Made available in DSpace on 2014-06-11T19:24:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-02Bitstream added on 2014-06-13T18:47:36Z : No. of bitstreams: 1 aquino_plm_me_sjrp.pdf: 528292 bytes, checksum: 19f5670b2dc7ea09762a3927055f84a8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Not available

<p>Sve veća potreba za hranom kao posledica povećanja broja ljudi &scaron;irom sveta dovela je do intenzifikacije poljoprivrednih aktivnosti usmerenih na direktnu proizvodnju hrane i hrane za životinje. U poslednjih nekoliko decenija sve vi&scaron;e se govori o problemu prekomerne upotrebe hemijskih pesticida, &scaron;to je je rezultovalo degradacijom kvaliteta zemlji&scaron;ta, kao i sve većim zagađenjem podzemnih i povr&scaron;inskih voda. Osim toga, nedovoljno naučnih podataka o uticaju hemijskih jedinjenja koja ulaze u sastav pesticida na zdravlje ljudi izaziva jo&scaron; veći stepen zabrinutosti. Stoga se iznalaženje novih načina za kontrolu biljnih &scaron;tetočina i prevenciju i tretman bolesti biljaka postavlja kao imperativ održive poljoprivredne proizvodnje, a samim tim i održivog razvoja. Jedna od alternativa koje su pokazale najveći potencijal jeste primena biopesticida, koji po svojoj naj&scaron;iroj definiciji predstavljaju žive organizme ili njihove proizvode koji pokazuju sposobnost suzbijanja &scaron;tetočina. Kada su u pitanju mikrobiolo&scaron;ki biopesticidi, bakterije roda <em>Bacillus</em> predstavljaju najzastupljenije mikroorganizme koji se mogu sresti u komercijalno dostupnim proizvodima. Proizvodnja mikrobiolońkih biopesticida, iako u konstantnom porastu, i dalje čini samo mali deo ukupnog svetskog trži&scaron;ta sredstava za za&scaron;titu bilja, najvi&scaron;e zbog visokih tro&scaron;kova i komplikovanog procesa proizvodnje ovog tipa biokontrolnih agenasa. Stoga je kao osnovni cilj istraživanja ove doktorske disertacije postavljena optimizacija uslova umnožavanja i primarnog izdvajanja biomase soja <em>Bacillus velezensis</em> sa primenom za sprečavanje pojave i &scaron;irenja bakterioza povrtarskih useva koje izazivaju fitopatogene vrste roda<em> Xanthomonas</em>, koji u Republici Srbiji izazivaju velike ekonomske gubitke u proizvodnji kupusa, paprike i paradajza. Prvi korak optimizacije bioprocesa podrazumevao je odabir optimalnog izvora ugljenika i organskog izvora azota za biotehnolo&scaron;ku proizvodnju biokontrolnih agenasa, pri čemu su najbolji rezultati dobijeni primenom glicerola i ekstrakta kvasca, &scaron;to je otvorilo mogućnost ispitivanja primene sirovog glicerola iz proizvodnje biodizela kao izvora ugljenika. Nakon toga je usledilo modelovanje sastava hranljive podloge na bazi komercijalnog i sirovog glicerola u pogledu ispitivanja uticaja komponenata podloge (glicerola, ekstrakta kvasca, (NH₄)₂SO₄ i K₂HPO₄) na odabrane odzive (prečnik zona inhibicije i rezidualne koncentracije nutrijenata &ndash; glicerola, ukupnog azota i ukupnog fosfora). Dobijeni matematički modeli posmatranih odziva, za koje je dokazano da su statistički značajni na nivou značajnosti od 95%, dalje su kori&scaron;ćeni za optimizaciju sastava hranljivih podloga. Optimizovan sastav hranljive podloge na bazi komercijalnog glicerola je sledeći: glicerol 10 g/l, ekstrakt kvasca 2,83 g/l, (NH₄)₂SO₄ 3 g/l, K₂HPO₄ 1,07 g/l i MgSO₄∙7H₂O 0,3 g/l. Sa druge strane, optimizovan sastav hranljive podloge na bazi sirovog glicerola dobija se primenom sledećih koncentracija nutrijenata: glicerol 10 g/l, K₂HPO₄ 4,66 g/l i MgSO₄∙7H₂O 0,3 g/l, &scaron;to ukazuje na mogućnost smanjenja tro&scaron;kova pripreme ove hranljive podloge zbog izostavljanja izvora azota. Validacija dobijenih matematičkih modela izvr&scaron;ena je eksperimentima kultivacije proizvodnog mikroorganizma u uvećanim razmerama - u Vulfovim bocama (2 l) i laboratorijskom bioreaktoru (3 l), čiji su rezultati pokazali zadovoljavajuću korelaciju sa modelima predviđenim vrednostima odziva bioprocesa, pri čemu su bolji rezultati dobijeni primenom hranljive podloge na bazi komercijalnog glicerola i kultivacijom proizvodnog mikroorganizma u laboratorijskom bioreaktoru. Unapređenje procesa unakrsne mikrofiltracije kultivacionih tečnosti dobijenih nakon kultivacije proizvodnog mikroorganizma <em>Bacillus velezensis</em> na hranljivoj podlozi na bazi komercijalnog glicerola primenom različitih hidrodinamiĉkih metoda pokazalo je značajan potencijal primene Kenics statičkog me&scaron;ača, uduvavanja vazduha i kombinacije ovih metoda za pobolj&scaron;anje fluksa permeata u stacionarnom stanju.<em> In planta</em> ispitivanje antimikrobne aktivnosti preparata na bazi <em>Bacillus velezensis</em> protiv fitopatogenih sojeva vrste <em>Xanthomonas euvesicatoria</em>, koji izazivaju bakterioznu pegavost paprike, pokazalo je značajan potencijal primene bikontrolnih agenasa na bazi <em>Bacillus velezensis</em> za suzbijanje ovog oboljenja paprike. Ispitivanje &scaron;ireg spektra delovanja preparata na bazi<em> Bacillus velezensis </em>pokazalo je značajan potencijal za suzbijanje oboljenja izazvanih fitopatogenim sojevima roda Fusarium, koji izazivaju truljenje jabuka u toku skladi&scaron;tenja. Rezultati proistekli iz okvira ove doktorske disertacije predstavljaju osnovu za dalje unapređenje i povećanje razmera biotehnolońkog postupka proizvodnje biokontrolnih agenasa na bazi proizvodnog mikroorganizma <em>Bacillus velezensis</em>, uz predlog re&scaron;enja za iskori&scaron;ćenje sirovog glicerola iz proizvodnje biodizela u biotehnolońkom postupku dobijanja proizvoda sa dodatom vredno&scaron;ću.</p> / <p>The raise of human population worldwide requires higher amount of available groceries, which has led to intensification of agricultural activities directed at production of food and feed. In the last few decades the problem of chemical pesticides‟ overuse has resulted in soil deterioration and contamination of surface and underground water reservoirs. Furthermore, insufficient scientific data concerning the chronic effect of chemicals used in pesticides on human health contribute to the global concern when it comes to pesticides‟ usage. Therefore finding new ways to control plant pests and to prevent or suppress plant diseases has become an imperative for sustainable agricultural production and consequently for sustainable development. One of the alternatives showing great potential is usage of biopesticides, comprising of living organisms or their products able to suppress plant pathogens. When it comes to microbial biopesticides, bacteria of the genus Bacillus are the most often used in commercial biocontrol products. Microbial biopesticides, although being a raising alternative, still represent only small share of the global pesticides‟ market, mostly due to expensive and complicated production process. Hence the main goal of the research in the framework of this PhD thesis was to optimize conditions of cultivation and separation processes for production of Bacillus velezensis biomass intended for application in prevention and suppression of plant diseases caused by phytopathogenic species of the genus Xanthomonas, which cause significant economic losses during agricultural production of cabbage, pepper and tomato in the Republic of Serbia. The first step of bioprocess optimization was to choose optimal carbon and organic nitrogen sources for biocontrol agents‟ production, where the best results have been achieved by using glycerol and yeast extract, which opened a new chapter of possibilities to utilize raw glycerol from biodiesel production as carbon source. After that, modeling of cultivation medium composition based on commercial and raw glycerol was performed in order to investigate the effect of cultivation medium components (glycerol, yeast extract, (NH4)2SO4 and K2HPO4) on the selected responses (inhibition zone diameter and residual concentration of nutrients &ndash; glycerol, total nitrogen and total phosphorus). The obtained mathematical models for the selected responses, proved to be statistically significant at the significance level of 95%, were further used for optimization of cultivation media composition. Optimized composition of the cultivation medium based on commercial glycerol was: glycerol 10 g/L, yeast extract 2.83 g/L, (NH4)2SO4 3 g/L, K2HPO4 1.07 g/L and MgSO4∙7H2O 0.3 g/L. On the other hand, optimized composition of the cultivation medium based on raw glycerol was: glycerol 10 g/L, K2HPO4 4.66 g/L and MgSO4∙7H2O 0.3 g/L, which has opened a possibility to reduce cost of cultivation medium preparation by excluding nitrogen sources. Validation of the obtained mathematical models was carried out by cultivating the producing microorganism at a larger scale &ndash; in Woulff bottles (2 L) and in a laboratory-scale bioreactor (3 L). Validation results were in good correlation with the model predicted values of the selected responses, where better results were achieved by using cultivation medium based on commercial glycerol and cultivation of the producing microorganism in the laboratory-scale bioreactor. Application of different hydrodynamic methods, such as Kenics static mixer, air sparging and their combination, has showed a significant potential for improvement of cross flow microfiltration of Bacillus velezensis cultivation broth based on commercial glycerol in terms of permeate flux enhancement. Also, significant potential of the Bаcillus velezensis cultivation broth for suppression of phytopathogenic Xanthomonas euvesicatoria strains, which cause bacterial spot of pepper, has been proven during in planta experiments. Investigation of wider spectra of antimicrobial activity expressed by preparation based on Bаcillus velezensis cultivation broth has revealed a significant potential for suppression of Fusarium phytopathogenic species responsible for apple rot during storage. The results arising from the research performed in the framework of this PhD thesis represent solid basis for further improvement and scale-up of biotechnological process for production of biocontrol agents based on Bacillus velezensis, with a proposal for utilization of raw glycerol from biodiesel production in biotechnological production of value-added products.</p>

Les pertes alimentaires mondiales se chiffrent à environ un tiers des aliments destinés à la consommation humaine, soit environ 1,3 milliards de tonnes par an (FAO). Une large fraction de ces pertes est due aux altérations microbiologiques des denrées alimentaires. L’utilisation de produits phytosanitaires reste aujourd’hui la solution la plus largement utilisée en agriculture pour limiter ces pertes. Cependant, avec le plan EcoPhyto 2, le gouvernement français a pour objectif de réduire de 50% l’usage des pesticides chimiques d’ici 2025, en particulier en promouvant l’émergence du biocontrôle. Pour développer cette approche, il est cependant nécessaire de comprendre, pour mieux les maitriser, les mécanismes sous-jacents. Les différents modes d’action de biocontrôle par les microorganismes décrits sont la stimulation des défenses naturelles des plantes, la production de substances antimicrobienne et la compétition nutritionnelle. L'originalité de ce projet est d'intégrer le mode de vie en biofilm dans les mécanismes de bioprotection (compétition spatiale et nutritionnelle, libération de principes antimicrobiens). Dans la filière Française des champignons de couche (Agaricus bisporus), l’agent de biocontrôle utilisé depuis 2008 par plus de 80 % de la filière, est Bacillus subtilis QST713. Ce biofongicide montre une nette efficacité contre Trichoderma aggressivum, la principale moisissure à l’origine de pertes économiques lors de la culture d’A. bisporus. Afin d’accompagner la filière dans cette voie biologique, nous avons entrepris de séquencer et étudier le génome de cette souche, afin de déterminer son potentiel de biocontrôle et sa capacité à former des biofilms. Nous avons également évalué l’impact de ce biofongicide sur la dynamique des communautés microbiennes du compost de culture d’A. bisporus exposé ou non à T. aggressivum. Enfin, l'étude de la reprogrammation cellulaire de cet agent de biocontrôle lors de sa culture en micromodèles axéniques, nous a permis une meilleure compréhension des phénomènes de colonisation des substrats et d'inhibition des flores indésirables. Ce projet a permis d’enrichir les connaissances vis-à-vis des mécanismes de biocontrôle dans la filière des champignons et pourra permettre une possible application à d’autres filières agricoles. / Worldwide, food losses amount for about one-third of food for human consumption, 1.3 billion tons per year (FAO). A large fraction of these losses are due to microbiological alterations. The use of phytosanitary products remains today the most widely used solution in agriculture to limit these losses. However, with the EcoPhyto 2 plan, the French government aims to reduce the use of chemical pesticides by 50% by 2025, in particular by promoting the emergence of biocontrol. To develop this approach, it is necessary to understand the underlying mechanisms. The different modes of action of biocontrol by the microorganisms described are the stimulation of the natural defenses of the plants, the production of antimicrobial substances and the nutritional competition. The originality of this project is to integrate the biofilm mode of life into bioprotection mechanisms (spatial and nutritional competition, release of antimicrobial principles). In the French sector of the button mushrooms (Agaricus bisporus) culture, the biocontrol agent used since 2008 by more than 80% of the sector, is Bacillus subtilis QST713. This biofungicide shows a clear efficacy against Trichoderma aggressivum, the main mold causing economic losses during the cultivation of A. bisporus. To accompany the sector in this biological pathway, we have sequenced and studied the genome of this strain, in order to determine its biocontrol potential and its ability to form biofilms. We also evaluated the impact of this biofungicide on the dynamics of microbial communities in A. bisporus culture compost exposed or not to T. aggressivum. Finally, the study of the cellular reprogramming of this biocontrol agent during the culture in axenic micromodels allowed us a better understanding of the substrates colonization phenomenon and the inhibition of undesirable flora. This project will enrich the knowledge of the biocontrol mechanisms used in the mushroom industry and may allow a possible application to other agricultural sectors.

No description available.

Il a été établi que la majorité des plantes vasculaires abritent des micro-organismes endophytes bactériens et fongiques, qui peuvent coloniser les tissus végétaux et former des associations allant du mutualisme à la pathogénèse. Les symbioses végétales mutualistes les plus communes impliquent les champignons endo-mycorhiziens arbusculaires (AMF). Ces champignons s’associent aux racines des plantes et leur permettent d’améliorer leur nutrition minérale, tandis qu’ils bénéficient des composés produits par l'hôte. Toutefois, les plantes de la famille Ericaceae s’engagent plutôt dans des associations mutualistes avec les champignons mycorhiziens éricoïdes (ErMF). Ces derniers sont morphologiquement et taxonomiquement mal définis, en apparence distribués aléatoirement parmi les espèces issues des grandes divisions taxonomiques des Ascomycota et Basidiomycota. En raison de cette incohérence taxonomique et de l'absence d'une histoire évolutive explicative, la diversité réelle de ces champignons est mal caractérisée. De ce fait, ce projet vise à étudier le microbiote associé à la plante Ericaceae Vaccinium macrocarpon Aït (canneberge), axant la recherche sur les angles morphologiques, génomiques et transcriptomiques des champignons de type ErMF et autres endophytes capables de contrôler la croissance des agents phytopathogènes et de stimuler la croissance des plantes. Notre première démarche présentée dans le chapitre 2 s’est focalisée sur la caractérisation du microbiote endophyte bactérien et fongique de la canneberge, une plante vivace principalement produite en Amérique du Nord, notamment au Québec. Nous avons isolé et identifié 180 micro-organismes à partir de plantes de cultivars variés, collectées de champs différents, et avons démontré l'existence d'une variabilité dans le microbiote selon les tissus, les cultivars, et même entre les champs d'une même ferme. Parmi les endophytes d’intérêt identifiés, l’isolat fongique Lachnum sp. EC5 a stimulé la croissance des cultivars de canneberge Stevens et Mullica Queen et a formé des structures intracellulaires similaires à celles des ErMF à l’intérieur des cellules racinaires de la canneberge. De plus, l’isolat EB37 identifié Bacillus velezensis s’est révélé être un puissant agent antifongique, montrant cependant une tolérance particulière au champignon Lachnum sp. EC5, lors des tests de confrontation. Ce volet sera détaillé avec plus de précision dans le chapitre 4. Le chapitre 3 a porté sur l’analyse génomique comparative de l’isolat fongique Lachnum sp. EC5 avec plusieurs espèces de champignons Leotiomycetes ErMF, saprophytes et pathogènes. Nous avons analysé le sécrétome protéique prédit de ces champignons et mis en évidence que les gènes codant pour les enzymes de dégradation des parois végétales ne sont pas corrélés au mode de vie fongique (mycorhizien, pathogène ou saprophyte). A l’inverse, 10 protéines effectrices de Lachnum sp. EC5 prédites pour cibler spécifiquement un compartiment intracellulaire chez les cellules végétales ont des similarités avec celles d’espèces mutualistes comme Meliniomyces variabilis et Oidiodendron maius. Aussi, la protéine effectrice putative Zn-MP, prédite pour cibler, potentiellement, les chloroplastes végétaux nous permet de proposer un rôle dans le renforcement de l’immunité végétale. Le chapitre 4 s’est intéressé aux mécanismes de régulation d'expression de gènes induits lors de l’interaction entre le champignon Lachnum sp. EC5 et la bactérie B. velezensis EB37. Ces mécanismes ont été comparés à ceux activés chez la bactérie en présence de champignons pathogènes. Nous avons démontré une physiologique cellulaire bactérienne distincte en présence de Lachnum sp. EC5, dénotée par une faible expression des gènes induits lors du stress nutritif associé aux processus de sporulation, de formation du biofilm, de secretion de CAZymes et de lipopeptides. Nous avons suggéré que la sous-régulation de ces mécanismes serait essentiellement explicable par une disponibilité plus importante en glucose ou en d’autres sources de carbone préférentielles pour la bactérie. En réponse, le champignon Lachnum sp. EC5 a vécu différents changements morphologiques. Il aurait détoxifié ses environnements intra et extra-cellulaires et surexprimé sa voie de production de carbone dépendante du cycle du glyoxylate, générant ainsi des conditions favorisant un contact physique entre les deux micro-organismes. En conclusion, nous avons argumenté et documenté que la définition des ErMF basée uniquement sur des critères morphologiques est mal adaptée à catégoriser ces champignons. Notre approche multidisciplinaire a mis en évidence la diversité du microbiote de la canneberge, a étendu la notion d’ErMF à d'autres champignons jusqu'ici exclus de ce groupe, et a souligné l'importance des associations interspécifiques sur l’interaction ErMF-plantes. Ces avancées permettront d’améliorer nos connaissances sur le microbiote des plantes éricacées contribuant, au développement de solutions environnementales éco-responsables pour l’industrie de la canneberge. / It has been established that the majority of vascular plants harbour bacterial and fungal endophytes that colonize plant tissues, and thus form associations that range from mutualism to pathogenesis. Mycorrhizal fungi are a particular class of endophytes that associate with plant roots and enhance plant mineral uptake. The most common type of mutualistic plant symbiosis involves arbuscular mycorrhizal fungi (AMF), whereas plants of the Ericaceae family instead engage in mutualistic associations with ericoid mycorrhizal fungi (ErMF). The ErMF group, in its current definition, includes both ascomycete and basidiomycete species, yet is morphologically, taxonomically and evolutionarily poorly defined, which implies that the group’s true diversity is not well understood. The objective of this project is to complement morphological information with genomic and transcriptomic data to better understand the role of ErMF in 1) controlling the negative effects of pathogenic infections, and 2) the potential plant growth stimulation for the Ericaceous plant Vaccinium macrocarpon Ait. Our first approach presented in Chapter 2 focused on the characterization of the bacterial and fungal endo-symbiotic microbiota of the Ericaceous plant, Vaccinium macrocarpon Ait (cranberry), a perennial plant mainly in North America, particularly in Quebec. We isolated ~180 distinct bacterial and fungal endophytes collected from roots, stems, and leaves of cranberry plants cultivated in Quebec, Canada. We show that the cranberry microbiome varies substantially between tissues, cultivars, and across fields of the same farm. Among the isolated endophytes, the fungus Lachnum sp. EC5 was found to promote the growth of cranberry cultivars Stevens and Mullica Queen, and to form intracellular structures resembling those other ErMF inside the cortical root cells. In addition, the bacterium Bacillus velezensis (EB37) has been found to be a potent antifungal agent. Interestingly, a confrontation test between EB37 and the fungus Lachnum sp. EC5 revealed a mutual tolerance, which we will describe later in chapter 4. In chapter 3, our project focused on the comparative genomic analysis of the fungus Lachnum sp. EC5 with several Leotiomycete ErMF, saprophytes and pathogens. We analyzed fungal secretomes and demonstrated that genes encoding plant cell wall degradation enzymes are conserved between the tested fungi which suggests that such proteins are not indicative of a particular fungal lifestyle. On the other hand, 10 effector proteins identified in Lachnum sp. EC5 were also only found in mutualistic fungi, such as Meliniomyces variabilis, Oidiodendron maius and have been reported to target the plant intracellular compartments. Also, the identification of the putative effector protein Zn-MP, specific to Lachnum sp. EC5 and predicted to target plant chloroplasts, suggest a role in the reinforcement of plant immunity. Chapter 4 focuses on the patterns of gene expression regulation induced in the biocontrol bacterium B. velezensis EB37 in interaction with the potentially mutualistic fungus Lachnum sp. EC5. These mechanisms were then compared to those activated when the bacterium is in the presence of pathogenic/saprophytic fungi. We demonstrated that in co-culture with Lachnum sp. EC5, EB37expresses fewer genes related to stress, and fewer related to the stationary phase which often involves production of bacterial biofilms and lipopeptides, such as mycosubtilin. We suggest that the lessened response to stress is related to an increased availability of glucose or other preferential sources of carbons for the bacterium. Conversely, Lachnum sp. EC5 in the presence of EB37 underwent morphological changes by a higher lateral branching., detoxified its external and internal environment by expressing both a catalase activity and efflux pumps, and overexpressed its glyoxylate cycle-dependent carbon production pathway, and thus promoting favourable conditions for close physical contact with the bacterium. In conclusion, we demonstrated that the morphological-based definitions are poorly adapted to the categorization of ErMF fungi. Our multidisciplinary approach highlighted the diversity of the cranberry microbiota, extended the notion of ErMF to other fungi hitherto excluded from this fungal group and underlined the importance of interspecific associations on the ErMF-plant interaction. These advances enhance our understanding of the Ericaceous plant microbiota and contributes to the development of sustainable solutions for the cranberry industry.