Dissertations / Theses on the topic 'Endophytes'

La physiologie et les traits d'histoire de vie des organismes ectothermes dépendent largement de la température de leur microclimat. Dans certaines relations insecte – plante, le phytophage manipule physiquement et/ou chimiquement son environnement végétal. Cependant, les effets de ces transformations sur le microclimat de l'herbivore sont encore inconnus. Nous avons détaillé précisément les modifications physiques induites par un lépidoptère mineur de feuille (Phyllonorycter blancardella, Gracillariidae) sur son environnement végétal (le pommier). Les impacts sur l'écologie thermique de la larve ont été quantifiés. La larve se nourrit et se développe au sein même des tissus de la feuille, dans une structure appelée mine.
Des mesures de spectrométrie optique ont démontré que la larve modifie profondément les propriétés optiques de la surface de la feuille au cours de son nourrissage. La structure mine absorbe bien plus de radiations dans le proche infrarouge que les tissus foliaires intacts. De plus, une quantité importante de radiations est transmise à l'intérieur de la mine par le tégument supérieur dans les zones prélevées par la larve. Ces radiations induisent une élévation importante de son activité respiratoire (rejet de CO2). En utilisant un analyseur de gaz par infrarouge, nous avons pu montrer par ailleurs que les stomates localisés dans le tégument inférieur de la mine réagissent à la présence de la larve en se fermant. Un modèle de diffusion de CO2 a révélé que les stomates réagissent directement aux variations d'émission de CO2 par la larve. Le budget thermique de la mine a ensuite été modélisé. Le modèle permet de prédire la température à l'intérieur de la mine à partir des modifications des propriétés optiques et de la physiologie des stomates, et à partir des variables climatiques. Ce modèle biophysique a été validé en comparant ses prédictions avec des mesures expérimentales de température de mines réalisées en environnement contrôlé. Le modèle à une précision de 0,8 °C dans l'intervalle de 12 °C à 42 °C. Le modèle prédit un important excès de température dans la mine, atteignant 10 °C au dessus de la température de l'air et 5 °C au dessus de la température des tissus foliaires intacts. Les deux types de modifications – propriétés optiques et comportement stomatiques – ont un impact équivalent sur l'excès de température. Cette approche démontre clairement que la larve contrôle son environnement physique en modifiant son environnement. Nos résultats sont finalement discutés dans une perspective d'écologie évolutive. Plus particulièrement, le rôle du microclimat des insectes endophages dans l'évolution de leurs sensibilités thermiques et de celles de leurs parasitoïdes est détaillé.

Endophytic fungi live within tissues of plant hosts without causing symptoms of disease. These fungi are broadly split into the taxonomically and ecologically cohesive Clavicipitaceous endophytes, which infect grasses, and the taxonomically diverse non-Clavicipitaceous endophytes, which are found in nearly all plants and have diverse ecological strategies. My dissertation has two sections: Section A investigates the intersection of Clavicipitaceous endophyte ecology with other ecological theory, including invasion ecology (Chapter II) and community ecology and climate change (Chapter III); Section B investigates the ecology of one group of non-Clavicipitaceous endophytes, the Xylariaceae, using a culture-based study in Ecuador (Chapter IV) and a next-generation sequencing based endophyte survey in Taiwan (Chapter V). Section B is centered on testing the Foraging Ascomycete (FA) hypothesis—the idea that some decomposer fungi may adapt an endophytic lifestyle to escape limitations in primary substrate in both time and space. In Chapter II, I utilized a host-specific Epichloë endophyte present ubiquitously in the European native range of the Pacific Northwest (PNW) invasive grass Brachypodium sylvaticum to test theories of invasion. In Chapter III, I examined the grass Agrostis capillaris in the context of a climate manipulation experiment in prairies in the PNW to elucidate patterns of interaction between multiple symbionts (Epichloë endophytes, dark septate root endophytes, and arbuscular mycorrhizal fungi) within single hosts across climatic variation. In Chapter IV, I began to test the FA hypothesis by examining spatial relationships of Xylaria endophytic fungi in the forest canopy with Xylaria decomposer fungi on the forest floor in a remote Ecuadorian cloud forest. In Chapter V, I build on the results from the previous study, using a novel technique to examine spatial ecology of the Xylariaceae, pairing traditional mycological collection with the preparation of a next-generation sequencing metabarcode library of endophytes over a much greater area. This dissertation includes previously published and unpublished coauthored material.

A significant challenge of the 21st century is the growing health threat stemming from drug-resistant infectious diseases. There is an undeniable need to discover new, safe and effective drugs with novel mechanisms of action to combat this threat. A study of drugs currently on the market showed that natural products account for approximately 75% of new anti-infective drugs, either as new agents or analogs based upon their structure. Unfortunately, major pharmaceutical companies have cut back tremendously in natural products research in part due to the frustrating obstacle of frequent rediscovery of compounds. Fungi in particular are difficult to work with in that they do not always produce the same variety and quantities of secondary metabolites under laboratory conditions. One of the groundbreaking discoveries evolving from genomics research is the observation that many fungi possess more gene clusters encoding for the production of secondary metabolites than the reported number of natural products isolated from those organisms. Simple epigenetic modifications such as DNA methlytransferase or histone deacetylase inhibition can activate silenced genes leading to the genesis of novel chemistry from the fungus. The work presented herein is a study of the isolation and characterization of anti-infective compounds from Floridian mangrove endophytes. In addition, epigenetic modifications were explored in order to increase the production of secondary metabolites as well as for the purpose of generating new analogs not found in the controls. Finally, structure activity relationship studies were performed in order to maximize the anti-malarial and antibiotic activity of cytosporone E.

Fungal endophytes associated with leaves of woody plants are thought to be diverse and abundant, especially in tropical forests. However, associations of endophytes with tropical woody angiosperms have not been characterized in detail. In this dissertation, I assess: (1) the scale of endophyte biodiversity in a tropical forest; (2) ecological, temporal, and host-mediated factors influencing endophyte abundance in leaves of tropical trees; (3) utility of endophyte morphospecies as functional taxonomic units; and (4) the nature of endophyte-host interactions. To characterize endophyte biodiversity, I measured endophyte richness in asymptomatic leaves of two distantly related, but co-occurring, host species in lowland forest in central Panama. I found that endophytes are highly diverse within leaves, plants, and host species, and that they demonstrate both host preference and spatial heterogeneity. To determine factors influencing abundance of tropical endophytes, I assessed effects of inoculum abundance, duration of exposure to inoculum, phylogenetic position of host, and leaf traits (e.g., chemical and structural defenses) on rates of endophyte infection. I found that endophyte abundance is sensitive to inoculum abundance in the short term, but to duration of exposure over the long term. In turn, inoculum abundance is sensitive to canopy cover, time of day, and time since precipitation. Generally, neither leaf traits nor phylogenetic position of hosts is related to rates of endophyte infection: endophytes occur with consistently high abundance and diversity among representatives of 14 orders of angiosperms. To assess utility of morphospecies as taxonomic units, I compared diversity and taxonomic composition among morphospecies of endophytes as delineated using several criteria, and among species as inferred using nrDNA data. I found that conservatively designated morphospecies can approximate species boundaries as defined by nrDNA data for diverse Ascomycota. Finally, I examined effects of endophytes on (1) minimum leaf conductance, a measure of water loss from leaves under drought conditions; and (2) leaf mortality and necrosis in the presence of a foliar pathogen for a focal host. I found that endophytes may cost their hosts by increasing water loss under conditions of severe drought, but that they also may play an important role in host defense against pathogens.

Le but de ce travail de thèse était de rechercher de nouvelles molécules inhibitrices de la SOD dans des extraits de substances naturelles. Cette enzyme majeure du stress oxydant étant impliquée dans de nombreux mécanismes de défense des cellules cancéreuses contre l'apoptose représente une voie thérapeutique d'avenir. Nous en avons recherché dans les champignons endophytes de plantes péruviennes, ces micro-organismes produisant de nombreux métabolites de défense des plantes hôtes. Ce travail de thèse a d'abord porté sur l'isolement, la culture, l'identification, l'extraction et la caractérisation des souches de champignons endophytes. Les analyses statistiques effectuées sur ces extraits avec les résultats obtenus en HPLC ont confirmé les problèmes de variabilités qualitative et quantitative pouvant être rencontrés au cours de la culture des endophytes et décrites dans la littérature. La seconde partie expérimentale a porté sur la recherche de furocoumarines dans les extraits obtenus, en raison de leur potentiel effet inhibiteur sur la SOD. Nous avons dérépliqué ces composés dans les extraits d'endophytes au cours de deux stratégies de spectrométrie de masse réalisées en mode d'ionisation négatif. La première approche, à l'aide d'un QTOF, a abouti à l'identification de deux furocoumarines, la 5-Methyl-4H-furo[2,3-b][1]benzopyran-4-one et la déhydropachyrrhizone. La seconde, à l'aide d'un OrbiTrap, a vu l'identification de quatre autres furocoumarines : l'Ochrocarpine A, la Moellendorffiline, l'Anisolactone et l'Anhydrorutarétine .Afin de compléter cette approche métabolomique et identifier les molécules inhibitrices de la SOD, nous avons tenté de mettre au point un test d'activité de cette enzyme, rapide, peu coûteux et réalisable en routine. Nous avons sélectionné le test au pyrogallol, mais celui-ci n'a pas démontré les qualités recherchées tant au niveau de la sensibilité que de la reproductibilité. Nous orientons à présent les recherches vers des approches différentes, la recherche directe d'adduits sur la SOD par LC-MS
Superoxide dismutase is one of the major proteins controlling the oxidizing stress and cellular homeostasis. It is involved in numerous cancer cells proliferation processes. This protein is considered as major anti-cancer target for the development of new anti-cancer drugs. The goal of this work, was to research and identify an inhibitor of the SOD in endophytic fungi from Peruvian plants. These micro-organisms are known to produce numerous metabolites for host plants protection. During the preparation of endophytic extracts and their characterization by HPLC and statistical analyzes, we have pointed out a high quantitative and qualitative variability of the chemical content of endophytic extracts inside a same strain. Considering the literature we have focused our work on the identification of furocoumarins because of their potential inhibitory effect on the SOD. For this purpose two mass spectrometry strategies using negative ionization mode were carried out. With the QTOF mass spectrometer we have identified Methyl-4H-furo [2,3-b] [1] benzopyran-4-one and the déhydropachyrrhizone. With the OrbiTrap, the Ochrocarpine A, Moellendorffiline the Anisolactone and the Anhydrorutarétine were identified. Biological evaluation of the different extracts was performed using pyrogallol test. This investigation did not allow us to identify an inhibitor of the SOD. In the future we may consider seeking SOD inhibitors by looking at the formation SOD-chemical compound adducts using an LC-MS investigation

Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.

Endophytes are microorganisms which colonize the internal tissues of the plants without causing any harm. Generally, endophytes are described as presenting beneficial functions to the plant host, such as plant growth promoting activity and protection against pathogens. The interaction between endophytes and plants is so close, that the term holobiont has been proposed to define the plant and its microbiome, as if they formed a unique organism. In the last decades, endophytes have gained more attention since there is growing evidence of their capacity to influence the plant’s physiology and to produce bioactive compounds. For example, it has been suggested that endophytes could synthesize compounds similar to the ones produced by the plant host, and modulate the production of plants secondary metabolites. In this context, endophytes appear as a powerful tool. On one hand, the exploration of endophytes allows to deepen our knowledge on the plant-microbiome interaction; on the other hand, the better comprehension of the holobiont can lead to a biotechnological application of endophytes. Thus, we decided to explore bacterial endophytes from different plants, to better comprehend the structure of their communities, to explore their phenotypic features, prospecting a possible biotechnological application. In this work, we have analyzed bacterial endophytes from medicinal plants tissues and seeds, and hyperaccumulator plants. Bacterial communities associated to the medicinal plants Echinacea purpurea and E. angustifolia were already described in our laboratory and showed very specific communities in association to different tissues of the plants (stem/leaves, roots) and rhizosphere. Then, we decided to furtherly explore such communities, mainly focusing on the possible forces that could determine the presence of different communities in different districts of the plants. The performed analysis indicated that bacterial strains themselves could be implied in determining the structure of their own community by means of antimicrobial production, which in turn determine the colonization by adaptive phenotypes. Endophytes exhibiting intriguing phenotypes, such as the inhibition of human opportunistic pathogens, were furtherly analyzed, by determining their genome sequences. The analysis of such sequences showed that the endophytic strains harbor many gene cluster which might be implied in inhibitory activities, confirming that endophytic bacterial strains are a promising field to investigate. Endophytic bacterial strains were also analyzed within E. purpurea seeds. In this case, endophytes were observed within the plant’s seeds, extracted, taxonomically and phenotypically characterized. More recently, we decided to explore endophytes which live in association with Phragmites australis plants, which are largely used in phytodepuration constructed wetlands. This part of the work was possible thanks to the collaboration with G.I.D.A. S.p.A., Gestione Impianti Depurazione Acque. The characterization of such endophytes has shown that they are able to grow in presence of critical elements for wastewater treatment, suggesting their possible application to obtain an enhancement of phytodepuration efficiency. Overall, the analyses performed in this work increased our knowledge on bacterial endophytes from different sources, their communities structure, and interaction with the host. As a consequence, it represents the starting point to a possible biotechnological application of endophytes, in order to address issues, such as antibiotic resistance and environmental cleanup.
Gli endofiti sono microrganismi che colonizzano i tessuti interni delle piante senza causare nessun danno. Generalmente, gli endofiti sono in grado di compiere funzioni come la promozione della crescita della pianta e la protezione contro patogeni. Il termine olobionte indica l’insieme costituito dalla pianta e dal suo microbiota, come se questi costituissero un unico grande organismo. Nelle ultime decadi, gli endofiti sono stati sempre più studiati, poiché è diventata sempre più evidente la loro capacità di influenzare la fisiologia della pianta e la produzione di molecole bioattive. Per esempio, è stato proposto che gli endofiti possono sintetizzare composti simili a quelli sintetizzati dalla pianta ospite e che possono modulare la produzione dei metaboliti secondari della pianta. In questo contesto, gli endofiti appaiono come strumenti di grande potenziale in campo biotecnologico. Per questi motivi, abbiamo deciso di analizzare batteri endofiti da piante diverse, allo scopo di capire meglio la struttura delle loro comunità e di esplorare le loro caratteristiche fenotipiche, prospettando così una possibile applicazione in campo biotecnologico. In questo lavoro, sono state analizzati batteri endofiti da piante medicinali, da semi di piante medicinali e da piante iperaccumulatrici di sostanze tossiche. Recentemente, comunità batteriche associate alle piante medicinali Echinacea purpurea and E. angustifolia sono state analizzate nel nostro laboratorio e hanno mostrato una struttura e composizione molto specifica nei vari tessuti delle piante (foglie/stelo, radici) e nella rizosfera. Successivamente, abbiamo deciso di studiare in modo più approfondito tali comunità, concentrandoci soprattutto nei fattori che potrebbero determinare la presenza di comunità così specifiche in diversi compartimenti delle piante. Le analisi effettuate hanno mostrato che le comunità batteriche stesse potrebbero prendere parte nella strutturazione delle proprie comunità, attraverso la produzione di molecule antimicrobiche, che determinano a loro volta la colonizzazione da parte di ceppi resistenti. Inoltre, alcuni ceppi batterici estratti da Echinacea purpurea e E. angustifolia sono stati ulteriormente analizzati dal punto di vista genetico, attraverso il sequenziamento dei loro genomi. Lo studio delle sequenze ha mostrato che i ceppi endofiti presentano diversi cluster genici presumibilmente coinvolti in attività antibiotica, confermando che tali ceppi potrebbero trovare un’applicazione in campo biotecnologico. Ceppi batterici endofiti sono stati analizzati anche all’interno di semi di E. purpurea. In questo caso, oltre ad osservare la presenza di endofiti all’interno dei semi, endofiti batterici sono stati estratti dai semi e caratterizzati dal punto di vista tassonomico e fenotipico. Recentemente, la nostra analisi degli endofiti si è estesa verso le piante iperaccumulatrici. Sono stati analizzati batteri endofiti in associazione alle piante di Phragmites australis, largamente utilizzate in impianti di fitodepurazione. Questa parte del lavoro è stata possibile grazie alla collaborazione con G.I.D.A. S.p.A, Gestione Impianti Depurazione Acque. La caratterizzazione di tali endofiti ha evidenziato la loro capacità di crescita in presenza di elementi, quali ferro, boro e selenio, che sono critici nel processo di depurazione delle acque. Queste evidenze rendono possibile ipotizzare l’applicazione di tali ceppi per un incremento dell’efficienza depurativa. In conclusione, le analisi effettuate in questo lavoro di tesi hanno permesso di appronfondire le nostre conoscenze riguardo batteri endofiti provenienti da diverse fonti. Questo lavoro rappresenta, inoltre, il punto d’inizio per un possibile utilizzo di tali batteri in campo biotecnologico, al fine di rispondere a precise richieste, quali la resistenza agli antibiotici e la necessità di diminuire l’inquinamento ambientale.

>Magister Scientiae - MSc
The term endophyte is used to define all microorganisms that, during a part of their life cycle, colonize the internal tissues of a plant host. Many endophytes have been found to promote plant growth by acting either as biocontrol agents, biofertilizers or phytohormone producers. This study aimed to characterise the endophytic microbial community diversity associated with sorghum farmed in South Africa. Members of any common endophytic bacterial species identified during the study might in future studies be developed to improve sorghum production. Sorghum tissues (roots, shoots, stems) were sampled in three South African provinces (Free State, Limpopo and North West), each site being characterised by the use of different agricultural practices. Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses were used to characterise the endophytic bacterial communities. The analysis clearly demonstrated that the endophytic bacterial community structure in the three sorghum tissue types differed, suggesting that endophyte colonization is tissue-specific. The endophytic bacterial community structure is quite similar in each tissue when comparing the populations present in the sampling sites. In the sorghum endophytic microbial communities, common bacterial species were identified using molecular tools: The cyanobacterium Synechococcus and Staphylococcus saprophyticus were identified in the root samples. Pantoea sp., Erwinia sp., Enterobacter sp. and Klebsiella sp. were found in all shoot samples. Nocardia fluminea, Bacillus cereus and Microbacterium sp. were isolated as common shoot endophytic bacteria. This study defines, for the first time, the endophytic bacterial species associated with South African sorghum plants. These common endophytic bacterial species can be used to enhance the yield of sorghum crops.

This thesis describes the isolation and structure elucidation of secondary metabolites from a number of endophytic Xylaria fungi. Six Xylaria endophytes were surface cultured on an aqueous malt extract-glucose medium. The fungus A311R, from a palm tree in Thailand, produced nonane-1,2,3-tricarboxylic acid, which was isolated for the first time as a natural product. Also isolated from the same fungus was spiculisporic acid; the first instance of isolation from a Xylaria fungus. The fungus 6RD12 produced cycloepoxydon, which was isolated for the first time from a Xylaria fungus, and 4,5,6-trihydroxy-3-propyl-3,4,6,7-tetrahydro-l//-isochromen- 8(5//)-one, which is a novel compound. The fungi A217R and A517R produced cytochalasin D, (S)-mellein and (3S,4S)-4-hydroxymellein as main secondary metabolites suggesting that the two fungi are the same species. The fungus X04 (Xylaria cf. juruensis) produced 2-Hydroxy-5-ethoxy-3-methylcyclohexa-2,5-dien- 1,4-dione as a novel compound, coriloxin as the main secondary metabolite in addition to (R)-mellein and a mixture of two stereoisomers of the 4-Hydroxymellein. The fungus 6RD8 produced (S)-Omethylmellein as the main secondary metabolite. l

Thesis (Ph. D.)--University of Kentucky, 2008.
Title from document title page (viewed on May 12, 2008). Document formatted into pages; contains: xvi, 221 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 214-219).

Loline alkaloids (LA) are secondary metabolites produced by Epichloandamp;euml; (anamorph, Neotyphodium) grass endophytes. They are toxic and deterrent to a broad range of herbivorous insects but not to livestock. This protective bioactivity has spurred considerable research into the LA biosynthetic pathway. LOL, the gene cluster containing nine genes, is required for LA biosynthesis. The regulation of LOL genes during LA production in culture and in symbio is of interest. In this study, coordinate regulation between LOL gene expression and LA production level was investigated in both MM culture and symbiota. Results showed that expression of LOL genes in N. uncinatum MM culture were tightly correlated with each other (p andamp;lt; 0.0005), and all presented a significant temporal quadratic pattern during LA production. Gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached before the highest amounts of LA were detected, and gene expression level declined to a very low level after amounts of LA plateaued. Observations suggested that the hierarchical clusters based on the correlation coefficient could help to predict the roles of LOL genes in the LA pathway. In symbiota, coordinate coregulation of LOL gene expression with LA was found in E. festucae-meadow fescue inflorescences and stromata, whereby lower LOL gene expression corresponded with the lower LA level in stromata. In N. uncinatum (or N. siegelii)-meadow fescue vegetative tissues, dramatically higher LA levels were found in younger leaf tissue than in older leaf tissue, yet no evidence was found to relate this difference to LOL gene expression differences. Instead, substrate availability may regulate the LA level. In particular, asparagine was more than 10-fold higher in young leaf tissue than in old tissue, although proline was significantly lower in young tissue. Therefore, different regulatory mechanisms underlie LOL gene expression and LA production in different circumstances. The GUS activity of Pro-lolC2-GUS and Pro-lolA2-GUS in Neotyphodium species was almost undetectable in culture, though the activity could be detected in symbiota. The mRNA of GUS did not exhibit the same pattern as lolC2 or lolA2 in culture during LA production time course. A Pro-lolC2-cre transgene was expressed in complex medium, in which lolC2 mRNA was not detectable. These results suggest that proper regulation of LOL genes in culture or symbiota is dependent on the LOL cluster.

Nous avons montre pour la premiere fois que des rhizobiums, bacteries qui forment habituellement des nodules fixateurs d'azote sur les racines des plantes de la famille des legumineuses etaient egalement des bacteries endophytes associees aux racines du riz sauvage oryza breviligulata. Cette espece pousse dans les mares temporaires d'afrique de l'ouest du senegal a la guinee et est consideree comme l'ancetre du riz africain cultive o. Glaberrima. En utilisant des methodes de caracterisation moleculaire, ces rhizobiums endophytes du riz ont ete identifies comme des bradyrhizobium photosynthetiques, une espece bacterienne qui etait jusqu'a present connue pour former des nodules de tiges sur deux especes de legumineuses aquatiques, aeschynomene indica et a. Sensitiva qui poussent dans les meme biotopes qu'o. Breviligulata. Ces bradyrhizobium photosynthetiques sont capables d'utiliser la lumiere comme source d'energie, et nous avons caracterise et sequence leurs genes de fixation de l'azote (gene nif). Nous avons egalement evalue la capacite de colonisation in vitro des racines du riz par les bradyrhizobium en utilisant une souche lacz marquee de la souche type ors278. Les resultats de cette etude cytologique montrent un attachement specifique precoce du bradyrhizobium aux cellules de la coiffe suivie d'une colonisation intense de la surface et des espaces intercellulaires. Le cytoplasme de quelques cellules du cortex de la racine est envahi par les bacteries mais, ces cellules ne se multiplient pas. Les bradyrhizobium photosynthetiques ont ete inocules a plusieurs especes de riz cultives (o. Sativa, o. Glaberrima) en serre et au champ afin de mesurer l'effet de l'inoculation sur le riz. Dans des rizieres de guinee, on observe un effet positif tres important de l'inoculation sur la croissance du riz en pepiniere, en particulier du systeme racinaire qui gagne 55% en longueur et de la largueur de la base de la tige (+ 25%). L'augmentation de la croissance du riz semble etre le resultat d'une production de phytohormones par le bradyrhizobium photosynthetique, et non d'un apport d'azote par la fixation biologique de l'azote.

Des algues brunes endophytes envahissent les tissus des laminariales, avec des effets potentiellement négatifs sur leur hôte. Des études moléculaires ont permis d'identifier deux genres, Laminarionema et Laminariocolax, dominant la diversité de ces endophytes. Une étude épidémiologique par qPCR a montré une forte prévalence de l'endophyte Laminarionema elsbetiae chez Saccharina latissima, avec des variations saisonnières et locales. En laboratoire, la présence de L. elsbetiae induit des réponses physiologiques différentes chez S. latissima, son hôte principal, et chez Laminaria digitata, un hôte occasionnel. Une approche transcriptomique a révélé des réponses moléculaires différentes chez les deux hôtes et l'endophyte, en lien avec les mécanismes de reconnaissance et de défense des deux partenaires. Ces spécificités du dialogue moléculaire lors des premières étapes de l'interaction pourraient expliquer la variabilité des profils d'infection observés dans les populations naturelles
Endophytic brown algae invade stipes and fronds of kelps with potential negative effects for their hosts. The molecular diversity of kelp endophytes was investigated and a majority of the isolated endophytes belonged to the genera Laminarionema and Laminariocolax. Using a qPCR approach, a high prevalence of the endophyte Laminarionema elsbetiae was detected in natural Saccharina latissima populations, but with seasonal and geographical variations. Co-cultivation experiments showed different physiological responses of the main host, S. latissima, and an occasional host, Laminaria digitata, to L. elsbetiae. A transcriptomic approach revealed important differences between the molecular responses of the two kelps, related to the recognition of the endophyte and subsequent defence reactions. These specific differences in the molecular cross-talk during the early steps of the interaction could explain the variability of natural infection patterns in kelp species

This PhD project aims to study the interactions between endophytic bacteria and phytoplasmas associated with grapevines to clarify the role of endophytic bacteria in grapevine yellows (GY) recovery and discuss the possibility to apply these bacteria for GY management. Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. To identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Library analysis of a PCR-amplified 16S rDNA fragment showed best sequence matches with Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Nucleotide sequences of 16S rDNA from bacteria isolated through cultivation showed best sequence matches with Curtobacterium, Stenotrophomonas, Methylobacterium, Pectobacterium, Enterobacter, Brevundimonas, Agrobacterium, Brevibacillus, Staphylococcus, Sphingomonas, Acaricomes, and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Furthermore, composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. LH-PCR of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure is different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed that three LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR database were used to monitor the distribution of bacterial endophytes in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Observed low bacterial richness and different microbial composition in infected and recovered plants suggest that phytoplasma infection could directly and/or indirectly restructure bacterial community selecting endophytic strains that are able to elicit plant defense response. Moreover, we investigated the influence of Flavescence dorée phytoplasmas (FDp) on endophytic bacterial community by studying the seasonal fluctuation of bacterial species associated with healthy, FDp-diseased and recovered plants during phytoplasma infection process. Statistical analysis indicated that, before phytoplasma titre inside diseased plant tissues becomes detectable, endophytic bacterial community is similar to that associated with healthy plants and differs from that associated with recovered plants. Moreover, it seems that a change in microbial composition could be determined when phytoplasmas start to replicate. LH-PCR database showed a seasonal fluctuation of some bacteria identified in the analyzed grapevines; these fluctuations are also related to the presence/absence of the pathogen. On the basis of these evidences we hypothesized that phytoplasma replication determines a change in microbial composition selecting few endophytes that could induce recovery mediated by priming response. Finally, in order to study in which way phytoplasmas interact with endophytes and host plant we developed and tested two different microscopy techniques. Fluorescence in situ hybridisation (FISH) allowed to localise, as expected, phytoplasmas in Catharanthus roseus L. (G. Don) phloem tissues, and also to visualize endophytes in phloem, xylem and parenchyma in the same leaf tissues. On the other hand, immune-confocal laser scanning microscopy was applied for demonstrating that SAP11 (a phytoplasma effector protein) can be unloaded from the phloem by itself. These techniques will be used in combination for clarifying if phytopasmas co-localise with ISR bacterial-inducers and for studying proteins involved in phytoplasmas-endophytes-host cross-talk.

Dans les conditions naturelles, les plantes interagissent avec une grande diversité de microorganismes, entretenant avec elles des types d’interactions variées allant du mutualisme à la pathogénie. Quelque soit le type d’interaction mis en place, les plantes sont capables de reconnaitre des motifs moléculaires microbiens conservés qui induisent l’activation d’une réponse immunitaire dite « non-hôte ». Cette réponse immunitaire basale a largement été étudiée dans le cas des interactions avec des microorganismes mutualistes et pathogènes. Cependant, dans le cas des « symbioses associatives » avec des rhizobactéries ou des bactéries endophytes bénéfiques, regroupées sous le terme de Plant Growth-Promoting Rhizobacteria (PGPR), les réponses immunitaires et physiologiques des plantes ont très peu été décrites. Dans ce contexte, ce projet de thèse a eu pour objectif de décrire les régulations transcriptionnelles de la monocotylédone modèle, le riz, en réponse à l’interaction avec des bactéries bénéfiques -rhizosphériques ou endophytes- et pathogènes du genre Burkholderia sensu lato (s.l.). Ce genre ubiquiste de béta-protéobactéries a la particularité d'avoir été subdivisé en deux genres aux écologies distinctes : le genre Paraburkholderia regroupant des espèces environnementales et bénéfiques des plantes, et le genre Burkholderia sensu stricto (s.s.) qui regroupe des espèces opportunistes et pathogènes de l'homme ainsi que des pathogènes de plantes, mais aussi des espèces phytobénéfiques (comme B. vietnamiensis). L’analyse par RNA-Seq de la réponse transcriptomique du riz à trois souches endophytes, Paraburkholderia kururiensis M130, Burkholderia vietnamiensis TVV75 et Paraburkholderia phytofirmans PsJN, ont permis de mettre en évidence des régulations physiologiques contrastées en fonction de la souche inoculée. De plus, des analyses comparatives de la colonisation des tissus racinaires par ces souches ont pu associer certaines de ces régulations à différents modes de colonisation. Enfin, l’expression de gènes impliqués dans la réponse immunitaire des plantes, identifiés par l’analyse fonctionnelle des transcriptomes, a été mesurée au cours de cinétiques d’interaction avec une plus large diversité de souches. Pour cela, dix souches de Burkholderia s.l., comprenant trois souches pathogènes, ainsi que trois souches PGPR modèles du riz des genres Azospirillum, Herbaspirillum et Pseudomonas ont été choisies. Cette dernière approche a permis de mettre en évidence des régulations transcriptionnelles associées à des types de colonisation, rhizosphérique et endophytique, ou d’interactions, bénéfiques et délétères. Ces travaux s’intègrent dans la caractérisation des bases moléculaires de la réponse des plantes aux microorganismes bénéfiques qui représentent un potentiel important pour le développement de solutions agronomiques durables favorisant la nutrition et la résistance des plantes aux maladies
In natural conditions, plants interact with a large diversity of microorganisms maintaining with them various types of interaction ranging from mutualism to pathogenesis. Whatever the type of interaction established, the plants are able to recognize conserved microbial molecular motifs which trigger a so-called “non-host” immune response when perceived. This basal immune response has been extensively studied in the case of interactions with mutualistic and pathogenic microorganisms. However, in the case of “associative symbiosis” with beneficial rhizobacteria or bacterial endophytes, grouped under the term Plant Growth-Promoting Rhizobacteria (PGPR), the immune and physiological responses of plants have been scarcely described. In this context, this thesis project aimed at describing the transcriptional regulations of the model monocotyledonous rice, in response to the interaction with beneficial -rhizospheric or endophytic- and pathogenic bacteria of the genus Burkholderia sensu lato (s.l.). This ubiquitous genus of beta-proteobacteria has the particularity of having been subdivided into two genera with distinct ecologies: the genus Paraburkholderia, which groups together environmental and plant-associated species, and the genus Burkholderia sensu stricto (s.s.), which groups together human opportunistic and pathogenic species but also phytobeneficial species such as B. vietnamiensis. RNA-Seq analysis of the transcriptomic response of rice to three endophytic strains, Paraburkholderia kururiensis M130, Burkholderia vietnamiensis TVV75 and Paraburkholderia phytofirmans PsJN, revealed contrasting physiological regulations depending on the inoculated strain; in addition, comparative analyses of root tissue colonization by these strains enabled to associate some of these regulations with different colonization patterns. Finally, the expression of genes involved in the immune response of plants, identified by the functional analysis of transcriptomes, was measured during interaction kinetics with a wider diversity of strains. For this, ten strains of Burkholderia s.l., comprising three pathogenic strains, as well as three model rice PGPR strains of the genera Azospirillum, Herbaspirillum and Pseudomonas were selected. This last approach highlighted transcriptional regulations associated with the types of colonization, rhizospheric and endophytic, or interaction, beneficial and deleterious.This work is part of the characterization of the molecular bases of plants’ response to beneficial microorganisms which represent an important potential for the development of sustainable agronomic solutions favoring nutrition and plant resistance to diseases

En raison de la nature symbiotique des micro-organismes endophytes, cette étude visait à étudier l'activité antibactérienne, antifongique et cytotoxique chez les métabolites secondaires des extraits endophytes foliaires de plantes de l'Amazonie brésilienne et du Cerrado. Dans ce travail de thèse 147 micro-organismes cultivables ont été isolés (130 champignons, 3 bactéries e 14 champignons non-identifiés ou inconnus) à partir de 28 plantes (4 espèces collectées au Brésil et 24 en Guyane Française). Tous les micro-organismes furent identifiés par analyse moléculaire des régions spécifiques de l'ADNr en utilisant des techniques de séquençage génomique. Des champignons endophytes de l’ordre des Xylariales furent ceux de plus importante fréquence d’isolation dans cette étude, représentés par 25 isolats. Des extraits bruts à l’AcOEt furent produits à partir de cultures de chaque micro-organisme isolé. Une proportion relative signifiante (23,1%) des extraits démontra une activité sur Candida albicans ATCC 10213, pendant que 4% furent actifs sur Staphylococcus aureus ATCC 29213. Le potentiel cytotoxique des extraits fut évalué pour des lignées cellulaires humaines KB (carcinome cervical utérin), MDA-MB-435 (mélanome), et MRC-5 (fibroblastes de poumon normal), résultant en proportion signifiante avec activité d’inhibition de la prolifération cellulaire (24,4%, 23,1% e 16,3%, respectivement).Dix-huit métabolites secondaires furent isolés et identifiés à partir du fractionnement des extraits bruts de huit endophytes. Dix-sept de ces substances furent déjà décrites précédemment dans la littérature: l’acide piliformique (24) et la griséofulvine (25) isolés à partir de Xylaria cubensis SNB-GCI02; La phomopirone (26), la pyrenocine (27), l’alterpérilenol (28) et la novae-zelandine (29), isolés de Lewia infectoria SNB-GTC2402; 5-métilmelleina (31) et la dihidrosporothrioride (32) isolées à partir de Xylaria sp. SNB-GTC2501; La mycoleptodiscine (34) et la mycoleptodiscine B (35) isolées à partir de Mycoleptodiscus sp. SNB-GTC2304; le mycoepoxidiène (36) et la altiloxina A (37) isolés à partir de Diaporthe pseudomangiferae SNB-GSS10; L’acremonisol (40), le semicochliodinol (41) et le cochliodinol (42) isolés à partir de Chaetomium globosum SNB-GTC2114; la colletofragarone A2 (43) isolée à partir de Colletotrichum sp. SNB-GTC0201; la flavoglaucine (44) isolée à partir de Eurotium rubrum BBS01. Parmi ces substances, la flavoglaucine (44) isolée à partir de E. rubrum BBS01 démontra une activité comparable au contrôle fluconazol en C. albicans (CIM de 4 µg.mL-1). La substance (44) présenta IC50 >10 µM sur cellules normales MRC-5, ce qui en fait un candidat pour des études ultérieures. Dans ce travail, fut identifié pour la première fois l’activité de la colletofragarone A2 (43) isolée à partir de Colletotrichum sp. SNB-GTC0201. La substance inédite nommée pyrrocidine C (30) fut isolée à partir de L. infectoria SNB-GTC2402 et identifiée par des analyses spectroscopiques (Casella et al., 2013). La pyrrocidine C (30) fut active en S. aureus ATCC 10213 (CIM de 2 µg.mL-1) et ne fut pas considérées cytotoxique pour les cellules normales MRC-5 (IC50 >10 µM), démontrant une sélectivité dans l’action antimicrobienne. Ces résultats démontrent la grande diversité des endophytes fongiques chez les plantes de l'Amazonie brésilienne et du Cerrado et la grande chimiodiversité associée aux métabolites secondaires de ces micro-organismes. Des champignons endophytes tropicaux, comme ceux présentés dans cette étude, peuvent apparaître comme une nouvelle source de substances antimicrobiennes et cytotoxiques
Because of the symbiotic nature of endophytes, this survey aims to investigate the probality of discovering antibacterial, antifungal and cytotoxic activities in secondary metabolites of leaf endophytes isolated from plants of Amazon and Cerrado biomes. In this study, 147 cultivable microorganisms were isolated (130 fungi, 3 bacteria and 14 unidentified or unknown microbes) from 28 plant species (4 species collected in Brazil and 24 in French Guyana). All endophytes were identified by molecular analyses of specific rDNA regions, with genomic sequencing techniques. Fungal endophytes belonging to Xylariales order were the most frequently isolated in this study, represented by 25 isolates. Crude AcOEt extracts were produced from cultures of each isolated endophyte. A significant relative proportion (23,1%) of extracts showed activity in Candida albicans ATCC 10213, while 4% were active in Staphylococcus aureus ATCC 29213. The cytotoxic potencial of the extracts was evaluated for human cell lines KB (uterin cervical carcinome), MDA-MB-435 (melanoma) and MRC-5 (normal lung fibroblasts), and a significant proportion of them showed cellular proliferation inhibition (24,4%, 23,1% e 16,3%, respectively).Eighteen secondary metabolites were isolated by the fractionation of eight endophytic extracts. Seventeen of these substances had already been previously described in the literature: piliformic acid (24) and griseofulvin (25) isolated from Xylaria cubensis SNB-GCI02; phomopiron A (26), pyrenocin A (27), alterperilenol (28) and novae-zelandin A (29) isolated from Lewia infectoria SNB-GTC2402; 5-metilmellein (31) and dihidrosporothriorid (32) isolated from Xylaria sp. SNB-GTC2501; mycoleptodiscin A (34) and mycoleptodiscin B (35) isolated from Mycoleptodiscus sp. SNB-GTC2304; mycoepoxidien (36) and altiloxin A (37) isolated from Diaporthe pseudomangiferae SNB-GSS10; acremonisol A (40), semicochliodinol A (41) and cochliodinol (42) isolated from Chaetomium globosum SNB-GTC2114; colletofragaron A2 (43) isolated from Colletotrichum sp. SNB-GTC0201; flavoglaucin (44) isolated from Eurotium rubrum BBS01. Among these substances, flavoglaucin (44), isolated from E. rubrum BBS01, showed comparable antifungal activity with the positive control fluconazol in C. albicans (MIC of 4 µg.mL-1). Flavoglaucin (44) also showed IC50 >10 µM in normal MRC-5 cells, becoming a good candidate for further studies. In this work, the cytotoxic activity of colletofragaron A2 (43), isolated from Colletotrichum sp. SNB-GTC0201 was described for the first time. The unpublished substance named pyrrocidin C (30) isolated from L. infectoria SNB-GTC2402 was identified by spectroscopic analyses (Casella et al., 2013). The pyrrocidin C (30) was active in S. aureus ATCC 10213 (MIC of 2 µg.mL-1), and was not considered cytotoxic for normal MRC-5 cells (IC50 >10 µM), showing selectivity in antimicrobial activity. These results demonstrate the great endophytic fungal diversity in plants of Amazon and Cerrado biomes, along with the chemodiversity associated to the secondary metabolites of these endophytes. Tropical fungal endophytes, like those seen in this work, may emerge as a new source of antimicrobial and cytotoxic substances
Devido à natureza simbiótica dos micro-organismos endofíticos, este estudo teve como objetivo investigar a atividade antibacteriana, antifúngica e citotóxica em metabólitos secundários de extratos de fungos endofíticos foliares de plantas do bioma Amazônia e Cerrado. Neste trabalho de tese foram isolados 147 micro-organismos cultiváveis (130 fungos, 3 bactérias e 14 fungos não-identificados ou desconhecidos) a partir de 28 plantas (4 espécies coletadas no Brasil e 24 na Guiana Francesa). Todos os micro-organismos foram identificados por análise molecular de regiões específicas de DNAr, com uso de técnicas de sequenciamento genômico. Fungos endofíticos da ordem Xylariales foram os de maior frequência de isolamento neste estudo, representados por 25 isolados. Extratos brutos em AcOEt foram produzidos a partir de culturas de cada micro-organismo isolado. Uma proporção relativa significante (23,1%) dos extratos demonstrou atividade em Candida albicans ATCC 10213, enquanto 4% foram ativos em Staphylococcus aureus ATCC 29213. O potencial citotóxico dos extratos foi avaliado para as linhagens celulares humanas KB (carcinoma cervical uterino), MDA-MB-435 (melanoma), e MRC-5 (fibroblastos de pulmão normal), resultando em proporção significante com atividade de inibição da proliferação celular (24,4%, 23,1% e 16,3%, respectivamente). Dezoito metabólitos secundários foram isolados a partir do fracionamento de oito extratos brutos endofíticos. Dezessete destas substâncias já tinham sido descritas anteriormente na literatura: ácido pilifórmico (24) e griseofulvina (25) isoladas de Xylaria cubensis SNB-GCI02; phomopirona A (26), pyrenocina A (27), alterperilenol (28) e novae-zelandina A (29) isoladas de Lewia infectoria SNB-GTC2402; 5-metilmelleina (31) e Dihidrosporothriolida (32) isoladas de Xylaria sp. SNB-GTC2501; mycoleptodiscina A (34) e mycoleptodiscina B (35) isoladas de Mycoleptodiscus sp. SNB-GTC2304; mycoepoxidieno (36) e altiloxina A (37) isoladas de Diaporthe pseudomangiferae SNB-GSS10; acremonisol A (40), semicochliodinol A (41) e cochliodinol (42) isoladas de Chaetomium globosum SNB-GTC2114; colletofragarona A2 (43) isolada de Colletotrichum sp. SNB-GTC0201; flavoglaucina (44) isolada de Eurotium rubrum BBS01. Dentre estas substâncias, a flavoglaucina (44) isolada de E. rubrum BBS01, demonstrou atividade comparável ao controle fluconazol em C. albicans (CIM de 4 µg.mL-1). Esta substância (44) apresentou IC50 >10 µM em células normais MRC-5, tornando-se candidata para estudos posteriores. Neste trabalho foi identificado pela primeira vez a atividade citotóxica da colletofragarona A2 (43), isolada de Colletotrichum sp. SNB-GTC0201. A substância inédita nomeada pyrrocidina C (30) foi isolada a partir de L. infectoria SNB-GTC2402 e identificada através de análises espectroscópicas (Casella et al., 2013). A pyrrocidina C (30) foi ativa em S. aureus ATCC 10213 (CIM de 2 µg.mL-1), e não foi considerada citotóxica para as células normais MRC-5 (IC50 >10 µM), demonstrando seletividade na ação antimicrobiana. Estes resultados demonstram a grande diversidade fúngica endofítica em plantas do bioma Amazônia e Cerrado, e a quimiodiversidade associada aos metabólitos secundários destes micro-organismos. Fungos endofíticos tropicais, como os vistos neste trabalho, podem emergir como uma nova fonte de substâncias antimicrobianas e citotóxicas

Pediomelum esculentum (commonly prairie turnip) is a perennial legume of the Great Plains, consisting of a deep taproot and large edible tuber, and has served as a nutritious staple in Native American diets. The tuber is capable of storing up to 20 percent protein by weight. P. esculentum is a legume, but not a prominent nodule former; instead, it grows in nitrogen-limited soils and produces large amounts of protein. This suggests the involvement of biological nitrogen fixation. We have investigated the presence of diazotrophic endophytes in P. esculentum. Bacteria were isolated from wild plants on nitrogen free media, identified with their partial 16S rRNA gene sequences, and screened for the presence of the nitrogen fixation gene nifH. Select isolates were applied as a co-inoculum to seedlings grown under gnotobiotic conditions in a growth chamber with no nitrogen source. Seedlings in both the inoculated and uninoculated group developed nodules and showed no signs of nitrogen stress. Bacteria isolated from the nodules and tubers of both groups were closely related to the same Bacillus bacterium isolated from seeds germinated under sterile conditions, according to partial 16S rRNA sequences. Bright field and fluorescence imaging revealed bacteria present in the intercellular space of four-week-old tubers and in the sterile germinated seeds. Sectioning and imaging of the nodules show a central nodule vasculature and infected cells extending inwards to the main root vasculature. Nitrogen fixation in the plants was indirectly confirmed by acetylene reduction. Our results suggest P. esculentum has formed a unique symbiosis with a nitrogen fixing Bacillus bacterium that transmits vertically in the seeds and induces nodules.

Magister Scientiae - MSc (Biotechnology)
The yearly increase of global population will result in a greater demand for crop production, but with the climates changes and a lack of available agricultural land it will become increasingly more difficult to provide sufficient crops to feed everyone adequately. Application of the PGPE has proven over the past researches to be able enhance growth of plants via various growth promoting mechanisms. To identify suitable growth promoting bacteria candidate, E. plantagineum plant was used to isolate endophytes from the root after surface sterilization. The isolates bacteria were used to inoculate Brassica napus L seeds. The effects of isolate's ability to promote growth were evaluated based on the certain growth parameters after 42 days in the green house. Isolate CP5 produced highest results in all growth parameter. Isolates CP5 was selected as potential candidate as significant improvement was shown by this isolate. This isolate was tested for the ability to produce ACC deaminase, solubilize phosphate, synthesize IAA and siderophore production. Furthermore isolate CP5 growth promotion abilities was tested on Brassica napus L under antimony stress.
2021-08-31

Prairies of the Pacific Northwest are threatened systems, with only ~2% of historic land remaining. The combined risk of global climate change and land use change make these systems a high conservation priority. However, little attention has been paid to the microbiota. Fungal endophytes are ubiquitous in plants and are important in ecosystem functioning and host dynamics. To understand fungal community assembly, we used high-throughput sequencing to investigate the composition of fungal foliar endophyte communities in two native, cool-season (C3) bunchgrasses along a natural latitudinal gradient. We quantified the importance of host, host traits, climate, edaphic factors, and spatial distance in microbial community composition. We found that spatial distance was the strongest predictors of endophyte community, while host traits (e.g., plant size, density) and abiotic environment were less important for community structure. These findings underline the importance of dispersal in shaping microbial communities. This thesis includes previously unpublished, co-authored material.

Abstract Although microbes are generally found as endophytes in many plant tissues, the plant shoot meristems have been considered virtually sterile. Plant tissue culture, which utilizes mostly the meristems, has nevertheless given numerous references to microbial existence in these tissues. Since the bud-derived tissue cultures of Scots pine (Pinus sylvestris L.) also become easily occupied by microbes, microbial presence was considered to be one potential cause of the low regeneration capacity of tissues from mature trees. The origin of the microbes occurring in tissue cultures of buds of Scots pine was established in this study. One of the microbes, classified as Hormonema dematioides (Ascomycota), was localized in the scale tissues of a bud. Several other microbes were detected, and were associated especially with the meristematic tissues of Scots pine buds. This group was comprised of the yeast Rhodotorula minuta, and bacteria belonging to Methylobacterium spp., the Pseudomonas fluorescens subgroup, and a Mycobacterium sp. These endophytes were located particularly in the outermost cells of meristems, in the epithelial cells of resin ducts, and in the cells of the developing stem, in the vicinity of the meristems. These endophytes were less frequently found in the vascular tissue or in the intercellular spaces of cells, which are typical locations for the previously known endophytic bacteria. The meristem-associated endophytes were discovered to affect growth of pine tissues, and some of them produced substances that are suitable as precursors for phytohormone synthesis. Additionally, pure cultures of both bacterial and fungal endophytes showed antagonism in vitro against pathogens. When the correlation between the presence endophytes and the degeneration of bud-derived tissue cultures of Scots pine was studied, it was discovered that the endophytes grew uncontrollably once a tissue culture was initiated from the bud. A high level of chitinase production was also detected in these tissue cultures, which was considered to be indicative of a defense reaction. However, the endophytes were not found to colonize excessively in every tissue, but all bud-derived tissue cultures of Scots pine eventually degenerate. Therefore, it was concluded that the endophytes may not exclusively be considered responsible for the degeneration of the cultures.

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

It has been established that perennial grasses harbour different types of endophytic bacteria and fungi. Switchgrass (Panicum vergatum L.) is identified as a model perennial energy crop. This study was conducted to explore fungal and bacterial endophyte communities inhabiting switchgrass cultivars of Quebec. The primary focus of this study was to isolate the endophytes, and provide taxonomic identifications based on comparative analysis of ITS rDNA gene sequences. A total of 145 endophytes isolates were recovered (52 bacteria and 93 fungi) from whole plant samples collected at early vegetative, and full reproductive stages. Five and nine different taxa of bacteria and fungi were identified, respectively. We evaluated the antagonistic activity of some endophytes against several fungal pathogens and selected candidate endophytes for future introduction into commercial switchgrass cultivars for biomass enhancement. We demonstrate the vertical transmission ability of some endophyte from one switchgrass generation to the next using species-specific primers. Artificial inoculation of young switchgrass seedlings with selected bacterial endophytes hold promise as a method of reinfection switchgrass seedlings.
Le panic érigé (Panicum vergatum L.) est reconnu comme une des plantes modèles pour la production de biomasse végétale. Il est connu que la plupart des plantes vasculaires étudiées à ce jour sont colonisées par des champignons et bactéries endophyte. Cette étude avait pour but d'explorer les communautés d'endophytes présentes dans différents cultivars de panic érigé au Québec, pour ensuite isoler les endophytes et effectuer leur identification taxonomique en comparant leur séquence ITS rADN. Nous avons obtenu un total de 145 isolats (52 bactéries et 93 champignons) venant de feuille de plante au stade végétative et au stade reproductive. Une fois les isolats identifiés, nous avons obtenu cinq différents groupes taxonomiques pour les bactéries et neuf pour les champignons. Nous avons évalué le potentiel antifongique des différents endophytes bactériens, avec pour objectif d'identifier les candidats potentiels à la ré-inoculation de cultivar de panic érigé commercial afin augmenter leur production de biomasse. À l'aide de séquences d'amorces spécifiques, nous avons pu démontrer la transmission verticale des endophytes.

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected.

Les graminees abritent souvent des champignons endophytes symbiotiques. La premiere partie de cette etude a ete consacree aux e-endophytes (proches des epichloe) du ray grass anglais (lolium perenne). 70% des populations naturelles de ray grass anglais sont porteuses de ces endophyte. La frequence de l'infection intra-population est generalement faible. Ce resultat surprenant a pu etre explique par un modele mutation (perte de l'endophyte) - selection conduisant a un etat d'equilibre polymorphe. De plus, nos resultats montrent que l'infection a une structure spatiale expliquee par une selection exercee par le climat. La seconde partie a ete consacree a l'etude de la coevolution entre les endophytes et leurs hotes. Pour cela, nous disposons d'un ensemble d'especes hotes (toutes les especes du genre lolium - quelques especes de festuca a feuilles larges et fines - vulpia myuros - dactylis glomerata - poa trivialis) et de leurs endophytes. Les phylogenies ont ete realisees a partir de caracteres moleculaires. Les arbres realises sur les champignons et sur leurs hotes sont significativement differents ce qui conduit a ecarter l'hypothese de coevolution.

Les graminees de la sous-famille des festucoidees hebergent frequemment des champignons symbiotiques appartenant au genre neotyphodium (acremonium section albo-lanosa). Ces endophytes sont rencontres soit dans les parties vegetatives aeriennes (gaines, feuilles et tiges) soit dans les graines. Des endophytes ont ete isoles, aussi bien a partir des gaines foliaires et des tiges qu'a partir des graines, de 26 especes differentes de graminees. Les etudes morphologiques, physiologiques et isoenzymatiques effectuees sur ces isolats ont montre l'existence de trois groupes d'endophytes chez les graminees europeennes. Les e-endophytes : la plupart des isolats obtenus peuvent appartenir a une des especes deja decrites de neotyphodium et epichloe. Cependant, un seul isolat issu de f. Arundinacea semble representer un nouveau groupe taxonomique (fatg-4) chez la fetuque elevee. L. Perenne heberge probablement un nouveau groupe taxonomique (lptg-3). L'endophyte de koeleria cristata a ete identifie comme e. Festucae. Par contre, l'endophyte de melica ciliata represente probablement une nouvelle espece. L'epichloe de bromus erectus serait tres proche de l'e. Baconii issu d'agrostis capillaris. Les p-endophytes : il semble que la coexistence des p-endophytes avec les e-endophytes soit frequente, gliocladium-like et phialophora-like ont ete isoles de f. Gigantea et l. Perenne respectivement. Les a-endophytes : un nouveau groupe d'endophytes a ete isole essentiellement des especes des lolium annuels et de f. Paniculata, il est caracterise par des petites conidies bacilliformes et une sporulation abondante et continue. Ces endophytes appartiennent au genre acremonium sensu stricto. L. Temulentum heberge egalement un deuxieme endophyte qui a ete identifie comme verticillium lecanii. La possibilite d'introduire les endophytes dans des especes hotes autres que leurs hotes naturels a ete confirmee. Dix-huit associations ont ete creees dans la presente etude, parmi lesquelles quatre associations sont nouvelles : l'association de l. Perenne avec les endophytes v. Lecanii et acremonium sp. Provenant respectivement de l. Temulentum et l. Persicum, et de l. Perenne et f. Arundinacea avec l'epichloe de b. Erectus n'avaient jamais ete signalees. Les observations microscopiques de graines issues de 10 associations ont montre que l'endophyte avait ete transmis aux graines, bien que n. Coenophialum et gliocladium-like soient transmis respectivement dans les graines de l. Perenne var. Vigor et f. Arundinacea var. Clarine a une frequence tres faible. Le critere employe dans cette etude, pour estimer le degre d'acceptation de l'endophyte par une autre plante hote (outre le critere de transmission a la graine), a ete l'activite peroxydasique (po). L'endophyte gliocladium-like provoque une augmentation significative de l'activite po de vigor et clarine infectes artificiellement par cet endophyte.

Microbes are leading producers of numerous active natural products, many of which have been developed as antibiotics, anticancer agents, and immunosuppressants. The importance and significance of microbial natural products was recognized by the Nobel Prizes in Physiology or Medicine awarded for discovery of penicillin, streptomycin, and avermectin in 1945, 1952 and 2015, respectively. The high occurrence of drug resistance and the high rate of re-isolation represent new challenges. In order to respond to the current challenges and meet the urgently clinical need, new approaches are required to exploit new sources from under-explored habitats and their biosynthetic potentials. This thesis presents efforts to investigate antimicrobial natural products from fungi and endophytes with the combination of genome mining, NMR fingerprint HRMS, and HRMS/MS. The thesis begins with a review on recent advances on anti-TB natural products (NPs) and their potential molecular targets (Chapter 1). 53 natural products showing antimycobacterial activity with defined molecular targets were summarized. Compounds with activity against drug-resistant TB (thiolactomycin, pyridomycin, novobiocin, lipiarmycin A3, gladiolin, kanglemycin A, (S)-(−)-acidomycin, ecumicin, lassomycin, and cyclomarin A) and novel targets (Mtb biotin synthase, ATP synthase, 1,4-dihydroxy-2-naphthoate prenyltransferase, biofilm) were highlighted. Chapter 2 reports on genome- and MS-based investigation of chlorinated compounds from the phytopathogenic fungus Bipolaris sorokiniana 11134 (BS11134). MS- and UV-guided isolation and purification led to the identification of six new compounds together with seven known xanthones. The biosynthetic gene cluster and putative biosynthetic pathway of isolated compounds were proposed. New chlorinated compound methyl 4-chloro-3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate showed activity against Staphylococcus aureus, methicillin-resistant S. aureus, and three clinical-resistant S. aureus strains with a shared minimum inhibitory concentration (MIC) of 12.5 μg/mL. In chapter 3, MS- and UV-guided isolations led to the isolation of ten meroterpenoids from BS11134, including two new compounds 19-dehydroxyl-3-epiarthripenoid A and 12-keto-cochlioquinone A. The stereochemistry difference of aliphatic side chain C-1–C-5 in isocochlioquinone F was proposed to result from stereospecificity of enoyl reductase (ER) of highly reducing polyketide synthase CcqF. Four compounds showed activity against S. aureus and methicillin-resistant S. aureus, with MIC values of 12.5–100 μg/mL. Cochlioquinone B exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5–25 μg/mL. Chapter 4 describes the genome-inspired chemical exploration of the marinederived fungus Aspergillus fumigatus MF071. Two new compounds 19S,20-epoxy-18-oxotryprostatin A and 20-hydroxy-18-oxotryprostatin A, in addition to 28 known compounds, were isolated from MF071. NMR data of monomethylsulochrin-4-sulphate and pseurotin H as naturally occurring compounds were firstly reported. The antibacterial activities of these compounds were evaluated. Helvolinic acid and helvolic acid exhibited strong activity against S. aureus (MIC: 6.25 and 3.13 μg/mL, respectively) and E. coli (MIC: 6.25 and 3.13 μg/mL, respectively). Genomic data analyses revealed the putative biosynthetic gene clusters ftm for fumitremorgins, pso for pseurotins, fga for fumigaclavines, and hel for helvolinic acid. Finally, chapter 5 describes the analytical method using 1H NMR and HR-LCMS to investigate the chemical profile of microbial co-cultures. A microbial single culture extract and co-culture extract library was constructed and subjected to antimycobacterial activity screening. The chemical investigation of Talaromyces sp. MF059 and Bacillus sp. LF017 co-culture led to the discovery of bacillibactin, one siderophore with catecholic trilactone structure. In summary, this thesis reports on the isolation and characterization of 67 secondary metabolites from fungi and endophytes, including 10 new compounds. 20 out of 67 showed antimicrobial activities against different bacteria. The structures of isolated compounds were elucidated based on NMR, HRESIMS, density functional theory (DFT) calculation, and DP4 probability analysis. The results show the great biosynthetic capacity of fungi and endophytes to produce diverse active metabolites. This thesis also demonstrates that the unexplored “biosynthetic dark matter” can be discovered using genome mining, HRMS, NMR, HRMS/MS, and co-culture.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Endophytic fungi of most woody trees are poorly studied due to their cryptic existence, the complexity of the community and the poor ability of traditional tools to characterize them. This study characterised the endophytic community present in commercially important Eucalyptus grandis trees in South Africa. Two different experimental approaches were used. Firstly environmental bar-coding, or metagenetics, was done using 454-pyrosequencing parallel sequencing of the barcode amplicons of all the fungal isolates present in the plants from the total DNA of the plant. Secondly, conventional DNA bar-coding of was done of fungal endophyte isolates. Isolates of the Botryosphaeriaceae family of latent endophytic pathogens were further characterised using a multi-gene phylogenetic approach from both E. grandis and related native S. cordatum that grew in close proximity. The endophytes within the three E. grandis trees were hyper diverse. A total of 1 281 Molecular Operational Taxonomic Units (MOTU) was identified based on 454- pyrosequencing of the E. grandis fungal endophyte infections. Only 85 fungal endophytic species were identified amongst isolates from one of these trees, using the conventional DNA bar-coding approach. Fifteen times more species/MOTU was thus recovered using a metagenetics compared to an isolation approach. Despite this high diversity the species accumulation curves indicate that more endophytic diversity is to be discovered. The multigene analysis of Botryosphaeriaceae isolates obtained from the E. grandis and S. cordatum trees show that three species co-infect both these hosts. Two novel host associations are also reported. This approach of verifying identities of cryptic species with appropriate multigene analyses is most likely needed for other diverse species complexes associated with these trees. A very thorough sampling strategy is required to adequately characterize the endophyte diversity in trees. The experimental approach, the 454-pyrosequening identification workflow and database described in this study will be useful to study these endophyte communities over time and space in future. Using these techniques and workflows described, questions related to host association, diversity and spatial distribution within hosts, and geographical delimitation of endophytes can be addressed.
Dissertation (MSc)--University of Pretoria, 2012.
Genetics
MSc
Unrestricted

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018.
Global demand for environmentally-friendly grapevine cultivation and pest control has necessitated an improved understanding of the relationship between soil properties and beneficial naturally occurring antagonists like entomopathogenic fungi (EPF). This group of fungi presents a viable alternative for the control of destructive pests such as the grapevine mealybug. Sixty-six soil samples were collected from 22 vineyards in the Western Cape, South Africa. The association between soil nutrient status and EPF prevalence was then examined. Fungi were isolated with methods of insect baiting and selective media. Fungal strains were identified and characterized using light microscopy and DNA analysis (ITS and BTub). In addition, fungal isolates were tested against a key grapevine pest, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) using an immersion bioassay at a concentration of 1 x 108 conidia ml-1. Twenty-three fungal strains were isolated and correspondence analysis (CA) of data indicated a positive association between fungal occurrence and moderate to high levels of soil-based macronutrients. Binomial logistic regression analysis revealed that soil N, K, Ca, Mg and S concentrations and C/N ratio were correlated with at least one EPF species. This study showed that some soil nutrient properties correspond to greater occurrence of EPF in grapevine soils. Strains of Beauvaria bassiana (Hypocreales: Clavicipitaceae) caused the highest mortalities (82% to 87%). In chapter three, I examined the effect of B. bassiana inoculation of grape plants on the infestation level of P. ficus, and the growth and volatile constituents of potted grape plants. The grapevines were inoculated with 1 x 108 conidia ml-1 of B. bassiana by drenching before experimentally infesting them with thirty P. ficus adult females. At four weeks post treatments, the fungus was re-isolated from leaves of 50% of the fungus exposed plants. No significant difference (P > 0.05) was observed in all the plant growth parameters measured in the fungus treated and control plants. Plant tissue analysis revealed markedly higher contents of Ca and Mg in leaf tissue of plants exposed to the B. bassiana relative to the control. GC-MS analyses showed that a significantly (X2=5.1; P<0.02) higher number of known anti-insect volatile compounds (9) including napthtalene were present among fungus treated plants compared to the control plants (5). However, B. bassiana did not have any significant effect on total polyphenol, alkaloid and flavonoids. Overall, treatment with fungus did not offer any protection against infestation of P. ficus. In conclusion, this is the first study to report on the isolation of indigenous entomopathogenic fungal (EPF) strains within vineyards of the Western Cape. The study revealed that inoculating grapevine plants during cultivation had a net positive effect on the production of volatile compounds in grapevines. These findings shed light on the mechanisms involved in endophytic fungus-plant-insect interactions. This study contributes valuable information to future development of ecological approaches involving EPF for insect control in vineyards and in general, agricultural settings.

Depuis le milieu du XXième siècle, le recours à la chimie de synthèse a permis de disposer d’un ensemble de solutions efficaces pour améliorer la santé des plantes (produits phytosanitaires) et la santé humaine (médicaments). Cependant, l’utilisation généralisée de certaines de ces solutions (produits phytosanitaires et antibiotiques), a eu des effets non intentionnels sur l’environnement et a conduit à la sélection de populations d’agents pathogènes résistants. Ces effets mettent en péril le recours à ces solutions pour maitriser les agents pathogènes des plantes et de l’homme. Il est donc devenu nécessaire d’identifier de nouvelles solutions, plus durables, pour la protection des cultures et le développement de médicaments, en explorant de nouvelles ressources biologiques et notamment des ressources microbiennes. Ma thèse s'est concentrée sur des bactéries qui effectuent tout ou partie de leur cycle dans les tissus de la plante et qui sont appelées des bactéries endophytes. Les bactéries endophytes présentent des potentialités intéressantes pour le développement de solutions de biocontrôle et production de bioactifs pharmaceutiques. Dans les deux premiers chapitres de cette thèse, j’ai exploré, par des approches de métabarcoding et/ou isolement des souches bactériennes, la diversité taxonomique et fonctionnelle des communautés endophytes de deux espèces végétales aux propriété remarquables, Chrysopogon zizanioides (le vétiver) et Leucojum aestivum (la nivéole d’été). J’ai ainsi constitué des banques de souches de bactéries endophytes dont les activités antifongiques vis-à-vis de champignons pathogènes du blé et, en particulier vis-à-vis de Fusarium graminearum, ont été analysées. Certaines souches affiliées aux genres Bacillus, Serratia, Janthinobacterium ou Streptomyces ont montré une excellente inhibition de la croissance mycélienne du phytopathogène en co-culture in vitro. Des métabolites spécialisés produits par ces souches ont également été étudiés, en ciblant deux groupes de molécules connues pour leurs activités antimicrobiennes ou leur intérêt thérapeutique : les alcaloïdes et les lipopeptides. Ainsi, un précurseur des alcaloïdes indoliques a été identifié dans deux souches bactériennes isolées de L. aestivum. Des lipopeptides, déjà connus pour leurs activités antimicrobiennes, ont également été identifiés dans des souches de Bacillus subtilis isolées des racines de vétiver. Ce travail, en explorant la diversité microbienne associée à des plantes aux propriétés particulières, a fourni un matériel biologique très riche qui pourra être utilisé pour caractériser de nouvelles solutions de biocontrôle ou pour la production de composés bioactifs. Les travaux réalisés dans les 2 premiers chapitres m’ont amené à isoler de nombreuses souches de bactéries dont seulement certaines sont potentiellement intéressante. Dans le troisième chapitre j’ai réalisé un travail préliminaire et innovant visant à enrichir un sol en bactéries potentiellement intéressantes. Ce travail apporte la preuve de concept de l'intérêt de s'appuyer sur le génotype de l'hôte et la suppressivité du sol pour guider le recrutement de microorganismes bénéfiques par la plante. Cette thèse pourrait ainsi fournir de nouvelles perspectives en termes de stratégies de sélection de solutions microbiennes de biocontrôle
Since the mid-21st century, the use of synthetic chemistry has provided a range of effective solutions for plant health (pesticides) and human health (medicines). However, the widespread use of some of these solutions has raised concerns for the environment and for human health and has led to the selection of resistant populations of pathogens. These effects call into question the use of these solutions to control plant and human pathogens. It is, therefore, necessary to identify innovative and more sustainable solutions for crop protection and drug development. One strategy is to explore new biological resources such as microbial resources.My thesis focused on bacteria that realize part of their life cycle in plant tissues and are called endophytic bacteria. These micro-organisms have interesting potential for the development of biocontrol solutions for agriculture and for the production of pharmaceutical drugs. In the two first chapters of this thesis, I explored the taxonomic and functional diversity of the endophytic communities of two plant species with remarkable properties, Chrysopogon zizanioides, and Leucojum aestivum, using metabarcoding approaches and/or isolation of bacterial strains. In particular, I have established strain libraries whose activities against wheat pathogenic fungi and, in particular, against Fusarium graminearum have been analyzed. Some strains affiliated to the genera Bacillus, Serratia, Janthinobacterium, or Streptomyces showed excellent inhibition of the mycelial growth of the plant pathogen in in vitro co-culture. Specialized metabolites produced by these strains were also studied, targeting two groups of molecules known for their antimicrobial activities or their therapeutic interest, alkaloids, and lipopeptides. Thus, a precursor of indole alkaloids was identified in two bacterial strains isolated from L. aestivum. Lipopeptides, already known for their antimicrobial activities, were also identified in Bacillus subtilis strains isolated from vetiver roots.Both approaches led to the identification of numerous strains but only a few interesting candidates. In the third chapter, I investigated an innovative strategy to easier target microorganisms of interest, and I focused on the protection of wheat against F. graminearum. The preliminary results provided a fundamental part to test the concept of the host-mediated engineering of soil and endosphere microbiota toward a resistant microbial community, leading to more efficient isolation of functional endophytes.This work, by exploring the endophyte diversity associated with plants with particular properties, provides very rich biological material that might be used as biocontrol solutions or for the production of bioactive compounds

Algae are increasingly being used in human affairs. Alongside with higher volumes, threats for seaweed natural reservoirs and seaweed farming are starting to appear. Epidemics seems a key complication, similar to agriculture and animal aquaculture. However, phycopathologies are much more under documented, and future diseases are currently difficult to foresee. Thus, efforts should be stressed on biodiversity of algal pathogens and algal defense strategies against diseases. This thesis is divided in two parts: the first section comprises Chapters Two and Three, which describe novel algal pathosystems from cell biology and molecular phylogeny perspectives. Chapter Two characterizes the obligate parasite Maullinia braseltonii sp. nov., a phytomyxid infecting Durvillaea (bull kelp) in the Southern hemisphere. Chapter Three reports new records, cell plasticity and epidemiology of the endophyte Laminariocolax aecidioides infecting Lessonia berteroana (huiro negro) in northern Chile. The two hosts aforementioned have commercial importance, and remarkable roles in coastal ecology in temperate marine habitats in the Southern hemisphere. Chapters Four, Five and Six correspond to a second section and describe cellular processes in novel brown algal pathosystems, related with infection and immunity responses. In Chapter Four, using the Macrocystis/Anisolpidium as a model, microscopy evidence indicate that autophagy is a key cellular process used either to help starving pathogen syncytia to survive (controlled) or to kill themselves (abortive), probably induced by the host. Likewise, host autophagy was also widely observed and may be involved in the neutralization of early-stage oomycetes or nutrient recycling to mount defences. Chapter Five shows the results on a screening of ca. 40 strains from different brown algal orders, to phenotype cellular defense markers against two phylogenetic distant pathogens (Anisolpidium ectocarpii and Maullinia ectocarpii). Overall, responses such as cell wall re-arrangements, programmed cell death, autophagy and ROS accumulation were confirmed to be at least partially conserved across different Phaeophycean lineages. Chapter 6 reports a boost on the phlorotannin metabolism in the model brown alga Ectocarpus (e.g. physode formation and polyketide synthase gene expression) after pathogen challenging, suggesting a similar protective role of this molecule as against grazers and deleterious light regimes. Phlorotannins role however needs to be elucidated.

The Neotropical cloud forest inhabiting orchid Dracula felix has long been postulated to be a fungal mimic due to the form of its lower labellum and attraction to it by drosophilid flies that are often found feeding on fungal fruiting bodies in the surrounding area. The low number of co-occurring flowers in the area combined with the high number of fruiting fungi appears to have driven the evolution of the orchid genus Dracula to mimic these co-occurring fungi so that pollinators may be recruited. Over several years of working with these orchids we have noticed a particular lapping behavior by the pollinating flies on the labella and sepals of the Dracula flowers. In this study we have first surveyed floral yeasts and molds associated with Dracula flowers and then investigated the role of these fungi in attracting pollinators and offering a food reward to retain them for pollination purposes. In addition to the floral yeasts, leaf endophytes and root associated fungi were cultured and identified, and their frequencies were determined.

Dans cette étude, le palmier Astrocaryum sciophilum a été choisi comme modèle pour l'étude de ses endophytes foliaires. Du fait de sa longévité, nous avons cherché à mettre en évidence une communauté compétitive d’endophytes en fonction de l’âge de ses feuilles. Afin d’évaluer si les métabolites produits par ces endophytes pourraient être utilisés en santé humaine, les extraits de chaque endophyte ont été testés contre Staphylococcus aureus résistant à la méticilline (SARM) ainsi que pour leur activité quorum quenching (QQ). En parallèle, afin d’identifier un rôle écologique de protection de la plante par ces endophytes, des co-cultures ont été réalisées avec le phytopathogène Fusarium oxysporum. Plusieurs extraits de souches ont été sélectionnés afin d’isoler et d’identifier le ou les métabolites responsables des activités biologiques observées. Différents outils analytiques ont permis de guider le processus d’isolement (LC-MS/MS, réseaux moléculaires et imagerie par spectrométrie de masse). L’étude de la communauté d’endophytes isolée des feuilles âgées n’a pas mis en évidence un arsenal chimique plus compétitif. Toutefois, deux souches bactériennes du genre Luteibacter sp. ont montré un extrait actif sur SARM et de nombreux extraits de bactéries présentent une activité QQ. Par la suite, le métabolome secondaire du genre Colletotrichum a été étudié à l’aide des réseaux moléculaires et un champignon de la famille des Xylariaceae a été étudié pour son activité contre F. oxysporum. Dans le cadre de cette thèse, sept souches endophytes ont été étudiées chimiquement permettant l’isolement et l’identification de 42 molécules dont dix sont nouvelles
The palm Astrocaryum sciophilum is the host plant model chosen in this work. Indeed, due to the longevity of its leaves, we expected to highlight a competitive community of endophytes within the oldest leaves. Thus, 197 endophytes have been isolated and identified from different leaves of six palm specimens. In order to evaluate whether the compounds produced by these microorganisms could be used for the treatment of human disease, the ethyl acetate extracts of each endophyte were tested against methicillin-resistant Staphylococcus aureus (MRSA) as well as for a quorum quenching (QQ) activity. Simultaneously, co-culture were carried with the fungi Fusarium oxysporum in order to highlight endophytes providing plant protection against phytopathogens. We selected extracts in order to isolate and identify the bioactive metabolites. Various analytical tools have been used to improve the isolation process (LC-MS/MS, molecular networking or MS imaging).The study of the endophytic community isolated from older leaves did not show a more competitive chemical arsenal. However, two Luteibacter strains exhibited an ethyl acetate extract active against MRSA and several bacteria provide quorum quenching extracts. The metabolome of Colletotrichum genus was studied using molecular networking and a fungus from the Xylariaceae family was studied for its capacity to inhibit F. oxysporum’s growth. In our study, seven endophyte strains were chemically investigated leading to the isolation and identification of 42 molecules whose ten are new

Pour la première fois, l’étude de la flore fongique endophytique de cinq plantes médicinales soudanaises : Calotropis procera (Ait.), Catharanthus roseus (L.), Euphorbia prostrata (Ait.), Trigonella foenum-graecum (L.), and Vernonia amygdalina (Del.) a été réalisée. Un total de 23 souches de champignons endophytes ont été isolées à partir des plantes après la stérilisation de surface puis les différentes analyses biologiques ont été effectuées. Les extraits bruts d’acétate d’éthyle de 21 endophytes ainsi que de leurs plantes hôtes ont été évalués pour leur teneur en phénols totaux et leur activité antioxydante en utilisant respectivement la méthode colorimértrique Folin-Ciocalteu et le piégeage des radicaux libres par la méthode 1,1,-diphényl-2-picrylhydrazil (DPPH) in vitro. Une évaluation générale de la cytotoxicité de 16 endophytes sélectionnés ainsi que de leurs plantes hôtes a été réalisée selon le test MTT sur trois types de cellules cancéreuses : carcinome du sein humain (MCF7), adénocarcinome du côlon (HT29 et HCT116). Ces extraits ont été aussi testés, selon la méthode de dilution en bouillon, sur deux souches bactériennes représentatives, Escherichia coli et la souche résistante à la méthicilline de Staphylococcus aureus. La teneur en phénols totaux (89,9 ±7,1 mg Equivalent d’Acide Gallique EAG/g) ainsi que l’activité antioxydante (IC50: 18±0,1 µg/mL) les plus élevées ont été observées pour l’endophyte, Aspergillus terreus 2 isolé à partir des graines de T. foenum-graecum. Byssochlamys spectabilis a montré l’activité cytotoxique la plus importante (1,51 ± 0,2 µg/mL), suivi par Cladosporium cladosporioides 2 (10,5 ± 1,5 µg/mL), puis par Alternaria sp. (13,5 ± 1,8 µg/mL). Seules six souches ont montré une activité contre S. aureus avec des valeurs de MIC qui se situent entre 0,125 et 2 mg/mL dont: Alternaria alternata (0,125 mg/mL), Alternaria sp. (0,250 mg/mL), Byssochlamys spectabilis (0,5 mg/mL). 10 composés purs (0,3 à 40 mg) ont été isolés à partir des extraits bruts d’acétate d’éthyle de Curvularia papendorfii. Le nouveau composé pur (AFB) 3,7,11,15-Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid (acide Khartomique) a montré une activité antibactérienne modérée contre S. aureus avec une CIM de 62,5 µg/mL et une faible activité cytotoxique sur les cellules MCF7 avec une IC50 > 100 µM. Le composé pur AF1 a montré une activité cytotoxique modérée sur les cellules HT29 avec une IC50 de 29,78 µM et une très faible activité antibactérienne contre S. aureus. Ces deux composés ne présentent pas d’activité antioxydante
This study investigated, for the first time, the endophytic fungi flora of five Sudanese medicinal plants: Calotropis procera (Ait.), Catharanthus roseus (L.), Euphorbia prostrata (Ait.), Trigonella foenum-graecum (L.) and Vernonia amygdalina (Del.). A total of 23 endophytic fungal strains were isolated from the plants after surface disinfection and different biological tests were performed. Total phenolic content (TPC) and total antioxidant activity of ethyl acetate crude extracts of 21 endophytes and their host plants were estimated using respectively the Folin-Ciocalteu colorimetric method and 1,1,-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging in vitro method. General evaluation of the cytotoxicity of 16 selected endophytes and their host plants was performed by the MTT assay using cancer cells type: Human breast carcinoma (MCF7) and Colon adenocarcinoma (HT29 and HCT116). Preliminary antibacterial screening was done for the 16 endophytes. These extracts were also tested against two representative bacterial strains, Escherichia coli and methicillin-resistant Staphylococcus aureus, by broth dilution tests. The endophyte, Aspergillus terreus 1 from T. foenum-graecum seeds had the highest TPC in term of Gallic Acid Equivalent (89.9 ± 7.1 mg GAE/g) and antioxidant activity (IC50: 18±0.1µg/mL). Byssochlamys spectabilis showed strong cytotoxicity (1.51 ± 0.2 µg/mL) followed by Cladosporium cladosporioides 2 (10.5 ± 1.5 µg/mL), then Alternaria sp. (13.5 ± 1.8 µg/mL). Only six strains showed activity against methicillin-resistant S. aureus with MIC values ranging between 0.125-2 mg/mL, Alternaria alternata (0.125 mg/mL) Alternaria sp. (0.250 mg/mL) and Byssochlamys spectabilis values (0.5 mg/mL). Ten pure compounds (0.3 to 40 mg) were isolated from ethyl acetate crude extract of Curvularia papendorfii .The new pure compound (AFB) 3,7,11,15-Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid (Khartoumic acid) revealed moderate antibacterial activity against S. aureus with MIC value 62.5 µg/mL and weak cytotoxicity with a IC50 > 100 µM against MCF7 cells. The pure compound AF1 showed moderate cytotoxic activity with IC50 value of 29.78 µM against HT29 and weak antibacterial activity with MIC 250 µg/mL against S. aureus. Both compounds displayed no antioxidant activity

Drug discovery from natural sources including plants, marine organisms and endophytes is still pivotal in pharmaceutical research due to their ability of producing diverse and unique secondary metabolites. Discovery of secondary metabolites from leaves of Vitex pinnata, a medicinal plant from Malaysia and its associated endophytic fungi, Lasiodiplodia theobromae, Nigrospora sp and Pestalotiopsis olivacea afforded nineteen compounds and several of these compounds exhibited good anticancer, antitrypanosomal and anti-mycobacterial activities. Secondary metabolites isolated from leaves of V. pinnata previously reported to exhibit good anti-inflammatory activity and interestingly the leaves were used by local Malay community to treat cuts and wounds. In recent years new strategy using metabolomics in natural products is trending among scientists and found to be efficient, intelligent and robust. Implements of metabolomics including hyphenated HR-LCMS, NMR and integrated with in-house database, AntiMarin were utilised to exploit endophytic fungal natural products from V. pinnata. Preliminary exploration of secondary metabolites production of all three endophytic fungi was simply achieved by applying metabolomics strategy for medium-scale fermentation. Metabolomics also was used as decision-making strategy in mining active metabolomes of endophytic fungus, Lasiodiplodia theobromae against Trypanosoma brucei brucei. Dereplication approach using metabolomics for identification of compounds from Pestalotiopsis olivacea was easily accomplished.

In this dissertation, I explore the diversity of endophytic fungi associated with above-ground tissues of neotropical ferns, assess factors shaping their community structure in three forests, and explore the contributions of endophytes and related strains to our understanding of the diversity and ecology of a representative genus of Ascomycota. In Appendix A, I report a survey of endophytic fungi from seven species of ferns at La Selva, Costa Rica. Using both species-level and phylogenetic analyses, I compare culturable endophytes as a function of fern taxonomy, frond tissues (blades vs. stalks), habits (terrestrial vs. epiphytic), and vegetation types (arboretum vs. primary and secondary forest) during two consecutive years. Analysis of>500 strains provides a first taxonomic overview of fern endophytes at a community scale and reveals high diversity, host preference, and interannual variation in fungal assemblages. However, when variation due to host species is taken into account, community structure is similar among fern tissue types, habits, and sampling sites over a small spatial scale. In Appendix B, I expand my work to evaluate the abundance, species-level and phylogenetic diversity, and distributions of fern-affiliated endophytes in three neotropical forests (La Selva, Costa Rica; Barro Colorado Island, Panama; Los Tuxtlas, Mexico). My analysis of > 2000 isolates reveals that communities differ significantly among fern species within sites, and among ferns in different sites. Intersite differences in communities are correlated with geographic distance and environmental dissimilarity. My work suggests that extrapolative estimates of fungal diversity should take into account not just host species, but locality and environmental variation as well. In Appendix C I focus on a representative genus of Ascomycota (Lecythophora) to evaluate how multi-locus phylogenetic analyses of endophytes can detect new species. Lecythophora is a geographically widespread genus that includes opportunistic human pathogens, produces novel secondary metabolites, and consists presently of six described species. Multi-locus analysis of 33 newly collected strains suggested seven phylogenetic species that are new to science and highlighted their capacity to inhibit growth of two plant pathogenic fungi. This work shows how recognition of cryptic species even in well-characterized genera has major implications for estimating fungal biodiversity.