Colletotrichum gossypii var. cephalosporioides, fungo causador da ramulose do algodoerio, é ocorre disperso em quase todo Brasil. Sem um eficiente esquema de aplicação de fungicidas, a ramulose pode provocar severos danos. Outra importante doença para o algodoeiro, em muitas regiões do mundo, é a mancha angular, causada por Xanthomonas axonopodis pv. malvacearum. O objetivo deste trabalho foi desenvolver um índice de favorabilidade baseado em variáveis meteorológicas para estimar a ocorrência da ramulose e da mancha angular (MA) do algodoeiro. O período de incubação, a freqüência de infecção e a severidade das doenças foram avaliados em câmaras de crescimento mantidas a 15, 20, 25 e 30&#176;C e com períodos de câmara úmida de 0, 2, 4, 8, 16, 32, e 64 horas após inoculação com suspensão inóculo de 105 conídios mL-1 e 106 IFC mL-1. Severidade da doença. Severidade (MA) e número de lesões por área (Ramulose) foram modelados como função da duração do período de molhamento (DPM) e da temperatura (T). Em experimentos de campo em Piracicaba, SP, parcelas de algodão foram inoculadas com ramulose e mancha angular, separadamente, a fim de se avaliar semanalmente o progresso das doenças ao longo do tempo. Os dados de temperatura do ar e molhamento foliar no campo foram utilizados no modelo obtido com os resultados de câmaras de crescimento e assim calcular o índice de favorabilidade. As variáveis meteorológicas radiação solar, T, umidade relativa do ar, DPM, chuva e velocidade do vento foram avaliadas como possíveis variáveis explanatórias às taxas de crescimento da doença no campo. Testou-se também, como variável explanatória, o índice de favorabilidade (0,0<IF<1,0), calculado com os modelos de superfície utilizando os dados de T e DPM, obtidos dos experimentos de campo. Em câmaras de crescimento, a temperatura ótima foi de 27&#176;C para ramulose e 22&#176;C para mancha angular, com período de incubação de 10 dias para ramulose e entre 5 e 6 dias para mancha angular. A máxima severidade de ramulose ocorreu entre 25 e 30&#176;C e a máxima severidade de ALS ocorreu entre 20 e 25&#176;C. A severidade de ambas diminui rapidamente em temperaturas maiores ou menores que nessa faixa. A severidade de ramulose aumentou no intervalo de 4 até 32 horas de molhamento enquanto que ocorreu o mesmo com ALS no intervalo de 0 a 8 horas de molhamento. A severidade de ambas foi melhor ajustada por um modelo exponencial logarítmico em função do molhamento que, pela substituição dos parâmetros por funções de temperatura, descreveu a superfície de resposta com elevada precisão e exatidão. No campo, a taxa de crescimento da ramulose foi melhor relacionada ao índice de favorabilidade e à chuva, por regressão não linear, com coeficientes de determinação de 0,89 e 0,91, respectivamente. A taxa de crescimento da mancha angular apresentou razoável relação linear com as variáveis temperatura durante o período de molhamento e vento máximo, com R2 de 0,75 e 0,84 respectivamente. / Colletotrichum gossypii var. cephalosporioides, the fungus that causes ramulosis in cotton crops, is widespread in Brazil. Without an efficient fungicide schedule, ramulosis disease could provoke severe yield losses on cotton. Other important disease to cotton crop in several regions of the world is the bacterial blight also known as angular leaf spot (ALS), caused by Xanthomonas axonopodis pv. malvacearum. The objective of this work was develop a weather based favorability index to each of this diseases. The incubation period, the infection frequency and the severity of both diseases were evaluated in growth-chamber experiments, incubated at 15, 20, 25, 30, and 35&#176;C while exposed to wetness periods of 0, 2, 4, 8, 16, 32 and 64 h after inoculation with 105 conidium mL-1 and 106 CFU mL-1. Disease severity (ALS) and number of lesions per leaf area (ramulosis), was modeled as a function of leaf wetness duration (LWD) and temperature (T). At the field experiments in Piracicaba, cotton plots were inoculated with both pathogens separately, and disease severity was evaluated weekly. LWD and T data from the field trials were used as input to the model from growth chambers results and output the disease favorability index. Weather variables as solar radiation, temperature, relative humidity, wetness duration, rain, and wind speed were also evaluated as possible explanatory variables to the disease growth rate in the field. Was also tested the favorability index (0.0<FI<1.0), as explanatory variable, calculated with the surface models using T and LWD from the field. In growth chambers, the optimum temperature for ramulosis was 27&#176;C and 22&#176;C for ALS and incubation of 10 days to ramulosis and 5 to 6 days do ALS. Maximum ramulosis severity occurred from 20 to 30&#176; C, and between 20 and 25&#176;C to bacterial blight, and both decreased sharply at warmer or cooler temperatures. Ramulosis severity increased as wetness periods were increased from 4 to 32 h. Bacterial blight severity increased from 0 to 8 h of wetness period. The severity of both diseases, were best fitted by a logarithmic exponential model as function of LWD which, by the substitution of parameters for temperature functions, described the surface response with high precision and accuracy, even being a very flexible model. In the field, ramulosis growth rate was strongly correlated with values of the disease favorability index and rainfall with R2=0.89 and R2=0.91, respectively. Bacterial blight growth rate had best linear correlation to temperature during the wetness period and maximum wind with R2=0.75 and R2=0.84, respectively.

Colletotrichum gossypii var. cephalosporioides, fungo causador da ramulose do algodoerio, é ocorre disperso em quase todo Brasil. Sem um eficiente esquema de aplicação de fungicidas, a ramulose pode provocar severos danos. Outra importante doença para o algodoeiro, em muitas regiões do mundo, é a mancha angular, causada por Xanthomonas axonopodis pv. malvacearum. O objetivo deste trabalho foi desenvolver um índice de favorabilidade baseado em variáveis meteorológicas para estimar a ocorrência da ramulose e da mancha angular (MA) do algodoeiro. O período de incubação, a freqüência de infecção e a severidade das doenças foram avaliados em câmaras de crescimento mantidas a 15, 20, 25 e 30&#176;C e com períodos de câmara úmida de 0, 2, 4, 8, 16, 32, e 64 horas após inoculação com suspensão inóculo de 105 conídios mL-1 e 106 IFC mL-1. Severidade da doença. Severidade (MA) e número de lesões por área (Ramulose) foram modelados como função da duração do período de molhamento (DPM) e da temperatura (T). Em experimentos de campo em Piracicaba, SP, parcelas de algodão foram inoculadas com ramulose e mancha angular, separadamente, a fim de se avaliar semanalmente o progresso das doenças ao longo do tempo. Os dados de temperatura do ar e molhamento foliar no campo foram utilizados no modelo obtido com os resultados de câmaras de crescimento e assim calcular o índice de favorabilidade. As variáveis meteorológicas radiação solar, T, umidade relativa do ar, DPM, chuva e velocidade do vento foram avaliadas como possíveis variáveis explanatórias às taxas de crescimento da doença no campo. Testou-se também, como variável explanatória, o índice de favorabilidade (0,0<IF<1,0), calculado com os modelos de superfície utilizando os dados de T e DPM, obtidos dos experimentos de campo. Em câmaras de crescimento, a temperatura ótima foi de 27&#176;C para ramulose e 22&#176;C para mancha angular, com período de incubação de 10 dias para ramulose e entre 5 e 6 dias para mancha angular. A máxima severidade de ramulose ocorreu entre 25 e 30&#176;C e a máxima severidade de ALS ocorreu entre 20 e 25&#176;C. A severidade de ambas diminui rapidamente em temperaturas maiores ou menores que nessa faixa. A severidade de ramulose aumentou no intervalo de 4 até 32 horas de molhamento enquanto que ocorreu o mesmo com ALS no intervalo de 0 a 8 horas de molhamento. A severidade de ambas foi melhor ajustada por um modelo exponencial logarítmico em função do molhamento que, pela substituição dos parâmetros por funções de temperatura, descreveu a superfície de resposta com elevada precisão e exatidão. No campo, a taxa de crescimento da ramulose foi melhor relacionada ao índice de favorabilidade e à chuva, por regressão não linear, com coeficientes de determinação de 0,89 e 0,91, respectivamente. A taxa de crescimento da mancha angular apresentou razoável relação linear com as variáveis temperatura durante o período de molhamento e vento máximo, com R2 de 0,75 e 0,84 respectivamente. / Colletotrichum gossypii var. cephalosporioides, the fungus that causes ramulosis in cotton crops, is widespread in Brazil. Without an efficient fungicide schedule, ramulosis disease could provoke severe yield losses on cotton. Other important disease to cotton crop in several regions of the world is the bacterial blight also known as angular leaf spot (ALS), caused by Xanthomonas axonopodis pv. malvacearum. The objective of this work was develop a weather based favorability index to each of this diseases. The incubation period, the infection frequency and the severity of both diseases were evaluated in growth-chamber experiments, incubated at 15, 20, 25, 30, and 35&#176;C while exposed to wetness periods of 0, 2, 4, 8, 16, 32 and 64 h after inoculation with 105 conidium mL-1 and 106 CFU mL-1. Disease severity (ALS) and number of lesions per leaf area (ramulosis), was modeled as a function of leaf wetness duration (LWD) and temperature (T). At the field experiments in Piracicaba, cotton plots were inoculated with both pathogens separately, and disease severity was evaluated weekly. LWD and T data from the field trials were used as input to the model from growth chambers results and output the disease favorability index. Weather variables as solar radiation, temperature, relative humidity, wetness duration, rain, and wind speed were also evaluated as possible explanatory variables to the disease growth rate in the field. Was also tested the favorability index (0.0<FI<1.0), as explanatory variable, calculated with the surface models using T and LWD from the field. In growth chambers, the optimum temperature for ramulosis was 27&#176;C and 22&#176;C for ALS and incubation of 10 days to ramulosis and 5 to 6 days do ALS. Maximum ramulosis severity occurred from 20 to 30&#176; C, and between 20 and 25&#176;C to bacterial blight, and both decreased sharply at warmer or cooler temperatures. Ramulosis severity increased as wetness periods were increased from 4 to 32 h. Bacterial blight severity increased from 0 to 8 h of wetness period. The severity of both diseases, were best fitted by a logarithmic exponential model as function of LWD which, by the substitution of parameters for temperature functions, described the surface response with high precision and accuracy, even being a very flexible model. In the field, ramulosis growth rate was strongly correlated with values of the disease favorability index and rainfall with R2=0.89 and R2=0.91, respectively. Bacterial blight growth rate had best linear correlation to temperature during the wetness period and maximum wind with R2=0.75 and R2=0.84, respectively.

Doctor of Philosophy / Plant Pathology / Frank F. White / Rice and wheat are two major crops that suffer losses from the diseases of bacterial blight and bacterial leaf streak, which are caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas translucens pv. undulosa (Xtu), respectively. Transcriptional-Activator Like (TAL) effectors, a special family of type III effector proteins from Xanthomonas, have been demonstrated as critical virulence factors that act by inducing corresponding susceptibility (S) genes in several disease complexes of plants. In this study, I analyzed the contributions of TAL effectors from Xoo and Xtu to virulence and in modulating host gene expression to enhance susceptibility. Specifically, the TalC effector from the African Xoo strain AXO1947 was identified as a critical virulence factor, which functions by promoting expression of the gene OsSWEET14 in rice. TalC is interchangeable with other major TAL effectors from Asian strains of Xoo on the basis of functional complementation. The TAL effector PthXo2 from the Asian Xoo strain JXO1 is a major virulence factor and contains 21.5 repeats in the central repetitive region that targets OsSWEET13 in indica rice varieties but not in japonica rice varieties. A one repeat deletion in the PthXo2 effector enabled effector specificity to switch from indica rice to japonica rice. TAL effector genes from a genomic analysis of the Xtu strain XT4699 and related strains were characterized with regards to their involvement in virulence and the modulation of host gene expression in the Chinese Spring wheat cultivar. The identification of TAL effectors with virulence contributions and their target S genes is important for understanding the virulence mechanisms of Xanthomonas bacteria and promises to provide new strategies for disease control.

Um dos principais fatores limitantes da produção de algodão é a ocorrência de doenças e, entre os patógenos mais importantes estão às bactérias, que causam danos significativos à cultura. A doença conhecida como mancha angular, causada pela bactéria Xanthomonas axonopodis pv. malvacearum (Xam), é uma doença grave, que tem gerado grande preocupação dos produtores. Porém, muitos cultivares com boas características agronômicas apresentam suscetibilidade ao patógeno e, poucos são aqueles que apresentam resistência à doença. O controle da mancha angular é realizado basicamente com uso de resistência genética ao patógeno. Uma das principais fontes de inóculo para esta bactéria é a semente infectada. Este trabalho objetivou o desenvolvimento de um meio semi-seletivo para o isolamento de Xam em sementes de algodão, visando a utilização em análises de rotina em laboratórios de patologia de sementes. O meio de cultura semi-seletivo possuí a seguinte constituição: 3 g de extrato de carne; 5 g de peptona; 10 g de amido solúvel; 10 mL de Tween 80; 0,25 g de cloreto de cálcio; 150 µL de solução de cristal violeta a 1%; 50 mg de cefalexina; 10 mg de clorothalonil; 10 mg de tiofanato metílico e água destilada por 1000 mL. Este meio de cultura possui baixa repressividade a Xam e permite isolar este patógeno de sementes de algodão. / One of the main limiting factors of the cotton production is the occurrence of diseases. The bacteria are among the most important pathogens causing significant losses in the production. Cotton bacterial blight is caused by the bacterium Xanthomonas axonopodis pv. malvacearum (Xam). Is a serious disease that affects cotton and has worried world producers. The main source of inoculum for this bacterium is the infected seed. This work had as objective the development of a semi-selective medium to detect Xanthomonas axonopodis pv malvacearum in cotton seeds for routine tests in seed pathology laboratories. By fungitoxicity tests, basead on qualitative and quantitative antibiograms, it was idealized a semi-selective medium with the folloing composition: peptone (5.0 g/L); agar (15.0 g/L); meat extract (3.0 g); starch (10.0 g/L); violet crystal (150.0 µL violet crystal solution at 1%); water (1,000 mL); CaCl2 (0.25 g); Tween 80 (10.0 mL/L). This medium has small effect on Xam and allows to isolate cotton seeds pathogens.

La RH est une réaction de défense. L’interaction entre G.hirsutum et Xcm repose sur le concept gène-à-gène. L’infection du cultivar Réba B50 possédant les gènes R B2B3 par Xcm18 conduit à une RH associée à une activité LOX, responsable de la peroxydation des lipides, et à la transcription du GhLOX1. Premièrement, 6 génotypes de G.hirsutum contenant divers gènes R ont été retenus pour analyser la variabilité de la réponse LOX suite à l’infection par Xcm1, 18 ou 20. Notre étude a porté sur plusieurs critères, le phénotype, la perte en eau, l’activité LOX et la transcription du GhLOX1. Les résultats montrent une variabilité du phénotype RH en fonction des sources de résistances. Pour chaque type d’interaction incompatible, l’activité LOX et la transcription du GhLOX1 révèlent une augmentation significative corrélée à l’apparition des symptômes RH et à la diminution de la teneur en eau. La réponse LOX est conservée lors de la RH, quelle que soit la race de Xcm ou le génotype. Le GhLOX1 considère comme un marqueur moléculaire de la résistance spécifique du cotonnier à Xcm. Deuxièmement, le rôle du GhLOX1dans la mise en place de la RH en analysant sa fonction potentielle par surexpression. Des cotylédons ont été transformés avec la séquence codante GhLOX1 fusionnée au CaMV35S. Ces cotylédons transformés ont révélé (i) une activité LOX significativement supérieure à celle des cotylédons témoins montrant que le GhLOX1 code pour une protéine active et (ii) un phénotype sans modifications apparentes par rapport à celui des cotylédons non transformées, sauf dans certains contextes d’interactions cotonnier/Xcm où la surexpression de ce gène induit l’apparition de symptômes de type RH. L’effet de l’agro-infiltration sur l’expression de certains gènes pendant la transformation a révélé l’induction précoce et non spécifique de l’expression de gènes de défense. Ces travaux constituent une première étape dans l’analyse fonctionnelle du GhLOX1 dans la résistance spécifique du cotonnier à Xcm / The HR is a defense strategy. The interaction between G.hirsutum and Xcm is governed by the gene-for-gene concept. The infection of the cultivar Reba B50 that contains B2B3 R genes by race Xcm18 leads to a HR associated with a LOX activity response involved in peroxidation of lipids and with transcription of GhLOX1. First, 6 genotypes of G. hirsutum containing various R genes were tested to analyze the variability of the LOX response following the infection by Xcm1, 18 or 20. Several criteria were investigated including the phenotype, the water loss, the LOX activity and GhLOX1 transcription. The results showed variation in HR phenotype according to the tested R genes. For each type of the incompatible interaction, LOX activity and transcription of GhLOX1 were always significantly increased paralleled the apparition of the HR symptoms and the decrease in the water content. LOX response (enzymatic activity and GhLOX1 transcription) is associated with HR whatever the genotype of both Xcm races and cotton plant. Thus, the GhLOX1 consider as a molecular marker of the cotton specific resistance to Xcm. Second, the role of the GhLOX1 gene in the execution of the cotton HR to Xcm by analyzing its possible function by over-expression, the cotyledons were transformed with the GhLOX1 coding sequence fused to the CaMV35S. These transformed cotyledons revealed (i) a LOX activity significantly higher than that detected in the control, showing that the GhLOX1 encodes for an active protein and (ii) that the phenotype of these cotyledons was indistinguishable as compared to the non transformed cotyledons, except when the HR symptoms were induced in some GhLOX1-over-expressed cotyledons. The effect of agro-infiltration on expression of some plant genes during the transformation revealed early and nonspecific induction of the expression of defense genes. This work constitutes a preliminary investigation for the functional analysis of the GhLOX1 in order to assess its role in the cotton specific resistance to Xcm

A doença crestamento bacteriano comum do feijoeiro, causada pela bactéria Xanthomonas axonopodis pv. phaseoli, ocorre principalmente nas regiões quentes e úmidas do globo. A resistência induzida consiste no aumento da resistência da planta por meio da utilização de agentes externos, sem qualquer alteração em seu genoma. Os objetivos do trabalho foram: (i) verificar a indução de resistência em feijoeiro com o uso do indutor abiótico acibenzolar-S-metil (ASM) contra X. axonopodis pv. phaseoli em casa de vegetação e em campo; (ii) avaliar os parâmetros bioquímicos (carboidratos, proteínas, fenóis, clorofila e atividade da enzima peroxidase) e fisiológico (potencial hídrico) nas interações entre plantas de feijão induzidas com o indutor contra X. axonopodis pv. phaseoli; (iii) avaliar os custos da indução de resistência em função da aplicação do indutor contra X. axonopodis pv. phaseoli em plantas de feijão com base nos parâmetros bioquímicos, fisiológicos e de produção. Para isso, realizaram-se dois experimentos. Os tratamentos foram dispostos da seguinte maneira: Tratamento A Controle água, Tratamento B Controle ASM, Tratamento C Água com inoculação do patógeno ao 7º dias após a emergência das plântulas (dae) (experimento 1) e ao 21º dae (experimento 2), Tratamento D Induzida com ASM e desafiada com o patógeno ao 7º dae (experimento 1) e ao 21º dae (experimento 2). As pulverizações ocorreram, para o experimento 1, aos 0, 7, 14 e 28 dae, a inoculação foi realizada no 7º dae e foram realizadas quatro coletas das amostras aos 0, 7, 21 e 35 dae. Entretanto para o experimento 2, as pulverizações foram feitas aos 0, 7, 14, 28 e 42 dae, a inoculação foi realizada no 21º dae e as cinco coletas foram conduzidas aos 0, 7, 21, 35 e 49 dae. Foram observadas diferenças estatísticas tanto entre tratamentos quanto entre épocas de coleta, bem como na interação dos tratamentos com as diferentes épocas, dependendo do parâmetro observado. De maneira geral, as plantas tratadas com ASM quando comparadas com plantas controle água, bem como as plantas pré-tratadas com ASM e desafiadas com a bactéria quando comparadas com as plantas pulverizadas com água e inoculadas com o patógeno, apresentaram: a) teores de clorofila próximos; b) aumentaram os teores de fenóis, proteínas totais e peroxidase e c) diminuíram os valores de carboidratos totais. As plantas que apresentaram os valores mais negativos de potencial hídrico foram as pulverizadas com água e inoculadas com o patógeno, seguidas das plantas induzidas com ASM e desafiadas com o patógeno. O mesmo padrão ocorreu com relação à produtividade, onde água+patógeno apresentou a maior redução de massa seca, número de vagens/planta e peso de 100 grãos, seguidos de plantas induzidas com ASM e desafiadas com o patógeno, ASM e água, respectivamente. Porém o número de grãos/vagem não sofreu alteração. Houve indução de resistência nas plantas em função da aplicação com ASM, visto que a severidade da doença diminuiu nas mesmas. / The common bacterial blight disease of bean caused by the bacterium Xanthomonnas axonopodis pv. phaseoli, occurs mainly in hot and humid regions of the globe. The induced resistance consists in the increase of the plant resistance by the use of external agents, without any change in its genome. The objectives of this study were: (i) to verify the induction of resistance in bean plants by using the abiotic inducer acibenzolar-S-methyl (ASM) against Xanthomonas axonopodis pv. phaseoli in greenhouse and field conditions, (ii) to evaluate the biochemical parameters (carbohydrates, proteins, phenols, chlorophyll and peroxidase activity) and physiological (water potential) in the interactions between bean plants treated with the inducer against X. axonopodis pv. phaseoli; (iii) to evaluate the costs of the induction of resistance by on the basis of biochemical, physiological and production parameters. Thus, to achieve this purpose, two experiments were carried out. The treatments were arranged as follows: Treatment A - Control water; Treatment B - ASM Control; Treatment C - Water and inoculation 7 days after emergency (dae) of the seedlings (experiment 1) and 21 dae (Experiment 2); Treatment D - Induced with ASM and challenged with the pathogen 7 dae (experiment 1) and 21 dae (experiment 2). The treatments with ASM were made for experiment 1, at 0, 7, 14 and 28 dae, the inoculation was done 7 dae and four samples were collected at 0, 7, 21 and 35 dae. However, for the experiment 2, the treatments with ASM were made at 0, 7, 14, 28 and 42 days after seedling emergence, the inoculation was performed 21 dae and five samples were collected 0, 7, 21, 35 and 49 dae. Statistical differences were observed between treatments, among harvest dates, as well as the interaction of treatments with different harvests, depending on the parameter observed. In general, plants treated with ASM when compared to plants control, as well as plants pretreated with ASM and challenged with the bacteria when compared to plants sprayed with water and inoculated with the pathogen, showed: a) the amount of chlorophyll was close; b) increased levels of phenolics, proteins and peroxidase; c) decreased values of carbohydrate. Plants that showed the more negative water potential were plants sprayed with water and inoculated with the pathogen, followed by plants induced with ASM and challenged with the pathogen. The same pattern occurred with productivity, plants sprayed with water and inoculated with the pathogen exhibited the higgest reduction in dry weight, number of pods per plant and 100 grain weight, followed by plants induced with ASM and challenged with the pathogen, ASM and water, respectively. In all treatments, the number of grains per pods did not change. Finally, there was induction of resistance in plants by ASM application since the disease severity decreased.

A doença crestamento bacteriano comum do feijoeiro, causada pela bactéria Xanthomonas axonopodis pv. phaseoli, ocorre principalmente nas regiões quentes e úmidas do globo. A resistência induzida consiste no aumento da resistência da planta por meio da utilização de agentes externos, sem qualquer alteração em seu genoma. Os objetivos do trabalho foram: (i) verificar a indução de resistência em feijoeiro com o uso do indutor abiótico acibenzolar-S-metil (ASM) contra X. axonopodis pv. phaseoli em casa de vegetação e em campo; (ii) avaliar os parâmetros bioquímicos (carboidratos, proteínas, fenóis, clorofila e atividade da enzima peroxidase) e fisiológico (potencial hídrico) nas interações entre plantas de feijão induzidas com o indutor contra X. axonopodis pv. phaseoli; (iii) avaliar os custos da indução de resistência em função da aplicação do indutor contra X. axonopodis pv. phaseoli em plantas de feijão com base nos parâmetros bioquímicos, fisiológicos e de produção. Para isso, realizaram-se dois experimentos. Os tratamentos foram dispostos da seguinte maneira: Tratamento A Controle água, Tratamento B Controle ASM, Tratamento C Água com inoculação do patógeno ao 7º dias após a emergência das plântulas (dae) (experimento 1) e ao 21º dae (experimento 2), Tratamento D Induzida com ASM e desafiada com o patógeno ao 7º dae (experimento 1) e ao 21º dae (experimento 2). As pulverizações ocorreram, para o experimento 1, aos 0, 7, 14 e 28 dae, a inoculação foi realizada no 7º dae e foram realizadas quatro coletas das amostras aos 0, 7, 21 e 35 dae. Entretanto para o experimento 2, as pulverizações foram feitas aos 0, 7, 14, 28 e 42 dae, a inoculação foi realizada no 21º dae e as cinco coletas foram conduzidas aos 0, 7, 21, 35 e 49 dae. Foram observadas diferenças estatísticas tanto entre tratamentos quanto entre épocas de coleta, bem como na interação dos tratamentos com as diferentes épocas, dependendo do parâmetro observado. De maneira geral, as plantas tratadas com ASM quando comparadas com plantas controle água, bem como as plantas pré-tratadas com ASM e desafiadas com a bactéria quando comparadas com as plantas pulverizadas com água e inoculadas com o patógeno, apresentaram: a) teores de clorofila próximos; b) aumentaram os teores de fenóis, proteínas totais e peroxidase e c) diminuíram os valores de carboidratos totais. As plantas que apresentaram os valores mais negativos de potencial hídrico foram as pulverizadas com água e inoculadas com o patógeno, seguidas das plantas induzidas com ASM e desafiadas com o patógeno. O mesmo padrão ocorreu com relação à produtividade, onde água+patógeno apresentou a maior redução de massa seca, número de vagens/planta e peso de 100 grãos, seguidos de plantas induzidas com ASM e desafiadas com o patógeno, ASM e água, respectivamente. Porém o número de grãos/vagem não sofreu alteração. Houve indução de resistência nas plantas em função da aplicação com ASM, visto que a severidade da doença diminuiu nas mesmas. / The common bacterial blight disease of bean caused by the bacterium Xanthomonnas axonopodis pv. phaseoli, occurs mainly in hot and humid regions of the globe. The induced resistance consists in the increase of the plant resistance by the use of external agents, without any change in its genome. The objectives of this study were: (i) to verify the induction of resistance in bean plants by using the abiotic inducer acibenzolar-S-methyl (ASM) against Xanthomonas axonopodis pv. phaseoli in greenhouse and field conditions, (ii) to evaluate the biochemical parameters (carbohydrates, proteins, phenols, chlorophyll and peroxidase activity) and physiological (water potential) in the interactions between bean plants treated with the inducer against X. axonopodis pv. phaseoli; (iii) to evaluate the costs of the induction of resistance by on the basis of biochemical, physiological and production parameters. Thus, to achieve this purpose, two experiments were carried out. The treatments were arranged as follows: Treatment A - Control water; Treatment B - ASM Control; Treatment C - Water and inoculation 7 days after emergency (dae) of the seedlings (experiment 1) and 21 dae (Experiment 2); Treatment D - Induced with ASM and challenged with the pathogen 7 dae (experiment 1) and 21 dae (experiment 2). The treatments with ASM were made for experiment 1, at 0, 7, 14 and 28 dae, the inoculation was done 7 dae and four samples were collected at 0, 7, 21 and 35 dae. However, for the experiment 2, the treatments with ASM were made at 0, 7, 14, 28 and 42 days after seedling emergence, the inoculation was performed 21 dae and five samples were collected 0, 7, 21, 35 and 49 dae. Statistical differences were observed between treatments, among harvest dates, as well as the interaction of treatments with different harvests, depending on the parameter observed. In general, plants treated with ASM when compared to plants control, as well as plants pretreated with ASM and challenged with the bacteria when compared to plants sprayed with water and inoculated with the pathogen, showed: a) the amount of chlorophyll was close; b) increased levels of phenolics, proteins and peroxidase; c) decreased values of carbohydrate. Plants that showed the more negative water potential were plants sprayed with water and inoculated with the pathogen, followed by plants induced with ASM and challenged with the pathogen. The same pattern occurred with productivity, plants sprayed with water and inoculated with the pathogen exhibited the higgest reduction in dry weight, number of pods per plant and 100 grain weight, followed by plants induced with ASM and challenged with the pathogen, ASM and water, respectively. In all treatments, the number of grains per pods did not change. Finally, there was induction of resistance in plants by ASM application since the disease severity decreased.

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Le manioc (Manihot esculenta L. Crantz) est cultivé dans toute la zone intertropicale. Compte tenu de ses utilisations potentielles, le développement de la culture du manioc a considérablement augmenté au niveau mondial, mais il est encore considéré comme une culture de subsistance en particulier dans les régions pauvres où il est cultivé par les petits producteurs. La production du manioc est fortement contrainte par des facteurs biotiques et abiotiques, dont la bactériose vasculaire du manioc (CBB) et la nécrose bactérienne du manioc (CBN) qui sont deux maladies causées par les bactéries Xanthomonas axonopodis pv. les manihotis (Xam) et X. cassavae (Xc), respectivement. CBB est considérée comme la principale maladie bactérienne, notamment au Venezuela où la CBB a été signalée pour la première fois dans les années 70. Dans les années 90, des études ont été menées pour élucider la variabilité génétique de Xam dans différentes régions du pays, au moyen d’outils moléculaires, mettant en évidence un degré élevé de polymorphisme parmi les souches analysés.Le sujet de cette thèse porte sur la situation de CBB au Venezuela 20 ans après. Quelle est la diversité génétique actuelle de Xam au Venezuela? Quelle est la structure génétique des populations de Xam dans ce pays, et comment diffèrent-elles des souches de Xam recueillies dans les années 90? En outre, étant donné que Xam et Xc provoquent des symptômes semblables sur feuilles de manioc et présentent des caractéristiques physiologiques et morphologiques similaires, nous avons également visé à développer un nouvel outil de diagnostic moléculaire permettant une détection rapide et fiable de Xam et de Xc tout étant capable de les discriminer l’une vis à vis de l’autre.Pour atteindre nos objectifs, nous avons d'abord établi une PCR-duplex comme outil de détection moléculaire des xanthomonades infectant le manioc. Sur la base de l'analyse in silico des séquences du génome de 66 souches de Xam et une de Xc, nous avons pu sélectionner 6 paires d'amorces candidates spécifiques de Xam et six autres pour Xc. Nous avons pu développer un test de PCR-duplex qui a été validé en testant 53 souches de Xam et 25 souches de Xc issues de différents pays, 18 souches non cibles contrôles et cinq souches épiphytes associées au manioc. Cette technique représente un outil utile pour détecter et différencier Xam et Xc provenant de cultures in vitro mais aussi fonctionnelle à partir de tissues de plantes infectés.Deuxièmement, nous avons donc évalué la diversité des populations de Xam à l’aide de l’étude de microsatellites (ou MLVA pour « Multiple loci VNTR analysis »). Une enquête de terrain menée dans six États du Venezuela a permis d'évaluer la présence de la maladie, son statut et nous a permis d'établir une collection de souches de Xam dont l’analyse détaillée de la diversité a pu être réalisée. Au total nous avons isolé 202 souches de Xam issues de six localités situées dans quatre états. À l'aide d'un schéma 14-MVLA, nous avons analysé 12 populations et mis en évidence un indice élevé de la diversité génétique au sein de ces populations et entre elles, et principalement dans l'est du pays.Le développement de ce type de recherche est essentiel pour une gestion raisonnée des cultures dans le monde. Conjugué aux politiques agricoles existantes, il nous permettra de mieux comprendre les agents pathogènes d'importance agricole et les mécanismes impliqués dans leur établissement dans le temps et à travers l’espace. L'objectif à long terme est d'appliquer des mesures de contrôle efficaces et durables, ce qui conduira à des mesures de quarantaine plus efficaces pour prévenir la propagation de la maladie. / Cassava (Manihot esculenta L. Crantz) belongs to the group of roots and tubers and is cultivated in the tropics worldwide. This species is a nutritional alternative in many populations where no optimal crop production, general conditions nor technological support exist. Considering its potential uses, the global development of cassava crop has increased significantly but it is still considered a subsistence crop in poor regions lead by smallholder producers. Cassava is affected by biotic and abiotic factors during its life cycle, which heavily limiting its optimal performance. A variety of pests and diseases are known to affect cassava production. Among them are those caused by Xanthomonas axonopodis pv. manihotis (Xam) and Xanthomonas cassavae (Xc), causal agents of Cassava Bacterial Blight (CBB) and Cassava Bacterial Necrosis (CBN) diseases, respectively. CBB is considered the major bacterial disease that affects cassava crop worldwide which is also the case in Venezuela where it was first reported in the 70s. Since the 90s, studies were conducted to elucidate Xam genetic variability in different regions in the country, by means of different molecular tools available at that time. A high degree of polymorphism among the isolates was reported, whether collected from the same or different fields. The Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin.Our questions deal with the situation of CBB in Venezuela 20 years later : what is the current genetic diversity of Xam populations in Venezuela? what is the genetic structure of Xam populations and how do they differ with respect to Xam strains collected in 90s. Moreover, because Xam and Xc cause similar symptoms on cassava leaves and display similar physiological and morphological characteristics, we also aimed at developing a new molecular diagnostic tool allowing for fast and reliable detection of Xam and able to discriminate with Xc.To achieve our goals, we first established a duplex-PCR as a molecular detection tool of cassava-infecting xanthomonads. Based on in silico analysis of the genome sequences of 66 Xam and 1 Xc strains, we were able to select 6 Xam and 6 Xc primers pairs candidates, of which one set of primers for each was selected for further studies. We were able to develop a duplex-PCR assay that was validated upon testing 53 Xam strains and 25 Xc strains from different countries, 18 non-target strains, and 5 epiphytic strains associated to cassava, proving this technique a useful tool to detect and differentiate Xam and Xc from in vitro cultures and in planta.Secondly we assessed the diversity of Xam populations through a variable number of tandem repeat analysis (MLVA). A field survey conducted in six states in Venezuela enabled to evaluate the occurence of the disease, its status and allowed us to establish a strain collection for detailed diversity analysis. We isolated 202 Xam strains from six localities, localized in four states. Using a MVLA14-scheme, we analyzed 12 populations highlighting a high index of genetic diversity among and within populations, mainly in the east of the country.The development of this type of research is essential in the management of crops in the world and coupled with the existing agricultural policies, it will allow us to have a deeper understanding of pathogens of agricultural importance and the mechanisms involved in their establishment over time and across regions. The long-term objective of this is to apply control measures that are effective in time, thus establishing more stringent quarantine measures to prevent the spread of the disease.

Made available in DSpace on 2017-07-10T17:37:39Z (GMT). No. of bitstreams: 1 Roberto_Luis_Portz.pdf: 1085899 bytes, checksum: 3efb6c9ddc743240ff09e54b5f1b5d9a (MD5) Previous issue date: 2003-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial blight of cassava (Xanthomonas axonopodis pv. manihotis) is the most important disease in the culture of the cassava and its occurrence is generalized in all the places where it is cultivated. The intensity of the disease is related, mainly, with the resistance and age of the plant and with the soil and weather conditions. With the objective of obtaining information about the genetic variability of these bacteria in the West region of Paraná, a research was accomplished, throughout sampling of stems with similar symptoms to the caused by the disease, at Entre Rios do Oeste, Marechal Cândido Rondon, Mercedes, Missal, Nova Santa Rosa and Pato Bragado. Stems of cassava varieties destined for industry and varieties for human consume were sampled. The isolates obtained were characterized to "in vitro" amylase activity, eletrophoretic analysis of isoenzymes α and β-esterase and aggressiveness. From 61 collected materials, it was possible to obtain 19 isolated of X. axonopodis pv. manihotis. Materials of human consume varieties showed larger incidence than those for industry. Stems from Pato Bragado, Entre Rios do Oeste and Mercedes showed incidences of 10, 27 and 10%, respectively, values smaller than those of Marechal Cândido Rondon (50%) and Nova Santa Rosa (58%). The isolates could be contained in three, six and 12 different groups to amylolytic activity, aggressiveness and esterase isoenzimes, respectively. There was not relationship between the amylase activity and aggressiveness of the isolates. Based on the origin, isolates of Marechal Cândido Rondon were more aggressive than those of other sampled areas. The clustering for esterase allowed to verify that isolates from Entre Rios do Oeste, Nova Santa Rosa and Mercedes showed high similarity degree. These results indicate there to be differentiation among the isolates of the bacteria presents in the studied regions / A bacteriose ou murcha bacteriana (Xanthomonas axonopodis pv. manihotis) é a doença de maior importância econômica na cultura da mandioca e sua ocorrência está generalizada em todos os locais onde esta é cultivada. A intensidade da doença está relacionada, principalmente, com a resistência e idade da planta e com as condições edafoclimáticas. Com o objetivo de obter informações sobre a variabilidade genética desta bactéria em municípios produtores da região Oeste do Paraná, foi realizado um levantamento, através de coletas de ramas com sintomas semelhantes aos causados pela doença, em Entre Rios do Oeste, Marechal Cândido Rondon, Mercedes, Missal, Nova Santa Rosa e Pato Bragado. Foram coletadas ramas de variedades de mandioca destinadas para indústria e para mesa. Os isolados obtidos foram caracterizados quanto à atividade in vitro de amilase, análise eletroforética de isoenzimas de α e β-esterase e quanto à agressividade. A partir de 61 materiais vegetais coletados, foi possível obter 19 isolados de X. axonopodis pv. manihotis, sendo que, materiais provenientes de variedades de mesa apresentaram maior incidência em relação àquelas para indústria. Manivas provenientes de Pato Bragado, Entre Rios do Oeste e Mercedes apresentaram incidências de 10, 27 e 10%, respectivamente, valores inferiores aos de Marechal Cândido Rondon (50%) e de Nova Santa Rosa (58%). Os isolados puderam ser agrupados em três, seis e 12 grupos distintos com relação à capacidade amilolítica, agressividade e isoenzimas de esterase, respectivamente. Não houve relação entre a atividade de amilase e agressividade dos isolados. Baseado na procedência, isolados de Marechal Cândido Rondon foram mais agressivos que os provenientes das outras regiões amostradas. O agrupamento com base em esterase permitiu verificar que isolados provenientes de Entre Rios do Oeste, Nova Santa Rosa e Mercedes apresentam, entre si, alto grau de similaridade. Estes resultados indicam haver diferenciação entre os isolados da bactéria presentes nos municípios amostrados