Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T15:11:31Z No. of bitstreams: 1 Claudeana Souza da Conceicao.pdf: 783432 bytes, checksum: fd59b60ffc3266d8d18f15005f8c14cc (MD5) / Made available in DSpace on 2017-02-17T15:11:31Z (GMT). No. of bitstreams: 1 Claudeana Souza da Conceicao.pdf: 783432 bytes, checksum: fd59b60ffc3266d8d18f15005f8c14cc (MD5) Previous issue date: 2013-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The combined effect of the antagonistic yeasts Rhodotorula aurantiaca LMA1, R. glutinis LMS and Pichia anomala CC-2 and silicon (Si) was evaluated in relation to the control of bacterial fruit blotch (Acidovorax citrulli) in seedlings and plants, and possible mechanisms of action involved in the control were analyzed. The incorporation of 1.41 g Si kg-1 (calcium silicate) into the substrate, together with foliar spraying with yeast (1.5 x 107 cells mL-1) and foliar spraying with 17 mM Si (potassium silicate) and the yeasts, separately or in combination, reduced the severity of the disease and protected seedlings and plants. These plants were inoculated with A. citrulli by foliar spraying (3.4 x 107 UFC mL-1) 24 h following the treatment. However, no additive or synergistic effects of the combined treatments were observed. The spraying of LMA1+Si and LMA1 resulted in the highest levels of control of bacterial fruit blotch in plants, and this level of control was higher than that provided by acibenzolar-S-methyl. Foliar spraying with LMA1 and Si, either separately or in combination, protected melon plants from infection by A. citrulli for 29 days. Increases in the activity of polyphenol oxidase (PFO) after foliar spraying with Si and LMA1, and increases of ascorbate peroxidase (APX) activity after foliar spraying with LMA1+Si and LMA1 are likely related to the induction of resistance to bacterial fruit blotch. / O efeito combinado de leveduras antagonistas (Rhodotorula aurantiaca LMA1, R. glutinis LMS e Pichia anomala CC-2) e silício (Si) foi avaliado em relação ao controle da mancha aquosa (Acidovorax citrulli) pela proteção de plântulas e plantas; e analisados possíveis mecanismos de ação envolvidos no controle. A incorporação de 1,41g Si kg-1 (silicato de cálcio) ao substrato e pulverização foliar com as leveduras (1,5 x 107 cels mL-1), assim como a pulverização com 17 mM Si (silicato de potássio) e leveduras, em combinação ou isoladamente, reduziram a severidade da doença, protegendo plântulas e plantas. As plantas foram inoculadas com A. citrulli por pulverização (3,4 x 107 UFC mL-1), 24 h após os tratamentos. No entanto, não foi verificado efeito aditivo ou sinergístico das combinações. A pulverização de LMA1+Si e LMA1 proporcionou os maiores níveis de controle da mancha aquosa em plantas, sendo superior ao acibenzolar-S-methyl. LMA1 e Si pulverizados em combinação ou não, protegeram as plantas de meloeiro da infecção por A. citrulli por 29 dias. Aumentos nos níveis das enzimas PFO pela pulverização de Si e LMA1 e APX por LMA1+Si e LMA1 estão provavelmente relacionados à indução de resistência a mancha aquosa.

Bacterial secretion systems have been playing essential roles in modulating the microbiota of most ecological niches. Among a variety of secretion systems, the Type VI Secretion System (T6SS), a nanomachine widely distributed in Gram-negative bacteria, is gaining increasing attention due to its involvement in microbe-microbe and microbe-host interactions through secreting toxins into host cells, microbial competitors, and the extracellular milieu. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising biological control agents (BCAs). The main body of this dissertation will focus on the characterization of the T6SS of a phytopathogen, Acidovorax citrulli (strain AAC00-1), and the secreted T6 effectors, and will also discuss the possible application of AAC00-1 as a BCA. The seed-borne, gram-negative A. citrulli is able to cause bacterial fruit blotch (BFB) disease and then result in devastating decrease in yields of important cucurbits including watermelon, melon, squash and cucumber. Our inter-microbial competition assays demonstrate that AAC00-1 contains an active T6SS and presents a dramatic antimicrobial activity against a variety of microbes, including Gram-negative bacteria, Gram-positive bacteria, and yeast, dependent upon its T6SS. A group of novel non-enzymatic effectors, Hyde1 proteins, delivered into prey cells through the T6SS, are responsible for this broad-spectrum antimicrobial activity. Expressing Hyde1 or its N-terminal transmembrane domain shows significant toxicity in both E. coli and AAC00-1, and the toxicity of Hyde1 can be counteracted by its immunity protein, Hyde2. A non-pathogenic AAC00-1 strain suppresses the growth of multiple deleterious phytopathogens in planta and protects plant host. Transgenic plants expressing either full-length Hyde1 or its transmembrane domain demonstrate improved resistance against both bacterial and oomycete pathogens. Altogether, we characterize the T6SS killing of AAC00-1, identify the determinant effectors and discuss the application of both AAC00-1 and its T6SS effector in plant disease management. Additionally, in order to develop molecular tools better serving our T6SS-related studies, we successfully generate a series of salicylic acid (SA)-inducible vectors, functioning in A. citrulli, that can be used for inducible gene expression, protein purification and other applications. The core regulatory component that we employ, is a transcriptional regulator, Sal7AR-V295F, due to its responsiveness to salicylate. By cloning this fragment to a broad-host-range plasmid, in this study, we establish multiple SA-inducible vectors that may be used in most Gram-negative bacteria. When using the E. coli strain C41(DE3) as the expression host, protein purification can be conducted routinely, upon the addition of affinity tags to our vectors, such as the maltose-binding protein (MBP) tag. Combining the modified vectors with the robust NanoLuc binary Technology (NanoBiT), we are able to devise a novel bacteria two-hybrid system as an effective method to detect protein-protein interaction. Two complementary fragments of the NanoLuc protein, LgBiT and SmBiT, with extremely low affinity, are fused to potential interactors, and they will be brought into proximity and reconstitute NanoLuc bioluminescence upon the occurrence of interaction. This system is used in our T6SS study to validate the interaction between Hyde1 toxin and its cognate immunity protein. Another fragment, HiBiT, which automatically interacts with LgBiT and reconstitutes NanoLuc, is cloned to the SA-inducible vector as well, enabling us to generate a split-NanoLuc-based method, for the purpose of detecting secretion of tagged T6 toxins into the prey bacterial cells expressing LgBiT. Overall, our SA-inducible vectors and their further modifications enrich the molecular tool repertoire for T6SS-related studies. / Doctor of Philosophy / Effective crop disease management is critical for agricultural production. Chemical spray has been practiced as one major approach to control plant diseases for more than a decade. However, increase of pesticide application could threaten public health and the environment. Biological control has been considered as one of the effective and environmental-friendly alternative approaches for disease control. In this dissertation, we identify that, Acidovorax citrulli (strain AAC00-1), a Gram-negative pathogen causing bacterial fruit blotch (BFB) disease in Cucurbitaceae, could be a potential biological control agent (BCA), because it carries an active Type VI Secretion System (T6SS), and the T6SS has been shown to contribute to the protective effects of many plant-associated BCAs. T6S is believed to mediate inter-bacterial competition through secreting toxins into microbial competitors. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising BCAs. We demonstrate that AAC00-1 suppresses the growth of multiple phytopathogens, depending upon its T6SS. Expressing ten out of eleven microbial toxins, encoded by Hyde1 genes, in E. coli shows significant toxicity. The wild type AAC00-1 strain inhibits the growth of multiple Arabidopsis leaf bacterial isolates, while an AAC00-1 Hyde1 mutant loses this capacity. The antimicrobial activity of AAC00-1 is proven to be broad-spectrum since this strain also shows inhibitory effect on the growth of Gram-positive bacteria and yeast. In planta disease assay suggests that a non-pathogenic AAC00-1 mutant defective in Type III secretion system (T3SS) maintains its capacity to suppress disease symptoms and pathogen growth on plants infected with different phytopathogens. Our study demonstrates the viability of the employment of non-pathogenic A. citrulli as an effective BCA in plant disease management.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-19T14:09:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) / Made available in DSpace on 2016-09-19T14:09:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) Previous issue date: 2016-01-25 / CAPES / A formação de biofilme é uma característica importante para as bactérias, por ser uma formação natural e altamente influenciada pelo ambiente circundante, confere aos microrganismos alta tolerância às adversidades e torna-se importante na virulência para patógenos. Sendo assim, apresentamos nesta tese uma investigação da adesão bacteriana e desenvolvimento de biofilme das fitobactérias Ralstonia solanacearum (Rsol) e Acidovorax citrulli (Acc), agronomicamente importantes, sobre superfícies hidrofóbicas, foi investigado também o emprego de extratos vegetais de plantas oriundas da Caatinga, na inibição da adesão bacteriana e sua capacidade bactericida contra R. solanacearum, e foi determinado o método mais eficiente na preparação amostras proteicas para R. solanacearum, A. citrulli e Pectobacterium carotovorum subsp. carotovorum (Pcc) para aplicação em estudos futuros de investigação molecular da formação de biofilmes fitopatogênicos. A formação de biofilme por diferentes isolados bacterianos após 24h de incubação em diferentes meios de cultura foi quantificado pelo método de cristal violeta e suas estruturas observadas por microscopia eletrônica de varredura e microscopia confocal. Foram avaliados também 22 extratos aquosos de 16 plantas coletadas na Caatinga quanto a capacidade de inibição da formação de biofilme de Rsol. Quanto à eficiência na obtenção de proteínas, foram testados os métodos de Trizol, Fenol, Centrifugação e Lise e avaliados através de eletroforese uni e bidimensional. Quanto a formação de biofilme os resultados obtidos indicam que, nas condições testadas, isolados de Rsol se mostrou diferente entre os isolados tanto quantitativa quando morfologicamente onde os isolados B5-5 CGH26 CGH8 e SCN 21 foram moderados ou fortes produtores de biofilme. Já os isolados de Acc não foram bons produtores de biofilme, apresentando apenas os isolados Acc1.43 e Acc 1.73 como fortes formadores de biofilme com quantidade e morfologia semelhantes. No screening de atividade antibiofilme, dentre os extratos testados apenas ramos de Harpochilus neesianus e folhas de Myroxylon peruiferum apresentaram atividade antibiofilme superior a 83% e 50%, respectivamente, e Jacaranda rugosa apresentou atividade antimicrobiana contra todos os isolados de Rsol testados. Quanto à extração de proteínas de alta qualidade o método de Lise foi o mais eficiente para Rsol e Pcc, apresentando respectivamente 369 ± 4 e 212 ± 3 diferentes spots de proteínas, contudo para Acc o método de centrifugação foi o mais indicado com 224 ± 8 spots. De acordo com os resultados deste estudo conclui-se que a formação de biofilme pode ser quantitativa e estruturalmente distinta entre isolados da mesma espécie. O screening das propriedades antimicrobianas das plantas fornece base de dados para o desenvolvimento de novos agentes antibacterianos naturais contra fitopatógenos seguros para o meio ambiente e para o desenvolvimento de estudos moleculares da formação de biofilme faz-se necessária uma prévia determinação de métodos para obtenção das macromoléculas a serem analisadas, sendo assim a seleção de métodos de extração é um ponto crucial para obtenção de amostras de qualidade para analises confiáveis. / Biofilm formation is an important feature for bacteria due to its naturally occurring and highly influence by the surrounding environment, giving to the microorganisms high tolerance to adversity and becoming essential in virulence for pathogens. Thus, we present in this thesis an investigation about bacterial adhesion and biofilm development of the phytobacteria Ralstonia solanacearum (Rsol) and Acidovorax citrulli (Acc), agronomically important, on hydrophobic surfaces; it was also investigated the use of plant extracts from the Caatinga region through the inhibition of the bacterial adhesion and its bactericidal activity against R. solanacearum. The most efficient method to prepare protein samples for R. solanacearum, A. citrulli and Pectobacterium carotovorum subsp. carotovorum (Pcc) was determined to be applied in future studies of molecular investigation of the formation of pathogenic biofilms. The biofilm formation by different bacterial strains after 24 h incubation in distinct culture media was quantified by the crystal violet method and its structures were observed by scanning electron microscopy and confocal microscopy. There were also evaluated 22 aqueous extracts from 16 plants collected in the Caatinga as its potential of inhibition of Rsol biofilm formation. In what concerns the efficiency in obtaining proteins, Trizol, Phenol, centrifugation, and Lyse were the methods evaluated by one- and two-dimensional electrophoresis. The results for biofilm formation demonstrate that, under the tested conditions, Rsol strains were different, both quantitatively and morphologically, and the strains namely B5-5, CGH26, CGH8, and SCN 21 were moderate or strong biofilm producers. Regarding the results for Acc strains, it is possible to note that they were not good biofilm producers, unless the strains Acc1.43 and Acc1.73 that were considered strong biofilm producers with similar quantity and morphology patterns. In relation to the screening of antibiofilm activity, only branches of Harpochilus neesianus and leaves of Myroxylon peruiferum presented antibiofilm activity with values higher than 83% and 50%, respectively, and Jacaranda rugosa showed activity antimicrobial against all the tested Rsol strains. The extraction of high quality proteins was performed most efficiently by the Lysis method for Rsol and Pcc, respectively with 369 ± 4 and 212 ± 3 different spots of proteins, however the centrifuge method was better for Acc with 224 ± 8 spots. According to the results of this study it is possible to conclude that biofilm formation can be quantitatively and structurally distinct from strains of the same species. The screening of the antimicrobial properties of the plants provides data as a basis for the development of new natural antibacterial agents against safe phytopathogens for the environment; in addition, for the development of molecular studies about the biofilm formation it is necessary a preliminary determination of the methods suitable for obtaining the macromolecules to be analyzed, so the selection of extraction methods is a crucial point for obtaining quality samples for reliable analysis.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T14:34:25Z No. of bitstreams: 1 Elias Inacio da Silva.pdf: 496927 bytes, checksum: d6971c4e240310675bba44bb481b6833 (MD5) / Made available in DSpace on 2017-03-10T14:34:25Z (GMT). No. of bitstreams: 1 Elias Inacio da Silva.pdf: 496927 bytes, checksum: d6971c4e240310675bba44bb481b6833 (MD5) Previous issue date: 2002-09-30 / Melon is one of the most popular cucurbits in the world. The bacterial blotch of melon, caused by Acidovorax avenae subsp. citrulli has caused 40 to 50% losses in the production and made the fruits inappropriate for commercialization in the State of Rio Grande do Norte. This study aimed to survey the bacterial blotch incidence in 18 melon planting areas of the counties Mossoró and Baraúna in Rio Grande do Norte, and to determine the ideal sample size for disease assessment. Prevalence of 100% was reported in the studied fields. The incidence of the bacterial fruit blotch ranged from 4.30 to 47.29%. Incidence levels were under 20% in 50% of the areas and higher than 40% in 17% of the areas. There was significant difference for the variables melon type (P=0.01) and years of melon culture in the area (P=0.05). The t test showed the melon Piel de sapo was more susceptible than the yellow type and the disease incidence in areas with less than 10 years of melon cropping was higher than those with more than20 years. However, there were no significant differences (P=0.05) for disease incidence averages among areas planted with corn (18%) and corn plus other grasses (20%) between melon seasons. Using the incidence data from 18 areas as pilot-samples, the sample size for disease assessment was determined according to the mean variability coefficient. The Pierson test showed no significant correlations (P=0.05) between the levels of disease incidence and sample sizes. Based on the data obtained andconsidering 10% of acceptable error, it is recommended that in future surveys of melon bacterial blotch incidence in fields in Rio Grande do Norte, a sample composed by 12 subsamples comprising 100 m2/ha and 20 fruit evaluated per subsample must be analyzed. / O meloeiro é uma das cucurbitáceas mais populares do mundo. A mancha-aquosa do melão, causada pela bactéria Acidovorax avenae subsp. citrulli, tem causado perdas de 40 a 50% na produção e depreciação no valor comercial do fruto no estado do Rio Grande do Norte. Os objetivos deste estudo foram realizar o levantamento da incidência da mancha-aquosa em 18 áreas de plantio de meloeiro dos municípios Mossoró e Baraúna, no Rio Grande do Norte e determinar o tamanho ideal das amostras para quantificação da incidência da doença no campo. Foi registrada a prevalência da doença em 100% dos campos. A incidência da mancha-aquosa variou entre 4,30% e 47,29%. Em 50% das áreas foram constatados níveis de incidência da doença inferiores a 20%, enquanto em 17% foram registrados valores superiores a 40%. Houve diferença significativa na incidência da doença em relação aos tipos de melão (P=0,01) e aos anosde plantio na mesma área (P=0,05). Pelo teste t o melão tipo Pele de sapo foi mais suscetível que o Amarelo e a incidência da doença foi maior em áreas com menos de 10 anos de plantio em relação àquelas com mais de 20 anos de plantio dessa cucurbitácea. Não foram constatadas diferenças significativas (P=0,05) pelo teste t, para médias de incidência da mancha-aquosa entre áreas com plantio de milho (18%) e milho com outras gramíneas (20%) na entressafra. Utilizando os dados de incidência da doença nas 18 áreas como amostragens-piloto, o tamanho das amostras para quantificação da doença foi determinado com base no coeficiente de variação da média. O teste de Pierson não constatou correlações significativas (P=0,05) entre os níveis de incidência da doença e o tamanho das amostras. Considerando os resultados obtidos e um erroaceitável de 10%, em futuros levantamentos da incidência da mancha-aquosa em plantios de meloeiro no Rio Grande do Norte, recomenda-se a utilização de uma amostra de 12 subparcelas com 100 m2/ha e 20 frutos avaliados por subparcela

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-16T13:22:52Z No. of bitstreams: 1 Edilaine Alves de Melo.pdf: 641010 bytes, checksum: 2a8032c2676a99782e8fd581d977a950 (MD5) / Made available in DSpace on 2017-03-16T13:22:52Z (GMT). No. of bitstreams: 1 Edilaine Alves de Melo.pdf: 641010 bytes, checksum: 2a8032c2676a99782e8fd581d977a950 (MD5) Previous issue date: 2012-07-27 / The bacterial fruit blotch caused by Acidovorax citrulli is one of the most severe diseases of melon (Cucumis melo), and a major problem in the Northeast, the main melon producing region of Brazil. Strategies for control of bacterial blotch include chemical and physical treatments of seeds and chemical sprays of the plant canopy. Since these treatments are not efficient and resistant melon cultivars do not exist, other strategies have been studied, including biological control. Our objectives were to analyze the efficiency of yeasts in the biocontrol of this disease by protecting seedlings and plants, and by treating melon seeds; and to verify the in vitro activity against the pathogen and the growth promotion of melon plants. None of the 60 yeasts inhibited the growth of the pathogen, but the isolates LMA1 (Rhodotorula aurantiaca), LMS (R. glutinis) and CC-2 (Pichia anomala) stood out as the most effective in protecting seedlings. When tested in plants and seeds, LMA1 and CC-2 maintained effectiveness. In plants, the reductions in disease index (ID) and area under the disease progress curve (AUDPC) compared to the control reach 58.6 and 47.2%, respectively, while seed treatments reduced ID and AUDPC up to 34.3 and 45.5%. These isolates did not promote the growth of melon plants and did not produce killer toxins in vitro. R. aurantiaca (LMA1) and P. anomala (CC-2) were effective in protecting plants and seedlings and for seed treatment of melon. Therefore, the use of these yeasts jointly with other control methods, such as resistant varieties and copper compounds, is important in integrated management of bacterial fruit blotch. / A mancha aquosa causada por Acidovorax citrulli, é uma das doenças mais severas do meloeiro (Cucumis melo) e um dos principais problemas para o Nordeste, a principal região produtora de melão do Brasil. Estratégias para o controle da mancha aquosa incluem tratamentos químicos e físicos das sementes e químico da parte aérea da planta. Uma vez que esses tratamentos não são eficientes e cultivares resistentes de meloeiro inexistem, outras estratégias têm sido investigadas, dentre elas o controle biológico. Os objetivos deste trabalho foram analisar a eficiência de leveduras no biocontrole dessa doença pela proteção de plântulas e plantas e pelo tratamento de sementes de meloeiro, além de verificar a atividade in vitro contra o patógeno e a promoção do crescimento de plantas de meloeiro. Nenhuma das 60 leveduras testadas inibiu o crescimento do patógeno, porém os isolados LMA1 (Rhodotorula aurantiaca), LMS (R. glutinis) e CC-2 (Pichia anomala) destacaram-se como os mais eficientes na proteção de plântulas. Quando testadas em plantas e sementes, LMA1 e CC-2 mantiveram a eficácia. Em plantas, as reduções de índice de doença (ID) e área abaixo da curva de progresso da doença (AACPD) em relação à testemunha foram de até 58,6 e 47,2%, respectivamente, enquanto que o tratamento de sementes reduziu o ID e AACPD em até 34,3 e 45,5%. Esses isolados não promoveram o crescimento do meloeiro e não produziram toxinas killer in vitro. R. aurantiaca (LMA1) e P. anomala (CC-2) foram eficazes na proteção de plântulas e plantas e no tratamento de sementes de meloeiro. Portanto, a utilização dessas leveduras junto a outros métodos de controle, tais como cultivares resistentes e utilização de compostos cúpricos, será importante no manejo integrado da mancha aquosa.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-16T14:38:17Z No. of bitstreams: 1 Elineide Barbosa de Souza.pdf: 390725 bytes, checksum: 343f1ce1b17f2c507a8bc4587f0077ac (MD5) / Made available in DSpace on 2017-03-16T14:38:17Z (GMT). No. of bitstreams: 1 Elineide Barbosa de Souza.pdf: 390725 bytes, checksum: 343f1ce1b17f2c507a8bc4587f0077ac (MD5) Previous issue date: 2002-06-28 / The variability of 20 Acidovorax avenae subsp. citrulli isolates in relation to melon fruit blotch components, hypersensitive reaction, and the bacterial transmission by seeds from inoculated fruits were studied. The influence of duration (0, 6, 24 and 48 h), the onset of leaf wetness period (0, 6, 24 and 48 h after inoculation), and inoculum concentration of A. avenae subsp. citrulli (3.4 x 101 to 3.4 x 107 CFU mL-1) on severity of fruit blotch in melon plants were also evaluated. The effects of temperature (15, 20, 25, 30, 35 and 40° C), humidity (0 and 6 h of moist chamber), inoculum concentration (3.4 x 101 to 3.4 x 107 CFU mL-1) and fruit age (40, 50, 60 and 70 days) on the development of melon fruit blotch were also verified. Seeds, plants and fruits were inoculated through vacuum infiltration, atomization and sub-epidermal injection, respectively. Seedlings and plants were assessed in relation to incubation period, diseaseindex, area under disease progress curve and disease progress rate; incubation period, diameter of external lesion and lesion depth were assessed on the fruits. The data were submitted to mean comparison tests, clustering tests or regression analysis. The Euclidian distance–single linkage confirmed the variability among the A. avenae subsp. citrulli strains allowing their separation in four similarity groups. Seed transmission ranged from 30 to 64 % and all strains induced hypersensitive reaction on tobacco and tomato leaves. The regression equations for the analyzed variables in melon plants were better adjusted by the quadratic or logarithmic models. The incubation period ranged from 1.3 to 2.7 days and was higher in plants without leaf wetness, although the disease index and area under disease progress curve increased as the duration of leaf wetness increased. The beginning of the leaf wetness period at 48 h after inoculation elevatedthe incubation period and disease progress rate in relation to the other periods. The disease progress rate, disease index and area under disease progress curve increased as the inoculum concentration increases, reaching maximum values of 4.4 infection units/day, 73.7 % and 18.9 at 3.4 x 107 UFC mL-1, respectively, at 3.4 x 101 CFU mL-1. The temperature and humidity influenced significantly (P=0,05) the severity of melon fruit blotch, however, the incubation period was not affected. The larger external lesions were observed in the fruits incubated at 35 and 30° C without moist chamber, and at 30° C in moist chamber for six h. In relation to lesion depth, those lesions in fruits incubated without moist chamber were deeper at 25 and 30° C. However, with moist chamber the lesions at 30° C were deeper than the others. No disease symptoms were observed onfruits incubated at 15 and 20° C. The humidity significantly (P=0,05) influenced the development of external lesions and lesions depth at 35 and 25° C, respectively. The diameter and depth of lesions increased as the inoculum concentration was higher and were reduced as the fruit age increased. No external or internal lesions were detected on fruits with 60 and 70 days inoculated with the pathogen at 3.4 x 101 CFU mL-1. / Foi estudada a variabilidade de 20 isolados de Acidovorax avenae subsp. citrulli quanto aos componentes da mancha-aquosa do melão e reação de hipersensibilidade, e analisada a transmissão da bactéria pelas sementes dos frutos inoculados. Foi também avaliada a influência da duração (0, 6, 24 e 48 horas) e início do período de molhamento foliar (0, 6, 24 e 48 horas após a inoculação), bem como da concentração de inóculo de A. avenae subsp. citrulli (3,4 x 101 a 3,4 x 107 UFC mL-1) na severidade da mancha-aquosa em meloeiro. A influência da temperatura (15, 20, 25, 30, 35 e 40° C), da umidade (0 e 6 horas de câmara úmida), da concentração de inóculo ( 3,4 x 101 a 3,4 x 107 UFC mL-1) e da idade do fruto (40, 50, 60 e 70 dias) no desenvolvimento da mancha-aquosa em melão, foram ainda verificadas. Sementes, plantas e frutos foram inoculados pelos métodos de infiltração a vácuo, pulverização e injeção subepidérmica, respectivamente. Plântulas e plantas foram avaliadas quanto ao período de incubação,índice de doença, área abaixo da curva do progresso da doença e taxa de progresso da doença, e os frutos, quanto ao período de incubação, diâmetro da lesão externa e profundidade da lesão. Os dados obtidos foram submetidos a testes de comparação de médias, testes de agrupamento ou análises de regressão. A análise da distância Euclidiana por ligações simples, confirmou a variabilidade entre os isolados de A. avenae subsp. citrulli, permitindo a separação destes em quatro grupos de similaridade. A transmissão da bactéria por sementes variou de 30 a 64 % e todos os isolados induziram reação de hipersensibilidade em folhas de fumo e tomate. As equações de regressão para as variáveis analisadas em meloeiros foram melhor ajustadas pelos modelos quadrático ou logarítmico. O período de incubação variou de 1,3 a 2,7 dias, emaior nas plantas sem molhamento foliar, contudo o índice de doença e a área abaixo da curva de progresso da doença aumentaram com o incremento da duração do molhamento foliar. O início do período de molhamento foliar às 48 horas após a inoculação elevou o período de incubação e a taxa de progresso da doença em relação aos demais períodos. O incremento da concentração de inóculo elevou a taxa de progresso da doença, índice de doença e área abaixo da curva de progresso da doença, os quais atingiram valores máximos de 4,4 unidades de infecção/dia, 73,7 % e 18,9, respectivamente, na concentração 3,4 x 107 UFC mL-1. A temperatura e umidade influenciaram significativamente a severidade da mancha-aquosa nos frutos, embora o período de incubação não tenha sido afetado. As maiores lesões externas foram observadas em frutos incubados a 35 e 30 ° C sem câmara úmida, e a 30° C em câmaraúmida por seis horas. Com relação à profundidade, as lesões nos frutos incubados sem câmara úmida foram maiores às temperaturas de 25 e 30° C. Em câmara úmida, as lesões a 30° C foram maiores que as demais. Não foi observado desenvolvimento da mancha-aquosa em frutos incubados a 15 e 20° C. A umidade influenciou significativamente (P=0,05) o diâmetro e profundidade da lesão, exceto às temperaturas de 35 e 25° C, respectivamente. O diâmetro e profundidade das lesões aumentaram com a elevação da concentração de inóculo e foram reduzidos com o aumento da idade do fruto. Na concentração 3,4 x 101 UFC mL-1 não foi observada a presença de sintomas da doença na casca e na polp

Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit. / Ph. D.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T15:33:36Z No. of bitstreams: 1 Francisco Conrado Queiroz Carvalho.pdf: 693930 bytes, checksum: 0e5f734618ab807f4c62e74984f62954 (MD5) / Made available in DSpace on 2017-03-10T15:33:36Z (GMT). No. of bitstreams: 1 Francisco Conrado Queiroz Carvalho.pdf: 693930 bytes, checksum: 0e5f734618ab807f4c62e74984f62954 (MD5) Previous issue date: 2012-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Bacterial fruit blotch caused by Acidovorax citrulli occurs in different parts of watermelon plant, at different stages of development but symptoms are more conspicuous and easy to diagnose in fruit. BFB has caused significant economic losses to melon production in Brazil and is a major threat to the watermelon fields. This justifies the research for resistance sources to be used in breeding programs aiming to obtain varieties resistant to BFB, since they do not yet exist. To select genotypes with potential use in the management of fruit blotch, the resistance level of watermelon genotypes belonging to the Cucurbits Germplasm Active Bank for the Brazilian Northeast (Banco Ativo de Germoplasma de Cucurbitáceas para o Nordeste Brasileiro - BAG) of Embrapa Semiárido was evaluated at different plant developmental stages: seeds (74 genotypes), seedlings and plants before flowering (29 genotypes) as well as plants during flowering and fruiting (7 genotypes). The genotypes were evaluated for the incidence or severity of the disease, which was estimated with the aid of descriptive scales. Additionally, A. citrulli transmission was determined in seeds derived from symptomatic and asymptomatic fruits. No watermelon genotype was immune to fruit blotch, and the majority showed variations in resistance responses. However, the genotypes BGCIA 979, BGCIA 34 and Sugar Baby showed high levels of resistance at most stages of plant development, thereby suggesting that these genotypes possess fruit blotch resistance genes that could be used in breeding programs. Seeds from symptomatic and asymptomatic fruits of the seven tested genotypes showed transmission rates of A. citrulli up to 35.3% and 8.7%, respectively. These results confirm that asymptomatic fruits can harbor contaminated seeds that are responsible for the transmission of the bacteria. / A mancha aquosa, causada pela bactéria Acidovorax citrulli, ocorre em distintos órgãos de melancia, em diferentes estádios de desenvolvimento, sendo os sintomas mais comuns e de fácil diagnose nos frutos. Essa doença é responsável por elevadas perdas econômicas na cultura do meloeiro no Brasil e uma grande ameaça para a melancia. Isto justifica a busca de fontes de resistência a serem utilizadas em programas de melhoramento visando à obtenção de variedades dessa olerícola com resistência a doença. Com o objetivo de selecionar genótipos com potencial de utilização no manejo da mancha aquosa, avaliou-se o nível de resistência de 74 genótipos de melancia pertencentes ao Banco Ativo de Germoplasma de Cucurbitáceas para o Nordeste Brasileiro (BAG) da Embrapa Semiárido, em diferentes estádios de desenvolvimento da planta: sementes (74 genótipos), plântulas e plantas antes da floração (29 genótipos), plantas durante a floração e frutificação (7 genótipos). Os genótipos foram avaliados quanto a incidência ou severidade da doença, esta estimada com auxílio de escalas descritivas. Adicionalmente, foi determinada a transmissão de A. citrulli em sementes oriundas de frutos sintomáticos e assintomáticos. Nenhum genótipo de melancia foi imune à mancha aquosa, e a maioria apresentou variação nas reações de resistência. Porém, os genótipos BGCIA 979, BGCIA 34 e ‘Sugar Baby’ mostraram altos níveis de resistência na maioria dos ensaios realizados, indicando possuírem genes para resistência à mancha aquosa que poderão ser utilizados em programas de melhoramento. Sementes de frutos sintomáticos e assintomáticos dos sete genótipos apresentaram transmissão de A. citrulli de até 35,3% e 8,7% respectivamente, confirmando que frutos assintomáticos podem abrigar sementes contaminadas responsáveis pela transmissão da bactéria.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-23T15:39:15Z No. of bitstreams: 1 Valter Alexandre Vieira da Silva.pdf: 281801 bytes, checksum: 0bb776ffcea4e506c269e4726b3eafa8 (MD5) / Made available in DSpace on 2017-03-23T15:39:15Z (GMT). No. of bitstreams: 1 Valter Alexandre Vieira da Silva.pdf: 281801 bytes, checksum: 0bb776ffcea4e506c269e4726b3eafa8 (MD5) Previous issue date: 2005-02-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / One of the main diseases occurring in melon fields in Brazilian Northeastern is the bacterial fruit blotch caused by the bacterium Acidovorax avenae subsp. citrulli. This study aimed to survey the bacterial fruit blotch incidence in 12 melon planting areas in Juazeiro (BA), in two consecutive plantings, and determine the ability of A. avenae subsp. citrulli to survive epiphytically and endophytically on the leaves and roots, and also in the rhizosphere of melon by using a mutant resistant to rifampicin (Aac1Rif). The surveys were conducted to determine the disease incidence and prevalence in 12 commercial melon areas chosen randomly with plants at harvesting time. In each area 25 sub-plots with 100 m2 were marked and 20 fruits evaluated in a diagonal line. In the first survey performed in January 2004, the prevalence of bacterial blotch was 33.33% and there was low incidence in the four areas where the diseaseoccurred, ranging from 0.2 to 4.4%. In the second survey performed in the same areas in April 2004 the disease was not detected. For the survival study on leaves, melon plants with 18-day, grown in greenhouse and field, were sprayed with mutant suspensions at concentrations 3.4 x 102, 3.4 x 103 and 3.4 x 104 CFU mL-1. To determine survival on roots and rhizosphere, seeds of melon type Yellow were sown in soil infested with suspensions of Aac1Rif at 3.4 x 105, 3.4 x 106 and 3.4 x 107 CFU mL-1. At 6-day intervals samples of leaves, roots and rhizosphere soil were collected and processed for isolation on NYDA medium amended with refampicin. Bacterial populations were then determined as UFC g-1 of sample and the data were transformed to log10 for regression analysis. On melon leaves, in greenhouse and field Aac1Rifsurvived epiphytically for 54 days. These epiphytic bacterial populations increasedinitially and decreased after certain time. The final populations were similar in the two conditions and ranged from 103 to 104 CFU g-1 leaf. On the roots and rhizosphere in greenhouse bacterial populations decreased according to the inoculum concentration and at 60 days after planting ranged from 102 to 103 CFU g-1 roots and 101 to 102 CFU g-1 soil. AacRif was not detected as endophytic in leaves or roots of melon. / Uma das principais doenças que vem ocorrendo em campos de meloeiro do Nordeste brasileiro é a mancha-aquosa, causada pela bactéria Acidovorax avenae subsp. citrulli. Este trabalho teve por objetivos realizar o levantamento epidemiológico da incidência da mancha-aquosa em 12 áreas comerciais de meloeiro em Juazeiro (BA), em dois plantios sucessivos, e determinar a capacidade de sobrevivência de A. avenae subsp. citrulli como epifítica e endofítica nas folhas e raízes, e na rizosfera de meloeiro, através da utilização de um mutante resistente a rifampicina (Aac1Rif). Levantamentos foram conduzidos para determinar a prevalência e a incidência da mancha-aquosa em 12 áreas comerciais de meloeiro, escolhidas ao acaso, com plantas em estádio de colheita. Em cada área foram demarcadas 25 subparcelas de 100 m2 e avaliados 20 frutos/subparcela ao longo da diagonal. No primeiro levantamento, realizado em janeirode 2004, a prevalência da mancha-aquosa foi de 33,33%, com baixa incidência nas quatro áreas em que foi registrada, variando de 0,2 a 4,4%. No segundo levantamento, realizado nas mesmas áreas em abril de 2004, a doença não foi constatada. Para o estudo de sobrevivência em folhas, meloeiros com 18 dias, cultivados em casa de vegetação e no campo, foram pulverizados com suspensões do mutante nas concentrações (3,4 x 102, 3,4 x 103 e 3,4 x 104 UFC mL-1). Para determinar a sobrevivência em raízes e na rizosfera, sementes de melão Amarelo foram semeadas em solo infestado com suspensões de Aac1Rif a 3,4 x 105, 3,4 x 106 e 3,4 x 107 UFC mL-1. A intervalos de seis dias, amostras de folhas, raízes e solo rizosférico foram coletadas e processadas para isolamento em meio ágar nutritivo-extrato de levedura-dextrosecontendo rifampicina. As populações bacterianas foram determinadas em UFC g-1 deamostra e os dados transformados em log10 para análise de regressão. Nas folhas de meloeiro, em casa de vegetação e campo, o mutante Aac1Rif sobreviveu epifiticamente durante 54 dias, observando-se inicialmente aumento da população bacteriana epifítica, com posterior declínio, sendo as populações finais semelhantes nas duas condições estudadas, com valores variando de 103 a 104 UFC g-1 de folha. Nas raízes e rizosfera, em casa de vegetação, a população bacteriana decresceu variando em função da concentração utilizada até atingir aos 60 dias após o plantio, níveis de 102 a 103 UFC g-1 de raiz e 101 a 102 UFC g-1 de solo. AacRif não foi detectado sobrevivendo endofiticamente em folhas ou raízes de meloeiro.

Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background. / Doctor of Philosophy / Melon and watermelon crops both belong to the gourd family. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies and detection methods are very limited. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate disease resistance genes, and optimization of virus induced gene silencing methodology for melon and watermelon crops. There are currently no nondestructive assays available to detect BFB in seeds. We evaluated the potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds. It was possible to detect BFB inside layers of seeds that were naturally inoculated through the flowers via x-ray imaging and seedcoat inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea and tea constituents as seed treatments to sanitize seeds infected with BFB. Green tea unlike black tea inhibited growth of BFB. Eighty one melon and forty four watermelon disease resistance genes were reidentified and genes that have potential resistance against BFB on melon plants were screened based on host selectivity of the pathogen. Finally, the virus induced gene silencing method was optimized for melon and watermelon plants for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate resistance genes were identified that might confer stable resistance in the right genetic background.