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

Invasive weeds are threatening ecosystem function and productivity all over the world by outcompeting desirable vegetation and reducing species diversity. One option for long-term control of such weeds is biological control using natural insect enemies. Such a program has been developed for management of Dalmatian toadflax (Linaria dalmatica (L.) Miller (Plantaginaceae)) in North America using a stem-mining weevil, Mecinus janthiniformis Toševski and Caldara (Coleoptera: Curculionidae). Although widely effective in northern regions, such as in British Columbia and in the American northwest, this insect has been slow to suppress Dalmatian toadflax in southern most regions of their current range, including areas in Utah and Colorado, and little is known of the limiting factors leading to slow weed suppression in these areas. Using field assessments of insect and plant activity over two growing seasons at several sites, this study aimed to provide degree-day and calendar-date descriptions of insect phenology. In addition, dead, overwintered Dalmatian toadflax stems were dissected to determine overwintering mortality of weevil adults before spring emergence, and living stems were dissected to determine development stages and mortality of weevils during summer development. Degree-day and calendar-date based models independently resulted in consistent trends in weevil phenology between sites and between years in this study, although the degree-day model is likely to be most useful for purposes of predicting weevil life cycle timing. Interestingly, the sexes differed in their phenology in that males consistently emerged from overwintering sites and were found on Dalmatian toadflax stems considerably earlier than females in the spring. Females as well as males tended to peak in abundance on stems in late-May when Dalmatian toadflax stems reached full maturity. Overall mortality of M. janthiniformis during a lifecycle was low for all samples; approximately 83% of adults successfully emerged from overwintered stems in the following spring, and greater than 65% of larvae survived to adulthood before overwintering. This resulted in >50% of weevils surviving larval development, overwintering, and spring emergence as adults. The majority of M. janthiniformis deaths (51%) resulted from parasitism by chalcidoid wasps during summer development to adulthood. These parasitoid wasps, and also M. janthiniformis adults, likely created the peculiar exit holes that were observed in live Dalmatian toadflax stems during the summer. Although M. janthiniformis populations were slow to provide effective control of Dalmatian toadflax at sites in Utah, this study indicates that the phenology and survivorship of M. janthiniformis individuals in Utah are well suited for successful biocontrol. If given enough time to build populations, M. janthiniformis appears to be capable of providing effective Dalmatian toadflax control in southern regions, but other limiting factors, such as precipitation and host plant quality, should be considered in future studies to explain slow weed suppression. Phenology models and estimates of mortality of M. janthiniformis generated by this study at sites in Utah may be helpful in implementing future biocontrol programs of Dalmatian toadflax.

Os grãos de milho são expostos, no campo e no armazenamento, à ação de fatores físicos, químicos e biológicos, que interagem entre si, favorecendo a contaminação fúngica, principalmente pelos gêneros Aspergillus, Fusarium e Penicilllium, considerados os mais importantes do ponto de vista toxigênico. O presente trabalho teve como principal objetivo investigar a eficácia do agente de biocontrole Afla-guard® no controle de fungos e de micotoxinas em grãos de milho, no campo, em diferentes estágios de maturidade da planta cultivada no município de Cássia dos Coqueiros, Estado de São Paulo. Visou, também, como objetivos adicionais, a determinação da atividade de água das amostras de milho, a pesquisa de fungos do ar, do solo e do milho, e a influência dos fatores climatológicos (temperatura e precipitação pluvial), no crescimento fúngico e na produção de micotoxinas. A micobiota do milho foi determinada pela técnica da semeadura direta utilizando meio de Ágar Dicloran Rosa Bengala Cloranfenicol (DRBC) e a determinação de micotoxinas por Cromatografia Líquida de Alta Eficiência. A identificação dos fungos foi realizada utilizando metodologia clássica (morfológica) e molecular (sequenciamento do DNA ribossomal). Nas amostras de milho, constatou-se a predominância de Fusarium verticillioides, em todas as coletas. Entretanto, a presença de Aspergillus flavus foi detectada, nas áreas tratadas com produto, a partir da 4ª coleta. No ar atmosférico, os fungos mais frequentes foram: Cladosporium spp., Trichoderma spp. e Fusarium solani. A. flavus foi isolado, principalmente, na 3ª coleta, do tratamento. No solo, os gêneros Penicillium, Trichoderma e Rhizopus foram os mais isolados em todas as coletas. A. flavus foi isolado a partir da 3ª coleta, somente nas áreas tratadas. Das amostras analisadas, a presença de fumonisinas foi detectada em 33,3 % das amostras tratadas com o agente de biocontrole e em 66,6% das amostras do grupo controle (não tratadas). Em relação às aflatoxinas, a presença da toxina foi detectada em apenas 2 amostras (1 tratamento e 1 controle) na 4ª coleta. Quanto à análise do potencial aflatoxigênico determinado, encontramos em 4 dos 13 isolados de A. flavus, aflatoxinas B1 e B2. Os demais isolados não foram potencialmente produtores, podendo ser, portanto, oriundos do Afla-guard®. Já em relação à temperatura, 26 °C foi a média encontrada e 6,3 mm foi a média detectada em relação à precipitação pluvial. Embora não tenha apresentado resultados estatisticamente significativos, o emprego do agente de biocontrole foi relevante no que diz respeito a diminuição da contaminação por Fusarium verticillioides e fumonisinas. Entretanto, outros estudos são necessários para uma melhor avaliação do produto / Maize grains are exposed, in the field and in storage, the action of physical, chemical and biological agents that interact with each other, encouraging fungal contamination, mainly by Aspergillus, Fusarium and Penicillium, considered the most important from the point of view toxigenic. This study aimed to investigate the effectiveness of Afla-guard® biocontrol agent in control of fungi and mycotoxins in corn grain in the field, at different stages of maturity of the plant cultivated in the city of Cassia dos Coqueiros, state São Paulo. Aimed also as additional objectives, determining the water activity of the maize samples, the research air fungi, soil and maize, and the influence of climatic factors (temperature and rainfall) in fungal growth and mycotoxin production. The mycobiota maize was determined by the technique of direct seeding using means of Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and the determination of mycotoxins by high-performance liquid chromatography. The identification of fungi was carried out using classical methods (morphological) and molecular (sequencing of ribosomal DNA). In samples of maize, there was a predominance of Fusarium verticillioides in all samplings. However, the presence of Aspergillus flavus was detected in the areas treated with product samplings from the 4th. In atmospheric air, the most common molds were Cladosporium spp, Trichoderma spp.. Fusarium solani and. A. flavus was isolated mainly in 3rd samplings, treatment. On the ground, Penicillium, Trichoderma and Rhizopus were the most isolated in all samples. A. flavus was isolated from the 3rd collects only in the treated areas. Of the samples tested, the presence of fumonisin was detected in 33.3% of the samples treated with the biocontrol agent and 66.6% of the control group samples (untreated). Regarding the aflatoxins, the presence of the toxin was detected in just 2 samples (1 treatment and 1 control) in 4th collect. The analysis of the potential aflatoxigenic, found in 4 of 13 isolates of A. flavus, aflatoxins B1 and B2. The remaining isolates were not potentially producers and can therefore be derived from the Afla-guard®. Regarding the temperature 26 ° C was found to average 6.3 mm and the average was detected in relation to rainfall. Although not shown statistically significant results, the use of biocontrol agent is relevant as regards the reduction of contamination by Fusarium verticillioides and fumonisins. However, other studies are needed to better evaluate the product

Os grãos de milho são expostos, no campo e no armazenamento, à ação de fatores físicos, químicos e biológicos, que interagem entre si, favorecendo a contaminação fúngica, principalmente pelos gêneros Aspergillus, Fusarium e Penicilllium, considerados os mais importantes do ponto de vista toxigênico. O presente trabalho teve como principal objetivo investigar a eficácia do agente de biocontrole Afla-guard® no controle de fungos e de micotoxinas em grãos de milho, no campo, em diferentes estágios de maturidade da planta cultivada no município de Cássia dos Coqueiros, Estado de São Paulo. Visou, também, como objetivos adicionais, a determinação da atividade de água das amostras de milho, a pesquisa de fungos do ar, do solo e do milho, e a influência dos fatores climatológicos (temperatura e precipitação pluvial), no crescimento fúngico e na produção de micotoxinas. A micobiota do milho foi determinada pela técnica da semeadura direta utilizando meio de Ágar Dicloran Rosa Bengala Cloranfenicol (DRBC) e a determinação de micotoxinas por Cromatografia Líquida de Alta Eficiência. A identificação dos fungos foi realizada utilizando metodologia clássica (morfológica) e molecular (sequenciamento do DNA ribossomal). Nas amostras de milho, constatou-se a predominância de Fusarium verticillioides, em todas as coletas. Entretanto, a presença de Aspergillus flavus foi detectada, nas áreas tratadas com produto, a partir da 4ª coleta. No ar atmosférico, os fungos mais frequentes foram: Cladosporium spp., Trichoderma spp. e Fusarium solani. A. flavus foi isolado, principalmente, na 3ª coleta, do tratamento. No solo, os gêneros Penicillium, Trichoderma e Rhizopus foram os mais isolados em todas as coletas. A. flavus foi isolado a partir da 3ª coleta, somente nas áreas tratadas. Das amostras analisadas, a presença de fumonisinas foi detectada em 33,3 % das amostras tratadas com o agente de biocontrole e em 66,6% das amostras do grupo controle (não tratadas). Em relação às aflatoxinas, a presença da toxina foi detectada em apenas 2 amostras (1 tratamento e 1 controle) na 4ª coleta. Quanto à análise do potencial aflatoxigênico determinado, encontramos em 4 dos 13 isolados de A. flavus, aflatoxinas B1 e B2. Os demais isolados não foram potencialmente produtores, podendo ser, portanto, oriundos do Afla-guard®. Já em relação à temperatura, 26 °C foi a média encontrada e 6,3 mm foi a média detectada em relação à precipitação pluvial. Embora não tenha apresentado resultados estatisticamente significativos, o emprego do agente de biocontrole foi relevante no que diz respeito a diminuição da contaminação por Fusarium verticillioides e fumonisinas. Entretanto, outros estudos são necessários para uma melhor avaliação do produto / Maize grains are exposed, in the field and in storage, the action of physical, chemical and biological agents that interact with each other, encouraging fungal contamination, mainly by Aspergillus, Fusarium and Penicillium, considered the most important from the point of view toxigenic. This study aimed to investigate the effectiveness of Afla-guard® biocontrol agent in control of fungi and mycotoxins in corn grain in the field, at different stages of maturity of the plant cultivated in the city of Cassia dos Coqueiros, state São Paulo. Aimed also as additional objectives, determining the water activity of the maize samples, the research air fungi, soil and maize, and the influence of climatic factors (temperature and rainfall) in fungal growth and mycotoxin production. The mycobiota maize was determined by the technique of direct seeding using means of Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and the determination of mycotoxins by high-performance liquid chromatography. The identification of fungi was carried out using classical methods (morphological) and molecular (sequencing of ribosomal DNA). In samples of maize, there was a predominance of Fusarium verticillioides in all samplings. However, the presence of Aspergillus flavus was detected in the areas treated with product samplings from the 4th. In atmospheric air, the most common molds were Cladosporium spp, Trichoderma spp.. Fusarium solani and. A. flavus was isolated mainly in 3rd samplings, treatment. On the ground, Penicillium, Trichoderma and Rhizopus were the most isolated in all samples. A. flavus was isolated from the 3rd collects only in the treated areas. Of the samples tested, the presence of fumonisin was detected in 33.3% of the samples treated with the biocontrol agent and 66.6% of the control group samples (untreated). Regarding the aflatoxins, the presence of the toxin was detected in just 2 samples (1 treatment and 1 control) in 4th collect. The analysis of the potential aflatoxigenic, found in 4 of 13 isolates of A. flavus, aflatoxins B1 and B2. The remaining isolates were not potentially producers and can therefore be derived from the Afla-guard®. Regarding the temperature 26 ° C was found to average 6.3 mm and the average was detected in relation to rainfall. Although not shown statistically significant results, the use of biocontrol agent is relevant as regards the reduction of contamination by Fusarium verticillioides and fumonisins. However, other studies are needed to better evaluate the product

Choisya ternata est une plante ornementale souvent touchée par la maladie de la pourriture racinaire provoquée par Phytophthora. Cette maladie peut induire de pertes allant jusqu’à 80 %, ce qui implique l’utilisation intensive de produits phytosanitaires. Afin de limiter l’utilisation de ces produits toxiques, une meilleure connaissance des acteurs de la défense des plantes est nécessaire. Pour cela, les objectifs de cette thèse sont I) d’identifier les espèces du genre Phytophthora pathogènes de C. ternata, II) d’étudier des acteurs de défense au niveau racinaire et III) de développer une approche de lutte alternative aux phytosanitaires. L’identification des espèces de Phytophthora par l’amplification et le séquençage de la région ITS, montre la présence de Phytophthora parasitica dans la production de C. ternata en pépinière. Ils mettent aussi en évidence, pour la première fois en France et chez Choisya, la présence de Phytophthora tropicalis. Deux cultivars de C. ternata sont utilisés pour étudier les acteurs de la défense racinaire contre P. parasitica, Aztec pearl (moins sensible à la pourriture racinaire) et Goldfinger (plus sensible). Les arabinogalactane-protéines (AGPs) sont des glycomolécules de la paroi cellulaire qui constitue une barrière physique face aux pathogènes. Des études ont montré le rôle des AGPs dans l’interaction plante-pathogène et plus particulièrement avec les oomycètes. Toutefois, le rôle des AGPs racinaires de Choisya dans l’interaction avec P. parasitica n’est pas étudié. Nos résultats montrent des différences biochimiques au niveau de la composition monosaccharidique des AGPs racinaires d’Aztec pearl en comparaison avec les AGPs de feuilles de ce cultivar et les AGPs de feuilles et de racines de Goldfinger. Contrairement aux autres fractions, ces AGPs n’ont pas augmenté la croissance du mycélium de P. parasitica. Ces résultats suggèrent que l’oomycète n’est pas capable de dégrader cette fraction pour l’utiliser comme une source d’énergie. Pendant l’infection de la plante par P. parasitica, ces AGPs peuvent freiner la pénétration du pathogène, et par conséquent diminuer la sévérité de la maladie. Les plantes ont aussi développé des molécules chimiques nommées métabolites secondaires (MS) capables de les protéger contre leurs agresseurs. Des études ont montré que la partie foliaire de Choisya est riche en MS tels que les alcaloïdes dont plusieurseffets pharmacologiques sont connus. Cependant, leur composition racinaire et leur rôle dans la protection de la plante contre P. parasitica ne sont pas élucidés. Nos résultats montrent que les racines des deux cultivars sont riches en alcaloïdes furoquinoliques. Certains alcaloïdes sont présents en plus grande quantité chez Aztec pearl, mais suite à l’inoculation de zoospores de P. parasitica cette différence n’est plus détectable. De plus, l’extrait contenant les alcaloïdes totaux d’Aztec pearl ont été capables d’inhiber la croissance du mycélium de l’oomycète, contrairement à l’extrait issu de Goldfinger. Ces résultats montrent le rôle potentiel des alcaloïdes furoquinoliques dans la protection de la plante contre cet oomycète. / Choisya ternata is an ornamental plant that suffers from root rot disease due to Phytophthora. This disease can lead to severe production losses (up to 80 %), which require intensive use of phytosanitary products. A better understanding of plants defenses in required in order to reduce the use of these products. Therefore, the objectives of this thesis are I) identifying Phytophthora spp. pathogens of C. ternata, II) studying roots defense actors and III) developing an alternative control approach. Phytophthora spp. identification by ITS region amplification and sequencing highlighted the presence of Phytophthora parasitica on Choisya. Furthermore, Phytophthora tropicalis was identified for the first time in France and on Choisya culture. Two C. ternata cultivars were used to study the plants root defense actors against P. parasitica, Aztec pearl (less susceptible to root rot) and Goldfinger (more susceptible to root rot). Arabinogalactan proteins (AGPs) are glycomolecules of the cell wall which constitutes a physical barrier to pathogens. Studies showed the role of AGPs in the plant-pathogen interaction and more specifically in the case of oomycetes. However, Choisya root AGPs role in the interaction with P. parasitica is not studied. Our results showed biochemical differences in the monosaccharide composition of Aztec pearl root AGPs and the other AGPs fractions (Aztec pearl leaves and Goldfinger roots and leaves). Contrary to other fractions Aztec pearl root AGPs did not increase P. parasitica mycelium growth. These results suggest that oomycete was not able to degrade this fraction and use it as energy source. When P. parasitica infects the plant, these AGPs may be able to slow the infection and reduce disease severity. Plants have also developed chemical molecules known as secondary metabolites (SM) capable of protecting them against attackers. Studies showed that C. ternata leaves are rich with SM as alkaloids that have many pharmacological activities. Nevertheless, roots alkaloids composition and role in plant protection are not studied. Our results showed that roots of the two cultivars are riche with furoquinoline alkaloids. Some these alkaloids were more concentrated in Aztec pearl. But after inoculation with P. parasitica zoospores, the difference was not detectable anymore. Moreover, the total alkaloids extract of Aztec pearl inhibited P. parasitica mycelium growth, unlike the extract of Goldfinger. Another strategy of plants protection is the use of beneficial soil microorganisms that limits pathogens development and stimulate plant defenses. These microorganisms are known as biological control agents (BCA) and are sometimes used in horticulture as an alternative control strategy. In our study, treatments of C. ternata by different BCA were evaluated by a developed real time PCR (qPCR) targeting ypt1 and by symptoms annotation. This evaluation showed that combined treatments by Glomus intraradices with Gliocladium catenulatum and G. intraradices with Trichoderma atroviridae (respectively mycorrhizal fungi and filamentous fungi) offer a better protection against P. parasitica.