molécula química. B. bassiana foi eficiente no controle de T. urticae em crisântemo (Dendranthema grandiflora) cultivado em estufa, quando pulverizado na concentração de 2x10 8 conídios/mL. O controle microbiano foi superior ao proporcionado pelo controle químico utilizado na propriedade agrícola. Efetuando-se quatro pulverizações do fungo em um período de 14 dias, a densidade reduziu de 1,8 para 0,1 ácaro/folha. Na cultura do morango (Fragaria spp.) a eficiência de B. bassiana foi inferior ao crisântemo, com densidade média de ácaros ao longo de 21 dias de avaliação para as concentrações 1x10 8 e 5x10 7 conídios/mL de 13 ácaros/folíolo, contra 43 ácaros/folíolo nas parcelas não tratadas. As variedades de morango Campinas e Princesa Isabel foram as que apresentaram as menores densidades do ácaro, contudo, não houve evidência de que estas variedades interferiram na eficiência de controle da praga por B. bassiana. Assim, M. anisopliae, B. bassiana e Hirsutella sp. foram os fungos mais promissores para serem formulados como micoacaricidas para o controle de T. urticae. / Among 45 isolates of hyphomycetes tested against Tetranychus urticae, 8 Beauveria bassiana and 4 Metarhizium anisopliae isolates caused mortality > 80 and 90%, respectively, 5 days after inoculation with 5x10 7 conidia/mL. Hirsutella sp. caused 73% mortality at a concentration of 1.7x10 7 conidia/mL. Eighty to 100% of cadavers infected by B. bassiana or M. anisopliae isolates had calcium crystals inside their bodies. Conidia, blastospores and yeastlike cells of five B. bassiana isolates were pathogenic against this pest. Significant differences (P ³ 0.05) were observed among the LC50's and slopes of dose-mortality lines for the different isolates and infective structures. LC50 values ranged from 4.95x10 6 to 8.21x10 7 cells/mL. There were no significant differences among the infective structures in the two tested variables. However, there were significant differences among the LC50's with different infective structures within the same B. bassiana isolate. Three fungicide formulations and 24 insecticides and/or mitecides were compatible with B. bassiana including those with the following active ingredients: propamocarb hydrochloride, sulphur, abamectin, acephate,acetamiprid, betacyfluthrin, bifenthrin, cyromazine, deltamethrin, diafentiuron, diflubenzuron, dimethoate, fenpropathrin, fenpyroximate, fenvalerate, imidacloprid, methamidophos, propargite, tebufenozide and trichlorfon. There was large variability in the toxicity of products withing a chemical group and products containing the same active ingredient. B. bassiana was an efficient T. urticae mite control in chrysanthemum (Dendranthema grandiflora), when applied at a concentration of 2x10 8 conidia/mL. Microbial control was better than that provided by chemical pesticides normally used in the greenhouses. With four fungal sprays within 14 days, the mite density was reduced from 1.8 to 0.1 mite/leaf. In strawberry (Fragaria sp.), T. urticae control was lower than in chrysanthemum, with mean density of mites for 21 days after application of 1x10 8 or 5x10 7 conidia/mL at 13 mites/leaflet, compared to 43 mites/leaflet in control plots. The strawberry varieties 'Campinas' and 'Princesa Isabel' had the lowest mite densities, however, these varieties did not affect the efficacy of mite control by B. bassiana. Thus, M. anisopliae, B. bassiana and Hirsutella sp. were the most promissing fungi to be formulated as mycomiticides for T. urticae control.

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No. of bitstreams: 2 Tese - Míriam de Almeida Marques - 2015.pdf: 2581091 bytes, checksum: fc6fddbab5faac3a1db80e1bbb9a905d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-08-07 / Vegetable oils and the fungus Beauveria bassiana has been tested on Bemisia tabaci, resulting in median controls of nymphs at field level. Several studies have shown that the efficiency of B. bassiana for control of Bemisia tabaci nymphs can be increased by combination with other control agents. Thus, this study was conducted to evaluate: the effect of the combination of B. bassiana with sublethal doses of castor oil in the mortality of B. tabaci nymphs; the compatibility of castor oil with B. bassiana and Trichogramma pretiosum, parasitoid of caterpillar eggs most used in the world. The effect of the castor oil at 100% (v/v) on conidia germination, vegetative growth and sporulation of B. bassiana after 24 h and seven, 15, 60, 90, 120 days of storage at 4°C and 26°C was evaluated in two experiments at laboratory. The effect of castor oil (Rícino®) at 1%, 2%, 3%, 4%, 5% and 6% (v/v) and neutral detergent at 2% (v/v) (emulsifier for the oil) on immature stages of T. pretiosum (egg, larva, pre-pupae and pupae) of parasitoid was also tested at laboratory. the chlorpyrifos insecticide 0.5 L a.i./ha and distilled water In the first stage of the study several experiments were conducted at screenhouse to determine the sublethal dose of commercial castor oil Azevedo® and Rícino® at 0.25%, 0.5%, 1.0%, 1.5 %, 2.0%, 3.0%, 4.0%, 5.0% and 6.0% (v/v) on mortality of different instars of B. tabaci. The castor oil was emulsified with Silwet® L-77 (0.01%, v/v), Zupp® neutral detergent (1%, 2%, 3% and 4%, v/v) and Solub'oil® (0.005%, 0.015%, 0.045%, 0.09%, 0.20% and 0.27%, v/v). The insecticide thiamethoxam 250 WG, was included as the chemical standard tested at label rate recommended by the manufacturer of 0.17 g a.i./L. Distilled water was used as the untreated control. In the second stage, B. bassiana CG1229 at 1×106 and 1x107 conidia/mL was tested alone or in combination with castor oil at 1% (v/v) emulsified in Silwet® L-77 0.01% (v/v) or Solub'oil® at 0.05% and 0.1% (v/v) on second instar nymphs of B. tabaci at screenhouse. Mortality of first instar to the fourth nymphs increased with an increase in concentration of castor oil and emulsifiers. Nymphs of the first and second instar were more susceptible to castor oil and 0.01% Silwet (v/v) than the third and fourth instars.The neutral detergent at 1%, 2%, 3% and 4% (v/v) killed 28.6%, 52.7%, 64.4% and 69.3% of second instar nymphs, respectively. When neutral detergent at 2% (v/v) was combined with castor oil 1% (v/v), killed 88.03% and 79.34% nymphs of first-second and third-fourth instar, respectively. The Solub'oil at 0.005%, 0.015%, 0.045%, 0.09% and 0.27% (v/v) killed 34.9%, 26.1%, 30.9%, 38.1% and 70.1% of second instar nymphs, respectively. When the Solub'oil at 0.2% (v/v) was combined with castor oil Azevedo at 1% (v/v), mortality of second instar nymphs was 84.71%. Reductions in the vegetative growth of B. bassiana by castor oil and Solub'oil were observed after 120 days of storage at 26°C when compared to pure conidia of the fungus. Conidia viability and conidiogenesis of B. bassiana was not affected by Castor oil at 26 ° C in all dates. Mortality of B. tabaci nymphs was significantly lower when castor oil + Silwet® L-77 was combined with B. bassiana (16.93%) compared to the fungus alone (57.94%). It was observed that Silwet® L- 77 at 0.01% did not emulsify efficiently the castor oil, not allowing conidia to germinate (9.82%). The combination of conidia of B. bassiana + castor oil + solub'oil and B. bassiana + Solub'oil significantly increased mortality of nymphs of B. tabaci (91.66% and 85.46%, respectively) compared to B. bassiana, castor oil or Solub'oil tested alone (75.57%, 59.12% and 53.92%, respectively). The Solub'oil at 0.5% or 1.0% did not affect the germination of conidia of B. bassiana (96.5%). Egg-larval stage of development of T. pretiosum was the least affected by different concentrations of castor oil + neutral detergent at 2% (v/v), with 81.9% of adult emergence of than the stages of pre-pupae and pupae, with 24.88% and 18.30% of emergency, respectively. Castor oil at 1% and 2% (v/v) was selective and innocuous to the egg-larval stage. All oil concentrations were considered mildly or moderately harmful to prepupae and pupae stages of T.pretiosum and caused significant reductions in adults emergency of parasitoid. The neutral detergent at 2% (v/v) did not affect immature stages of T. pretiosum, with emergency of adults ≤ 95.8% and it was classified as harmless to this parasitoid. Castor oil and emulsifiers have the potential to control B. tabaci biotype B. Castor oil when combined with conidia of B. bassiana + Solub'oil or only with Solub'oil increases mortality of nymphs of B. tabaci. Castor oil is selective to all T. pretiosum stages of development at concentrations ≤ 2%. / Os óleos vegetais e o fungo Beauveria bassiana tem sido testados sobre Bemisia tabaci, resultando em controles medianos de ninfas em nível de campo. Vários estudos tem demonstrado que a eficiência de B. bassiana no controle de ninfas de B. tabaci pode ser aumentada pela combinação com outros agentes de controle. Desta forma, este trabalho foi conduzido para avaliar: o efeito da combinação de B. bassiana com doses subletais do óleo de mamona na mortalidade de ninfas de B. tabaci; a compatibilidade do óleo de mamona com o B. bassiana e com o Trichogramma pretiosum, parasitoide de ovos de lagartas mais utilizado no mundo. Na primeira etapa dos estudos, foram instalados em casa telada vários experimentos para determinar a dose subletal do óleo comercial de mamona Azevedo® e o Óleo de Rícino® a 0,25%, 0,5%, 1,0%, 1,5%, 2,0%, 3,0%, 4,0%, 5,0% e 6,0% (v/v) na mortalidade de diferentes ínstares de B. tabaci. Como emulsionantes para o óleo de mamona foram testados o Silwet® L-77 (0,01%, v/v), o detergente neutro Zupp® (1%, 2%, 3% e 4%, v/v) e o Solub’oil® (0,005%, 0,015%, 0,045%, 0,09%, 0,20% e 0,27%, v/v). Como padrão de comparação com inseticida químico foi utilizado o thiamethoxam (Actara® 250 WG) a 0,17 g i.a./L. Na testemunha foi utilizada água destilada. Na segunda etapa dos estudos, em condições de em casa telada, o B. bassiana CG1229 a 1x106 e 1x107 (conídios/mL) foi testado sozinho ou em combinação com o óleo de mamona a 1% (v/v) emulsionado em Silwet® L-77 a 0,01% (v/v) ou em Solub’oil® a 0,05% e 0,1% (v/v) na mortalidade de ninfas de segundo ínstar da B. tabaci. Em laboratório, foram realizados dois experimentos que testaram a compatibilidade do óleo de mamona a 100% (v/v) na germinação, crescimento vegetativo e esporulação de B. bassiana as 24 h e 7, 15, 60, 90, 120 dias de armazenamento a 4º C e 26º C. No estudo de compatibilidade com T. pretiosum, foi avaliado o efeito do Óleo de Rícino® a 1%, 2%, 3%, 4%, 5% e 6% (v/v), do detergente neutro a 2% (v/v) (emulsificante para o óleo), do inseticida clorpirifós a 0,5 L i.a./ha e da água destilada sobre estágios imaturos (ovo-larva, pré-pupa e pupa) do parasitoide. A mortalidade de ninfas de primeiro ao quarto ínstares aumentou significativamente com o aumento da concentração do óleo de mamona e dos emulsionantes. Ninfas de primeiro e segundo ínstares foram mais suscetíveis ao óleo de mamona e ao Silwet a 0,01% (v/v) do que as de terceiro e quarto ínstares, com mortalidades de 71,32% e 55,01%, em comparação a 26,38% e 48,15%, respectivamente. O detergente neutro a 1%, 2%, 3% e 4% (v/v) causaram mortalidades de ninfas de segundo íntar de 28,6%, 52,7%, 64,4% e 69,3%, respectivamente. Quando este produto a 2% v/v foi aplicado com o óleo de mamona a 1% (v/v), causaram mortalidade de 88,03% e 79,34% em ninfas de primeiro-segundo e terceiro-quarto ínstares, respectivamente. O Solub’oil a 0,005%, 0,015%, 0,045%, 0,09% e 0,27% (v/v) causou mortalidade de ninfas do segundo íntar de 34,94%, 26,14%, 30,91%, 38,14% e 70,15%, respectivamente. Quando o Solub’oil a 0,2% (v/v) foi combinado com o óleo de mamona a 1% (v/v), causaram mortalidade de ninfas do segundo ínstar de 84,71%. Reduções significativas do crescimento vegetativo de B. bassiana foram observadas aos 07 e 120 dias de armazenamento a 4ºC e 26°C, respectivamente, pela presença do óleo de mamona e Solub’oil em relação a conídios puros do fungo. O óleo de mamona não afetou a viabilidade dos conídios de B. bassiana e nem a conidiogênese na temperatura de 26°C em todas as datas avaliadas. A mortalidade de ninfas de B. tabaci foi significativamente menor na combinação óleo de mamona + B. bassiana + Silwet® L-77 (16,93%) em relação ao fungo sozinho (57,94%), pois o Silwet® L-77 a 0,01% não emulsificou eficientemente o óleo de mamona, não permitindo a germinação dos conídios (9,82%). A combinação de conídios de B. bassiana + óleo de manona + Solub’oil e B. bassiana + Solub’oil aumentou significativamente a mortalidade de ninfas de B. tabaci (91,66% e 85,46%, respectivamente), em relação ao B. bassiana, óleo de mamona ou Solub’oil testados sozinhos (75,57%, 59,12% e 53,92%, respectivamente). O Solub'oil a 0,5% ou 1,0% não interferiu na germinação dos conídios de B. bassiana (96,5%). O estágio de desenvolvimento de ovo-larva de T. pretiosum foi o menos afetado pelas diferentes concentrações do óleo de mamona + detergente neutro a 2% (v/v), com 81,94% de emergência de adultos, do que os estágios de pré-pupa e pupa, com 24,88% e 18,30% de emergência, respectivamente. O óleo de mamona a 1% e 2% (v/v) foi seletivo e inócuo a fase de ovo-larva. Todas as concentrações do óleo foram consideradas levemente ou moderadamente nocivas aos estágios de pré-pupa e pupa de T. pretiosum e causaram significativas reduções na emergência de adultos do parasitoide. O detergente neutro a 2% (v/v) não afetou as fases imaturas de T. pretiosum, com percentagens de emergência de adultos ≤ 95,77% e foi classificado como inócuo a este parasitoide. O óleo de mamona e emulsionantes apresentam potencial no controle de B. tabaci biótipo B. O óleo de mamona quando combinado com conídios de B. bassiana + Solub’oil ou somente com Solub’oil aumenta a mortalidade de ninfas de B. tabaci. O óleo de mamona é seletivo a todas as fases de desenvolvimento do T. pretiosum em concentrações ≤ 2%.

Made available in DSpace on 2017-07-10T17:37:48Z (GMT). No. of bitstreams: 1 Ana_Paula_Mamprim.PDF: 1025073 bytes, checksum: a4585bdee440fbcff474cdb4a0e84089 (MD5) Previous issue date: 2011-08-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to evaluate the compatibility of aqueous and alcoholic plant extracts, alternative products and basidiocarps of the fungus Pycnoporus sanguineus on Metarhizium anisopliae. These plants and the basidiocarps were collected and transferred to drying oven at 40°C for 7 days and then ground into a fine powder. The extracts and basidiocarps were used in 10% concentration and the alternative products in 3 concentrations, being established on product labels (CR), half (0,5CR) and twice (2CR). All treatments were sprayed on the already inoculated fungus in PDA culture medium. Viability was evaluated by direct counting of viable and unviable conidia after incubation for 16 h at 26 ± 1 º C, 12h photoperiod. For Colony Forming Unit (CFU), it was counted the number of colonies after 5 days of incubation. To vegetative growth and production of conidia, the fungus was inoculated in three points on the culture medium surface of each petri dish, staying for 7 days for subsequent colonies measurement and counting conidia. For the viability was found that all aqueous extracts, except the chinaberry differed from the control. For the CFU, only cinnamon and laurel extracts and P.sanguineus not differed from the control. In diameter, lemon grass, rue, chinaberry, cinnamon, citronella and rosemary extracts differed from the control. For conidiogeneous, lemon grass, rue, cinnamon and rosemary extracts and P. sanguineus also had significance in relation to control. All the alcoholic extracts, for viability, differed from the control. However, for CFU these same extracts showed no significance. For the diameter, it was a significant difference to the alcoholic extracts of rue, castor, neem and chinaberry. For the conidia production there were significant difference between alcoholic extracts of rue, castor, chinaberry, cinnamon, neem, rosemary and laurel. For the alternative products, in the viability parameter, all products showed significant difference in relation to control, except Forth (0,5 CR and CR) and Agro-Mos® in CR. For CFU, there was significant difference at all concentrations tested. For the diameter, only 0,5CR Pironim® differed at the control. For the conidia production, Plant Clean® (0.5 CR and CR), Pironim® (CR), Bordeaux Mixture (0.5 CR and CR) and Sulfur Mixture (CR and 2CR) showed significant differences compared to control. Despite of the biological parameters present percentage change (reduction or stimulation), there was compatibility for aqueous and alcoholic extracts. In relation to alternative products, only Sulfur Mixture was incompatible for the fungus M. anisopliae. / O objetivo deste trabalho foi avaliar a compatibilidade dos extratos vegetais aquosos e alcoólicos e de produtos alternativos, além de extratos de basidiocarpos de Pycnoporus sanguineus sobre o fungo entomopatogênico Metarhizium anisopliae. As plantas e os basidiocarpos foram coletados e transferidos para estufa de secagem em 40 ºC por 7 dias, sendo em seguida, moídos até se obter um pó fino. Os extratos e o basidiocarpos foram utilizados na concentração 10%, e os produtos alternativos em três concentrações, sendo a estabelecida nos rótulos dos produtos (CR), a metade (0,5CR) e o dobro da mesma (2CR). Em todos os tratamentos foram feitas pulverizações sobre o fungo já repicado no meio de cultura BDA. Avaliou-se viabilidade, por meio da contagem direta dos conídios viáveis e inviáveis após incubação por 16 h em 26±1ºC, fotofase 12h. Para Unidade Formadora de Colônias (UFC) contou-se o número de colônias após 5 dias de incubação. Em relação ao crescimento vegetativo e a produção de conídios, o fungo foi repicado em 3 pontos na superfície do meio de cultura de cada placa de Petri, permanecendo em incubação por 7 dias, para posterior medição das colônias e contagem de conídios. Verificou-se que todos os extratos aquosos, exceto o de cinamomo, diferiram da testemunha para viabilidade. Para o UFC, somente os extratos de canela, louro e P.sanguineus, não diferiram da testemunha. No diâmetro, os extratos de capim cidreira, arruda, cinamomo, canela, citronela, alecrim diferiram da testemunha. Assim como na conidiogênese onde os extratos capim cidreira, arruda, canela, alecrim e P sanguineus também apresentaram significância em relação à testemunha. Para os extratos alcoólicos observou-se que na viabilidade, todos diferiram da testemunha. Enquanto que na UFC esses mesmos extratos não apresentaram significância. Para o diâmetro ocorreu uma diferença significativa para os extratos alcoólicos de arruda, mamona, cinamomo e nim. Já para a produção de conídios verificou-se diferença significativa dos extratos alcoólicos arruda, mamona, cinamomo, canela, nim, alecrim e louro. Para os produtos fitossanitários alternativos, no parâmetro viabilidade, todos os produtos apresentaram diferença significativa em relação a testemunha, exceto Forth (0,5 CR e na CR) e Agro-mos® na CR. Na UFC, foi verificada diferença significativa em todas as concentrações testadas. Para o diâmetro apenas o Pironim® na 0,5CR diferiu da testemunha. Já para a produção de conídios o produto Planta Clean® (0,5 CR e na CR), Pironim® (CR), Calda Bordalesa (0,5 CR e na CR) e Calda Sulfocálcica (CR e no 2CR) apresentaram diferenças significativas dos tratamentos em relação à testemunha. Apesar dos parâmetros biológicos apresentarem variação percentual (estímulo ou redução) verificou-se a compatibilidade para os extratos aquosos e alcoólicos. Em relação aos produtos alternativos somente a Calda Sulfocálcica se mostrou incompatível para o fungo M. anisopliae.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.