Orientador: Antonio de Goes / Banca: Edson Luiz Furtado / Banca: Kátia Cristina Kupper / Banca: Rita de Cássia Panizzi / Banca: Jaime Maia dos Santos / Resumo: A mancha preta dos citros (MPC) doença causada pelo fungo Guignardia citricarpa Kiely [anamorfo: Phyllosticta citricarpa (McAlp.)Van der Aa], deprecia comercialmente os frutos, causando sua queda prematura e elevando o custo de produção. Até o presente momento, informações acerca da etiologia da doença são escassas. Portanto, o presente trabalho teve como objetivos: (i) criar metodologias alternativas de obtenção da estrutura reprodutiva do fungo em laboratório; (ii) observar a taxa de germinação in vitro de conídios do fungo; (iii) estudar a influência da poda de plantas cítricas no campo sobre a expressão de sintomas de falsa melanose; (iv) e, a relação entre a presença de sintomas de ácaro da falsa ferrugem e sintomas do tipo mancha trincada em frutos cítricos. De maneira geral, tanto pseudotécios quanto ascósporos foram obtidos em maior número quando da utilização da metodologia de discos foliares autoclavados, bem como a maior expressão dos sintomas mediante inoculações através desta metodologia. Com os resultados obtidos, pode-se afirmar que através da realização de práticas culturais como a poda dos ramos secos, bem como o controle do ácaro da falsa ferrugem, o progresso da MPC no campo é reduzido. / Abstract: Citrus black spot (CBS) disease caused by the fungus Guignardia citricarpa Kiely [anamorph: Phyllosticta citricarpa (McAlp.)Van der Aa], depreciates the fruit comercially, causing its premature fall and elevating production cost. Until the present moment, there is a lack of informations about the etiology of the disease. Therefore, the aim of the present work was: (i) create alternative methodologies to obtain reproductive structures on laboratory; (ii) observe the in vitro conidia germination rate; (iii) study the influence of the harvest of citric plants on the expression of false melanose symptoms; (iv) and the relation between the expression of symptoms of citrus rust mite on the presence of cracked spot symptoms. Both pseudothecia and ascospores were obtained in larger rate when using autoclavated leave discs, and also the larger expression of symptoms on inoculations made by this method. By the results obtained, it can be afirmed that the harvest of dead twigs, and the control of the citrus rust mite, the progress of CBS on field is reduced. / Doutor

Made available in DSpace on 2014-06-11T19:33:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-03-29Bitstream added on 2014-06-13T19:04:54Z : No. of bitstreams: 1 nozaki_mh_dr_jabo.pdf: 745023 bytes, checksum: 9d7c07f38a8943e8fa117a5e301b9c6f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A mancha preta dos citros (MPC) doença causada pelo fungo Guignardia citricarpa Kiely [anamorfo: Phyllosticta citricarpa (McAlp.)Van der Aa], deprecia comercialmente os frutos, causando sua queda prematura e elevando o custo de produção. Até o presente momento, informações acerca da etiologia da doença são escassas. Portanto, o presente trabalho teve como objetivos: (i) criar metodologias alternativas de obtenção da estrutura reprodutiva do fungo em laboratório; (ii) observar a taxa de germinação in vitro de conídios do fungo; (iii) estudar a influência da poda de plantas cítricas no campo sobre a expressão de sintomas de falsa melanose; (iv) e, a relação entre a presença de sintomas de ácaro da falsa ferrugem e sintomas do tipo mancha trincada em frutos cítricos. De maneira geral, tanto pseudotécios quanto ascósporos foram obtidos em maior número quando da utilização da metodologia de discos foliares autoclavados, bem como a maior expressão dos sintomas mediante inoculações através desta metodologia. Com os resultados obtidos, pode-se afirmar que através da realização de práticas culturais como a poda dos ramos secos, bem como o controle do ácaro da falsa ferrugem, o progresso da MPC no campo é reduzido. / Citrus black spot (CBS) disease caused by the fungus Guignardia citricarpa Kiely [anamorph: Phyllosticta citricarpa (McAlp.)Van der Aa], depreciates the fruit comercially, causing its premature fall and elevating production cost. Until the present moment, there is a lack of informations about the etiology of the disease. Therefore, the aim of the present work was: (i) create alternative methodologies to obtain reproductive structures on laboratory; (ii) observe the in vitro conidia germination rate; (iii) study the influence of the harvest of citric plants on the expression of false melanose symptoms; (iv) and the relation between the expression of symptoms of citrus rust mite on the presence of cracked spot symptoms. Both pseudothecia and ascospores were obtained in larger rate when using autoclavated leave discs, and also the larger expression of symptoms on inoculations made by this method. By the results obtained, it can be afirmed that the harvest of dead twigs, and the control of the citrus rust mite, the progress of CBS on field is reduced.

Made available in DSpace on 2014-06-11T19:28:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-05Bitstream added on 2014-06-13T19:37:04Z : No. of bitstreams: 1 ikeda_m_me_jabo.pdf: 375593 bytes, checksum: 6a24c080db13dd8a16ff7686dcbed145 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundecitrus / O objetivo deste trabalho foi avaliar a resposta do número de aplicações do fungicida pyraclostrobin no controle da Mancha Preta dos Citros (Guignardia citricarpa), assim como comparar o comportamento das diferentes estrobilurinas recomendadas no controle da doença. Também foi avaliada a influência dos tratamentos fungicidas sobre a produção e qualidade tecnológica dos frutos. Para tal foram realizados dois experimentos, em pomares de laranjeira „Valência‟ com histórico comprovado da ocorrência da doença em anos anteriores e com elevada intensidade e frequência na distribuição dos sintomas entre as plantas. Foram avaliados seis tratamentos, com três a quatro aplicações de fungicidas sistêmicos, isolado ou em mistura com cobre. Os tratamentos citados foram precedidos por duas aplicações de fungicida cúprico (CO), iniciando-se na fase de 2/3 de pétalas caídas e repetidas depois de quatro semanas. As aplicações de produtos sistêmicos (três ou quatro) foram realizadas quatro semanas após a segunda aplicação de fungicida cúprico, com intervalo de seis semanas entre elas. As estrobilurinas avaliadas foram pyraclostrobin (PY), azoxystrobin (AZ) e trifloxystrobin (TR) isolado ou em combinação com fungicida cúprico. Todos os tratamentos aplicados receberam um tratamento adicional com carbendazim (CA) como fechamento das pulverizações. Dessa forma, os tratamentos e as respectivas sequências foram:... / The objective of this study was to evaluate the number of applications of the fungicide pyraclostrobin for control of Citrus Black Spot (Guignardia citricarpa) and compare the action of the different recommended strobilurins for disease control. We also analyzed the influence of fungicide treatments on the production and technological quality of fruit. For this purpose two experiments were conducted in orchards of „Valencia‟ sweet orange with proven track records of disease occurrence in previous years and with high intensity and frequency distribution of symptoms among the plants. We evaluated six treatments, with three or four applications of systemic fungicides, alone or mixed with copper. The cited treatments were preceded by two applications of copper fungicide (CO), beginning on the phase 2/3 of petals fallen and repeated after four weeks. The applications of systemic products (three or four) were performed four weeks after the second application of cooper fungicide, with an interval of six weeks between them. The strobilurins tested were pyraclostrobin (PY), azoxystrobin (AZ) and trifloxystrobin (TR) alone or in combination with cooper fungicide. All treatments received an additional treatment with carbendazim (CA) as the closure of sprays. Thus, treatments and their sequences were:... (Complete abstract click electronic access below)

Citrus is a valuable fruit crop in world trade. Citrus Black Spot (CBS), caused by Guignardia citricarpa Kiely, is a fungal disease of citrus. It occurs in many citrus producing countries including parts of Australia and South Africa, but it does not occur in the countries of the European Union (EU) or the United States of America (USA). To prevent the introduction of CBS, the EU and the USA have phytosanitary regulations that restrict the import of citrus fruit from areas where CBS is found. This study uses two bioclimatic modelling approaches — CLIMEX and response surface modelling — to predict which areas have climates suitable for CBS to establish. The work focuses on the citrus growing areas of South Africa and Europe, but other parts of the world are also considered. As CBS is dependent on citrus, geographical areas of global citrus production are also mapped, and models are used to predict which areas of South Africa have climates suitable for citrus cultivation under current and future climates. The potential impacts of climate change on CBS distribution in South Africa are also estimated. Results indicate that under current and future climates many areas in South Africa where citrus is not currently grown have a climate suitable for citrus cultivation, but most of these areas are also climatically favourable for CBS. Of the current citrus producing areas in South Africa, only the Northern and Western Cape Provinces are predicted to be unsuitable for CBS. Under climate change scenarios, some citrus production areas of Western Cape are predicted to become suitable for CBS, but the greater part of the Northern Cape will remain climatically unsuitable for the establishment of CBS in future. The climates of several CBS-free citrus producing areas around the world, such as Mexico, and Florida and Texas (USA) are suitable for CBS. However, European climate is unfavourable for CBS establishment, and provided importing countries comply to minimum standards, phytosanitary restrictions on the import of fruit from CBS infected areas may be unnecessary. This study is the first of its kind in citriculture, and in South Africa it is one of the few studies that investigates the effects of climate change on the potential distribution of a plant pathogen. Bioclimatic modelling was found to be a very useful means to combine complex data in order to make predictions relevant to Pest Risk Assessments. / Thesis (PhD (Environmental Management))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Orientador: Antonio de Goes / Banca: Rita de Cássia Panizzi / Banca: Kátia Cristina Kupper / Resumo: O objetivo deste trabalho foi avaliar a resposta do número de aplicações do fungicida pyraclostrobin no controle da Mancha Preta dos Citros (Guignardia citricarpa), assim como comparar o comportamento das diferentes estrobilurinas recomendadas no controle da doença. Também foi avaliada a influência dos tratamentos fungicidas sobre a produção e qualidade tecnológica dos frutos. Para tal foram realizados dois experimentos, em pomares de laranjeira „Valência‟ com histórico comprovado da ocorrência da doença em anos anteriores e com elevada intensidade e frequência na distribuição dos sintomas entre as plantas. Foram avaliados seis tratamentos, com três a quatro aplicações de fungicidas sistêmicos, isolado ou em mistura com cobre. Os tratamentos citados foram precedidos por duas aplicações de fungicida cúprico (CO), iniciando-se na fase de 2/3 de pétalas caídas e repetidas depois de quatro semanas. As aplicações de produtos sistêmicos (três ou quatro) foram realizadas quatro semanas após a segunda aplicação de fungicida cúprico, com intervalo de seis semanas entre elas. As estrobilurinas avaliadas foram pyraclostrobin (PY), azoxystrobin (AZ) e trifloxystrobin (TR) isolado ou em combinação com fungicida cúprico. Todos os tratamentos aplicados receberam um tratamento adicional com carbendazim (CA) como fechamento das pulverizações. Dessa forma, os tratamentos e as respectivas sequências foram:... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the number of applications of the fungicide pyraclostrobin for control of Citrus Black Spot (Guignardia citricarpa) and compare the action of the different recommended strobilurins for disease control. We also analyzed the influence of fungicide treatments on the production and technological quality of fruit. For this purpose two experiments were conducted in orchards of „Valencia‟ sweet orange with proven track records of disease occurrence in previous years and with high intensity and frequency distribution of symptoms among the plants. We evaluated six treatments, with three or four applications of systemic fungicides, alone or mixed with copper. The cited treatments were preceded by two applications of copper fungicide (CO), beginning on the phase 2/3 of petals fallen and repeated after four weeks. The applications of systemic products (three or four) were performed four weeks after the second application of cooper fungicide, with an interval of six weeks between them. The strobilurins tested were pyraclostrobin (PY), azoxystrobin (AZ) and trifloxystrobin (TR) alone or in combination with cooper fungicide. All treatments received an additional treatment with carbendazim (CA) as the closure of sprays. Thus, treatments and their sequences were:... (Complete abstract click electronic access below) / Mestre

Citrus black spot (CBS), caused by Guignardia citricarpa Kiely, occurs in various citrus producing regions of the world. Due to the potential phytosanitary risk associated with the export of fruit from CBS positive production areas to CBS-free countries, restrictive trade barriers have been introduced. This study aimed to further elucidate some epidemiological aspects of CBS that can be used to address critical questions identified in the pest risk assessment submitted by South Africa to the World Trade Organisation to address phytosanitary trade restrictions. Results indicated that Eureka lemon leaf litter exposed to viable pycnidiospores under controlled conditions or in the field in different production regions of South Africa, were not infected and colonised by G. citricarpa. Symptomatic CBS fruit or peel lying on the ground underneath citrus trees therefore can not lead to infection and colonisation of freshly detached leaves or leaf litter, or represent a source of inoculum in citrus orchards. Symptomatic fruit therefore pose no danger for the establishment of the pathogen in CBS-free orchards and are not considered to be a pathway for the pathogen. The period of leaf susceptibility to G. citricarpa was indicated to be maximum eight and ten months from development, for Valencia orange and Eureka lemon, respectively, in a greenhouse study. The susceptibility period of citrus leaves to infection by the black spot pathogen could be longer than previously perceived. Ascospores were captured, using the newly developed Kotzé Inoculum Monitor (KIM), from natural Valencia orange and Eureka lemon leaf litter during October to March with peak ascospore availability between December to February. The KIM is the first sampler designed to capture fungal spores directly from plant material in the laboratory without environmental influences and was effectively used to confirm that ascospores production is seasonal. The KIM in combination with environmental data can be used to improve control through more targeted fungicide applications. Techniques such as isolations and DNA amplification with species-specific primers to detect the pathogen directly from symptomless green leaves have a low success rate due to the restricted growth of the pathogen in latently infected tissue. Artificial leaf wilting enhanced the detection of G. citricarpa from symptomless leaves. Leaf wilting is a reliable, fast and effective method to detect the CBS pathogen and can be applied to monitor citrus nurseries and orchards throughout the year. It can also be applied to monitor pest-free orchards to maintain its CBS pest-free status. This study confirmed that sanitation practices, such as leaf litter removal and mulching of leaf litter with wheat straw can decrease the primary inoculum, ascospores, of CBS and contribute to better management of the disease in a commercial orchard. Regardless of the prevailing climatic conditions each year, control achieved through leaf litter management resulted in >95% clean fruit and are equal to the control achieved with industry standard fungicides. This approach provided improved integrated disease control and an alternative to chemical control. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Citrus black spot (CBS) caused by Guignardia citricarpa is responsible for economic losses in Southern African countries such as South Africa, Mozambique, Swaziland, Zimbabwe and Namibia. Black spot is considered to be a phytosanitary disease for the European Union and the United States of America markets. Exporters to these countries incur losses throughout the supply chain due to phytosanitary restrictions. For these reasons, the occurrence and management practices of CBS and its impact on growers in Southern Africa were investigated through a survey using a questionnaire. In the study, it was found that when CBS was present it was primarily managed by using chemicals and general orchard sanitation. In addition, growers in some of the surveyed countries or production regions follow spraying programs that are based on disease forecasting models and this practice has proven very effective in managing the disease. Furthermore, furfural, a sugarcane waste product was assessed for its efficacy in controlling G. citricarpa. The efficacy of the product as a contact or a fumigant was demonstrated in vitro and in vivo on fresh leaves, leaf litter and fruit lesions as well as in soil. A molecular study, using a Polymerase Chain Reaction protocol was conducted to assess the survival of the pathogen in the soil after exposure to furfural. The product however only proved efficient under natural conditions. The non-target effect of furfural on the soil micro-flora was also assessed. The product proved suitable for soil applications as it is not phytotoxic and has minimal non-target effects on bacterial populations. Furfural proved to control G. citricarpa, by breaking the life cycle, thus reducing the disease incidence. The application of furfural on a larger scale (irrigation or spraying) will therefore improve the control of CBS in developing countries. / Dissertation (MInstAgrar)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Devido à percepção dos consumidores sobre o impacto da utilização de pesticidas sobre o ambiente e saúde humana, além da aquisição de resistência por parte dos fitopatógenos, a sociedade tem exercido pressões que levaram ao estabelecimento de políticas governamentais que restringem a utilização de fungicidas levando agricultores e pesquisadores a considerar a aplicação de técnicas de controle biológico de fitopatógenos fúngicos. Guignardia citricarpa é o agente causal da pinta preta dos citros, que é de grande importância econômica, pois interfere na produção e causa depreciação estética dos frutos, acarretando em prejuízos principalmente na comercialização de frutos in natura para o mercado externo. Neste contexto, o objetivo deste trabalho foi a avaliação in vitro do potencial de linhagens de Saccharomyces cerevisiae, utilizadas em processos fermentativos, como agentes de biocontrole contra G. citricarpa. Através de ensaio em placa, foi evidenciado que entre as linhagens de S. cerevisiae testadas (BG-1, CR-1, CAT-1, KD-1, K-1 e PE-2), a CR-1 demonstrou a maior atividade antagônica contra o fitopatógeno, causando 73% de inibição do crescimento micelial. Também foi demonstrado que as linhagens são capazes de produzir voláteis de ação fungistática inibindo em até 83% o desenvolvimento do patógeno. O filtrado de cultura autoclavado e não autoclavado, bem como as células inativadas termicamente, obtidas a partir do crescimento da linhagem CR-1 por 24 h em meio YEPD, não causaram redução do crescimento vegetativo do fungo. A produção de enzimas hidrolíticas extracelulares (quitinases, β-1,3-glucanases e proteases) pela levedura não foi detectada em meio YEPD contendo glicose ou preparação de parede celular de G. citricarpa, nos tempos de cultivo avaliados. Baseado nas informações obtidas, foi possível constatar que as linhagens de S. cerevisiae, em especial a linhagem CR-1, são potenciais antagonistas para o controle de G. citricarpa. O possível mecanismo utilizado para inibição pela levedura é a produção de voláteis, no entanto, outros mecanismos não podem ser descartados. Desta forma, o presente trabalho mostra o potencial de S. cerevisiae no controle de G. citricarpa em frutos de laranja na pós-colheita. / Due to the consumers perception about the impact caused by pesticides utilization over the environment and human health, besides the acquisition of resistance for part of the phytopathogens, the society has exercised pressures that had led to the establishment of governmental politics that restrict the use of fungicides leading agriculturists and researchers to consider the application of techniques of biological control of plant pathogenic fungi. Guignardia citricarpa is the causal agent of citrus black spot that has a great economic importance, therefore interfering in production and causing aesthetic depreciation of the fruits that can interfere with commercialization of fresh-fruit in the external market. In this context, the aim of this work was to evaluate in vitro the potential of Saccharomyces cerevisiae strains, used in fermentative process, as biocontrol agents against G. citricarpa. Through plate assay it was evidenced that among the tested strains of S. cerevisiae (BG-1, CR-1, CAT-1, KD-1, K-1 and PE-2), the strain CR-1 was the one that demonstrated the greatest antagonic activity against the phytopathogen, causing 73% of micelial growth inhibition. It was also demonstrated that the strains were able to produce volatile compounds with fungistatic action inhibiting up to 83% the development of the pathogen. The autoclaved and not autoclaved culture filtrate, as well as the termical inactivated cell obtained from the growth of strain CR-1 in YEPD medium for 24 h, did not cause reduction in the fungal vegetative growth. The production of extracellular hydrolytic enzymes (chitinases, β-1,3-glucanases and proteases) by the yeast was not detected in YEPD medium with glucose or cell wall preparation of G. citricarpa at the evaluated times. Based upon the obtained information it was possible to evidence that the strains of S. cerevisiae, specially the strain CR-1, are potentials antagonists for the control of G. citricarpa. The possible mechanism used for inhibition by yeast is the volatile production, however other mechanisms cannot be discarded. Thus, the present work shows the potential of S. cerevisiae to control G. citricarpa in orange fruits in postharvest.

O Brasil é líder absoluto no comércio internacional de suco de laranja concentrado congelado participando com 82% do volume comercializado no mundo. Guignardia citricarpa é um fungo Ascomiceto agente causal da Mancha Preta dos Citros (MPC), uma doença importante no contexto da Citricultura, causando lesões negras em frutos tornando-os impróprios para a exportação, já que não são aceitos na União Européia pois o patógeno é classificado como quarentenário. O presente trabalho teve como objetivo principal estabeler a metodologia de transformação genética de G. citricarpa para futuramente auxiliar no entendimento dos mecanismos de patogenicidade desta espécie, visando diminuir perdas na citricultura brasileira devido à MPC. Além disso, foi realizada uma análise do gene da enzima endopoligalacturonase, a qual está associada à capacidade de patógenos em colonizar plantas. Para elucidar esse fenômeno, foi realizada a busca de genes relacionados à patogenicidade em outros fungos fitopatogênicos previamente descritos e confirmados participarem no processo de doenças em diversas plantas. Considerando que esses genes possuem uma região conservada para esse fim, após o alinhamento dessas sequências foram construídos primers e o gene de endopoligalacturonase foi identificado no gênero Guignardia sp. Outra espécie pertencente a esse gênero é G. mangiferae, conhecida como endófito de citros, ou seja, coloniza os tecidos internos da planta hospedeira sem causar dano. Em análises enzimáticas foi observado que a quantidade de endopoligalacturonase produzida pela espécie patogênica é superior à da espécie endofítica, mostrando que essa enzima pode participar do processo de patogenicidade de G. citricarpa. Estudos para comprovar a participação de genes nos mecanismos de patogenicidade em diversas espécies utilizando reconhecimento de genes e genômica funcional, expressão e knockout de genes estão sendo realizados, permitindo uma visão geral da organização genômica do sistema patogênico. Pensando nisso, esse trabalho descreve pela primeira vez a metodologia de transformação genética de G. citricarpa via micélio e a obtenção de transformantes expressando a proteína verde fluorescente (GFP). Micélios do fungo foram transformados pelo sistema via Agrobacterium tumefaciens com o plasmídeo pFAT-gfp, contendo os genes de resistência à higromicina B (hgr) e da GFP. A otimização do protocolo de agrotransformação foi realizada a partir do teste de diferentes condições como: tipo de membrana, concentração de agente indutor e tempo de cocultivo. A melhor condição incluiu a utilização de membrana de éster de celulose; 200 PM de AS e 96 horas de co-cultivo. Os transformantes apresentaram alta estabilidade mitótica (82%) e tiveram a inserção do gene hgr confirmada por PCR e do gfp observada em microscopia óptica de epifluorescência. Além disso, foi acompanhado o desenvolvimento do fungo inoculado em frutos, mostrando a interação planta-patógeno. O estabelecimento do sistema de transformação por Agrobacterium para G. citricarpa possibilita o uso dessa ferramenta para estudos de mutagênese insercional e interrupção gênica visando a identificação de genes importantes, como os envolvidos com os mecanismos de patogenicidade utilizados por esse fungo. / Brazil is the world leader in the international trade of frozen orange juice concentrate, taking part with around 82% of the traded volume. Guignardia citricarpa (anamorph Phyllosticta citricarpa) is a fungal pathogen of citrus plants, being described as the causal agent of citrus black spot (CBS), one of the most important fungal diseases of citrus worldwide. Its symptoms are black lesions on fruit, making them unsuitable for the international fresh market, since they are included in the quarantine list of the European Plant Protection Organization (EPPO). Moreover, when the disease is severe it may cause extensive premature fruit drop that reduces yields of fruit for processing. Taking this into consideration, the current work aimed to improve the understanding on the pathogenic mechanisms of this fungus. Firstly, in an attempt to elucidate this phenomenon, it was performed a search for pathogenicity genes previously reported to some pathogenic fungi and confirmed to participate in the process of infection in many plants , especially endopolygalacturonase. Primers were designed using the conserved regions of the genes and allowed the identification of the Guignardia spp. endopolygalacturonase gene for the first time. This enzyme has been described as playing important role in the process of fungal diseases in plants. In the present work, enzymatic analysis showed that the pathogen G. citricarpa produced significantly greater amounts of endopolygalacturonase when compared to G. mangiferae, a closely related fungus described as a citrus endophyte. This result suggests that this enzyme may participate in the process of pathogenicity, characteristic of the pathogenic species. Genetic transformation methods have been used to prove the involvement of genes in pathogenic mechanisms, however, a suitable methodology for G. citricarpa has not been described yet. In this way, this study describes for the first time a methodology for genetic transformation of G. citricarpa via mycelia and the successful generation of transformants expressing the green fluorescent protein (GFP) and resistant to the hygromycin B antibiotic. Mycelia of the fungus were genetically transformed via Agrobacterium tumefaciens hosting the plasmid pFAT-gfp, which carries the genes for resistance to hygromycin B (hph) and for GFP (gfp). The protocol was optimized through different test conditions (type of membrane, concentration of the inducing agent acetosyringone and duration of the co-cultivation period). The higher transformation efficiencies were observed using cellulose ester membrane, 200 PM of acetosyringone and 96 hours of cocultivation. The transformants showed high mitotic stability (82%) and the insertion of the T-DNA was confirmed by PCR and GFP expression through epifluorescence microscopy observation. Moreover, it was observed the development of the fungus in inoculated oranges, showing the plant-pathogen interaction observed by epifluorescense microscopy. The establishment of the Agrobacterium-mediated transformation system for G. citricarpa represents an important step on the search for unveiling important genes of this fungus, such as those involved in the pathogenic mechanisms.

Devido à percepção dos consumidores sobre o impacto da utilização de pesticidas sobre o ambiente e saúde humana, além da aquisição de resistência por parte dos fitopatógenos, a sociedade tem exercido pressões que levaram ao estabelecimento de políticas governamentais que restringem a utilização de fungicidas levando agricultores e pesquisadores a considerar a aplicação de técnicas de controle biológico de fitopatógenos fúngicos. Guignardia citricarpa é o agente causal da pinta preta dos citros, que é de grande importância econômica, pois interfere na produção e causa depreciação estética dos frutos, acarretando em prejuízos principalmente na comercialização de frutos in natura para o mercado externo. Neste contexto, o objetivo deste trabalho foi a avaliação in vitro do potencial de linhagens de Saccharomyces cerevisiae, utilizadas em processos fermentativos, como agentes de biocontrole contra G. citricarpa. Através de ensaio em placa, foi evidenciado que entre as linhagens de S. cerevisiae testadas (BG-1, CR-1, CAT-1, KD-1, K-1 e PE-2), a CR-1 demonstrou a maior atividade antagônica contra o fitopatógeno, causando 73% de inibição do crescimento micelial. Também foi demonstrado que as linhagens são capazes de produzir voláteis de ação fungistática inibindo em até 83% o desenvolvimento do patógeno. O filtrado de cultura autoclavado e não autoclavado, bem como as células inativadas termicamente, obtidas a partir do crescimento da linhagem CR-1 por 24 h em meio YEPD, não causaram redução do crescimento vegetativo do fungo. A produção de enzimas hidrolíticas extracelulares (quitinases, β-1,3-glucanases e proteases) pela levedura não foi detectada em meio YEPD contendo glicose ou preparação de parede celular de G. citricarpa, nos tempos de cultivo avaliados. Baseado nas informações obtidas, foi possível constatar que as linhagens de S. cerevisiae, em especial a linhagem CR-1, são potenciais antagonistas para o controle de G. citricarpa. O possível mecanismo utilizado para inibição pela levedura é a produção de voláteis, no entanto, outros mecanismos não podem ser descartados. Desta forma, o presente trabalho mostra o potencial de S. cerevisiae no controle de G. citricarpa em frutos de laranja na pós-colheita. / Due to the consumers perception about the impact caused by pesticides utilization over the environment and human health, besides the acquisition of resistance for part of the phytopathogens, the society has exercised pressures that had led to the establishment of governmental politics that restrict the use of fungicides leading agriculturists and researchers to consider the application of techniques of biological control of plant pathogenic fungi. Guignardia citricarpa is the causal agent of citrus black spot that has a great economic importance, therefore interfering in production and causing aesthetic depreciation of the fruits that can interfere with commercialization of fresh-fruit in the external market. In this context, the aim of this work was to evaluate in vitro the potential of Saccharomyces cerevisiae strains, used in fermentative process, as biocontrol agents against G. citricarpa. Through plate assay it was evidenced that among the tested strains of S. cerevisiae (BG-1, CR-1, CAT-1, KD-1, K-1 and PE-2), the strain CR-1 was the one that demonstrated the greatest antagonic activity against the phytopathogen, causing 73% of micelial growth inhibition. It was also demonstrated that the strains were able to produce volatile compounds with fungistatic action inhibiting up to 83% the development of the pathogen. The autoclaved and not autoclaved culture filtrate, as well as the termical inactivated cell obtained from the growth of strain CR-1 in YEPD medium for 24 h, did not cause reduction in the fungal vegetative growth. The production of extracellular hydrolytic enzymes (chitinases, β-1,3-glucanases and proteases) by the yeast was not detected in YEPD medium with glucose or cell wall preparation of G. citricarpa at the evaluated times. Based upon the obtained information it was possible to evidence that the strains of S. cerevisiae, specially the strain CR-1, are potentials antagonists for the control of G. citricarpa. The possible mechanism used for inhibition by yeast is the volatile production, however other mechanisms cannot be discarded. Thus, the present work shows the potential of S. cerevisiae to control G. citricarpa in orange fruits in postharvest.