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The endophytic fungus Aspergillus niger isolated from specie plant Hancornia speciosa, commonly known as mangabeira, was cultivated in different culture media and used for identification of six substances in their extracts, such as oxalic, citric, tartaric, p-acetylbenzoic, hexylitaconic acids and phyrofen. For quantification of pyrophen in different extracts, it was developed a method by High Performance Liquid Chromatography that was efficient for both extract analysis and fermented broth. The method allowed to evaluate the effect of different media ways and culture time on the pyrophen. The analyzed media for pyrophen production were mangaba juice, Czapeck and potato dextrose broth (PDB) and the highest yield was obtained using mangaba juice. The effect of culture time was evaluated by cultivating the fungus in the PDB medium, being the ideal period of fermentation for pyrophen production was 12 days. / O fungo endofítico Aspergilus niger isolado da espécie vegetal Hancornia speciosa, conhecida popularmente como mangabeira, foi cultivado nos meios suco de mangaba, Czapeck e Caldo de Batata Dextrose (CDB) e levou ao isolamento e identificação de seis substâncias em seus extratos, sendo elas os ácidos oxálico, cítrico, tartárico, p-acetil-benzóico e hexilitacônico e a substância pirofeno. Visando encontrar a melhor condição de cultivo para produção de pirofeno foi desenvolvido um método analítico por Cromatografia Líquida de Alta Eficiência para análise desta substância. O método desenvolvido foi eficiente tanto para análise dos extratos quanto para análise do caldo fermentado e permitiu avaliar o efeito dos diferentes meios e do tempo de cultivo na produção desta substância. A maior produção de pirofeno foi obtida utilizando suco de mangaba como meio de cultivo e o período ideal de fermentação para a produção de pirofeno foi de 12 dias, quando cultivado no meio líquido CDB.

The endophytic fungus Aspergillus niger isolated from specie plant Hancornia speciosa, commonly known as mangabeira, was cultivated in different culture media and used for identification of six substances in their extracts, such as oxalic, citric, tartaric, p-acetylbenzoic, hexylitaconic acids and phyrofen. For quantification of pyrophen in different extracts, it was developed a method by High Performance Liquid Chromatography that was efficient for both extract analysis and fermented broth. The method allowed to evaluate the effect of different media ways and culture time on the pyrophen. The analyzed media for pyrophen production were mangaba juice, Czapeck and potato dextrose broth (PDB) and the highest yield was obtained using mangaba juice. The effect of culture time was evaluated by cultivating the fungus in the PDB medium, being the ideal period of fermentation for pyrophen production was 12 days. / O fungo endofítico Aspergilus niger isolado da espécie vegetal Hancornia speciosa, conhecida popularmente como mangabeira, foi cultivado nos meios suco de mangaba, Czapeck e Caldo de Batata Dextrose (CDB) e levou ao isolamento e identificação de seis substâncias em seus extratos, sendo elas os ácidos oxálico, cítrico, tartárico, p-acetil-benzóico e hexilitacônico e a substância pirofeno. Visando encontrar a melhor condição de cultivo para produção de pirofeno foi desenvolvido um método analítico por Cromatografia Líquida de Alta Eficiência para análise desta substância. O método desenvolvido foi eficiente tanto para análise dos extratos quanto para análise do caldo fermentado e permitiu avaliar o efeito dos diferentes meios e do tempo de cultivo na produção desta substância. A maior produção de pirofeno foi obtida utilizando suco de mangaba como meio de cultivo e o período ideal de fermentação para a produção de pirofeno foi de 12 dias, quando cultivado no meio líquido CDB.

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Five new species of ophiostomatoid fungi, colon ising the infructescences of serotinous Protea species, have recently been discovered in South Africa. Prior to this, ophostomatoid fungi were thought to be restricted to the Northern Hemisphere. The discovery of these five species thus extensively expanded the known geographical range of these fungi, now also to include the Southern Hemisphere. Since this discovery, few studies have focused on the interesting ecology of this group, which is uniquely adapted for spore dispersal by arthropods. Studies focussed on ophiostomatoid fungi provide an unique opportunity to study inter-organism interactions between fungi, their host plants and the arthropods responsible for their spore dispersal. Very few similar studies have been undertaken, particularly in the Fynbos Biome, to which most of our economically important Protea species are confined. The current study provides insight into some ecological aspects of these complex interactions. The seasonal distribution of the ophiostomatoid fungi associated with the infructescences of members of the Proteaceae was investigated. Definite seasonal patterns were observed, with peak fungal colonisation occurring during the wetter winter months. While determining the host specificity of these fungi, a new Protea host for Ophiostoma splendens was identified, and a new species of Rhyncomeliola was discovered. All ophiostomatoid species native to Fynbos in the Stellenbosch region are exclusively associated with the infructescences of species in the plant genus Protea. By employing a multiplex Polymerase Chain Reaction (PCR), a set of group specific primers was developed for the identification of South African Ophiostoma and Gondwanamyces fungal DNA. This newly developed method was used to scan arthropods collected from the infructescences of P. repens for the presence of 0. splendens and G. proteae spores. By using these group-specific primers, three insects possibly responsible for long range dispersal of fungal spores were identified. Three further insect species, possibly contributing to the short-range dispersal of ophiostomatoid spores, were also implicated through this method. All arthropods associated with the fruiting structures of selected members of the Proteaceae were also investigated. A total of 62 pseudospecies (ca. 7500 individuals), belonging to 45 different arthropod families, were collected. Proteaceae species with larger fruiting structures housed more arthropod species and higher numbers of individuals than Proteaceae taxa with smaller fruiting structures. Some plant species housed similar arthropod communities, while others housed unique suites of arthropods. Seasonal patterns in arthropod numbers were observed, and it was found that, in most instances, arthropod numbers peaked during the autumn and winter months. Twenty-five fungal taxa were isolated from various Proteaceae arthropods, many of which are genera known to include Proteaceae pathogenic species. Ophiostomatoid fungi have a saprophytic relationship with their Protea hosts, and may deter some of these potentially harmful fungi from colon ising the infrutescences. Some fungi showed a high degree of specificity towards potential vectors, while others were found on a diverse range of arthropods. This study highlights the existence of complex inter-organismal interactions within the Fynbos Biome, a study area where the interactions between plants, fungi and insects have been grossly neglected. The few studies that have been conducted in this field have mostly focussed on a limited number of organisms, and no wide-scale attempts, such as presented here, have been published. It is important to obtain a holistic view in any ecological study that focuses on interactions between different suites of organisms. Ultimately this will aid in the development of better conservation strategies. This study thus provides a muchneeded start in studies on multi-organismal interactions in the Fynbos Biome. / AFRIKAANSE OPSOMMING: Vyf nuwe ophiostoma-agtige fungus spesies is onlangs in die vrug-liggame van Suid Afrikaanse Protea plante ontdek. Voor hierdie ontdekking, was die algemene opvatting dat ophiostoma-agtige fungi beperk is tot die Noordelike Halfrond. Die ontdekking van hierdie vyf spesies het dus die bekende geografiese verspreiding van hierdie tipe fungi vergroot om nou ook die Suidelike Halfrond in te sluit. Sederdien het min studies gefokus op die interesante ekologie van hierdie groep, wat aangepas is vir spoorverspreiding deur geleedpotiges. Studies wat fokus op die ophiostoma-agtige fungi voorsien 'n unieke geleentheid om die inter-organismiese-interaksies tussen fungi, hul gasheer plante en die geleedpotiges wat hul spore versprei, te bestudeer. Baie min soortgelyke studies is al voorheen in die Fynbos Bioom, waar die meeste van ons ekonomies-belangrike Protea spesies voorkom, onderneem. Die huidige studie verleen meer inligting oor sommige ekologiese aspekte van hierdie komplekse interaksies. Seisoenale verspreidingspatrone van ophiostoma-agtige fungi geassosieer met die vrug-liggame van lede van die Proteaceae, is ondersoek. Daar is defnitiewe seisoenale patrone in die aanwesigheid van fungi gevind, met 'n piek in fungi kolonisasie-getalle gedurende die vogtiger wintermaande. 'n Nuwe Protea gasheer vir Ophiostoma splendens en 'n nuwe fungus spesie (Rhyncomeliola sp.), is ontdek. Alle Fynbos ophiostoma-agtige spesies in die Stellenbosch area, is beperk tot Protea spesies. 'n Multipleks Polimerase Ketting Reaksie is ontwikkel om die spesifieke DNS van ophiostoma-agtige fungi te herken. Hierdie metode is gebruik om te toets vir die aanwesigheid van spore van hierdie fungi op die liggame van geleedpotiges (geassosieerd met P. repens). Hierdeur is drie insekte wat waarskynlik verantwoordelik is vir die langafstand verspreiding van spore geidentifiseer. Drie verdere insekte wat moontlik verantwoordelik is vir die kortafstand verspreiding van ophiostoma-agtige fungi is ook aangewys. Alle geleedpotiges geassosieer met die vrugstrukture van geselekteerde lede van die Proteaceae is ondersoek. 'n Totaal van 62 pseudo-spesies (omtrent 7500 individue), wat tot 45 families behoort, is versamel. Proteaceae spesies met groter vrugstrukture het meer geleedpotige spesies en individue gehuisves as taksa met kleiner vrugstrukture. Sommige plant spesies het soortgelyke geleedpotige gemeenskappe gehuisves, terwyl unieke groepe geleedpotiges in ander plante voorgekom het. Alhoewel die geleedpotiges seisonale variasies in getalle geopenbaar het, het die meeste groepe 'n piek in getalle getoon gedurende die herfs- en wintermaande. Vyf-en-twintig fungi taksa is geïsoleer vanaf verskeie Proteaceae geleedpotiges. Sommige van hierdie is genera wat Proteaceae patogeniese spesies insluit. Sommige fungi het 'n hoë graad van spesifisiteit teenoor potensiële vektore getoon, terwyl ander weer algemeen op verskeie geleedpotige spesies gevind is. Hierdie studie dui op die bestaan van komplekse inter-organismiese-interaksies in die Fynbos Bioom, in 'n area waar die studie van interaksies tussen plante, fungi en insekte nog baie min aandag geniet het. Die beperkte aantal studies wat tot dusver in hierdie veld onderneem is, fokus gewoonlik slegs op 'n paar organismes. Geen grootskaalse studies, soos die een hier voorgelê, is al gepubliseer nie. In enige ekologiese studie wat fokus op interaksies tussen verskeie organismes, is dit belangrik om 'n holistiese siening te probeer verkry. Uiteindelik sal dit bydra tot die ontwikkeling van beter bewarings-strategië. Hierdie studie verleen dus 'n noodsaaklike begin tot studies op multiorganismiese- interaksies.

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae, and their bacterial symbionts, have been studied intensively because of their role as natural mortality factor for soil-dwelling arthropods, and their potential as biological control agents for belowground insect pests. Moreover, EPN are recognized as key players in regulating soil food webs and triggering trophic cascades. However, most studies of interactions with EPN have been conducted under laboratory setting and simplified conditions, without consider the dynamic of the EPN and their interactions with other soil components in a wider context. In this respect, knowledge of the effect that other soil organisms or human induced factor may have on EPN dynamic and life cycle in the soil may contribute to improve tactics for their implementation and success as natural regulators of herbivores. The present investigation focused on the interactions of EPN with a selection of insecticides, and biotic (saprobic fungus and plant parasitic nematodes) elements that may be present in the soil, and may potentially interact with EPN. Specifically, I investigated how these factors may affect the life cycle (host search behavior, virulence and reproduction) of EPN. Appendix A shows the effect that a group of selected synthetic and biological insecticides have on EPN virulence and reproduction. The results obtained from this study revealed that most combinations of EPN and insecticides under study increased the mortality of the insect host. However, it was also found that some of these combinations reduced the nematode progeny production and emergence of IJs from the insect cadaver. In contrast in Appendix B, when examining the effect of the saprobic fungus Fusarium oxysporum in the life cycle of the EPN Heterorhabditis sonorensis, it was found that this fungus negatively affected the virulence and reproduction of the EPN in the insect host. In the third study of this dissertation (Appendix C) the interactions studied considered the effect of two EPN on an organism of a different trophic guild, the plant parasitic nematode Tylenchulus semipenetrans. This plant parasitic nematode causes serious diseases in citrus plants by infecting their roots and defoliating their branches. Previous studies have shown that some EPN species may negatively affect the life cycle of plant parasitic nematodes by reducing the damage produced by this plant parasite. Results from this study confirm the antagonistic effect between the selected EPN and the citrus nematode. Specifically, it was found that the presence in the soil of both EPN reduced the survival of infective juveniles of the citrus nematode and their penetration to the root. Moreover, the presence of EPN had an antagonistic effect in the production of eggs of T. semipenetrans females.

Orientador: Paulo Cezar Ceresini / Banca: Helvécio Della Coletta Filho / Banca: Celso Dornelas Fernandes / Banca: Antonio de Goes / Banca: Dilermando Perecin / Resumo: No início dos anos 90, o fungo Rhizoctonia solani emergiu como um patógeno importante associado à morte de pastagens do gênero braquiária na Colômbia. Inicialmente, esse estudo indicou que R. solani AG-1 IA predomina como patógeno associado à queima da folha da braquiária nos Llanos Colombianos. Para o estudo da estrutura genético-populacional do patógeno, um total de 198 isolados de R. solani AG-1 IA foram coletados de campos de B. brizantha cv. Toledo, de Brachiaria híbrido Mulato e do arroz. Os isolados foram genotipados usando dez loci microssatélites. Um sistema reprodutivo misto (reprodução sexuada e clones adaptados) caracterizou as populações de R. solani AG-1 IA que infectam a braquiária. A alta fração clonal e os desvios do equilíbrio de Hardy-Weinberg encontrados foram consistentes com o efeito Wahlund associado à mistura de populações. Padrões históricos de migração entre populações hospedeiro-distintas indicaram a origem provável das populações que infectam braquiária a partir de populações que infectavam arroz. Para determinar o efeito do estresse térmico na evolutibilidade para crescimento micelial, populações de R. solani AG-1 IA infectando braquiária ou arroz e R. oryzae-sativae do arroz, foram submetidas à temperatura ótima e de estresse (25 e 35°C). A herdabilidade para crescimento micelial sob condições de estresse foi considerada alta na população de R. oryzae-sativae do arroz, indicando que os patógenos têm potencial de adaptação à temperatura de 35°C / Abstract: In the early '90s, the fungus Rhizoctonia solani has emerged as an important pathogen associated with the death of Brachiaria pastures in the Colombian Llanos. Initially, this study indicated that R. solani AG-1 IA was the predominant pathogen associated with the leaf blight in the Colombian Llanos. To study the population genetic structure of the pathogen, a total of 198 isolates of R. solani AG-1 IA were collected from fields of B. brizantha cv. Toledo, Brachiaria Mulato hybrid and from rice. These isolates were genotyped using ten microsatellite loci. A mixed mating system (sexual reproduction and dispersal of adapted clones) characterized the populations of R. solani AG-1 IA infecting Brachiaria. The high clonal fraction and deviations from Hardy-Weinberg equilibrium found in three out of four populations were consistent with Wahlund effect associated with the mixing of populations. Historical patterns of migration between different host-populations indicated that the likely source of the current populations infecting Brachiaria in the Colombian Llanos was from populations that originally infected rice. To determine the effect of heat stress on the evolvability for mycelial growth, two host distinct populations of Rhizoctonia solani AG-1 IA infecting either brachiaria or rice and a rice-infecting population of R. oryzae-sativae, were submitted to optimal and stress temperatures (25°C and 35°C). The heritability values for mycelial growth under heat stress was considered high for rice-infecting population of R. oryzae-sativae, indicating that these pathogen's population have a high potential to adapt to 35°C temperature / Doutor

Neste trabalho realizamos a análise das variações na expressão gênica global do fungo aquático Blastocladiella emersonii submetido ao estresse de carência de oxigênio (hipóxia), utilizando a técnica de microarranjos de cDNA em lâminas contendo 3773 genes distintos. Nos experimentos de hipóxia gradual (diminuição gradual da concentração de oxigênio dissolvido, seguido de reoxigenação) e hipóxia direta (diminuição direta da concentração de oxigênio dissolvido, seguido de reoxigenação) observamos que 650 genes foram diferencialmente expressos em pelo menos uma das condições de estresse e que 534 deles mostraram-se afetados (direta ou indiretamente) pela disponibilidade de oxigênio, uma vez que apresentaram recuperação (ou tendência à recuperação) da sua expressão aos níveis normais, quando as células foram reoxigenadas. Além de modular a expressão de diversos genes sem função conhecida, B. emersonii responde à hipóxia reajustando a expressão de genes responsáveis pela produção e consumo de energia. Pelo menos transcricionalmente, este fungo favorece o metabolismo anaeróbico, através da indução de genes que codificam enzimas da via glicolítica e lactato desidrogenase, ao passo que no ciclo do ácido cítrico, a maioria dos genes encontram-se reprimidos ou não sofrem alteração na expressão. Processos dispendiosos em energia como síntese protéica, metabolismo de aminoácidos, enovelamento de proteínas e transporte por membrana apresentaram perfis predominantemente de repressão gênica quando em carência de oxigênio. Ainda utilizando a técnica de microarranjos, mostramos semelhanças entre os perfis transcricionais nos experimentos hipóxia e de carência de Fe2+ (tratamento com quelante de Fe2+ 2,2´-dipyridyl) sugerem que estes estresses estão de alguma forma relacionados, fornecendo bons indícios de que o íon Fe2+ possa ter um papel importante no mecanismo sensor de oxigênio e/ou de resposta a hipóxia em B. emersonii. Além disso, o tratamento prévio de células submetidas à hipóxia com o antibiótico geldanamicina, um conhecido inibidor da proteína de choque térmico HSP90, levou à diminuição da indução de certos genes de hipóxia, indicando que este fungo pode possuir algum mecanismo semelhante ao do fator de transcrição de hipóxia HIF1-α de mamíferos, uma vez que este fator também é afetado por geldanamicina. Adicionalmente, desenvolvemos um protocolo para transformação de B. emersonii mediada por Agrobacterium tumefasciens que se mostrou promissor. A transferência do T-DNA contendo um gene de resistência a higromicina B, presente no vetor binário pBINPLUS-Hph, foi evidenciada pelo crescimento normal e esporulação das células transformadas, na presença do antibiótico e pela amplificação do gene de resistência no DNA genômico de células transformadas. / In this work we analyzed global gene expression changes in the aquatic fungus Blastocladiella emersonii submitted to oxygen deprivation (hypoxia), using cDNA microarrays containing 3,773 distinct genes. In gradual hypoxia (gradual decrease in dissolved oxygen concentration, followed by reoxygenation) and direct hypoxia (direct decrease of dissolved oxygen concentration, followed by reoxygenation) we observed 650 differentially expressed genes in at least one of the stress conditions tested, 534 of them being affected (directly or indirectly) by oxygen availability, since they showed recovery of normal expression levels or a tendency to recover, when cells were reoxygenated. Besides modulating many genes with no previously assigned function, B. emersonii responds to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favour anaerobic metabolism through the induction of genes encoding glycolytic enzymes and lactate dehydrogenase, while in the TCA-cycle, most genes were repressed or unchanged. Energy-costly processes like protein synthesis, amino acid metabolism, protein folding and transport had their gene expression profiles predominantly repressed during oxygen deprivation. Microarray experiments also showed similarities between the transcriptional profile of genes in hypoxia and iron (II) deprivation (treatment with the iron (II) chelator 2,2\'-dipyridyl), suggesting that these stresses are somehow related, giving good evidence that Fe2+ ion could have a role in the mechanism of oxygen sensing and/or response to hypoxia in B. emersonii. Furthermore, pretreatment of cells subjected to hypoxia with the antibiotic geldanamycin, a known inhibitor of the heat shock protein HSP90, caused a significant decrease in the induction of certain hypoxic genes, indicating that this fungus could have a mechanism similar to that of the mammalian hypoxia transcription factor HIF-1α, which is also affected by geldanamycin. Additionally, we developed an Agrobacterium tumefasciens-mediated protocol for transformation of B. emersonii that has shown to be promising. The capacity to transfer the T-DNA containing a hygromycin B resistance gene, present in the pBINPLUSHph binary vector, was evidenced by the normal growth and sporulation of the transformed cells in the presence of antibiotic and by amplification of the resistance gene from the genomic DNA of transformed cells

O inseto vetor Diaphorina citri Kuwayama 1907 (Hemiptera: Liviidae) é considerado a principal praga da citricultura mundial. O controle desta praga é feito quase exclusivamente com inseticidas químicos, existindo a demanda por alternativas mais sustentáveis de controle. O desenvolvimento de um bioproduto a base de fungo entomopatogênico pode ser uma alternativa promissora para o manejo integrado de D. citri em pomares comerciais de citros. Assim, esse estudo teve como objetivo viabilizar a utilização do fungo entomopatogênico Isaria fumosorosea ESALQ-1296 para o controle de D. citri, avaliando: 1- a ação de adjuvantes na adesão e germinação de I. fumosorosea em ninfas e adultos de D. citri; 2- a relação entre a viabilidade dos conídios e a mortalidade de D. citri; 3- o efeito da umidade relativa do ar e da radiação ultravioleta na eficácia de I. fumosorosea em condições de laboratório; 4- o período entre a aplicação de fungicidas químicos e I. fumosorosea em campo; 5- a seletividade de I. fumosorosea aos inimigos naturais Tamarixia radiata e Ceraeochrysa cincta em condições de laboratório; 6- o volume de calda adequado para as aplicações de I. fumosorosea para o controle de D. citri em pomares comerciais; 7- a eficiência de I. fumosorosea no manejo de D. citri e seu efeito em outras espécies de artrópodes em dois sistemas de produção de citros. Análises por microscopia eletrônica de varredura revelaram que a adição dos adjuvantes KBRAdj e Silwet L-77 acelerou o desenvolvimento dos conídios de I. fumosorosea na cutícula de todos os estágios do inseto em comparação com o Tween 80. As formulações de I. fumosorosea com KBRAdj 0,075% apresentaram melhor desempenho no controle de D. citri em todos os níveis de umidade, mas especialmente em períodos de baixa umidade relativas (≤50%) e sob exposição à radiação UV-B (> 2 horas) em relação a aplicação com Tween. Os fungicidas Kumus® DF, Kocide® WDG, Nativo®, Flint 500® WG não influenciaram no desempenho do entomopatógeno quando esses foram pulverizados um dia antes ou após a aplicação do fungo sobre os adultos de D. citri. Nos estudos de seletividade foi demonstrado que a maioria das misturas de I. fumosorosea com os adjuvantes foi inócua para os adultos de T. radiata e as larvas neonatas de C. cincta. Em pomares comerciais, foi demostrado que as aplicações de I. fumosorosea foram mais eficientes no volume de 60 mL.m-3 de área de copa. Na área com produção orgânica de citros foi verificado que as aplicações de I. fumosorosea reduziram significativamente a população natural de D. citri e do ácaro da leprose (Brevipalpus spp.). Já na área com produção convencional de citros foi observado que as pulverizações de I. fumosorosea reduziram significativamente a população do ácaro da falsa ferrugem (Phyllocoptruta oleivora) e a população de D. citri foi extremamente baixa em todos os tratamentos. As aplicações de I. fumosorosea não apresentaram efeitos negativos nas populações dos predadores joaninhas, crisopídeos e hemerobídeos. Portanto, conclui-se que a utilização do fungo I. fumosorosea no manejo de D. citri pode ser uma tática sustentável para minimizar as aplicações sistemáticas de pesticidas, diminuindo seus impactos no agroecossistema. / The insect vector Diaphorina citri Kuwayama 1907 (Hemiptera: Liviidae) is the main citrus pest in the world. The control of this pest is done almost exclusively with chemical insecticides, and there is a demand for more sustainable control alternatives. The development of a bioproduct based on entomopathogenic fungi may be a promising alternative for the integrated management of D. citri in commercial citrus orchards. The objective of this study was to evaluate important parameters to increase efficiency of applications of the entomopathogenic fungus Isaria fumosorosea ESALQ-1296 for the control of D. citri. The following studies have been developed: 1- the action of adjuvants on adhesion and germination of I. fumosorosea on nymphs and adults of D. citri; 2- the relationship between the conidia viability and the mortality of D. citri; 3- the effect of relative air humidity and ultraviolet radiation on the efficacy of I. fumosorosea under laboratory conditions; 4 - the period between the application of chemical fungicides and I. fumosorosea in field; 5- the selectivity of I. fumosorosea to the natural enemies Tamarixia radiata and Ceraeochrysa cincta under laboratory conditions; 6 - the volume of syrup suitable for I. fumosorosea applications for the control of D. citri in commercial orchards; 7- the efficiency of I. fumosorosea to management of D. citri and its effect against other species of arthropods in two systems of citrus production. Scanning electron microscopy analysis revealed that the addition of the adjuvants KBRAdj and Silwet L-77 accelerated the development of I. fumosorosea conidia on the cuticle of all stages of the insect compared to Tween 80. The formulations of I. fumosorosea with KBRAdj 0.075% showed better performance in controling D. citri at all moisture levels, but especially in periods of low relative humidity (≤50%) and exposure to UV-B radiation (> 2 hours) compared to Tween application. The fungicides Kumus® DF, Kocide® WDG, Nativo®, Flint 500® WG did not influence the performance of the entomopathogen when they were sprayed the day before or after application of the fungus on D. citri adults. In the selectivity studies, it was shown that most of the mixtures of I. fumosorosea with the adjuvants were innocuous for the adults of T. radiata and the neonates of C. cincta. In commercial orchards, it was demonstrated that I. fumosorosea applications were more efficient in the volume of 60 mL.m-3 of leaves. In the area with organic citrus production it was verified that I. fumosorosea applications significantly reduced the natural population of D. citri and the false spider mite (Brevipalpus spp.). In the area with conventional citrus production it was observed that sprays of I. fumosorosea significantly reduced the population of the false rust mite (Phyllocoptruta oleivora) and D. citri population was extremely low in all treatments. The applications of I. fumosorosea did not present negative effects in the populations of the ladybirds, lacewings and hemerobide predators. Therefore, it is concluded that the use of I. fumosorosea fungus in the management of D. citri can be a sustainable tactic to minimize the systematic applications of pesticides, reducing their impacts on the agroecosystem.

Estudos em nosso laboratório caracterizaram camundongos B10.A e A/J, respectivamente, como susceptíveis e resistentes à infecção pulmonar pelo fungo Paracoccidioides brasiliensis. A imunidade inata desempenha papel fundamental no controle inicial dos patógenos e na regulação da resposta imune adquirida. Como os macrófagos alveolares são as primeiras células do hospedeiro a interagir com o fungo, propusemo-nos a estudar a capacidade fungicida e secretora dos macrófagos alveolares de camundongos susceptíveis e resistentes ao P. brasiliensis para melhor compreender a PCM pulmonar. Camundongos B10.A e A/J normais (n: 10-15) foram submetidos a lavagem bronco-alveolar (LBA) e a suspensão celular obtida (2x105 células/poço) foi pré-ativada durante a noite com IFN-γ, IL-12 ou a combinação destas duas citocinas (50.000, 10.000 e 2.000 pg/rnL) e desafiados in vitro com leveduras viáveis de P. brasiliensis (relação fungo-macrófagos de 1:50). Após 72h de incubação a atividade fungicida dos macrófagos foi avaliada através da contagem do número de fungos viáveis pelo método de unidades formadoras de colônias (UFC). A produção de nitrito e de citocinas foi avaliada no sobrenadante de cultivo através da reação de Griess e ELISA, respectivamente. Os dados foram expressos como média ± EP e analisados pelo teste T -Student. Nossos resultados mostraram que os macrófagos de camundongos B 10.A pré-ativados com IFN-γ, IL-12 ou ambas as citocinas nas diferentes concentrações ensaiadas apresentaram elevada capacidade fungicida (51-97%) acompanhada de produção aumentada de NO, IL-12 e MCP-l e baixa síntese de IL-10 e GM-CSF. Por outro lado, somente o tratamento com a mais alta concentração de IFN-γ foi capaz de induzir atividade fungicida em macrófagos alveolares de animais A/J. A síntese de NO ocorreu sempre em baixos níveis e nos sobrenadantes dos co-cultivos observamos altos níveis de IL-10 e GM-CSF associados a baixas concentrações de IL-12 e MCP-l. A inibição da síntese de NO por aminoguanidina demonstrou que a atividade fungicida dos macrófagos de animais B10.A, mas não de A/J, era mediada por este composto tratamento dos macrófagos com anticorpo monoclonal anti-IL-10 não alterou a capacidade fungicida dos macrófagos de ambas as linhagens, entretanto induziu franca produção de NO que, para animais A/J, não se traduziu em atividade microbicida aparente. Por outro lado. a neutralização de TGF-β induziu alta capacidade fungicida das células de animais A/J níveis aumentados de NO e TNF-α aliados a níveis reduzidos de IL-10. O mesmo tratamento não alterou a já elevada capacidade fungicida dos macrófagos alveolares de animais B10.A mas aumentou a produção de NO, IL-12 e TNF-α. Em conclusão, os macrófagos alveolares de camundongos B10.A são facilmente ativáveis por 1FN-γ e IL-12, apresentam elevada capacidade fungicida, NO-dependente, sobre leveduras do P. brasiliensis associada com a síntese de níveis elevados de NO e IL-12. Por outro lado, os macrófagos de camundongos A/J são pobremente ativados por 1FN-γ e IL-12, produzem baixos níveis de NO, a sua capacidade fungicida, que parece ser NO-independente, pode ser aumentada pela neutralização do TGF-β endógeno, mas não pelo tratamento com anti-IL-10. / Previous studies in our laboratory characterized B10.A and A/J mice as susceptible and resistant strains to pulmonary Paracoccidioides brasiliensis infection. Innate immunity plays a fundamental role on the control of the initial growth of pathogens as well as in the acquired immunity that subsequently develops. As alveolar macrophages are the first host cells to interact with P. brasiliensis we decided to study the fungicidal and secretory ability of alveolar macrophages from resistant and susceptible mice to P. brasiliensis to better understand the pulmonary model of paracoccidioidomycosis. Normal B10.A and A/J mice (n=10-15) were submitted to bronchoalveolar lavage (BAL) and cell suspensions (2x105 cells/well) were pre-activated ovemight with IFN-γ, IL-12 or the combination of these two cytokines (50,000, 10,000 and 2,000 pg/rnL). After that, macrophages were in vitro challenged with P. brasiliensis yeasts (1:50 fungus: macrophage ratio) and 72h later fungicidal activity was determined by colony forming units counts (CFU). Nitrite and cytokines production were determined in culture supematants by Griess reaction and ELISA, respectively. Data were expressed as means ± SE and analyzed by Student\'s t test. Our results showed that B10.A macrophages pre-activated with the different assayed concentrations of IFNγ, IL-12 or both cytokines presented elevated fungicidal ability (51¬-97%) concomitant with the presence of high levels of NO, IL-12 and MCP-l and low amounts of IL-10 and GM-CSF. NO synthesis occurred in low levels but high concentrations of IL-10 and GM-CSF associated to low amounts of IL-12 and MCP-1 were detected in the co-cultures supematants. NO synthesis inhibition by aminoguanidine clearly showed that the fungicidal ability of B10.A but not of A/J macrophages was NO¬ dependent. Treatment with anti-IL-10 monoclonal antibodies did not alter the fungicidal ability of macrophages from both mouse strains but enhanced NO synthesis which, however, did not alter the absent microbicidal ability of A/J macrophages. On the contrary, anti-TGF-β treatment induced an increased fungicidal ability of A/J cells associated with enhanced levels of NO and TNF-α besides diminished amounts of IL-10. The same treatment did not alter the high fungicidal ability of B10.A alveolar macrophages but increased NO, IL-12 and TNF-α production. In conclusion, alveolar macrophages from susceptible mice are easily activated by IFNγ and IL-12, present a high and NO-dependent ability to kill P. brasiliensis yeasts and secrete elevated levels of NO and IL-12. In contrast, alveolar macrophages from resistant mice are poorly activated by IFNγ and IL¬12, secrete low amounts of NO and high of IL-10. Their fungicidal ability which appears to be NO-independent can be restored by neutralization of endogenous TGF-β but not by anti-IL-10 treatment.