Spiroplasma citri est un mollicute phytopathogène transmis de plante à plante par des cicadelles du genre Circulifer selon un mode persistant circulant-multipliant. Les franchissements de l’épithélium intestinal et des glandes salivaires sont basés sur un mécanisme d’endocytose/exocytose. Ce processus d’invasion met en jeu, au sein de complexes protéiques, des protéines bactériennes spécifiques qui reconnaissent des motifs déterminés présents à la surface des cellules eucaryotes. La recherche de protéines de l’insecte vecteur interagissant avec S. citri a notamment conduit à l’identification de l’actine. Cette interaction a pu être confirmée à la fois in vitro et in vivo. L’interaction de l’actine avec son partenaire chez S. citri, qui s’est avéré être la phosphoglycérate kinase (PGK), est impliquée dans l’internalisation du spiroplasme dans les cellules de l’insecte. La région minimale de liaison à l’actine de la PGK a également été déterminée.La réalisation d’un mutant de S. citri dépourvu de PGK a été entreprise avec le plasmide navette pGOT mais n’a pas permis la sélection de spiroplasmes mutés dans ce gène essentiel. Néanmoins, la recherche de l’évènement de recombinaison chez les clones obtenus a permis de mettre en évidence la mutation de deux gènes chez ces derniers.L'ensemble des protéines impliquées dans la transmission du spiroplasme, identifiées au préalable chez ce dernier, n’étant pas essentielle au cours de ce processus, une étude préliminaire des complexes multi-protéiques contenant plusieurs de ces protéines a été réalisée. L’identification de complexes impliquant à la fois la PGK, la P32 et les ScARPs pourrait permettre de mieux comprendre les mécanismes régissant la vection de S. citri par son insecte. / Spiroplasma citri is a phytopathogenic mollicute transmitted from plant to plant by leafhoppers of the genus Circulifer in a persistent and propagative manner on condition to cross the intestinal and salivary glands barriers of the insect. These crossings are based on a endocytosis/exocytosis mechanism which involves bacterial protein complexes in order to recognize specific patterns on the surface of eukaryotic cells.Specific molecular interactions between S. citri proteins and those of C. haematoceps were investigated using far Western technology and had notably led to the identification of actin. This interaction has been confirmed both in vitro and in vivo. The interaction of actin with his partner in S. citri, which has been identified as the phosphoglycerate kinase (PGK), is involved in the internalization of spiroplasma into the insect cells. The minimal actin-binding region of PGK was also determined.The realization of a S. citri PGK mutant was carried out using the shuttle plasmid pGOT but the selection of spiroplasmas mutated in this essential gene failed. Nevertheless, findings in the localization of recombination events in S. citri chromosome, allowed us to identify mutations in two genes that could be tested in our experimental transmission system.The set of proteins involved in the spiroplasmal transmission, previously identified as non essential proteins in the invasion process, prompted us to study the role of multi-protein complexes containing several of these proteins. Identification of complexes involving both the PGK, the P32 and ScARPs should enable us to better understand mechanisms governing the transmission of S. citri by its insect.

Orientadores: Telma Teixeira Franco, Carlos Raimundo Ferreira Grosso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-07T12:03:40Z (GMT). No. of bitstreams: 1 OliveiraJunior_EnioNazarede_D.pdf: 5260922 bytes, checksum: 838753bbbf60bb35edf5aeb62dbbc33e (MD5) Previous issue date: 2006 / Resumo: Na primeira etapa deste trabalho foi feita uma revisão de literatura referente à atividade antifúngica de quitina, quitosana e seus derivados contra diferentes tipos de microrganismos, como bactérias, fungos e leveduras. Nesta revisão são descritos importantes desenvolvimentos relativos a aplicação de quitosana e seus derivados como substância antimicrobiana contra bactérias, fungos e leveduras, hipóteses envolvidas na suas atividades antimicrobianas e efeitos na qualidade e armazenagem de vegetais frescos tratados com quitosana e seus derivados. A segunda etapa deste trabalho se refere a produção e caracterização de quitosanas com diferentes graus de polimerização (DP) obtidas por tratamento térmico e quitosanas com diferentes graus de acetilação (FA) obtidas por desacetilação alcalina. A terceira etapa deste trabalho refere-se à avaliação dos efeitos da atividade antifúngica de quinze amostras de quitosana com diferentes graus de polimerização e diferentes graus de acetilação contra quatro fungos fitopatogênicos (Alternaria alternata, Botrytis cinerea, Penicillium expansum e Rhizopus stolonifer). Os crescimentos fúngicos foram avaliados em micro placas de 96 reservatórios e medidos em leitor de microplacas. A atividade antifúngica de quitosana aumentou com o decréscimo do FA. O efeito da combinação de quitosana com menor FA e maior DP teve a mais alta atividade fungistática contra os fungos A. alternata e B. cinerea. Os resultados tendem a indicar que os grupamentos amino protonados (NH3 +) foram um importante fator envolvido no efeito antifúngico. Os fungos mais resistentes foram Penicillium expansum e Botrytis cinerea e os mais sensíveis foram Alternaria alternata e Rhizopus stolonifer. Na quarta etapa deste trabalho foi investigado o efeito de quitoligômeros no crescimento dos mesmos fungos analisados na terceira etapa. Verificou-se que misturas de quitoligômeros de DP 5 a 8, DP 2 a 12 e de DP 2 a 11, apresentaram baixo ou nenhum efeito no crescimento fúngico na concentração máxima de 1,000 µg × mL-1. A última etapa deste trabalho refere-se às mudanças morfológicas nas hifas dos fungos tratados com quitosana. As micrografias obtidas por microscopia eletrônica de varredura com emissão de campo mostraram que os micélios fúngicos tratados com quitosana estavam agregados, excessivamente ramificados, inchados e de comprimentos reduzidos / Abstract: In the first step of this work, a literature review concerning antimicrobial activity of chitin, chitosan and their derivatives against different groups of microorganisms, such as bacteria, yeast and fungi was made. Important developments related to applications of chitosan and their derivatives, as an antimicrobial substance against bacteria, fungi and yeasts, hypotheses involved in the antimicrobial activities and effects on storability and quality of fresh vegetables treated with chitosan and its derivatives are described in this present review. The second step of this work, concerns the production of chitosans with different degrees of polymerization (DP) by thermal treatment and chitosans with different degrees of acetylation (FA) by alkaline deacetylation. The third step of this work, concerns the evaluation of antifungal effects of fifteen chitosans with different degrees of polymerization and different degrees of acetylation against four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum and Rhyzopus stolonifer) by using the 96-well microtiter plate and a microplate reader. Antifungal activity of chitosans increased with FA decreasing. Combination effect of chitosan with low FA and high DP showed the highest fungistatic activity against A. alternata and B. cinerea. The results indicated that free amino groups protonated (NH3 +) were an important factor for antifungal effect. The most resistant fungi were Penicillium expansum and Botrytis cinerea and the most sensitive fungi were Alternaria alternata and Rhizopus stolonifer. In the fourth step of this work, it was investigated the effect of chitooligomers in the fungal growth of the same target fungi analyzed in the third step. The results suggested that chitooligomer mixtures of DP 5 to 8, DP 2 to 12 and DP 2 to 11 showed low or no effect on the fungal growth at concentration of 1,000 µg × mL-1. The last step of this work, concerns the morphological changes on hyphae of fungi treated with chitosan. Mycelial aggregation and morphological structural changes as excessive branching, swelling of the cell wall and hyphae size reduction were observed in the micrographs obtained by scanning electron microscopy field emission / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Huanglongbing (HLB), considerada uma das mais graves doenças dos citros, está associada a Candidatus Liberibacter spp., bactérias endógenas restritas ao floema e de difícil cultivo em meio de cultura. Diferentemente de outras doenças que afetam plantas cítricas, ainda não foram encontradas dentro do gênero Citrus espécies resistentes ao HLB. Genes de interesse agronômico têm sido empregados na transformação genética de citros visando a resistência a doenças. Dentre estes, destacam-se os que conferem resistência a bactérias incluindo attA, que codificam os peptídeos antibacterianos atacinas, e hrpN, que ativam a produção de proteínas harpinas, relacionadas ao sistema de defesa. O objetivo deste trabalho foi avaliar a resistência de plantas de laranja doce contendo o gene da atacina A (attA) ou o gene da harpina (hrpN) à Candidatus Liberibacter asiaticus (CLas) utilizando duas formas de inoculação: borbulhões infectados e o inseto vetor da bactéria, Diaphorina citri. Para as plantas contendo o gene hrpN, apenas o segundo método foi utilizado. Os principais sintomas do HLB foram observados aos quatro e oito meses após a inoculação por borbulhões. A infecção das plantas foi confirmada com a detecção de CLas por PCR (quatro e oito meses) e Rt-qPCR (oito meses). Em plantas inoculadas por D. citri, os sintomas foram observados oito e doze meses após a inoculação, assim como a detecção da bactéria por PCR. Após 15, 17 e 18 meses, foi realizada uma nova avaliação por Rt-PCR a partir de spots (imprints em membrana) de folhas. Rt-PCR foi empregado também em spots dos psilídeos utilizados na inoculação. Não foi possível avaliar a resistência ao HLB em plantas contendo o gene attA ou hrpN a partir da inoculação por D. citri. Os resultados de detecção da bactéria nas plantas e nos psilídeos utilizados para inoculação indicam que, possivelmente, não ocorreu a inoculação devido ao baixo percentual de psilídeos que continham CLas utilizados. Dentre as plantas transgênicas contendo o gene attA inoculadas por borbulhões infectivos, oito eventos (cinco de laranja Pera, dois de laranja Hamlin e um de laranja Valência) apresentaram menores títulos bacterianos e algumas também demonstraram redução dos sintomas do HLB quando comparadas com plantas não transgênicas, oito meses após a inoculação, indicando uma possível ação do peptídeo atacina A contra o agente causal do HLB. / Haunglongbing (HLB), considered one of the most serious diseases of citrus, is associated to Candidatus Liberibacter spp., endogenous and phloem-inhabiting bacteria not easily grown in culture medium No species within the genus Citrus is known to resist this bacterial infection. The use of genes of agronomic interest for genetic transformation aiming disease resistance in citrus has been reported. Among these genes, attA that codes for the antibacterial peptides attacin, and hrpN, that codes for proteins harpin that activate the plant defense system may have potential in searching for HLB resistance. The objective of this study was to evaluate the resistance of sweet orange containing attA or hrpN to Candidatus Liberibacter asiaticus (CLas) inoculated through infected budstick grafting or the insect vector, Diaphorina citri. For the plants containing hrpN, only the second method was used. The most obvious HLB symptoms were observed four and eight months after inoculation by infected budstick when CLas also was detected by PCR (four months) and RT-qPCR (eight months). For those inoculated with D. citri, symptoms were observed and bacteria detected eight and twelve months after inoculation. Fifteen, 17 and 18 months after inoculation, a new attempt was made for CLas detection, now through Rt-PCR from leaf and psyllids imprinting spots on membrane. It was not possible to evaluate the HLB resistance in plants containing attA or hrpN gene from D. citri inoculation. The results of CLas detection in plants and psyllids indicate that possibly there was no inoculation due the low rate of psyllids contained CLas used. Among the plants containing attA, five, two and one event of, respectively, Pera, Hamlin, and Valencia sweet orange had lower bacterial titers than those non transgenic plants and some also showed milder HLB symptoms, eight months after inoculation, suggesting a possible effect of attacin A against the causal agent of HLB.

[ES] El complejo de especies de Ralstonia solanacearum es el responsable de la marchitez bacteriana, una enfermedad que afecta en todo el mundo a cultivos y plantas ornamentales de gran importancia económica. El filotipo (ft) II raza 3 biovar (bv) 2 produce la podredumbre parda de la patata y la marchitez bacteriana de las solanáceas en climas templados, y se ha introducido recientemente en algunas áreas de la Unión Europea y de Estados Unidos, donde este patógeno se considera un organismo de cuarentena. La presencia de R. solanacearum ft II raza 3 bv 2 en estas zonas ha hecho surgir ciertos interrogantes sobre la biología y la patogenicidad de esta bacteria, algunos de los cuales se han abordado en este trabajo. De esta manera, se han evaluado aspectos del comportamiento, la capacidad de supervivencia y la capacidad para la inducción de la enfermedad, de cepas de R. solanacearum ft II raza 3 bv 2 de origen europeo en diferentes especies vegetales y diversas muestras de agua medioambiental. El comportamiento en cuanto a la colonización de R. solanacearum ft II raza 3 bv 2 in planta ha permitido realizar una clasificación en huéspedes susceptibles o tolerantes y en no huéspedes, en base a la localización histológica del patógeno y su aislamiento. Se ha observado que los huéspedes susceptibles y tolerantes eran intensamente invadidos a nivel del xilema de las raíces, pero densamente o bien débilmente colonizados, respectivamente, a nivel del xilema del tallo. Ninguno de ellos debe ser considerado como candidato para la rotación de cultivos en suelos contaminados. Por otra parte, se ha observado que en los no huéspedes no se producía la invasión a nivel del xilema de la planta, aunque podría haber presencia esporádica del patógeno en el córtex de las raíces o en la superficie y, por lo tanto, algunos podrían actuar como reservorios. Los no huéspedes podrían seleccionarse para los sistemas de rotación tras su evaluación en condiciones de campo. La capacidad de supervivencia de R. solanacearum ft II raza 3 bv 2 en microcosmos de agua medioambiental se ha visto influida por factores tanto abióticos como bióticos. El patógeno ha mostrado una resistencia considerable frente a la exposición a la oligotrofía como único factor de stress. Así, desarrolló varias estrategias que le permitieron sobrevivir en microcosmos de agua medioambiental durante cuatro años bajo condiciones de limitación de nutrientes, manteniendo la capacidad de inducir la enfermedad en el huésped incluso a través de riego. Las estrategias adaptativas para superar la oligotrofía observadas durante este largo periodo han sido la respuesta de supervivencia frente a la privación de nutrientes, la entrada en el estado viable no cultivable, una transformación progresiva de las formas bacilares típicas en cocoides de reducido tamaño, los fenómenos de filamentación y gemación, y la agregación. El patógeno también ha mostrado estrategias eficientes cuando ha sido expuesto a diferentes temperaturas simultaneamente a las condiciones de escasez de nutrientes. De esta manera, a 4°C el frío indujo la entrada en un estado viable no cultivable que fue dependiente del contenido en nutrientes del agua, mientras que a 14°C y 24°C las respuestas de supervivencia frente a la privación de nutrientes, aunque aparentemente similares, revelaron un efecto de la temperatura en la formación de los cocoides. Por otra parte, la persistencia del patógeno se redujo de manera significativa en microcosmos de agua medioambiental por la actividad predadora, competitiva o lítica de los protozoos, las bacterias y/o los bacteriófagos presentes en el agua. Entre todos ellos, se demostró que los fagos líticos fueron los responsables principales del descenso en las poblaciones de R. solanacearum, si bien los protozoos y las otras bacterias contribuyeron en cierta medida. El efecto fue más apreciable a 24°C que a 14°C debido a que las interacciones bióticas fueron más lentas a la temperatura más baja. Esta bacteria ha mostrado una gran capacidad de adaptación, consiguiendo sobrevivir y mantener su poder patógeno en prácticamente todas las condiciones ensayadas. La finalidad de este trabajo ha sido contribuir al progreso del conocimiento de las interacciones entre R. solanacearum ft II raza 3 bv 2 y sus ambientes naturales, para mejorar las estrategias preventivas tanto de la diseminación del patógeno como de la propagación de la marchitez bacteriana en dichos hábitats. / [EN] The Ralstonia solanacearum species complex causes bacterial wilt, a plant disease affecting economically important crops and ornamentals worldwide. The phylotype (ph) II race 3 biovar (bv) 2 produces potato brown rot and bacterial wilt in solanaceous plants in temperate climates and has recently been introduced to several areas of the European Union and the USA, where the pathogen has a quarantine status. Presence of R. solanacearum ph II race 3 bv 2 in these zones raised questions on biological and phytopathological aspects of this bacterium, some of them being addressed in this work. Thus behaviour, ability for survival and disease inducing capacity of European R. solanacearum ph II race 3 bv 2 have been assessed in a range of different plant species and diverse surface run-off water samples. Behaviour of R. solanacearum ph II race 3 bv 2 in planta has led to a classification in susceptible or tolerant hosts and non-hosts, based on the pathogen histological localization and isolation. Susceptible and tolerant hosts were highly invaded in root xylem but, heavily or weakly colonized respectively in stem xylem. They are to be avoided as candidates for crop rotation. Non-hosts were not invaded in plant xylem but, occasional presence of the pathogen in root cortex or on surface might occur and so, some of them might act as reservoirs. They could be selected for crop rotation systems after being carefully tested in open field conditions. Ability for survival of R. solanacearum ph II race 3 bv 2 in environmental water microcosms has been influenced by abiotic and biotic factors. When faced to oligotrophy as the only stressing factor the pathogen displayed a considerable endurance. It resorted to a number of survival strategies which enabled it to survive in environmental water microcosms over four years under starvation, retaining disease inducing capacity in the host even by watering. Adaptations to overcome nutrient limitation during the long period were starvation-survival responses, the entrance into a viable but non-culturable state, progressive transformation from the typical bacillar shape into coccoid forms reduced in size, filamentation and budding phenomena and aggregation. Survival strategies were also successfully exhibited by the pathogen when exposed to environmental temperatures simultaneously to nutrient scarcity conditions. Thus, at 4°C a viable but non-culturable state dependent on water nutrient contents was cold-induced, whilst at 14°C and 24°C apparently similar starvation-survival responses revealed a distinct effect of temperature on coccoid formation. On the other hand, indigenous freshwater protozoa, bacteria and/or phages with predatory, competitive or lytic activity reduced significantly pathogen persistence. Among them, lytic bacteriophages were the main responsible for the decrease in R. solanacearum populations, although protozoa and other bacteria also contributed. The effect was more appreciable at 24°C than at 14°C because of slower biotic interactions at the lower temperature. The pathogen was able to adapt itself, succeeding in surviving and keeping pathogenic in almost all condition. This work intends to contribute to the progress in the knowledge of the interactions between R. solanacearum ph II race 3 bv 2 and natural environments, which may allow to improve the strategies to prevent pathogen dissemination and bacterial wilt spread in natural settings. / [FR] Le complexe d'espèces de Ralstonia solanacearum est l'agent causal du flétrissement bactérien, une maladie qui affecte des cultures et des plantes ornementales de grande importance économique dans le monde entier. Le phylotype (phyl) II race 3 biovar (bv) 2 produit la pourriture brune de la pomme de terre et d’une manière générale le flétrissement bactérien des Solanacées dans les climats tempérés. Ce phylotype II a récemment été introduit dans des certaines régions de l'Union Européenne et des États-Unis, où cette bactérie est considérée comme un pathogène de quarantaine. La présence de R. solanacearum phyl II race 3 bv 2 dans ces régions a soulevé quelques questions au sujet de sa biologie et de son pouvoir pathogène, questions qui ont été abordées dans ce travail. Nous avons notamment évalué, chez R. solanacearum phyl II race 3 bv 2 d'origine européenne, l'aptitude à survivre dans divers échantillons de l'eau environnementale de surface et la capacité à induire la maladie dans des différentes espèces de plantes cultivées. Notre étude de la capacité de colonisation de R. solanacearum phyl II race 3 bv 2 in planta a permis une classification en hôtes sensibles ou tolérants et non hôtes sur la base de la localisation histologique de l'agent pathogène et de son isolement in planta. Il a été noté que les hôtes sensibles et tolérants sont intensément envahis à niveau du xylème des racines, mais fortement ou faiblement colonisés à niveau du xylème de la tige chez les plantes sensibles ou tolérantes respectivement. Aucune de ces plantes ne doit être recommandée comme candidat dans une rotation culturale. En outre, nous avons noté que le xylème des plantes non hôtes n’est pas envahi, bien que l’on puisse noter la présence sporadique de l'agent pathogène dans le cortex des racines ou sur la surface racinaire. Cela signifie que certaines plantes non hôtes pourraient potentiellement être des réservoirs d’inoculum latent. En conséquence les plantes non hôtes qui pourraient être recommandées dans des systèmes de rotation culturale doivent être évaluées non seulement au laboratoire mais aussi au champ dans des conditions naturelles. Nous avons montré que la capacité de survie de R. solanacearum phyl II race 3 bv 2 dans des microcosmes de l'eau environnementale, préalablement filtrée ou autoclavée, est influencée par des facteurs abiotiques et biotiques. L'agent pathogène fait preuve d'une résistance considérable quand il est exposé à l'oligotrophie comme seul facteur de stress. Nous avons mis en évidence différentes stratégies utilisées par cette bactérie pour survivre plus de quatre ans dans des conditions de limitation des éléments nutritifs dans des microcosmes de l'eau environnementale tout en maintenant sa capacité d'induction de la maladie chez un hôte sensible à la suite de sa dissémination par irrigation. Les adaptations utilisées par cette bactérie pour surmonter l'oligotrophie au cours de cette longue période ont été l'entrée dans l'état viable non cultivable, une transformation progressive des typiques formes bacillaires vers des formes coccoïdes de petite taille, des phénomènes de filamentation et de bourgeonnement et d'agrégation. L'agent pathogène a également utilisé des stratégies efficaces quand il a été exposé à des différentes températures de l'environnement en même temps qu'à des conditions de limitation des éléments nutritifs. Ainsi à 4°C le froid a entraîné un état viable non cultivable qui a été dépendant de la teneur en éléments nutritifs de l'eau. Par contre à 14°C et 24°C les réponses de survie face à la privation des éléments nutritifs ont induit la formation de formes coccoïdes. En outre, la persistance de l'agent pathogène a été considérablement réduite dans des microcosmes de l'eau environnementale par l'activité prédatrice, compétitive ou lytique des protozoaires, des bactéries et/ou des bactériophages qui étaient contenus dans de l'eau. Notamment, il a été constaté que les phages lytiques sont les principaux responsables de la baisse des populations de R. solanacearum, bien que les protozoaires et d'autres bactéries aient également contribués dans une certaine mesure. L'effet a été plus sensible à 24°C qu'à 14°C en raison d'interactions biotiques plus lentes aux températures plus basses. En conclusion cette bactérie a démontré une grande capacité pour s'adapter, survivre et de maintenir son pouvoir pathogène dans pratiquement toutes les conditions couramment rencontrées dans les conditions naturelles. Le but de ce travail a été de contribuer à l'avancement de la connaissance des interactions entre R. solanacearum phyl II race 3 bv 2 et les milieux aquatiques naturels ou les plantes hôtes potentielles, dans l’espoir de développer les meilleures stratégies pour empêcher la propagation de l'agent pathogène et donc du flétrissement bactérien dans ces environnements. / [CA] El complex d'espècies de Ralstonia solanacearum és l'agent causal del pansiment bacterià, una malaltia vegetal que afecta a tot el món a cultius i plantes ornamentals de gran importància econòmica. El filotipo (fil) II raça 3 biovar (bv) 2 produeix la podridura marró de la patata i el pansiment bacterià en plantes solanàcies de climes temperats, i s'ha introduït recentment en algunes àrees de la Unió Europea i dels Estats Units, on aquest patogen es considera de quarantena. La presència de R. solanacearum fil II raça 3 bv 2 en aquestes zones ha fet sorgir certs interrogants sobre qüestions biològiques i fitopatològiques d'aquest bacteri, algunes de les quals s'han abordat en aquest treball. D'aquesta manera s'han valorat aspectes del comportament, la capacitat de supervivència i la capacitat per a la inducció de la malaltia, de R. solanacearum fil II raça 3 bv 2 de procedència europea en diferents espècies vegetals i diverses mostres d'aigua d'escorrentia superficial. El comportament pel que fa a la colonització de R. solanacearum fil II raça 3 bv 2 in planta ha proporcionat una classificació en hostes susceptibles o tolerants i en no hostes, en base a la localització histològica del patogen i el seu aïllament. S'observà que els hostes susceptibles i tolerants eren intensament envaïts a nivell del xilema de les arrels, però densament o bé feblement colonitzats respectivament a nivell del xilema de la tija. Cap d'ells ha de ser considerat com a candidat per a la rotació cultural. D'altra banda, es va observar que els no hostes no eren envaïts en el xilema de la planta, encara que podria haver presència esporàdica del patogen en el còrtex de les arrels o a la surperfície i, per tant, alguns podrien actuar com a reservoris. Els no hostes podrien ser seleccionats per als sistemes de rotació cultural després d'haver estat avaluats en condicions de camp. La capacitat de supervivència de R. solanacearum fil II raça 3 bv 2 en microcosmos d'aigua mediambiental s'ha vist influïda per factors abiòtics i biòtics. El patogen ha mostrat una resistència considerable quan ha estat exposat a l'oligotrofia com a únic factor d'estrès. Així, va recórrer a diverses estratègies que li van permetre sobreviure en microcosmos d'aigua mediambiental durant quatre anys sota condicions de limitació de nutrients, mantenint la capacitat per a la inducció de la malaltia en l'hoste fins i tot a través de reg. Les adaptacions per superar l'oligotrofia que s'han observat durant aquest llarg període han estat respostes de supervivència front a la privació de nutrients, l'entrada en l'estat viable no cultivable, una transformació progressiva de les típiques formes bacilars a cocoids de reduït tamany, els fenòmens de filamentació i gemmació, i l'agregació. El patogen també ha mostrat estratègies eficients quan ha estat exposat a diferents temperatures mediambientals simultàneament a les condicions d'escassetat de nutrients. D'aquesta manera, a 4°C el fred va induir un estat viable no cultivable que va ser dependent del contingut en nutrients de l'aigua, mentre que a 14°C i 24°C les respostes de supervivència front a la privació de nutrients, encara que aparentment similars, van revelar un efecte de la temperatura a la formació dels cocoids. D'altra banda, la persistència del patogen s'ha vist significativament reduïda en microcosmos d'aigua mediambiental per l'activitat predadora, competitiva o lítica dels protozous, els bacteris i/o els bacteriòfags que contenia l'aigua. Entre tots ells, es va determinar que els fags lítics eren els responsables principals del descens en les poblacions de R. solanacearum, si bé els protozous i els altres bacteris van contribuir en certa mesura. L'efecte va ser més apreciable a 24°C que a 14°C degut a que les interaccions biòtiques van ser més lentes a la temperatura més baixa. Aquest bacteri ha mostrat una gran capacitat d'adaptació, aconseguint sobreviure i mantenir el seu poder patogènic en pràcticament totes les condicions. La finalitat d'aquest treball és contribuir al progrés del coneixement de les interaccions entre R. solanacearum fil II raça 3 bv 2 i els seus ambients naturals, el que pot desenvolupar una millora de les estratègies per a la prevenció tant de la disseminació del patogen com de la propagació del pansiment bacterià en aquests ambients. / B. Álvarez specially thanks the Marie Curie Foundation for a Marie Curie Fellowship at the IFR40 in Toulouse (France) and the Instituto Valenciano de Investigaciones Agrarias for a predoctoral grant. This work has been funded by the European contract QLK3-CT-2000-01598 acronym “Potatocontrol” and FAIR 5-CT97-3632 of the European Union, FD 1997-2279 of the Ministerio de Educación y Ciencia of Spain and GV05/214 of the Generalitat Valenciana. / Álvarez Ortega, MB. (2009). Biology of Ralstonia Solanacearum Phylotype II in Host and Non-Host Environments [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191532

Thesis (M. Tech) --Central University of Technology, Free State, 2008 / Aphids (Hemiptera: Aphididae) are amongst the most destructive insects in agricultural crop production systems. This reputation stems from their complex life cycles which are mostly linked to a parthenogenetic mode of reproduction, allowing them to reach immense population sizes within a short period of time. They are also notorious as important and efficient vectors of several plant viral diseases. Their short fecund life cycles allow them to be pests on crops with a short growth period, e.g. lettuce (Lactuca sativa L.). It is common practice to provide this crop with some degree of protection from environmental extremes on the South African Highveld. Shadehouses are popular in this regard, but aphids are small enough to find their way into these structures, and their presence on lettuce is discouraged due to phytosanitary issues. In addition, the excessive use of insecticides is criticized due to the negative influence on human health, and because aphids can rapidly develop resistance. This necessitates the use of alternative control options in order to suppress aphid numbers. Biological control is popular in this regard and the use of predatory ladybirds (Coleoptera: Coccinellidae) is a popular choice. This study investigated the aphid and coccinellid species complex encountered under varying shadehouse conditions on cultivated head lettuce in the central Free State Province (South Africa). Their seasonality was also examined, along with variations in their population size throughout a one-year period. Finally, the impact of varying aphid populations on some physical characteristics of head lettuce was examined, and recommendations for aphid control (using naturally occurring coccinellid predators) were made. Two shadehouse structures were evaluated during this study. One was fully covered with shade netting and designed to exclude the pugnacious ant, Anoplolepis custodiens (Hymenoptera: Formicidae), while the other was partially covered with shade netting (on the roof area) allowing access to the ants. Six cycles of head lettuce were planted and sampled four times during each cycle. These were scheduled to monitor the seedling, vegetative and heading stage of lettuce. Four important aphid species were recorded on the lettuce, namely Acyrthosiphon lactucae, Nasonovia ribisnigri, Myzus persicae and Macrosiphum euphorbiae. Both structures harboured similar aphid and coccinellid species, but their population dynamics differed. A. lactucae dominated in the absence of A. custodiens in the fully covered structure (whole study), while N. ribisnigri dominated in the partially covered structure in the presence of these ants during the warmer months (December – January). M. euphorbiae replaced this species as the dominant species in the absence of A. custodiens (April – September). M. persicae occured during the winter (May – August) in the fully covered structure. Promising coccinellid predators were Hippodamia variegata and Scymnus sp. 1, and to a lesser extent, Exochomus flavipes and Cheilomenes lunata. However, the fully covered structure hampered the entrance of the larger adult coccinellid species, resulting in their lower occurrence. Aphid and coccinellid activity peaked during the summer months (October – January), and the fully covered structure attained the highest aphid infestation levels and coccinellid larval numbers during this time. On the other hand, aphid numbers were higher in the partially covered structure during the cooler months of the year (April – July) and this structure also harboured more adult coccinellids. In most cases, aphid infestation levels did not affect the amount of leaves formed. However, symptomatic damage in terms of head weight reduction did occur under severe infestation levels. Specific environmental conditions within a shadehouse structure concurrently contributed to this reduction, with less favourable conditions accelerating this condition. Results from this study have shown that even though the type of shadehouse structure does not influence the insect species complex found on lettuce, it does have an influence on detrimental and beneficial insect population dynamics. Aphid species infesting lettuce have been identified, along with coccinellid predators that could potentially be used in their control. Both types of structures had advantages and disadvantages, and therefore, decisions concerning shadehouses should not be focused on which type of structure to use, but rather which type of structure to use during different seasons of the year.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

Neem cake is a product of the cold pressing from the neem kernels to obtain neem oil. Bio-active substances from neem cake extracted solutions were evaluated for their potential to control the root knot nematodes and other pests of plants. In this study different concentrations of the solution extracted from neem cake was tested against the second stage juveniles of the plant parasitic nematode Meloidogyne spp. and four phytopathogenic fungi: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytopthora capsici. Toxicity of neem cake extractions is represented by the EC50 value for the second-stage juvenile (J2) of Meloidogyne spp. and the four phytopathogenic fungi via Probit analysis. A 5% dilution of the solvent extracting from neem cake already caused 100% larval mortality after 24 hours exposure. Undiluted neem cake extraction effectively inhibited the growth of the four phytopathogenic fungi. The EC50 value of neem cake on J2-larvae of Meloidogyne nematode and on the fungi Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici was 0.51, 0.74, 0.30, 0.51 and 4.33%, respectively. / Bánh dầu neem là sản phẩm của quá trình ép nhân hạt neem để lấy dầu. Các hoạt chất sinh học từ dịch chiết bánh dầu neem đã được đánh giá có tiềm năng lớn trong phòng trừ tuyến trùng nốt sưng và các loài dịch hại khác của nhiều loại cây trồng. Trong nghiên cứu này các nồng độ dịch chiết khác nhau của bánh dầu neem đã được thử nghiệm khả năng diệt tuyến trùng (ấu trùng tuổi 2 thuộc giống Meloidogyne spp.) và ức chế 4 loài nấm bệnh như: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. và Phytopthora capsici. Độc tính của dịch chiết bánh dầu neem được biểu diễn bởi giá trị EC50 đối với ấu trùng tuổi 2 của tuyến trùng Meloidogyne spp. và các loài nấm bệnh thông qua phân tích Probit. Dịch chiết bánh dầu neem ở nồng độ 5% đã làm chết 100% cá thể IJ2 của Meloidogyne spp sau 24 giờ phơi nhiễm. Dịch nguyên chất bánh dầu neem ức chế cả 4 loài nấm bệnh. Giá trị EC50 của bánh dầu neem lên ấu trùng tuổi 2 của Meloidogyne spp và các loài nấm bệnh Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici tương ứng là 0.51, 0.74, 0.30, 0.51 và 4.33%.

Thesis (PhD (Botany and Zology))--University of Stellenbosch, 2006. / The well-known, and often phytopathogenic, ophiostomatoid fungi are represented in South Africa by the two phylogenetically distantly related genera Ophiostoma (Ophiostomatales) and Gondwanamyces (Microascales). They are commonly associated with the fruiting structures (infructescences) of serotinous members of the African endemic plant genus Protea. The species O. splendens, O. africanum, O. protearum, G. proteae and G. capensis have been collected from various Protea spp. in South Africa where, like other ophiostomatoid fungi, they are thought to be transported by arthropod vectors. The present study set out to identify the vector organisms of Protea-associated members of mainly Ophiostoma species, using both molecular and direct isolation methods. A polymerase chain reaction (PCR) and taxon specific primers for the two Protea-associated ophiostomatoid genera were developed. Implementation of these newly developed methods revealed the presence of Ophiostoma and Gondwanamyces DNA on three insect species. They included a beetle (Genuchus hottentottus), a bug (Oxycarenus maculates) and a psocopteran species. It was, however, curious that the frequency of these insects that tested positive for ophiostomatoid DNA was very low, despite the fact that ophiostomatoid fungi are known to colonise more than 50% of Protea infructescences. Subsequent direct isolation methods revealed the presence of reproductive propagules of Ophiostoma spp. on four Protea-associated mite species (Oodinychus sp., two Tarsonemus spp. and Proctolaelaps vandenbergi). These mites are numerous within Protea infructescences and Ophiostoma spp. were isolated from a high frequency of these individuals. The Oodinychus sp. mite was found to vector most of the Protea-associated Ophiostoma species. It was thus postulated that the mites (in particular the Oodinychus sp.) act as primary vectors of the Protea-associated Ophiostoma species. The association between Oodinychus mites collected from P. repens and O. splendens proved to be mutualistic. Mites feeding on this fungus showed significantly higher population growth than mites feeding on any of the other fungal species tested. The short- and long-distance dispersal methods of these mites were also investigated. Firstly the ability of mites to move from drying infructescences to moist and sheltered areas such as provided by intact infructescences on the same plant was investigated experimentally. Significantly more mites were found to actively disperse from drying infructescences to artificially manufactured infructescences containing moistened filter paper shreds than to artificially manufactured infructescences containing dry filter paper shreds. The frequent fires associated with the habitat of these mites would, however, require movement over larger areas than what would be possible through self-dispersal. Dispersal of mites via air currents was thus investigated using sticky traps, but no Ophiostoma-vectoring mites were captured in this way. Self-dispersal aided by air currents could thus be ruled out, and our investigations shifted to vectored dispersal. Numerous insects emerging from Ophiostoma-containing P. repens and P. neriifolia infructescences were collected using specially designed emergence cages. Scanning electron microscopy and stereo-microscopy revealed that all three Ophiostoma-vectoring mite genera were phoretic on the beetle G. hottentottus. Tarsonemus spp. and P. vandenbergi were also phoretic on the beetles Trichostetha fascicularis and T. capensis associated with P. repens and P. neriifolia flowers. Mites collected from the surface of these beetles were found to vector reproductive propagules of various Ophiostoma spp. This thus seems to be the only method of long-distance dispersal of these mites and subsequently also the Protea-associated Ophiostoma species. Molecular phylogenetic reconstruction based on large subunit, ITS and beta-tubulin DNA sequence data suggests a polyphyletic origin for the Protea-associated members of Ophiostoma, which proposes multiple invasions of this unusual niche by these fungi. These studies also revealed the presence of four new species of Ophiostoma associated with Protea spp. The new species O. palmiculminatum, O. phasma, O. gemellus and Sporothrix variecibatus were thus described. Ophiostoma palmiculminatum is associated with P. repens infructescences and the Oodinychus mites collected from them. Ophiostoma phasma was collected from various Protea and mite species. Ophiostoma gemellus and Sporothrix variecibatus were initially only isolated from mites, but have subsequently also been isolated from Protea spp. The present study clarifies many aspects pertaining to the phylogeny and ecology of the interesting members of Ophiostoma associated with Protea hosts. As such this study will form the platform for further studies on the co-evolution of these insect / mite / fungi / plant associations.

O Greening ou Huanglongbing (HLB) é a doença mais devastadora dos citros na atualidade. Levantamentos mostram que a enfermidade está presente em cerca de 60% dos talhões do parque citrícola paulista, demostrando sua grande capacidade de disseminação. O psilídeo-asiático-dos-citros, Diaphorina citri Kuwayama (Hemiptera: Psylidae) é um importante vetor dos agentes causais associados ao HLB. Sua capacidade de dispersão está intimamente relacionada ao progresso da doença no campo. Não se tem conhecimento dos principais fatores que possam interferir no deslocamento do inseto até plantas cítricas. Sendo assim, a presente pesquisa teve como objetivo investigar a influência de alguns fatores ambientais sobre a dispersão do psilídeo D. citri, discutindo-os em relação à epidemiologia do HLB dos citros. Inicialmente foram realizados testes em laboratório para adaptação da metodologia de marcação, liberação e recaptura usando pó fluorescente. Grupos de psilídeos adultos foram marcados e sua sobrevivência avaliada, verificando a influência de quatro cores distintas de pó fluorescente (amarelo, azul, rosa e verde). Posteriormente realizaram-se estudos de campo com quatro liberações de psilídeos, com marcadores distintos, em quatro áreas experimentais circulares e adjacentes, sendo formadas por círculos concêntricos localizados a 18, 24 e 30 m de cada centro de liberação. Os insetos foram monitorados visualmente e por armadilhas adesivas amarelas. Nas 1ª e 2ª liberações, foi verificado o efeito das coberturas vegetais milho (Zea mays L.) e capim-braquiária (Brachiaria decumbens Stapf.) na dispersão de D. citri. Já nas 3ª e 4ª liberações, foi estudado o efeito de brotações no deslocamento do inseto. A influência dos ventos foi avaliada em ambas as situações. A análise de sobrevivência indicou que não houve interferência das quatro cores de pó fluorescente em D. citri, sendo a marcação visível no corpo dos insetos durante os 48 dias de avaliação. De acordo com as análises fatoriais, não foi observada qualquer interferência da cobertura vegetal milho, medindo 2 m de altura, na dispersão do inseto. Na presença de brotações, adultos de D. citri dispersaram menos em busca de novos hospedeiros, acumulando-se nas primeiras linhas de plantas cítricas. Após 6 horas da liberação, a capacidade de dispersão máxima de D. citri foi três vezes maior na condição de ausência de brotações. A dispersão média e a área total abrangida variaram assimptoticamente com o tempo, ou seja, o inseto possui inicialmente grande velocidade de dispersão, porém esta tende a se estabilizar com o incremento do tempo. Esses resultados deixam claro que D. citri realiza movimentos dependendo das condições fenológicas de seus hospedeiros, ressaltando a grande importância de se intensificar métodos de controle regionais em épocas em que o fluxo vegetativo das plantas cítricas não é tão intenso. / Greening or Huanglongbing (HLB) is the most devastating disease of citrus today. Surveys show that the disease is present in about 60% of the park stands citrus Sao Paulo, demonstrating its great ability to spread. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psylidae) is an important vector of the causative agents associated with HLB. Its ability to spread is closely related to disease progression in the field. There is no knowledge of the main factors that can affect the movement of the insect to citrus plants. Thus, the present study aimed to investigate the influence of some environmental factors on the dispersal of psyllid D. citri, discussing them in relation to the epidemiology of HLB of citrus. Initially tests were performed in the laboratory to adapt the methodology of marking, release and recapture using fluorescent powder. Groups of adult psyllids were marked and their survival assessed by checking the influence of four different colors of fluorescent powder (yellow, blue, pink and green). Subsequent studies were conducted with four field releases psyllids, with distinct markers on four experimental areas adjacent circular and being formed by concentric circles located at 18, 24 and 30 m for each center release. The insects were monitored visually and by yellow sticky traps. In the 1st and 2nd releases, was to determine the effect of cover crops maize (Zea mays L.) and grass (Brachiaria decumbens Stapf.) in the dispersal of D. citri. In the 3rd and 4th releases, were studied the effect of shootings in insect movement. The influence of the wind has been evaluated in both situations. Survival analysis indicated that there was no interference of the four colors of fluorescent powder on D. citri, and marking was visible on the body of insects during the 48 days of evaluation. According to the factor analysis, no interference of vegetation corn (measuring 2 m in height) was observed in the dispersal of the insect. In the presence of shoots, adult D. citri less dispersed looking for new hosts and it accumulate in the first lines of citrus plants. After 6 hours of release, the D. citri maximum dispersal was three times higher in the condition of absence of shoots. The dispersal medium and the total area covered varied asymptotically with time, ie the insect has initially high speed dispersal, but this tends to stabilize with increasing time. These results clearly show that D. citri performs movements depending on the phenology of its hosts, emphasizing the importance of intensifying regional control methods at times when the flow of plant citrus is not intense.

O presente trabalho teve por objetivos avaliar métodos de inoculação de Nectria haematococca e Phytophthora parasitica e idades de Passiflora edulis f. flavicarpa suscetíveis à infecção; avaliar a ocorrência de damping-off e podridão de colo do maracujazeiro em solo infestado; avaliar o comportamento de diferentes Passsifloraceas e genótipos de maracujazeiro amarelo aos respectivos patógenos; realizar testes de controle químico in vitro, tratamento químico erradicante em solo infestado e tratamento químico curativo em P. edulis f. flavicarpa para os respectivos patógenos. Inoculações no colo das plantas de P. edulis f. flavicarpa proporcionaram maiores níveis de doença comparadas às inoculações no sistema radicular, previamente ferido. Os resultados sugerem que N. haematococca seja um patógeno que penetra através de ferimentos. A mortalidade foi maior quando a inoculação foi realizada em plantas mais jovens e quando os patógenos N. haematococca e P. parasitica estavam em associação. Dentre as 17 espécies de Passiflora avaliadas para resistência aos patógenos, as espécies P. nitida, P. laurifolia e P. alata apresentaram as menores médias de lesões de N. haematococca, enquanto que para P. parasitica foram as espécies P. suberosa, P. foetida e P. morifolia as menos afetadas. Passiflora sidaefolia, P. edulis f. flavicarpa e P. edulis f. edulis foram as mais suscetíveis a ambos os patógenos, com sintomas que culminaram com a morte de plantas. Os genótipos de P. edulis f. flavicarpa mais resistentes a N. haematococca foram os procedentes de Morretes (PR) e a variedade Maguari e de Sapucaí (SP), enquanto que para P. parasitica foram os genótipos de Morretes (PR), Jaboticabal (SP) e LE13P2 (IAC) os menos afetados. A variedade Sul-Brasil e o genótipo de Livramento (BA) foram altamente suscetíveis a ambos os patógenos, com sintomas que culminaram com a morte de plantas. No teste de fungitoxidade in vitro avaliou-se a eficiência dos fungicidas na inibição do crescimento micelial de N. haematococca e P. parasitica. Na dose 100 ppm somente prochloraz inibiu totalmente o crescimento micelial de N. haematococca e nenhum produto inibiu acima de 82 % o crescimento de P. parasitica. Os fungicidas prochloraz, thiabendazole, thiram+thiabendazole, carbendazim, triflumizole e captan exerceram controle erradicante em solo infestado com N. haematococca, inibindo a incidência da doença em plantas com seis semanas pós-germinação. O mesmo foi observado com os produtos kif, dimethomorph, metalaxyl+mancozeb, mancozeb, cymoxanil+maneb e oxicloreto de cobre para P. parasitica. Os fungicidas testados em tratamento curativo inibiram o desenvolvimento da doença com melhores resultados quando aplicados dois dias após a inoculação, comparado a sete dias. Os fungicidas prochloraz e carbendazim destacaram-se por evitar a morte de plantas inoculadas com N. haematococca e os fungicidas kif, dimethomorph, metalaxyl+mancozeb e cymoxanil+maneb apresentaram eficiência semelhante entre si e superior a fosetyl- Al no controle de P. parasitica. / The objectives of the present work were to evaluate methods of inoculation of Nectria haematococca and Phytophthora parasitica and ages of Passiflora edulis f. flavicarpa which are susceptible to infection; to evaluate the damping-off and collar rot of passion fruit plant in infested soil; to evaluate the behavior of different Passsifloraceas and yellow genotypes of passion fruit to the respective pathogens; to carry out tests of chemical control in vitro, eradicative chemical treatment in infested soil and curative chemical treatment in P. edulis f. flavicarpa for the respective pathogens. Inoculations in the collar zone of P. edulis f. flavicarpa plants provided higher levels of disease when compared to the inoculations in the radicular system previously wounded. The results suggest that N. haematococca is a pathogen that penetrates through wounds. Mortality was higher when the inoculation was carried out in younger plants and when both pathogens were together. Amongst the 17 species of Passiflora tested for resistance to the pathogens, P. nitida, P. laurifolia, and P. alata showed the lowest average of N. haematococca lesions, while P. suberosa, P. morifolia, and P. foetida were the least affected species by P. parasitica. Passiflora sidaefolia, P. edulis f. flavicarpa, and P. edulis f. edulis were the most susceptible to both pathogens, showing symptoms that culminated with the death of the plants. The most resistant genotypes of P. edulis f. flavicarpa to N. haematococca were those from Morretes (PR), Maguari variety, and those from Sapucaí (SP); with respect to P. parasitica, the genotypes from Morretes (PR), Jaboticabal (SP), and LE13P2 (IAC) were the least affected. The Sul- Brasil variety and the genotype from Livramento (BA) had been highly susceptible to both pathogens, having symptoms that culminated with the death of plants. The in vitro efficiency of the fungicides in the inhibition of the mycelial growth of N. haematococca and P. parasitica was evaluated. At 100 ppm, only prochloraz inhibited totally the mycelial growth of N. haematococca and no product inhibited over 82 % the growth of P. parasitica. Prochloraz, thiabendazole, thiram+thiabendazole, carbendazim, triflumizole, and captan controlled eradicatively the soil infested by N. haematococca, inhibiting the incidence of the disease in plants which were six weeks old. The same was observed for the products kif, dimethomorph, metalaxyl+mancozeb, mancozeb, cymoxanil+maneb, and copper oxychloride for P. parasitica. The tested fungicides in curative treatment inhibited the development of the disease with better results when applied two days after the inoculation, compared to seven days. Prochloraz and carbendazim were outstanding for preventing the death of plants inoculated with N. haematococca. Kif, dimethomorph, metalaxyl+mancozeb, and cymoxanil+maneb showed similar efficiency and were superior to fosetyl-Al in the control of P. parasitica.