Dissertations / Theses on the topic 'Secondary metabolites of fungal origin'

Un projet visant à identifier des mycoherbicides pour lutter contre les adventices a été initié entre l’UMR Agroécologie de Dijon et la société DE SANGOSSE® (Agen). Trois volets ont structuré ce projet à l’issue d’une collecte de prélèvement de 475 plantes représentatives de 23 espèces d’adventices symptomatiques et asymptomatiques en Bourgogne et en Beauce. Le 1er volet reposait sur une approche de type metabarcoding (technologie Illumina), pour évaluer et comparer la diversité des communautés fongiques endophytes des plantes symptomatiques et asymptomatiques. 542 genres fongiques ont ainsi été identifiés. Des taxons associés aux plantes symptomatiques ont été identifiés. Parmi ceux-ci, certains sont des pathogènes connus, d’autres non et ils constituent des pistes à exploiter pour la recherche de candidats mycoherbicides. Le deuxième volet repose sur une approche conventionnelle de microbiologie et pathologie. Une collection de 194 champignons associés aux symptômes des adventices a été constituée. La pathogénicité de ces isolats a été testée grâce à une série de screenings de plus en plus sélectifs qui ont abouti à la sélection de cinq souches, identifiées par séquençage de l’ITS ou d’autres marqueurs taxonomiques. Une souche appartient à l’espèce Boeremia exigua var exigua, une autre à l’espèce Alternaria alternata, deux appartiennent à l’espèce A. penicillata et la dernière au genre Alternaria. Le troisième volet visait à identifier le mode d’action d’une souche par une double approche, métabolomique et microscopique. La souche de B. exigua var exigua secrète des métabolites phytotoxiques mais également infeste et semble détruire les tissus végétaux sous-épidermique de la plante hôte.Ce projet exploratoire a fourni des pistes de taxons fongiques associés à des symptômes observés sur adventices en analysant la diversité par une approche moléculaire et a fourni des souches fongiques, mycoherbicides potentiels, par une approche microbiologique dont on voit bien qu’elle reste une méthode incontournable, malgré ses limites, pour obtenir des candidats fongiques à action herbicide
A project aiming at identifying mycoherbicides to control weeds has been initiated between the UMR Agroécologie (Dijon) and the company DE SANGOSSE® (Agen, France). Three axes structured this project after a sampling collection of 475 plants representative of 23 species of symptomatic and asymptomatic weeds was carried out in Burgundy and Beauce. The first part was based on a metabarcoding approach (Illumina technology), to evaluate end compare the diversity of endophytic fungi communities of symptomatic and asymptomatic weeds. 542 fungal genera have been identified. Taxa associated with symptomatic plants have been identified. Of these, some are known pathogens, others are not, and both constitute avenues to exploit for the research of mycoherbicide candidates. The second axe is based on a conventional approach to microbiology and pathology. A collection of 194 fungi associated with weed symptoms was established. The pathogenicity of these isolates was tested through a series of increasingly selective screenings that resulted in the selection of five strains that were identified by sequencing of ITS or other taxonomic markers. One strain belongs to the species Boeremia exigua var exigua, another species Alternaria alternata, two belong to the species A. penicillata and the last to the genus Alternaria. The third axe aimed at identifying the mode of action of a strain by a dual metabolomics and microscopic approach. The strain of B. exigua var exigua produced phytotoxic secondary metabolites but also infested and apparently destroyed the sub-epidermal plant tissues of the host plant.This exploratory project provided tracks to exploit fungal taxa associated with observed weeds symptoms, by analyzing the diversity, by a molecular approach and provided fungal strains, potential mycoherbicides by a conventional microbiological approach that we can see it remains an unavoidable method, despite its limitations, to obtain fungal candidates with herbicidal action

Recentemente foram isolados da esponja marinha Axinella cf. corrugata dois compostos derivados da esculetina: o éster metílico do ácido 4-esculetínico e o éster etílico do ácido 4-esculetínico. Este último apresentou importante atividade contra o vírus da SARS. Este projeto teve como meta isolar e cultivar as linhagens de fungos associadas à esponja Axinella cf. corrugata, bem como analisar seus extratos por HPLC-PDA-MS, objetivando a possível detecção desses compostos (ou derivados) nesses extratos. Avaliações preliminares levaram à obtenção de 11 amostras potencialmente relacionadas a esses compostos. Dentre elas, uma apresentou espectros no UV e de massas muito similares aos obtidos para o aduto de sódio do éster metílico do ácido 4-esculetínico. No entanto análises espectroscópicas mais detalhadas por RMN - 1H, RMN - 13C, HSQC, HMBC e COSY da amostra purificada permitiram identificar o composto isolado, a 1,3,6-trihidroxi-8-metil-9H-xanten-9-ona. Simultaneamente às análises químicas, os extratos obtidos a partir das linhagens fúngicas isoladas da esponja Axinella cf. corrugata tiveram suas atividades biológicas avaliadas frente a microrganismos e células tumorais humanas, resultando em mais de 20% dos extratos com alguma atividade biológica.
Recently two compounds derived from esculetin have been isolated from the marine sponge Axinella cf. corrugata: the methyl ester of esculetin-4-carboxylic acid and the ethyl ester of esculetin-4-carboxylic acid. The latter displayed antiviral activity against the SARS virus. This project aimed the isolation and the growth of fungal strains associated to the sponge Axinella cf. corrugata, and the subsequent analysis of the fungal extracts by HPLC-PDA-MS, aiming the possible detection of the esculetin compounds (or derivatives) in those extracts. Preliminary analysis yielded 11 samples potentially related to these compounds. Among these extracts, one presented UV and MS spectra very similar to the spectra obtained for the sodium adduct of the methyl ester of esculetin-4-carboxylic acid. However, a detailed spectroscopic analysis of a pure compound isolated by RMN - 1H, RMN - 13C, HSQC, HMBC e COSY allowed the identification of the compound, which is 1,3,6-trihydroxy-8-methyl-9H-xanthen-9-one. Simultaneously to the chemical analysis of the fungal crude extracts, the biological activities of the obtained extracts were evaluated against microrganisms and human tumoral cell lines. More than 20% of the extracts displayed some biological activity.

La résistance aux antibiotiques ou la difficulté de plus en plus croissante à traiter les maladies actuelles avec les composés disponibles sur le marché ont contraint les chercheurs à trouver de nouvelles sources de molécules actives. Les lichens produisent divers composés biologiquement actifs en raison de la grande diversité de leur écosystème. Ils représentent ainsi une source prometteuse de composés bioactifs. Le profilage chimique de Nephroma laevigatum a été effectué. Une analyse LC-MS/MS avec des approches en réseaux moléculaires ont permis d’appréhender la diversité chimique de ce lichen et quatre composés différents ont été isolés et identifiés par RMN puis testés pour leur activité antiproliférative. Cependant, les ressources en lichens sont limitées, ce qui restreint leur utilisation. De plus, le thalle lichénique constitue une niche écologique de choix pour d’autres microorganismes, ce qui en fait une source potentielle de nouvelles molécules d’intérêts. La culture de champignons endolichéniques a été entreprise. Ainsi, 46 souches ont été isolées et identifiées par DNA barcoding (amorces ITS4 et ITS5). Les souches identifiées appartiennent au genre Nemania, Daldinia, Peziza et Coniochaeta. Une investigation biologique a été réalisée sur six souches sélectionnées appartenant à deux espèces (Nemania aenea var. aureolatum et N. serpens). Ainsi, deux souches se sont démarquées par leurs activités antiprolifératives et anti-biofilms. Des études chimiques et biologiques plus approfondies de ces dernières (Gir_20 N. aenea var. aureolatum et Cor_08 N. serpens) ont été par la suite effectuées et huit composés différents ont été isolés et identifiés par RMN 1D et 2D. L’étude de l’effet des extraits sur les lignées cancéreuses humaines HT- 29, HCT116, PC-3 et DU145 a permis de mettre en évidence des changements morphologiques au niveau cellulaire. L’analyse de l’expression de marqueurs protéiques pro- et anti-apoptotiques ainsi que la fragmentation de l’ADN mettent en évidence l’induction de l’apoptose. Le profilage chimique par LC-MS/MS de ces souches a ensuite été réalisé et comparé par des approches en réseaux moléculaires permettant ainsi de visualiser la diversité chimique entre les deux espèces de champignons endolichéniques
Antibiotics resistance or increase of difficulty to treat for current diseases with commercially available compounds has obligated researchers to find new sources of active molecules. Lichens produce various biologically active compounds due to the great diversity of their ecosystem. Thus, they represent a promising source of bioactive compounds. Chemical profiling of Nephroma laevigatum was performed. LC-MS/MS analysis with molecular network approach allowed understanding chemical diversity of this lichen and four different compounds were isolated and identified by NMR and tested for their antiproliferative activity. However, lichen resources are limited, which limits their use. In addition, lichen thalli are an ecological niche for other microorganisms and a wide reservoir for access to bioactive molecules. Cultivation of endolichenic fungi was undertaken. Thus, 46 strains were isolated and identified by DNA barcoding (primers ITS4 and ITS5). The isolated fungi belong to genus Nemania, Daldinia, Peziza and Coniochaeta. Biological investigation was carried out on six selected strains belonging to two species (Nemania aenea var. aureolatum and N. serpens). So, two strains distinguished by their antiproliferative and anti-biofilm activities. Further chemical and biological studies of these strains (Gir_20 N. aenea var. aureolatum and Cor_08 N. serpens) were subsequently performed and eight different compounds were isolated and identified by 1D and 2D NMR. Study of effect of the extracts on the human cancer lines HT-29, HCT116, PC-3 and DU145 made it possible to highlight morphological changes at the cellular level. Analyses of the expression of pro- and anti-apoptotic protein markers as well as DNA fragmentation demonstrate the induction of apoptosis. LC-MS/MS chemical profiling of these strains was performed and compared with molecular network approach, to visualize chemical diversity between the two species of endolichenic fungi

La globalizzazione del mercato agroalimentare ha determinato una crescente attenzione da parte dei consumatori verso i prodotti alimentari, non solo in termini di qualità e di sicurezza, ma anche di origine geografica. Infatti, il territorio d’origine ha un forte impatto sull’alimento, dovuto alle condizioni pedoclimatiche che ne determinano le caratteristiche. Poiché non esistono dei metodi analitici di routine per l’autenticazione della provenienza geografica, lo scopo del progetto di ricerca è quello di determinare l’origine geografica e l’autenticità degli alimenti mediante profiling dei composti fenolici e steroli, grazie all’applicazione di tecnologie omiche, tecniche statistiche e chemometriche. La componente fenolica e/o steroli dei campioni, viene analizzata tramite cromatografia liquida (UHPLC) accoppiata ad uno spettrometro di massa (Q-TOF-MS). I dati così ottenuti, vengono elaborati mediante statistica multivariata. L’applicazione combinata di avanzate tecnologie omiche e tecniche statistiche chemometriche ha portato come risultato l’effettiva identificazione della provenienza geografica e autenticità di numerose matrici alimentari. I dati ottenuti dimostrano che i metaboliti secondari possiedono proprietà discriminanti. L’approccio di metabolomica UHPLC/Q-TOF-MS combinato a una statistica multivariata risulta essere adeguato per identificare potenziali markers. Il lavoro attuale è focalizzato sulla ricerca di nuovi metaboliti che, insieme a fenoli e steroli possano confermare la potenza di questo approccio.
Nowadays, food traceability is a growing consumer interest worldwide. Food traceability could be considered a fundamental tool for ensuring safety and high quality of food. Food quality is based not only on the safety and integrity of food, but also on the authenticity, the genuineness of the raw material and the geographical origin. The aim of the work was to investigate the potential of untargeted metabolomics to ensure the authenticity and traceability of foods. Secondary metabolites, like polyphenols and sterols, could be conveniently used to meet this goal due to their chemical diversity and their responses to environmental stimuli. Samples were analyzed through UHPLC-ESI/QTOF-MS. The obtained data were subjected to multivariate statistical analysis. The obtained results showed that secondary metabolites can be efficiently used for authenticity and traceability purposes, with regards to cultivars and geographical origin. These information confirm the role of environmental factors in shaping the actual profile of secondary metabolites in plant foods. The markers found could be used for a target quantification method, a less expensive and less sophisticated analysis, in order to provide an efficient tool that could help to guarantee food quality on routine basis.

SUMMARY Soil organisms, in particular fungi and decomposer insects are primary drivers of organic matter recycling and energy fluxes (Swift et al. 1979; Cadish and Giller 1997; Bardgett et al. 2005). Fungi play a crucial role in the cycling of carbon, nitrogen and phosphorus in terrestrial ecosystems functioning while having to deal in the same time with relentless attacks from fungivores. Only few studies, however, investigated the structuring forces of the population dynamics of fungi and the abundant decomposer fungivores, such as Collembola, with whom they continuously interact. This thesis investigated the interactions between fungi and Collembola focussing particularly on the effects of fungal secondary metabolites from different perspectives. Fungal secondary metabolites are believed to be one of the main vectors driving this interaction. Aiming to get specific insights into the nature of the mechanisms driving this interaction I focused on testing three overarching hypothesis: H1. Fungal secondary compounds mediate the Collembola – fungi interaction H2. Collembola have evolved means to detect fungal toxicity H3. Genetic evidence (transcript regulation) can be used to understand the molecular nature of the Collembola – fungi interaction The above three overarching hypothesis have been addressed in three experimental studies, each with several pointed hypothesis. H1. The first experimental study consisted of a feeding choice experiment offering single and mixed fungal diets using labelled fungal species (C3 and C4; 13C and 15N) of different toxicity. Collembola fractionation and carbon/ nitrogen incorporation of fungal species were assessed via stable isotope analysis. Four knock out mutants of Aspergillus nidulans with the sterigmatocystin production blocked at different steps along the biosynthetic pathway were combined in mixed diets with either the high quality fungus Cladosporium cladosporioides or the low quality fungus A. nidulans (wildtype). This study aimed at understanding the impact of fungal secondary metabolites and more specifically sterigmatocystin (ST) on Collembola performance in single and mixed diets and stabile isotope fractionation. It was hypothesised that (i) presence of sterigmatocystin (ST) impairs Collembola performance with increasing fungal toxicity of the A. nidulans strains, (ii) mixed diets will be beneficial to Collemboal fitness due to toxin dilution and (iii) the fractionation of 13C and 15N it is more pronounced in more toxic diets. We found that ST generally but not uniformly diminished springtail SUMMARY v fitness partially supporting the idea that secondary compounds act as shield against fungivory. However, the use of knockout mutants A. nidulans of the ST pathway (S3-S6) led to rather idiosyncratic responses. Although Collembola fitness was not uniformly increased in mixed diets (suggesting a species specific response) the results still support the toxin dilution hypothesis since no correlation between fungal N content and ingestion could be found. Strong and specific responses of the two Collembola species to mixed diets, knock out mutants and toxins suggest the evolution of species specific strategies to cope with the constraints associated with living in different soil layers. The hypothesis suggesting a link between stable isotope fractionation and fungal toxins has been partially supported with the results suggesting that fungal toxin content may be more important than the nutrient content in controlling stable isotope fractionation of 13C and 15N. H2. The second study focused on the olfactory ability of Collembola to perceive fungal toxicity via olfactory/volatile cues. By means of an olfactometer approach this experiment hypothesized that (i) Collembola are able to olfactorily perceive and distinguish fungal species/strains differing in secondary metabolism, (ii) that Collembola are able to sense and respond to fungal grazing by avoiding to forage on grazed fungi and that (iii) grazing by Collembola triggers in secondary metabolite gene expression in one Basidiomycete and one Ascomycete fungal species using a custom made cDNA microarrays (Chapter 3). All investigated Collembola species recognized fungal olfactory cues and directed their movement to fungal patches and moreover towards fungal strains with suppressed secondary metabolites, in particular towards the mutant ΔlaeA with the main part of secondary metabolites silenced. The volatile cues of conspecifically grazed fungi provoked a movement from two of the three Collembola species (H. nitidus and S. furcifera) towards ungrazed fungi. However, the response of S. furcifera was restricted to fungi extensively exposed to grazing (5 days) suggesting that the response varies between Collembola species. Surprisingly, the investigated fungal gene spectrum did not significantly respond to grazing by Collembola. The results support the first and second hypothesis indicating that Collembola are able to olfactorily differentiate fungi of different toxicity, orientate their movement towards more palatable fungi and avoid movement towards fungi previously exposed to grazing. The lack of changes in fungal gene regulation by grazing suggests that refined methods need to be adopted to investigate the genetic response of fungi to grazing. SUMMARY vi H3. The third study investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of Folsomia candida, the Collembola species used as model species in ecotoxicology. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ΔLaeA) were combined with the high quality fungus C. cladosporioides as mixed diets or offered as single diets. I hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ΔlaeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ΔlaeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory. Overall, the work conducted in this thesis suggests that fungal secondary metabolites act as a structuring force in Collembola-fungi interactions and population dynamics. Using multiple approaches (food choice, olfactory and genetical) the results brings new insights supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory.

PART I. A deuterium exchange analysis of 2,5-dihydroxyacetanilide (5) in the absence and presence of DHAE II was performed to test the nucleophilicity of the substrate in the absence and presence of catalyst. In addition, inhibition studies using 1,4-dihydroxybenzene were performed to determine the role that the N-acetyl side chain group plays in the formation of a stable substrate-enzyme complex. 1,4-Dihydroxybenzene was found to be a weak inhibitor, indicating that the N-acetyl functionality may play a crucial role in forming stable enzyme-substrate interactions. The synthesis of dihydroquinoline 7 was pursued to investigate the enzyme substrate interactions between DHAE and a substrate where the N-acetyl side chain has been fixed to a particular orientation. Efforts towards formation of the C6-C7 bond as a key step in the synthesis of dihydroquinoline 7 using palladium couplings, organocuprates, Lewis acid catalysts, and aza-Claisen reactions were pursued. To complement the results obtained, the electron distribution in amide 21 was calculated using Semi Empirical methods. The results revealed that the electron density in the aromatic ring is centered around C4, suggesting that this is the most nucleophilic carbon in the ring. PART II. Slagenins A (1), B (2), and C (3) were synthesized by β-functionalization of olefin 14. The desired tetrahydrofuroimidazolidin-2-one system was achieved by intramolecular oxidative addition of alcohol 4 to the imidazolone ring. When this reaction was carried out in the presence of methanol slagenins B (2) and C (3) were obtained in good yield. Heating 2 and 3 in aqueous acid gave slagenin A (1) as the sole product. (Z)-debromoaxinohydantoin (17) was synthesized by intramolecular cyclization of α-methoxy imidazolone 11b under acidic conditions followed by a double oxidation reaction to furnish the hydantoin-lactam functionality. These conditions were originally developed for a practical synthesis of the related alkaloid (Z)-debromohymenialdisine (20). A series of acid and base catalyzed reactions of imidazoles bearing an α-β unsaturated system or a β-halogen functionality showed that cyclizations via an S[subscript N]2 path favor formation of an oxazoline ring system. Preliminary studies using pyrrolocarboxamideacetals suggest that β-ketone 73 would be an appropriate substrate for the formation of the pyrrolopyrazine system in the agelastatins.
Graduation date: 2002

M.Sc.
Plants have the ability to continuously respond to various stimuli which alter their physiology, morphology and development. These stimuli may be abiotic or biotic and range from essential to toxic in their effects. One of these stimuli is a steroid from fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol acts as a pathogen-associated molecular pattern molecule and triggers defence mechanisms in plants, characterised by highly regulated and interrelated events that include the elicitation of the oxidative burst and expression of a number of defencerelated genes. However, the ergosterol-induced global cellular reprogramming of the host has not been fully investigated in all aspects. No metabolomic study has previously been conducted to elucidate, for instance, the effect of ergosterol on plant metabolism. A clear and broader understanding of the molecular mechanisms involved in plant : ergosterol interactions is of paramount importance, for it would open up possibilities of developing novel, more effective and sustainable strategies to control or eradicate fungal diseases in plants. In plants, the metabolome is a compilation of all primary and secondary metabolites. The latter are the final recipients of genetic information, and their levels can influence gene expression and protein stability. Metabolite patterns reveal the actual cellular dynamic environment. Hence, qualitative and quantitative measurements of extra- and intracellular metabolites yield insights into the cellular processes that control the biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within a biological system under specific physiological conditions. In the present study a metabolomic approach was used to elucidate and analyse changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol treatment. Special attention is given to sesquiterpenoids since the antimicrobial compounds (phytoalexins) isolated from plants within the Solanaceae are mostly bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0 - 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5- triphenyltetrazolium chloride (TTC), was used to determine whether cell death occurred due to ergosterol treatment. No loss of cell viability was observed over the concentration range and time periods used in this study, indicating that the observed responses were due to the treatment alone and possible secondary responses due to cell death could be excluded. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro extraction and a general methanol extraction. Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS, UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques.

碩士
臺北醫學大學
醫學研究所
95
Pracparatum mungo is one of the Chinese traditional herbs. Since it shows the functions of heat-cleaning, detoxification and anti-inflammatory effects, it was popularly recognized as the saint medicine for detoxification. Our laboratory has previously isolated two fungal strains from Pracparatum mungo and designated the strains as MZK-P01 and MZK-P02, respectively. The fermentation products of either MZK-P01 or MZK-P02 in combination of germ brown rice exhibited a series of antioxidant activity. After the observation of their morphological and cultural characteristics, they were identified to belong to the genus of Scopulariopsis sp. Based on the above-mentioned background, the present study was setting at the discovery of their biological active metabolites as well as the strain identification. MZK-P01, a more promising one, was selected in this study for its superior biological ability In summary, our study has achieved the experimental results and could be divided into four parts as follows: Part 1 deals with the strain identification of MZK-P01. By the observation of morphological characteristics to the strain through light microscope, dissecting light microscope and scan electronic microscope, the hyphae, conidiophores, and conidia were clearly observed. The results led us to confirm the strain to belong to the species of Scopulariopsis flava. The evidence of the PCR product of strain MZK-P01 rDNA including its partial sequence also pointed out that the strain to be a species of Scopulariopsis sp. Part 2 describes the bioactive activity to the target metabolites. The fermentation of MZK-P01strain was carried out with a liquid culture of reciprocating shaking by using Czapek-Dox broth, Sabouraud dextrose broth, and potato dextrose broth as the cultivation source. When the fermentation condition was set at Czapek-Dox broth combined with 3% mung bean powder, the antimicrobial activity against Staphylococcus aureus could reach to the highest. Part 3 discusses the isolation and purification procedure of the active metabolites. Based on the acid-base conversion, an activity-guided fractionation was carried out to the fermentation filtrate. The acidic fraction containing the bioactive products was obtained accordingly. TLC and HPLC were successfully used as the main tools to isolate one of the target bioactive substances in a form of pure crystal needles. The substance was thus designated as MZK-P01-A. Part 4 explains the analytical results of the spectroscopic data to the bioactive product. The UV spectrum showed a maximal absorption at 287 nm. The IR spectrum showed characteristic at 3441(broad, m), 1699(sharp, s), 1406(broad, m) and 1450 (sharp, w) cm-1 respectively. These results could be explained that the compound MZK-P01-A possesses an acidic C=O group and an aromatic group in its molecular structure. The spectroscopic data of the compound indicated that it is a novel natural product with the new molecular structure differing from the fungal metabolites being reported. Keyword: Pracparatum mung, mung bean, antimicrobial activity, antioxidant activity, liquid fermentation, Scopulariopsis flava.

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