Atualmente, a videira Niagara Rosada é a variedade de uva de mesa mais cultivada no Estado de São Paulo, Brasil. Contudo, sua produtividade e custo de produção têm sido afetados pela ocorrência de doenças fúngicas, principalmente o míldio, causado pelo fungo Plasmopara viticola. Para o controle dessa doença, têm sido feitas pulverizações com fungicidas, as quais muitas vezes em excesso. Dentre as variáveis meteorológicas que influenciam a ocorrência de doenças fúngicas nas plantas, a duração do período de molhamento (DPM) é uma das mais importantes. A presença de molhamento sobre a superfície das plantas provê a água requerida pelos patógenos para o processo de germinação e infecção do tecido foliar. Visando a subsidiar sistemas agrometeorológicos de alerta fitossanitário, cuja finalidade é possibilitar a racionalização do uso de fungicidas, os objetivos do presente estudo foram: determinar a posição da videira com a DPM mais longa e sua relação com a DPM medida na posição padrão, a 30 cm de altura sobre o gramado; avaliar a estimativa da DPM sobre o gramado por diferentes modelos a partir de dados meteorológicos obtidos em uma estação meteorológica padrão e verificar as relações entre a DPM estimada para o gramado e a DPM medida no vinhedo; e correlacionar a epidemiologia do míldio (P. viticola) durante o ciclo da videira Niagara Rosada, sem controle químico, com as variáveis DPM medida no vinhedo (posição da videira com a DPM mais longa), DPM estimada na posição padrão sobre o gramado pelo melhor modelo, assim como com outras variáveis meteorológicas. A DPM foi medida em posição padrão sobre o gramado e em quatro diferentes posições da videira: topo da planta com a face superior do sensor voltada para sudoeste e nordeste (Topo-SW e Topo-NE) e altura dos cachos de uva com a face superior do sensor voltada para sudoeste e nordeste (Dossel-SW e Dossel-NE). A epidemiologia do míldio foi avaliada sem controle químico, utilizando-se escala de notas de 0 a 4, com nove níveis de severidade, para as folhas e cachos da videira. No estudo da variabilidade espacial da DPM, não houve diferença significativa entre a parte mais alta (1,6 m) e a parte mais baixa (1,0 m) da videira, assim como entre as faces sudoeste e nordeste das plantas. Ao se analisar a relação entre a DPM sobre o gramado e a DPM nas diferentes posições da videira obtida por meio de regressão linear simples, observou-se uma boa correlação, com R2 = 0,88. Na avaliação dos modelos de estimativa da DPM, o modelo CART foi o que teve o melhor desempenho sobre o gramado, sendo que esta estimativa também apresentou uma boa correlação com a DPM medida no interior do vinhedo. Isso permitiu concluir que é possível estimar a DPM no vinhedo de Niagara Rosada a partir de medidas ou estimativas da DPM na estação meteorológica padrão. Na modelagem do desenvolvimento do míldio, a DPM esteve presente nas melhores correlações com a severidade do míldio na videira Niagara Rosada, mostrando a grande importância desta variável para a ocorrência de doenças fúngicas nos vinhedos. / Nowadays, the \'Niagara Rosada\' grapevine is the most cultivated table grape variety in the State of São Paulo, Brazil. However, yield and production cost of this grapevine have been affected by fungal diseases, mainly downy mildew, caused by Plasmopara viticola fungus. For controlling this disease, producers have been applied an excessive number of sprays with fungicides. Among the meteorological variables that influence the occurrence of fungal plant diseases, leaf wetness duration (LWD) is one of the most important. The wetness presence on plant surface provides the water required by the phatogens to germinate and to infect leaf tissues. Aiming to subsidize the plant disease warning systems, which has as purpose to rationalize the use of fungicides in the vineyards, the objectives of the present study were: to determine the canopy position of the Niagara Rosada table grape with longer LWD and its correlation with measured standard LWD over turfgrass; to estimate LWD over turfgrass considering different models with data from a standard weather station, and to evaluate the correlation between estimated LWD over turfgrass and LWD measured in the vineyard; and to correlate downy mildew occurrence in the vineyard, without chemical control, with measured LWD at the vineyard (canopy position with longer LWD), with estimated LWD in standard condition over turfgrass for the best model, and with other meteorological variables. LWD was measured in standard condition over turfgrass and in four different canopy positions of the vineyard: at the top of the plants, with sensors facing southwest and northeast (Top-SW and Top-NE), and at the grape bunches height, with sensors facing southwest and northeast (Bottom-SW and Bottom-NE). The downy mildew epidemiology during the grapevine cycle was evaluated without chemical control, using scores ranging from 0 to 4, with nine severity levels, for leaves and bunches of grapevine. When the spatial variability of LWD was studied, no significant difference was observed between the top (1.6 m) and the bottom (1.0 m) of the canopy and also between the southwest and northeast face of the plants. The analysis of the relationship between standard LWD over turfgrass and crop LWD in different positions of the grape canopy showed a define correlation (R2 = 0.88). Among the LWD estimative methods, CART was the one with the best performance to estimate LWD over turfgrass. The results from this model also presented a good correlation with measured LWD inside the vineyard, showing that LWD can be estimated for this crop with data from a nearby standard weather station. For the downy mildew modeling, LWD also present the best correlations with disease severity in the \'Niagara Rosada\' vineyard, showing the great importance of this variable for fungal diseases occurrence in this crop.

Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. / Microsoft� Word 2010 / Adobe Acrobat 9.54 Paper Capture Plug-in

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases (endo-PGs) from phytopathogenic fungi. For proteins to confer resistance to invading plant pathogens, it is preferred that they are either associated with the plant cell wall or secreted into the intercellular spaces where they can act almost immediately upon pathogen attack. The bactericidal efficacy of the Hen Egg White Lysozyme (HEWL) has previously been unequivocally demonstrated in transgenic plants; however, most of the protein remains intracellular. It was hypothesized that bean PGIP1, that has previously been expressed correctly in transgenic tomato plants and was found to inhibit the endopolygalacturonase activity of Stenocarpella maydis in a reducing sugar assay, would deliver the HEWL protein to the intercellular spaces due to its inherent translocation to the plant cell wall by means of a translational fusion between bean pgip1 and hewl genes. In this study, the efficacy of such a translational fusion was determined. The bean pgip1-hewl fusion was inserted into the binary vector pCAMBIA2300 and transformed into Nicotiana tabacum cv. LA Burley 21 plants by Agrobacterium-mediated transformation. Phenotypically normal transgenic plants were produced. Stable transgene insertion into the transgenic N. tabacum genomes was verified by PCR and Southern blot analyses. To demonstrate the efficacy of the bean PGIP1-HEWL fusion, independent homogenate and intercellular fluid protein extracts were prepared from transgenic N. tabacum leaf material. Protein extracts prepared so as to enrich for PGIP activity were tested in vitro for inhibition of S. maydis endo-PGs whereas protein extracts for HEWL activity were tested for lysis of Micrococcus luteus cells. Biochemical assays showed that bean PGIP1-HEWL inhibited S. maydis endo-PGs and cleaved M. luteus cell walls sufficiently to suggest that the PGIP1- HEWL fusion was structurally and functionally stable. Total protein extracts from the PGIP-HEWL and HEWL transgenic plants showed similar levels of HEWL specific activity, whereas intercellular fluid samples from PGIP-HEWL transgenic plants showed high activity in contrast to HEWL plants. With the success of showing protein activity in vitro of HEWL in intercellular spaces, bean PGIP1 can be recommended as a vehicle for delivery of other proteins to cell surfaces. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Feltman, NR 2006, Development of the polygalacturonase inhibiting protein (PGIP) for delivery of foreign proteins to the surfaces of plant cells, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-07152008-111131 / > E416/gm / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

Os elementos genéticos móveis encontrados no genoma da bactéria Xylella fastidiosa (Xf) são representados principalmente por bacteriófagos (na forma de profagos inseridos no genoma) e ilhas genômicas. As integrases são responsáveis pelo processo de mobilização (integração e/ou excisão) destes elementos, através do mecanismo de recombinação sítio-específica. Bacteriófagos e ilhas genômicas estão associados a eventos de rearranjos genômicos e à aquisição e ou interrupção de genes importantes para bactéria, tendo implicação direta na diversidade e organização genômica e, por conseqüência, na diferenciação entre linhagens. A extensão e o impacto desses eventos é o foco deste trabalho, através da análise in-silico das integrases e sua associação com regiões de profagos e ilhas genômicas, no genoma de quatro linhagens de Xf. Os dados aqui apresentados corroboram o papel das integrases e seus elementos genéticos móveis associados como agentes chaves no processo de diversidade e evolução da organização genômica entre as quatro linhagens de Xf. / The mobile genetic elements found in the genome of the bacterium Xylella fastidiosa (Xf) are mainly represented by bacteriophages (as prophages inserted into the genome) and genomic islands. The integrases are responsible for the process of mobilization (integration and / or excision) of these elements through the mechanism of site-specific recombination. Bacteriophages and genomic islands are associated with events of genomic rearrangements and the acquisition and or interruption of important genes for bacteria, with direct involvement in diversity and genomic organization and, consequently, the differentiation between strains. The extent and impact of these events are the focus of this work, through in-silico analysis of integrase and association with regions of prophages and genomic islands in the genome of four strains of Xf. The data presented here support the role of integrases and their mobile genetic elements associated as key players in the process of evolution and diversity of genomic organization between the four strains of Xf.

Orientador: Edson Luiz Furtado / Resumo: A seringueira (Hevea brasiliensis) é a terceira cultura florestal mais plantada no Brasil, e o estado de São Paulo é responsável por mais de 60% da produção de látex do país. É uma espécie nativa da região amazônica que possui hábito caducifólio, e durante o período de reenfolha é suscetível ao ataque de fitopatógenos foliares, que prejudicam e até limitam seu estabelecimento em determinadas regiões. Dentre os principais patógenos foliares da seringueira, destacam-se os fungos causadores de oídios, microrganismos biotróficos que infectam tecidos jovens, e podem causar desfolha, principalmente em condições de viveiro e jardim clonal. O agente causal do oídio da seringueira é descrito como Erysiphe quercicola, porém, devido a insuficientes descrições taxonômicas, morfológicas e moleculares desta doença, seu agente causal permanece em discussão, além da fase sexual deste patógeno carecer de qualquer registro neste hospedeiro. Portanto, o objetivo deste trabalho é caracterizar o oídio da seringueira, utilizando caracterizações morfológicas, análises moleculares e testes de patogenicidade. No período de julho a dezembro de 2019, foram coletadas amostras foliares de seringueira em diferentes estágios fenológicos infectadas com oídio na região de Botucatu, São Paulo. Para a caracterização morfológica, foram registradas e mensuradas características das hifas, conídios e conidióforos, que correspondem à fase assexuada do fungo. A caracterização molecular foi realizada através de análi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rubber tree (Hevea brasiliensis) is the third most planted forest crop in Brazil, and the state of São Paulo is responsible for more than 60% of the country's latex production. It is a species native to the Amazon region that has a deciduous habit, and during the re-leafing period it is susceptible to the attack of leaf phytopathogens, which harm and even limit its establishment in certain regions. Among the main leaf pathogens of the rubber tree, there are the fungi that cause powdery mildew, biotrophic microorganisms that infect young tissues, and can cause defoliation, especially in nursery and clonal garden conditions. The causal agent of rubber mildew is described as Erysiphe quercicola, however, due to insufficient taxonomic, morphological and molecular descriptions of this disease, its causal agent remains under discussion, in addition to the sexual phase of this pathogen lacking any record in this host. Therefore, the objective of this work is to characterize powdery mildew mildew, using morphological characterizations, molecular analyzes and pathogenicity tests. From July to December 2019, leaf samples of rubber trees were collected at different phenological stages infected with powdery mildew in the Botucatu region, São Paulo. For morphological characterization, characteristics of hyphae, conidia and conidiophores, which correspond to the asexual phase of the fungus, were recorded and measured. Molecular characterization was performed through phylogenetic analyzes o... (Complete abstract click electronic access below) / Mestre

Les champignons phytopathogènes sont des parasites majeurs des espèces végétales, autant naturelles que domestiquées. Botrytis cinerea, l’agent de la pourriture grise, en infecte plus de 1400 et est ainsi considéré comme un pathogène généraliste. Pourtant, des données récentes ont mis en évidence une structure des populations liée à leur hôte d’origine. Cette observation soulève l’hypothèse d’une spécialisation à l’hôte, à l’œuvre chez une espèce généraliste. Ce modèle d’étude pourrait permettre de faire avancer la connaissance des mécanismes évolutifs en jeu dans la divergence précoce des populations et la formation de nouvelles espèces fongiques. Cette thèse de doctorat a pour objectifs : (i) de démontrer formellement la spécialisation à l’hôte dans les populations de B. cinerea et d’en déterminer la magnitude et (ii) d’identifier les déterminants génomiques de cette spécialisation. Ainsi j’ai étudié la structure des populations par l’analyse de microsatellites d’un échantillon de 683 isolats, que nous avons corrélé à des tests de pathogénicité croisés sur différents hôtes. Ces travaux ont permis de mettre en évidence la spécialisation de B. cinerea aux hôtes tomate et vigne. En complément de ces lignées sélectionnées, l’espère Botrytis cinerea est constituée d’individus généralistes capables de coloniser les autres hôtes. Des méthodes d’inférence de structure et de généalogies sur des données de polymorphisme issues du séquençage de 32 individus nous ont permis de mieux définir la structure des populations ainsi que d’identifier une lignée spécialisée à la tomate. Enfin, des tests de McDonald-Kreitman et des méthodes de scans génomiques pour détecter des balayages sélectifs ont permis de mettre en évidence des gènes soumis à la sélection divergente entre les populations spécialisées, révélant de possibles déterminants de cette spécialisation. Ces travaux sont ainsi une base pour la validation de plusieurs gènes impliqués dans la pathogénicité hôte-spécifique de B. cinerea, et ouvrent la voie à des améliorations de la gestion du réservoir d’hôtes et des pratiques culturales contre la pourriture grise. / Phytopathogenic fungi are major parasites to wild or domesticated plant species. The grey mold fungus, Botrytis cinerea, infects more than 1400 plant species and thus is considered a broad generalist. However, recent data have revealed population structure correlated to the host of origin of isolates. This observation raises the hypothesis of ongoing host specialization in a generalist species. Studying this question could greatly deepen our theoretical knowledge of the evolutionary mechanisms involved in the early stages of population divergence and subsequent speciation. This thesis aims (i) to formally demonstrate the host specialization in B. cinerea’s populations and determine its magnitude, and (ii) to identify the genomic determinants of this specialization. Thus, I studied population structure based on 683 isolates characterized using microsatellite markers. We compared the inferred genetic structure with variations in aggressiveness measured through cross-pathogenicity tests on multiple hosts. These experiments and analyses confirmed the specialization of B. cinerea to tomato and grapevine hosts. Besides these specialized lineages, the species B. cinerea is composed of generalist individuals capable of infecting multiple hosts. I sequenced the whole genome of 32 individuals and characterized nucleotide polymorphism. Structure inference and genomic genealogy methods allowed us to more accurately define the population structure and identify a lineage specialized on tomato. Lastly, McDonald-Kreitman tests and genomic scans methods allowed the identification of genes under divergent natural selection between populations, revealing possible genomic determinants of specialization. This work can serve as foundation for the validation of multiple genes involved in host-specific pathogenicity of B. cinerea, and pave the way for the implementation of efficient strategies for managing pathogen reservoirs and new agricultural practices for controlling grey mold.

The initial phase in the development of a biological control strategy is screening of biological control agents. Secondary to this phase is the establishment of accurate, effective application techniques. However, successful control requires a thorough understanding of all factors affecting the relationship between host plant, pathogen and other microbes. The purpose of this study was to screen and identify potential bacterial antagonists against Alternaria, a fungal citrus pathogen, attachment of the antagonists to bees, and bee dissemination of the antagonist to citrus flowers. A total of 568 bacterial epiphytes were screened on agar plates for antagonism against Alternaria. Only eight of these isolates, which were identified as Bacillus subtilis, B licheniformis, B. melcerons, B. polymyxa, B. thermoglycodasius, B. sphaericus, B. amiloliquefaciens, and B. coagulans, showed inhibitory effects on the growth of Alternaria. The most effective isolates were B. subtilis and B. licheniformis. Further screening was done with B. subtilis and B. subtilis commercial powder (Avogreen). These bacteria were sprayed on citrus flowers for colonisation studies. Mean populations of B. subtilis and the commercial powder recovered from the flowers were 104 and 103 cfu/stamen respectively. The organisms colonised the styler end and ovary of the flowers when observed under scanning electron microscope (SEM). Avogreen was placed in an inoculum dispenser, which was attached to the entrance of the hive. Honeybees emerging from the beehive acquired 104 cfu/bee. The powder attached to the thorax and thoracic appendages, as revealed by SEM. One active beehive was placed in an enclosure with fifteen flowering citrus nursery trees in pots for dissemination trials. Mean populations of commercial B. subtilis recovered from the flowers visited by bees were 104 cfu/stamen. Electron microscope studies revealed that the antagonist was colonising the styler end and ovary of the flowers. Field dissemination studies were unsuccessful due to low yields. / Dissertation (Magister Institutiones Agrariae)--University of Pretoria, 2003. / Plant Production and Soil Science / unrestricted

Thesis: Ph. D., Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Zooplankton, such as copepods, are highly abundant environmental reservoirs of many bacterial pathogens. Although copepods are known to support diverse and productive bacterial communities, little is understood about whether copepods are affected by bacterial attachment and whether they can regulate these associations through mechanisms such as the innate immune response. This thesis investigates the potential role that copepod physiology may play in regulating Vibrio association and the community structure of its microbiome. To this end, the intrinsic ability of oceanic copepod hosts to transcriptionally respond to mild stressors was first investigated. Specifically, the transcriptional regulation of several heat shock proteins (Hsps), a highly conserved superfamily of molecular chaperones, in the copepod Calanusfinmarchicus was examined and demonstrated that Hsps are a conserved element of the copepod's transcriptional response to stressful conditions and diapause regulation. To then investigate whether copepod hosts respond to and regulate their microbiota, the transcriptomic response of an estuarine copepod Eurytemora affinis to two distinct Vibric species, a free-living strain (V. ordalii 12B09) and a zooplankton specialist (V. sp. F10 9ZB36), was examined with RNA-Seq. Our findings provide evidence that the copepod E. affinis does distinctly recognize and respond to colonizing vibrios via transcriptional regulation of innate immune response elements and transcripts involved in maintaining cuticle integrity. Our work also suggests that association with E. affinis can significantly impact the physiology of Vibrio colonists. Finally, the inter-individual variability of the C.finmarchicus microbiome was examined to identify how specifically and predictably bacterial communities assemble on copepods and whether host physiology influences the bacterial community structure. Our findings suggest that copepods have a predictable "core microbiome" that persists throughout the host's entrance into diapause, a dormancy period characterized by dramatic physiological changes in the host. However, diapausing and active populations harbor distinct flexible microbiomes which may be driven by factors such including the copepod's feeding history, body size, and bacterial interactions. This thesis work highlights the role of copepods as dynamic reservoirs of diverse bacterial communities and implicates copepod host physiology as an important contributor to the activity, abundance, and community structure of its microbiome. / by Amalia Aruda Almada. / Ph. D.

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