Démarche d’ingénierie écologique en santé des plantes : gestion des couvre-sol des vergers pour lutter contre les bactérioses des arbres fruitiers provoquées par Pseudomonas syringaeIdentifier les réservoirs et sources d’inoculum des agents phytopathogènes est un enjeu majeur en pathologie végétale. Les systèmes agricoles pérennes, tels que les vergers, sont soumis aux attaques de nombreux ravageurs et agents microbiens pathogènes. P. syringae, agent phytopathogène responsable de l’émergence de maladies des arbres fruitiers, dont la récente épidémie du chancre bactérien du kiwi causée P. syringae pv. actinidiae (Psa), représente un enjeu économique important au niveau mondial. En France, les moyens de lutte sont constitués de traitements cupriques et de gestes préventifs visant à réduire la propagation de la bactérie au sein et entre les vergers. Avec la prise de conscience de la nécessité de la conservation de l’environnement par les consommateurs et producteurs, les méthodes de cultures actuelles ont tendances à s’orienter vers celles de l’agroécologie et l’usage de l’ingénierie écologique au service de la santé des plantes. En verger, la gestion des communautés de plantes des couvre-sols donne de bons résultats dans la lutte conte certaines espèces de ravageurs, tels que les arthropodes herbivores, mais les effets sur les communautés de microorganismes pathogènes restent inexplorés. Les plantes couvre-sol et adventices des vergers hébergent d’abondantes communautés de P. syringae, cependant le rôle de ces couverts végétaux dans l’émergence des maladies des arbres fruitiers reste incompris. Par conséquent, les travaux de recherche presentés ici focalisent sur l’étude simultanée des communautés de P. syringae associées aux plantes couvre-sol et aux arbres fruitiers de trois vergers d’abricotiers et de quatre vergers de kiwis du département de la Drôme, sud-est de la France, choisis pour leur état sanitaire (sain, malade ou émergence de la maladie), ainsi que des pratiques de gestion du couvre-sol différentes (sol nu, enherbement des inter-rangs, enherbement des inter rangs et rangs des arbres). En l’absence d’outils permettant une identification rapide et une affiliation à l’un des 13 phylogroupes actuellement décris pour l’espèce P. syringae, l’étude du génome complet d’une cinquantaine de souches de P. syringae a permis la mise au point de marqueurs moléculaires capables d’identifier 9 des 13 phylogroupes. L’étude des communautés de P. syringae hébergées par les couvre-sols végétaux montre un effet de la composition des communautés des plantes couvre-sol sur l’abondance et la structure des communautés de P. syringae. La présence de Prunella vulgaris, une plante de la famille des Lamiaceae, est corrélée avec une diminution de l’abondance des P. syringae. La reproductibilité de ce résultat est actuellement en cours d’investigation dans une parcelle expérimentale. Cependant, les résultats préliminaires montrent une absence d’effet de P. vulgaris sur l’abondance de P. syringae. L’étude simultanée des communautés de P. syringae des couvre-sols végétaux et des arbres fruitiers montre que des échanges se font entre les deux compartiments en raison de la présence de souches génétiquement proches. Chez le kiwi, lorsque Psa est présent il coexiste toujours avec d’autres P. syringae, soulevant la question des interactions entre ces souches et leur rôle dans l’émergence de la maladie. Enfin, les résultats mettent en avant un potentiel antagonisme entre les phylogroupes 1 et 2. / Process of ecological engineering in plant health: study of the role of orchard ground cover plants in the emergence of fruit tree diseases caused by Pseudomonas syringaeIdentification of reservoirs and inoculum sources of plant pathogenic microorganisms is a major issue in plant pathology. Perennial agricultural systems, such as orchards, are exposed to many pests and pathogenic microorganisms. P. syringae, a phytopathogenic bacterium responsible for the emergence of diseases of fruit trees, including the recent outbreak of bacterial canker of kiwifruit caused P. syringae pv. actinidiae (Psa), represent an important economic issue worldwide. In France, means of control of bacterial canker consist of copper treatments and preventive measures in order to reduce the spread of bacteria within and between orchards. With the awareness for environmental conservation by consumers and producers, current cultivation methods tend to be progressively replaced by more agroecological ones and the use of ecological engineering to improve plant health. Ecological engineering of orchard ground cover plant communities provides good results for the control of orchard pests, such as herbivorous arthropods, but the effects on pathogenic microbial communities remains unexplored. The ground cover plants and orchard weeds host abundant P. syringae communities, however the role of ground covers in the emergence of fruit tree diseases remains ignored. Therefore, the research presented here is focused on the simultaneous study of P. syringae communities associated with ground covers and fruit trees from three apricot and four kiwifruit orchards of Drôme county, southeastern France, chosen for their health status (healthy, diseased, or disease emergence), as well as different ground cover management practices (bare soil, ground cover in inter-rows, ground cover in inter-rows and tree rows). In the absence of tools for rapid identification and affiliation to one of 13 currently described phylogroups for the P. syringae species, the screening of whole genomes of more than fifty P. syringae strains has allowed the development of specific molecular markers able to identify 9 of the 13 phylogroups. Results show that ground cover P. syringae community abundances and structures are correlated to plant community composition. The presence of Prunella vulgaris, a plant of the Lamiaceae family, is correlated to a decrease in the P. syringae abundances. Reproducibility of this result is currently under investigation in an experimental field. However, preliminary results from the experimental field show that the presence of P. vulgaris in 1-year-old ground covers is not correlated to a decrease in P. syringae abundances. Simultaneous study of ground cover and fruit tree P. syringae communities highlight bacterial exchanges between these two compartments because of the presence of genetically correlated strains in both of them. When present, Psa coexist with other P. syringae, raising the question of the interaction between these strains and their role in the emergence of the disease. Finally, the results highlight a potential antagonism between phylogroups 1 and 2.

No description available.

Made available in DSpace on 2015-03-26T13:37:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4434052 bytes, checksum: e6bd53f46965b44a3dcdbb9a5a63b4ff (MD5) Previous issue date: 2008-02-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work aimed to select tomato prokaryotic phylloplane residents for biocontrol purposes, by using Xanthomonas campestris pv. vesicatoria and Alternaria solani as challenging pathogens. Additionally, to perform a screening test using detached leaves. Consequently, leaves from healthy tomato plants were collected at Viçosa, MG and epiphytic prokaryote populations were extracted by shacking them in PBS (0,85% NaCl in 0,1M phosphate buffer saline, pH 7.0) following sonication (60Hz, 20 minutes) and serial dilution and plating in culture medium. Three hundred bacterial isolates were obtained and preserved in refrigerator, with periodical tubetube transfer, in a refrigerator. The screening was performed in two selection cycles. In the first selection in greenhouse, with three replicates per isolate and Xanthomonas campestris pv. vesicatoria as challenging pathogen, 79 isolates were able to reduce disease by 82%. In a second selection cycle, using 6 replicates per isolate, the 33 isolates were able to reduce severity by 50% in average. The 33 isolates had their antagonistic potential tested against the tomato fungal and bacterial pathogens Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani and Corynespora cassiicola in vitro conditions. Out of the 33 isolates, the isolate RFK-24 inhibited growth of Xathomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis and Alternaria solani while isolate RFS-183 was able to inhibit Alternaria solani, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, but none of them inhibited Corynespora cassiicola. Being RFK-24 and RFS- 183 the ones with a wider antagonistic potential out of the 33 previously selected, they were chosen for continuing the research with detached leaves and the challenging pathogens Alternaria solani e Xanthomonas campestris pv. vesicatoria. There was a positive correlation between disease severity reduction in plants the greenhouse and in detached leaves, for both antagonists. For situations involving large number of isolates to undergo mass screening, the approach with detached organs may replace the laborious and time consuming greenhouse screening. / O presente trabalho teve como objetivo isolar procariotos residentes de filoplano de tomateiro como agentes de biocontrole contra o patógeno Xanthomonas campestris pv. vesicatoria. 300 isolados foram obtidos a partir de folhas sadias de tomateiros coletadas na micro-região de Viçosa-MG, as quais foram submetidas à extração das populações procarióticas epifíticas em solução de tampão fosfato (PBS) com emprego de ultra-som (60Hz, 20 min.) e semeadura de diluições em série em placas de Petri contendo meio 523. Em casa-de-vegetação, numa primeira etapa 79 isolados apresentaram capacidade de reduzir, em média, 82 % da severidade de doença. Desses, numa segunda etapa, 33 isolados apresentaram redução média da severidade de doença em 50 %. Os 33 isolados foram submetidos a testes de antibiose in vitro, em que foi avaliada a potencialidade em inibir o crescimento bacteriano e fúngico dos seguintes patógenos do tomateiro: Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani e Corynespora cassiicola. Dos 33 isolados pré- selecionados verificou-se que o isolado RFK-24 foi capaz de inibir o crescimento dos patógenos, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis e Alternaria solani e o RFS-183 em inibir o crescimento dos patógenos Alternaria solani, Xanthomons campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, mas ambos não inibiram o crescimento da Corynespora cassiicola. Os dois isolados foram selecionados para ensaios in vivo e em folíolos destacados, contra os patógenos desafiantes Alternaria solani e Xanthomons campestris pv. vesicatoria. Para ambos foi observada a correlação entre a severidade de doença apresentada em casa-de-vegetação e os resultados em folíolos destacados, como a inibição da germinação de conídios do patógeno fúngico e a supressividade em meio semi-seletivo dos antagonistas sob o patógeno bacteriano. Os resultados obtidos com o método com folíolos destacados e sua correlação com os dados de biocontrole experimental em casa-de-vegetação permitem deduzir que o uso de folíolos destacados pode ser utilizado como método para seleção massal.

Orientador: Suzete Aparecida Lanza Destéfano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T19:29:47Z (GMT). No. of bitstreams: 1 Goulart_MarcelaCristina_M.pdf: 1843141 bytes, checksum: 317126844277b642b5edc69d405b000b (MD5) Previous issue date: 2014 / Resumo: A soja é considerada uma das culturas mais importantes no Brasil, em função de seu alto valor sócio-econômico, determinado pelas inúmeras aplicações de seus produtos e subprodutos e consequente expressão no mercado interno e externo. No entanto, a cultura desta oleaginosa é frequentemente ameaçada com a ocorrência de um vasto número de doenças, que podem acarretar depreciação do produto, redução no rendimento e perdas econômicas para os produtores. Dentre as principais doenças bacterianas que afetam a cultura da soja, destacam-se a pústula bacteriana, causada por Xanthomonas axonopodis pv. glycines (Xag); o crestamento bacteriano, causado por Pseudomonas savastanoi pv. glycines (Psg); e a murcha de Curtobacterium causada por Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), ocasionando perdas na produção de até 40%. A condição sanitária das sementes é extremamente importante se considerarmos que elas são veículos desses agentes fitopatogênicos que nelas podem se alojar e serem levados ao campo, provocando redução na germinação e vigor, e originando focos primários de infecção de doenças. O presente trabalho teve por objetivo desenvolver nova metodologia de diagnóstico com o uso das técnicas moleculares que permitissem detectar e identificar a presença de Psg, Xag e Cff em sementes de soja por meio do desenvolvimento de primers espécie específicos. Os primers desenhados a partir de sequências da região espaçadora 16S-23S RNAr, mostraram-se altamente específicos e sensíveis. O par de primers Curto2f/p322anti gerou um fragmento de 675 pb e capacidade de detecção a partir de 0,01 ng de DNA genômico e aproximadamente 5x103 UFC/PCR; o par de primers Psgl/p322anti gerou um fragmento de 500 pb e o grau mínimo de sensibilidade foi de 1 pg de DNA genômico e cerca de 80 UFC/PCR; o par de primers Xanth2f/p322anti gerou um fragmento de 545 pb e capacidade de detecção a partir de 1 ng de DNA genômico e cerca de 700 UFC/PCR. Posteriormente, as sementes de soja foram infectadas artificialmente nas condições de 1; 0,5 e 0,1% de infecção. Nas amplificações com os primers espécie-específicos desenvolvidos, foi possível detectar as fitobactérias em todos os níveis de infecção testados diretamente do extrato bruto, e nas amplificações após o enriquecimento do extrato (BIO-PCR) o sinal positivo foi potencializado / Abstract: Soybean is considered one of the most important crops in Brazil, due to its high socio-economic value, determined by its several products and sub products and its significant expression on the internal and external market. However, this oleaginous plant is often affected by the occurrence of different diseases, which cause depreciation of the product, reduction in yield and substantial economic losses. Among the main bacterial diseases, the bacterial pustule, caused by Xanthomonas axonopodis pv. glycines (Xag), the bacterial blight caused by Pseudomonas savastanoi pv. glycines (Psg), and bacterial tan spot caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), producing yield losses of up to 40%. The seed health is extremely important since they are considered vehicles of pathogenic agents which can be led to the field, causing germination reduction and vigor; and yielding primary infection of the diseases. This study aimed to develop a new method of diagnosis using molecular tools to detect and identify Psg, Xag or Cff in soybean seeds, through species-specific primers. The primers were designed from sequences of the 16S-23S rRNA and they were highly specific and sensitive. The pair of primers Curto2f/p322anti generated a fragment of 675 bp and was able of detecting down to 0.01 ng of genomic DNA and about 5x103 CFU/PCR; the primer set Psgl/p322anti produced a fragment of 500 bp and reached a detection limit of 1 pg of genomic DNA and about 80 CFU/PCR; and Xanth2f/p322anti yielded a fragment of 545 bp and could detect up to 1 ng of genomic DNA and about 700 CFU/PCR. Subsequently, soybean seeds were artificially infected in the following conditions: 1, 0,5% and 0,1% infection. In the amplifications using the species-specific primers, it was possible to detect the three different phytobacteria at all tested levels of infection directly of the crude extract and in the amplifications after enrichment of the extract (BIO-PCR), the positive signal was enhanced / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular

Pour diversifier leur matériel génétique, s’adapter aux perturbations environnementales et coloniser de nouvelles niches, les bactéries utilisent plusieurs processus évolutifs dont l’acquisition de matériel génétique par transfert horizontal de gènes comme la conjugaison, la transduction et la transformation. À ces trois mécanismes naturels s’ajoute l’électrotransformation due aux phénomènes électriques liés à la décharge de foudre. La présence dans les nuages de bactéries aérosolisées capables de former des noyaux de glace à l’origine des précipitations et impliquées dans le déclenchement de la foudre, telles que la bactérie phytopathogène à répartition mondiale Pseudomonas syringae, nous a conduit à proposer que l’électrotransformation naturelle dans les nuages pouvait affecter les bactéries, contribuant ainsi à augmenter leur potentiel adaptatif. Dans un premier temps, nous avons déterminé si la bactérie glaçogène P. syringae pouvait survivre à des électroporations simulant des décharges de foudre et acquérir du matériel génétique exogène dans les nuages. Comparée à deux autres bactéries, P. syringae se révèle être mieux adaptée pour la survie et l’électrotransformation génétique, ce qui suggère qu’elle serait capable de survivre et d’évoluer durant son transport dans les nuages. Nous avons ensuite évalué l’impact d’électroporations simulant les décharges de foudre sur la survie, le potentiel d’électrotransformation et la diversité de bactéries présentes dans des échantillons de pluie comme substitut des communautés bactériennes des nuages. Ces dernières étaient plus résistantes que les souches de laboratoire et certaines étaient capables d’acquérir de l’ADN exogène par électrotransformation. Les bactéries de la pluie isolées provenaient de différentes origines et présentaient différents modes de vie, représentatifs des sources probables d’émissions de bactéries terrestres. Cette étude montre que les bactéries aérosolisées de divers écosystèmes terrestres sont susceptibles de se disséminer dans de nouveaux habitats grâce aux nuages tout en étant capable d’acquérir de nouveaux gènes par éléctrotransformation, et d’augmenter ainsi potentiellement leur diversité génétique. La dernière partie de mon travail a évalué si l’électrotransformation appliquée aux bactéries indigènes du sol pouvait être employée pour dépolluer les sols contaminés par un pesticide largement utilisé autrefois, le lindane. L’optimisation des expériences met en évidence l’incorporation par les bactéries indigènes d’un plasmide contenant le gène codant les premières déchlorinations du lindane au travers d’une combinaison de transformation naturelle et d’électrotransformation. En conclusion, nous avons montré que l’électrotransformation naturelle liée aux décharges électriques, comme celles se produisant dans les nuages ou atteignant le sol, peut être impliquée dans le transfert horizontal de gènes chez les bactéries et, considérant l’importance de la foudre à travers le monde, pourrait jouer un rôle dans l’adaptation et l’évolution de ces organismes. / To diversify their genetic material, allowing adaptation to environmental disturbances and colonization of new ecological niches, bacteria use various evolutionary processes, including the acquisition of new genetic material by horizontal transfer mechanisms such as conjugation, transduction and transformation. Electrotransformation mediated by lightningrelated electrical phenomena may constitute an additional gene transfer mechanism occurring in nature. The presence in clouds of bacteria capable of forming ice nuclei that lead to precipitations and are involved in the triggering of lightning, such as the global phytopathogen Pseudomonas syringae, led us to postulate that natural electrotransformation in clouds may affect bacteria, by contributing to increase their adaptive potential. We first determined if the ice nucleator bacterium P. syringae could survive when in clouds and acquire exogenous genetic material through lightning shock-simulating in vitro electroporation. In comparison to two other bacteria, P. syringae appears to be best adapted for survival and for genetic electrotransformation under these conditions, which suggests that this bacterium would be able to survive and evolve whilst being transported in clouds. Secondly, we evaluated the impact of lightning shock-simulating in vitro electroporation on the survival, the electrotransformation potential and the diversity of bacteria collected from rain samples. These isolates better resisted lightning than the laboratory strains and some were able to electrotransform exogenous DNA. The rain bacteria we isolated were of different origins and were representative of life modes of the various sources of bacterial emissions on Earth. Our study suggests that bacteria aerosolized from diverse terrestrial ecosystems can spread to new habitats through clouds whilst also being able to acquire new genetic material via lightning-based electrotransformation, thereby potentially enhancing their genetic diversity. The final part of our work consisted of evaluating whether electrotransformation could be applied to the engineering of indigenous soil bacteria in order to develop a tool for the bioremediation of lindane, a once widely used pesticide. Optimized experiments revealed that both natural and electrotransformation contributed to the incorporation of a plasmid harboring a gene encoding the first lindane dechlorination steps by indigenous soil bacteria. In conclusion, we showed that natural electrotransformation mediated by electrical discharges such as those occurring in clouds or reaching soils can be involved in the horizontal gene transfer process among bacteria and, considering the importance of lightning worldwide, may play a role in the adaptation and evolution of these organisms.

Background: Bacteriophage Φ6 is a lipid-enveloped dsRNA bacteriophage. The limitations in our knowledge of how this bacteriophage occurs in the environment are limited by non-selective isolation techniques. Research on finding phages in the environment in the past has employed the Double Agar Layer (DAL) plaque assay using Tryptic Soy Agar (TSA), a non-selective media. The bacterial host for bacteriophage Φ6 is Pseudomonas syringae. In this study, we tested Pseudomonas Agar, a selective media that suppresses the growth of bacteria except Pseudomonas species, in the standard double agar layer plaque assay for Φ6. Methods: DAL plaque assays were performed to determine the sensitivity of both Tryptic Soy Agar (TSA) and Pseudomonas Agar (PA) for determining the titer of pure bacteriophage Φ6 stocks. We used Pseudomonas syringae (HB10Y) as the host, and the plaque formation on both agars was compared. Following the evaluation of PA with pure Φ6 stocks, PA effectiveness for Φ6 isolation from environmental samples was tested in spiked waters obtained from irrigation ponds at an agricultural farm. Results: Comparison of TSA and PA using pure Φ6 cultured in the laboratory and spiked environmental samples showed that PA agar can detect bacteriophage Φ6 as well as the standard DAL assay using TSA. On PA, formation of clear visible plaques comparable to the plaques formed using TSA was observed. Conclusions: Pseudomonas Agar can be used for the isolation of bacteriophage Φ6 in environmental samples. This may enhance the detection of these phages in the environment.

No description available.

Plants are continuously exposed to different abiotic and biotic stresses in their natural environment. Their capacity to survive depends on the capacity to perceive external signal and quality amount a defence response for protection from the stress perceived. The purpose of this project was to study the impact of combined abiotic stress and biotic stress on the outcome of the disease inducing Arabidopsis thaliana – Pseudomonas syringae interaction. This study included a focus on the role of ABA in these interactions and also whether 3´-O-β D- ribofuranosyl adenosine (hereafter it called ‘400’ compound), a novel adenosine derived compound induced during compatible interactions, was involved. The later involved the targetted disruption of a putative 400 biosynthetic pathway involving analysis of knockout mutants of enzymes; APD-ribose diphosphatase NAD binding / hydrolases of the NUDIX class, glucosyl transferases, ribosyltransferases, a ribose-phosphate pyrophosphokinase3 and galactosyltransferases. Unfortunately, none of these targeted interventions modified the host response to Pseudomonas infection, nor altered levels of 400 in challenged leaves. The primary research investigated the interaction between abiotic and biotic stresses in Arabidopsis plants focussing on the modulation of plant defence against multiple, and possibly antagonistic, stress responses and the role plant hormones play in this process. We showed that high light caused enhanced susceptibility to the already virulent Pseudomonas syringae DC3000pvsp61. The pathways contributing to this enhanced susceptibility were largely ABA independent. Subsequent characterization of transgenic lines expressing the soluble Arabidopsis abscisic acid receptors, PYRABACTIN RESISTANCE1-LIKE4-6 provided compelling evidence for a role for these receptors in DC3000 virulence strategies, but they contribute to a lesser extent to the enhanced susceptibility under high light. This was corroborated genetically by using mutants of the immediately downstream targets of PYLs, the type two protein phosphatase, specifically the triple mutant hab1-1/abi2-1/abi1-2. A number of epitope and fluorescent constructs were generated to facilitate future studies of the role of ABA signaling. Targetted profiling suggested that SA dynamics were altered under DC3000 challenged Arabidopsis grown under high light. Furthermore, differential accumulation of flavonoids suggested these may also play a role in attenuating host defences under high light. Finally we provide evidence based on comparative analysis of that the photoreceptors phytochrome double mutant phyA-211/phyB-9 and cry1/cry2 behave antagonistically in Arabidopsis response to DC3000. Overall our studies support the conclusion that plants abiotic stress (HL) response takes precedence over biotic stress (DC3000) responses and that abiotic stress is detrimental to plant immunity. The luciferase transgenic PYL lines showed high level of expression of ClucP::PYL5 plant tissues challenged 2hpi of DC3000 (OD600: 0.15) in comparison with C1lucP::PYL6. This result opposes to what RT-PCR reported; which was that three PYLs genes display similar expression level at 6hpi of hrpA or 18hpi of DC3000. The epitope tags of CaMV::HA transgenic plants showed HA-tagged signal with stunted phenotype in a range of PYL4, 5 and 6 plants but none of the plants displayed any differences in susceptibility to DC3000. Although, RT-PCR assay showed high levels of expression in the three PYLs, 6hpi of hrpA but no signal was detected in B8eGFP::PYL5 transgenic line either followed the DC3000 and hrpA infection or by examined plant seedlings at early stages under confocal microscopy.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T13:39:56Z No. of bitstreams: 1 Camila Cristina Lage de Andrade.pdf: 845467 bytes, checksum: ef8d7e269cd9b79e14fc8dbcd17e8edd (MD5) / Made available in DSpace on 2017-02-17T13:39:56Z (GMT). No. of bitstreams: 1 Camila Cristina Lage de Andrade.pdf: 845467 bytes, checksum: ef8d7e269cd9b79e14fc8dbcd17e8edd (MD5) Previous issue date: 2012-02-16 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The bacterial speck caused by Pseudomonas syringae pv. tomato (Pst) (Okabe) Young, Dye & Wilkie has economic relevance to the tomato for industry in Brazil. The use of products that potentiate and/or induce plant defenses is an alternative that meets the requirements of integrated disease management. This study aimed to evaluate the effect of silicon (Si) and chemical inducers in some components of tomato resistance to bacterial speck and activity of enzymes involved in plant defense. In the first study, tomato plants were grown in: soil without calcium silicate (control) (T1), soil without calcium silicate and plants sprayed with Supa Silica® (SS) (2 mL/L SS) (T2) and soil with calcium silicate (0.38 g) (T3), being inoculated by spraying with a suspension of the pathogen. Our research evaluated the incubation period (IP), number of lesions (NL) per plant, severity estimated by the software QUANT 1.0 (SEQ) and leaf concentration of Si, as well as the activity of enzymes peroxidases (POX), polyphenoloxidases (PPO) β-1,3-glucanases (GLU), phenylalanine ammonia lyases (PAL), lipoxygenases (LOX) and concentration of malondialdehyde (MDA). It was assessed the effect of SS in the Pst growth in vitro. There was no significant difference between treatments for the IP and the foliar concentration of Si. No significant differences were found for the NL per plant and SEQ between T3 and control. T2 significantly reduced NL in 46.8 and 45.1% and SEQ in 61.5 and 56.2% when compared with control and T3, respectively. There was negative linear response of SS doses on the Pst growth in vitro. The activity of POX, PPO and GLU was significantly higher in T2 and T3. The activity of FAL and LOX was significantly higher in T3. The MDA concentration was significantly higher in T2 compared to control, in non-inoculated plants and at 7 days after inoculation (d.a.i.), being significantly lower at 10 d.a.i. In the second study, three experiments were conducted to evaluate the effect of jasmonic acid (JA; 0.1 mM), ethephon (ET, 0.5 mM) and acibenzolar-S-methyl (Bion®, ASM; 300 mg/L) sprayed 48 h before inoculation with Pst, evaluating the IP and NL per plant, and the activity of enzymes POX, PPO, GLU and LOX. It was also assessed the effect of JA and ET in the growth of Pst in vitro. Only in experiment 3 incubation period increased significantly by one day in the plants sprayed with ASM compared with control. In all experiments, NL per plant was significantly reduced by JA, ET e ASM in relation to the control reaching values of 38.9, 45.3 and 68.1%, respectively, in experiment 2. The growth of Pst in vitro was not significantly influenced. In some evaluation times JA has significantly raised the activity of POX, PPO and GLU; ASM has elevated the activity of PPO, GLU and LOX, while ET only significantly raised the activity of GLU and LOX compared to the control. The results of these studies suggest that spraying tomato plants with SS and the inducers JA, ET and ASM affected some components of tomato resistance to bacterial speck, and activated the enzymes POX, PPO, GLU, PAL and LOX, involved in defense responses of tomato plants to Pst. / A pinta bacteriana causada por Pseudomonas syringae pv. tomato (Pst) (Okabe) Young, Dye & Wilkie tem importância econômica para a cultura do tomateiro industrial no Brasil. O uso de agentes potencializadores e/ou indutores das defesas das plantas é uma alternativa que atende aos requisitos do manejo integrado de doenças. Este estudo avaliou o efeito do silício (Si) e de indutores químicos em alguns componentes de resistência do tomateiro à pinta bacteriana e na atividade de enzimas envolvidas na defesa das plantas. No primeiro estudo plantas de tomateiro foram cultivadas em: solo sem silicato de cálcio (controle) (T1), solo sem silicato de cálcio e plantas pulverizadas com Supa Sílica® (SS) (2 mL/L de SS) (T2) e solo com silicato de cálcio (0,38 g) (T3), sendo inoculadas por pulverização com suspensão do patógeno. Foram avaliados o período de incubação (PI), número de lesões (NL) por planta, severidade estimada pelo software QUANT 1.0 (SEQ) e concentração foliar de Si, bem como a atividade das enzimas peroxidases (POX), polifenoloxidases (PFO), β-1,3-glucanases (GLU), fenilalanina amônia-liases (FAL), lipoxigenases (LOX) e concentração de aldeído malônico (MDA). Também foi avaliado o efeito do SS no crescimento de Pst in vitro. Não houve diferença significativa entre os tratamentos para o PI e para a concentração foliar de Si. Não houve diferença significativa para o NL por planta e a SEQ entre o tratamento T3 e o controle. O tratamento T2 reduziu significativamente o NL 46,8 e 45,1% e a SEQ 61,5 e 56,2%, em relação ao controle e ao tratamento T3, respectivamente. Houve resposta linear negativa das doses de SS no crescimento da Pst in vitro. A atividade das POX, PFO e GLU foi significativamente maior nos tratamentos T2 e T3. A atividade das FAL e das LOX foi significativamente maior no T3. A concentração de MDA foi significativamente maior no tratamento T2 em relação ao controle, nas plantas não inoculadas com Pst e aos 7 dias após inoculação (d.a.i.); sendo significativamente menor aos 10 d.a.i. No segundo estudo, três experimentos foram realizados para avaliar o efeito dos indutores ácido jasmônico (AJ; 0,1 mM), ethephon (ET; 0,5 mM) e acibenzolar-S-metil (Bion®, ASM; 300 mg/L) pulverizados 48 h antes da inoculação da Pst, avaliando-se o PI e o NL por planta, além da atividade das enzimas POX, PFO, GLU e LOX. Também foi avaliado o efeito do AJ e do ET no crescimento da Pst in vitro. Apenas no experimento 3 o PI aumentou significativamente em 1 dia nas plantas pulverizadas com ASM em relação ao controle. O NL por planta foi significativamente reduzido pelos tratamentos AJ, ET e ASM em relação ao controle para todos os experimentos, atingindo valores de 38,9, 45,3 e 68,1%, respectivamente, no experimento 2. O crescimento da Pst in vitro não foi influenciado significativamente pelo AJ ou ET. Em determinadas épocas de avaliação, o AJ elevou significativamente a atividade das POX, PFO e GLU; ASM elevou a atividade das PFO, GLU e LOX e o ET elevou a atividade das GLU e LOX em relação ao controle. Os resultados desses estudos evidenciam que a pulverização com SS e com indutores AJ, ET e ASM afetaram alguns dos componentes de resistência do tomateiro à pinta bacteriana, além de potencializar as enzimas POX, PFO, GLU, FAL e LOX, relacionadas com a defesa das plantas em resposta à Pst.

To diversify their genetic material, allowing adaptation to environmental disturbances and colonization of new ecological niches, bacteria use various evolutionary processes, including the acquisition of new genetic material by horizontal transfer mechanisms such as conjugation, transduction and transformation. Electrotransformation mediated by lightningrelated electrical phenomena may constitute an additional gene transfer mechanism occurring in nature. The presence in clouds of bacteria capable of forming ice nuclei that lead to precipitations and are involved in the triggering of lightning, such as the global phytopathogen Pseudomonas syringae, led us to postulate that natural electrotransformation in clouds may affect bacteria, by contributing to increase their adaptive potential. We first determined if the ice nucleator bacterium P. syringae could survive when in clouds and acquire exogenous genetic material through lightning shock-simulating in vitro electroporation. In comparison to two other bacteria, P. syringae appears to be best adapted for survival and for genetic electrotransformation under these conditions, which suggests that this bacterium would be able to survive and evolve whilst being transported in clouds. Secondly, we evaluated the impact of lightning shock-simulating in vitro electroporation on the survival, the electrotransformation potential and the diversity of bacteria collected from rain samples. These isolates better resisted lightning than the laboratory strains and some were able to electrotransform exogenous DNA. The rain bacteria we isolated were of different origins and were representative of life modes of the various sources of bacterial emissions on Earth. Our study suggests that bacteria aerosolized from diverse terrestrial ecosystems can spread to new habitats through clouds whilst also being able to acquire new genetic material via lightning-based electrotransformation, thereby potentially enhancing their genetic diversity. The final part of our work consisted of evaluating whether electrotransformation could be applied to the engineering of indigenous soil bacteria in order to develop a tool for the bioremediation of lindane, a once widely used pesticide. Optimized experiments revealed that both natural and electrotransformation contributed to the incorporation of a plasmid harboring a gene encoding the first lindane dechlorination steps by indigenous soil bacteria. In conclusion, we showed that natural electrotransformation mediated by electrical discharges such as those occurring in clouds or reaching soils can be involved in the horizontal gene transfer process among bacteria and, considering the importance of lightning worldwide, may play a role in the adaptation and evolution of these organisms.