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.

Les endophytes sont des microorganismes (généralement des bactéries et des champignons) qui vivent dans les tissus végétaux mais n'activent pas le système immunitaire/défense des plantes, contrairement aux pathogènes végétaux qui activent généralement les réponses immunitaires des plantes. Des recherches récentes ont montré que pratiquement toutes les plantes cultivées en plein champ contiennent un certain nombre d'endophytes, et que certains endophytes stimulent la croissance des plantes et renforcent la résistance contre les agents pathogènes. Les endophytes sécrètent des composés chimiques (métabolites secondaires) qui suppriment la croissance des agents pathogènes, un processus connu sous le nom de biocontrôle. En raison de ces propriétés de biocontrôle, les endophytes sont une alternative potentielle aux pesticides chimiques pour lutter contre les maladies des plantes. En conséquence, le biocontrôle est devenu un domaine de recherche important. Mon projet de recherche comportait les objectifs spécifiques suivants : (i) isoler les endophytes des plants de canneberges acquis auprès de deux producteurs commerciaux de canneberges de la variété Stevens situés au Québec, Canada (Bieler Cranberries Inc, et Gillivert Inc.) ; (ii) tester l'activité de biocontrôle des endophytes contre une collection de champignons pathogènes et ensuite inoculer les endophytes les plus actifs dans des plants de canneberges obtenus par germination de la variété Stevens (Bieler Cranberries Inc. ) et Scarlet Knight (Daniele Landreville) ; et (iii) identifier des groupes de gènes de métabolites secondaires en séquençant, assemblant et annotant le génome d'un endophyte qui présentait de fortes caractéristiques de biocontrôle. Dans le cadre de ce projet de recherche, des tests antagonistes in vitro ont été réalisés avec des endophytes de la canneberge et un champignon pathogène, qui ont montré que Pseudomonas sp. CSWB3, Pseudomonas sp. CLWB12 et la souche fongique Lachnum sp. EFK28 étaient les plus actifs et ces souches ont donc été sélectionnées pour des études plus approfondies. Des expériences de germination de semis in vitro et d'inoculation d'endophytes ont montré que les souches bactériennes Pseudomonas sp. CSWB3 et Pseudomonas sp. CLWB12 amélioraient la croissance des semis de canneberges de la variété Stevens. Comme les Pseudomonas sp. CSWB3 et Pseudomonas sp. CLWB12 ont tous deux un effet antagoniste élevé sur les champignons pathogènes, un seul (Pseudomonas sp. CSWB3) a été soumis à une analyse du génome. Le séquençage, l'assemblage, l'annotation et l'analyse du génome de Pseudomonas sp. CSWB3 a révélé que cette souche possède cinq groupes de gènes biosynthétiques de métabolites secondaires qui codent pour les protéines responsables de la biosynthèse des composés antifongiques/antimicrobiens : pyrrolnitrine, pyoluteorine, putisolvine, 2,4-diacétylephloroglucinol, bicornutine A1 et bicornutine A2. Sur la base des résultats de ces travaux, nous concluons que certains endophytes de la canneberge qui possèdent des groupes de gènes codant pour des métabolites secondaires antifongiques peuvent supprimer les pathogènes fongiques et améliorer la croissance des plantes. / Endophytes are microorganisms (typically bacteria and fungi) that live within plant tissue but do not activate the plant defense/immune system, unlike plant pathogens that typically do activate plant immune responses. Recent research has shown that virtually all plants grown under field conditions contain a number of endophytes, and that certain endophytes stimulate plant growth and enhance resistance against pathogens. Endophytes secrete chemical compounds (secondary metabolites) that suppress pathogen growth, a process known as biocontrol. Because of these biocontrol properties, endophytes are a potential alternative to chemical pesticides for combatting plant disease. Accordingly, biocontrol has become an important field of research. My research project was comprised of the following specific aims: (i) isolate endophytes from cranberry plants that were acquired from two commercial producers of cranberries of the Stevens variety located in Quebec, Canada (Bieler Cranberries Inc, and Gillivert Inc.); (ii) test the biocontrol activity of endophytes against a collection of fungal pathogens and then inoculate the most active endophytes into cranberry seedlings that were obtained by germinating Stevens (Bieler Cranberries Inc.) and Scarlet Knight (Daniele Landreville) seeds; and (iii) identify secondary metabolite gene clusters by sequencing, assembling, and annotating the genome of one endophyte that exhibited strong biocontrol characteristics. As part of this research project, in vitro antagonistic tests were conducted with cranberry endophytes and fungal pathogen, which showed that Pseudomonas sp. CSWB3, Pseudomonas sp. CLWB12, and the fungal strain Lachnum sp. EFK28 were the most active and therefore these strains were selected for further studies. In vitro seedling germination and endophyte inoculation experiments showed that the bacterial strains Pseudomonas sp. CSWB3 and Pseudomonas sp. CLWB12 enhanced the growth of cranberry seedlings of the Stevens variety. Since Pseudomonas sp. CSWB3 and Pseudomonas sp. CLWB12 both had a high antagonistic effect on fungal pathogens, only one (Pseudomonas sp. CSWB3) was subjected to genome analysis. Sequencing, assembly, annotation, and analysis of the Pseudomonas sp. CSWB3 genome revealed that this strain possesses five secondary metabolite biosynthetic gene clusters that encode proteins responsible for the biosynthesis of the antifungal/antimicrobial compounds pyrrolnitrin, pyoluteorin, putisolvin, 2,4-diacetylephloroglucinol, bicornutin A1, and bicornutin A2. Based on the results of this work, we conclude that certain cranberry endophytes that possess gene clusters encoding antifungal secondary metabolites can suppress fungal pathogens and enhance plant growth.

Penicillium nordicum è un importante contaminante di salumi, rappresentanando il 10 % e il 26 % della popolazione di Penicillium spp . isolati , rispettivamente dall'aria e dai prodotti carnei stagionati in un'indagine gestita in Italia ( Battilani et al. , 2007). Diverse colonie di P. nordicum isolate dai salumi hanno dimostrato di essere importanti produttori di ocratossina A , OTA ( Sansom e Frisvad , 2004 . Pietri et al, 2006 ; . Battilani et al , 2010). Attualmente, l'impostazione appropriata delle condizioni ambientali (temperatura, umidità relativa e circolazione dell'aria ), è l'unico strumento accettato per impedire la crescita incontrollata di P. nordicum all'interno degli impianti di stagionatura attraverso una accurata analisi dei punti critici di controllo e l’ideazione di un relativo piano HACCP (Hazard Analysis and Critical Control Points) ben struttutato ( Asefa et al , 2011; Virgili et al , 2012). Anche se il sistema HACCP è stato applicato con successo nel settore alimentare ci sono rischi per la sicurezza alimentare non attentamente considerati. Questo è particolarmente vero per quanto riguarda i rischi micotossigeni associati ai prodotti alimentari di origine animale. Il termine "rischi micotossigeni" è utilizzato da Asefa et al. ( 2011) per descrivere lieviti patogeni e metaboliti secondari tossici prodotti da specie fungine tossigene che contaminano i prodotti alimentari e incidono sulla sicurezza alimentare. La maggior parte dei piani HACCP nelle attività di trasformazione alimentare, come ad esempio la produzione di formaggi e di prodotti carnei stagionati, tiene in considerazione principalmente il rischio derivante da agenti batterici (Arvanitoyannis e Mavropoulos, 2000; Barbuti e Parolari, 2002) anche se tali prodotti alimentari vengono spesso contaminati da funghi micotossigeni e dai loro metaboliti (Spotti et al 1989; Spotti et al , 2001a; Battilani et al 2007). Pertanto, dovrebbe essere cruciale definire un piano HACCP specificamente incentrato sui rischi micotossigeni. L'identificazione, il controllo e la standardizzazione della micoflora superficie dei salumi è fondamentale per preservare la sicurezza delle produzioni e la salute dei consumatori . Questo è il contesto in cui deve essere valutata l’efficacia e l’affidabilità per l’identificazione delle popolazioni di Penicillium spp di interessante per la produzione alimentare. In questo contesto , il progetto di ricerca di questa tesi di dottorato ha cercato di approfondire le conoscenze su tali tematiche con l'intento di limitare il rischio micotossigeno nella catena di produzione dei prodotti carnei stagionati. Sono stati affrontati i seguenti argomenti: 1 . studio della composizione e dinamica della microflora fungina presente sulla superficie dei salumi (prodotto testato, salame) e l'aria di ambienti di stagionatura tenendo conto dell'influenza di alcuni parametri di processo (inoculo starter, temperatura, fase produttiva). 2 . sviluppo di un metodo MALDI TOF MS per l'identificazione di Penicilium a livello di specie per le prospettive future di screening diretti della microflora presente sui salumi. 3 . confronto e integrazione di diverse tecniche, come l'analisi morfologica, l’analisi molecolare e l’analisi tramite spettrometria di massa, per l'identificazione delle specie di Penicillium presenti nei salumi. 4 . valutazione dei lieviti selezionati, isolati dalla superficie di prosciutto crudo, per competere con P. nordicum ed inibire l'accumulo di OTA nella prospettiva del loro uso come starter superficiali con funzione di agenti di biocontrollo. / Penicillium nordicum is an important contaminant of cured meat products, representing 10% and 26% of the Penicillium spp. isolated, respectively, from the air or the products in a survey managed in Italy (Battilani et al., 2007). Several P. nordicum cured meat isolates proved to be important producers of ochratoxin A, OTA (Sansom and Frisvad, 2004; Pietri et al., 2006; Battilani et al., 2010). Currently, the appropriate setting of environmental conditions (temperature, relative humidity and air circulation), is the only accepted tool to prevent the uncontrolled growth of P. nordicum inside dry-curing plants through a carefully structured Hazard Analysis Critical Control Point (HACCP) plan (Asefa et al., 2011; Virgili et al., 2012). Even if the HACCP system has been successfully applied in the food industry, there are food safety hazards not carefully considered. This is especially true with regard to mycotoxigenic hazards associated with animal food products. The term “mycotoxigenic hazards” is used by Asefa et al. (2011) to describe pathogenic yeasts and toxic secondary metabolites of toxigenic moulds that contaminate food products and affect food safety. Most HACCP plans in food processing activities, such as the production of cheese and dry-cured meat products, considered mainly bacterial agents (Arvanitoyannis and Mavropoulos, 2000; Barbuti and Parolari, 2002), even if such food products get often contaminated with mycotoxigenic fungi and their metabolites (Spotti et al 1989; Spotti et al., 2001a; Battilani et al 2007). Therefore, it should be crucial to define a HACCP plan specifically focused on the mycotoxigenic hazards. The identification, control and standardization of the surface mycoflora of cured meat products is mandatory to preserve the productions safety and the consumers health. This is the context of the effectiveness and reliability evaluation for the Penicillium spp. identification methods of interesting species for food production. In this context, the research project of this PHD thesis tried to fill some gaps of knowledge with the attempt to limit the mycotoxigenic risk in the cured meat products chain. The following topics were faced: 1. study of the composition and dynamic of fungal microflora present on the surface of cured meat products (salami) and the air of seasoning environments taking into account the influence of some process parameters (starter inoculum, curing temperature, stage of seasoning). 2. development of a MALDI TOF MS method for the identification of Penicilium at species level for future direct screening perspectives of the microflora present on cured meat products. 3. comparison and integration of different techniques, as morphological, molecular and mass spectral analysis, for the identification of Penicillium species in cured meat products. 4. evaluation of selected yeasts, isolated from dry-cured ham surface, to compete with P. nordicum and to inhibit OTA accumulation in the perspective of their use as surface starter biocontrol agents.