Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States. / Ph. D. / Some fungi produce dangerous toxins called mycotoxins that contaminate food and feed and cause adverse affects when consumed. The mycotoxin deoxynivalenol (DON) contaminates staple crops such as wheat, barley, and corn and when consumed by domesticated animals it can cause weight loss, feed refusal, vomiting, and even death. The goal of this research is to detoxify DON using miroorganisms such as bacteria or fungi as well as enzymes. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) utilize a cell free protein synthesis (CFPS) system to detoxify DON using known acetyltransferase genes, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food and strategies to mitigate them. For objective one, microorganisms were collected from plant and soil samples and incubated in solution containing 100 ppm DON. Two mixed cultures were discovered to convert DON to another metabolite, 3-keto-DON that is considered less toxic. In objective two, a cell free protein synthesis (CFPS) system was used to establish its functionality as a tool to screen for enzymes that will detoxify DON. Known acetyltransferase genes were expressed in the CFPS and DON was converted to the metabolite 3-acetyl-DON. The mixed cultures discovered in objective one were then utilized in objective three to determine what enzymes were responsible for the conversion of DON to 3-keto-DON. Objective four was established to shed light about the dangers of mycotoxins and how growers and scientists test for mycotoxins in food and feed.

<p dir="ltr">This research is about the innovative method for the control of the one of the fungal disease, Fusarium head blight, in wheat.</p><p dir="ltr"><i>Fusarium graminearum</i> is a devastating fungus that causes Fusarium head blight (FHB) disease in cereal crops, including wheat, rice, barley, and oats, as well as ear rot and stalk rot in maize. Currently, application of synthetic agrochemical fungicides remains the major mitigation strategy for FHB disease control. However, fungicide treatment carries risks to human health and the environment. Spray-induced gene silencing (SIGS) is suggested as an effective, sustainable, and environmentally friendly alternative for the control of fungal diseases. SIGS uses double-stranded RNA (dsRNA) to induce RNAi against eukaryotic pathogens and pest’s genes. Unlike fungicides, dsRNA can be engineered and carefully designed to target specific fungal pathogens, essentially limiting off-target effects and reducing harm to non-target organisms, including beneficial species.</p><p dir="ltr">In this study, we investigated the <i>in vitro</i> and <i>in vivo</i> effects of dsRNA application on growth and pathogenicity of <i>Fusarium graminearum</i> (<i>Fg</i>). We designed dsRNA against eight fungal genes <i>FgMGV1</i>, <i>FgRAS1</i>, <i>FgCOT1</i>, <i>FgPp2A</i>, <i>FgCAK1</i>, <i>FgTRI5</i>, <i>FgGMK1</i>, and <i>FgYCK1</i>, which have been previously reported to have a functional role in fungal growth and pathogenicity. I postulated that silencing the transcript expression of these selected fungal genes will lead to an attenuation of <i>F</i>. <i>graminearum</i> growth, development and virulence. For lab-scale <i>in vivo</i> dsRNA production, we first designed dsRNAs for each of the fungal genes using the pssRNAit webserver. These gene fragments were subsequently cloned into the L4440 plasmid and transformed into the RNAase III-deficient <i>E</i>. <i>coli</i> strain <i>HT115</i>(DE3). Induction of dsRNA production was mediated by the addition of Isopropyl-β-D-thiogalactoside (IPTG). Graphene Quantum Dots (GQDs) were used as nanocarrier, which were synthesized by pyrolyzing citric acid coupled with surface functionalization by using branched polyethyleneimine (bPEI).</p><p dir="ltr">The performance of ds<i>TRI5</i>, ds<i>MGV1</i>, ds<i>YCK1</i> and ds<i>COT1</i> in reducing fungal biomass in SNA medium was better than dsRNA of other genes, as evidence by lowering fungal biomass by almost half, while ds<i>CAK1</i> and ds<i>GMK1</i> being the least effective in controlling biomass. The inhibitory effect of dsRNA on fungal growth in plate was also investigated. The mycelial growth of <i>F. graminearum</i> on plates was highly inhibited with distinct inhibition zones when plate media was made by mixing the dsRNAs. When wheat spikes of two varieties i.e., ‘AL105’ and ‘Gilat’ were point inoculated with <i>F</i>. <i>graminearum</i>, and after three days sprayed with dsRNAs, the percent symptomatic spikelets (PSS) significantly reduced to 20-25% in both varieties while non-treated control spikes showed 100% symptomatic spikelets. We also evaluated the effect of dsRNA on lowering the accumulation of mycotoxin deoxynivalenol (DON) post-infection followed by the analysis of transcript abundance for two genes <i>FgRAS1</i> and <i>FgPP2A</i> after the application of dsRNA in the liquid culture.</p><p dir="ltr">In summary, we have provided the first example of utilizing graphene quantum dots, for the delivery of dsRNA in SIGS applications in wheat against <i>Fusarium graminearum</i>. In addition, exogenously applied dsRNA degrades naturally, leaving no harmful chemical residues on plants, soil, or water bodies, making it safer for human consumption and the environment. Further optimization of delivery systems for improving the uptake of pathogens efficiency should be done before commercial use of RNA-based disease management in fields.</p>

The global food supply is being stressed by climate change, a growing population, and harmful diseases. One risk to vital cereal crops such as wheat and barley is Fusarium head blight (FHB), caused by the fungal plant pathogen Fusarium graminearum. Ascospores of the fungus are released from perithecia on the residues of corn and small grains and can be transported long distances (>500 m) through the atmosphere. The overall objective of this work was to assess the influence of meteorological conditions and perithecial maturity on ascospore release. The research focuses on F. graminearum because of its damaging impact to staple crops and the global ubiquity of FHB. The first specific objective was to apply state-of-the-science techniques to identify causal meteorological variables of ascospore release. We analyzed field measurements of airborne ascospores against meteorological conditions at Virginia Tech's Kentland Farm, Blacksburg, Virginia, USA and used convergent cross mapping and multivariate state space reconstruction to identify significant causal agents within this complicated natural and dynamic system. We identified relative humidity, solar radiation, wind speed, and air temperature as predictors of ascospore release. Our second research objective was to understand the impact of varying meteorological conditions on ascospore release under controlled environmental conditions. We assessed ascospore release in a chamber with controlled temperature (15°C and 25°C) and relative humidity (60%, 75%, and 95%). Ascospores released from ascospore-producing structures (perithecia) were captured on microscope slides placed inside of 3D-printed ascospore discharge devices. Results showed the sensitivity of ascospore release to relative humidity and temperature, with cool temperature and high relative humidity resulting in greater quantities of ascospores released. Our third research objective was to determine the relationship between the maturity, the number of ascospores, and the hardness of perithecia. A mechanical compression testing instrument was used to investigate the hardness of perithecia at various stages of maturity, producing a mean perithecium compression constant quantifying the uniaxial compression force required to rupture a perithecium. Results indicated that old perithecia contain the greatest amount of ascospores and exhibit increased resiliency, requiring greater forces to rupture, compared to young perithecia. This research has illustrated the complexities of F. graminearum ascospore release by describing the impact of several meteorological conditions and perithecial maturity on the timing and quantity of released ascospores. Collectively, our results may inform wheat growers on the nature and timing of ascospore release, which could help inform FHB management decisions in the future. / Ph. D.

Essex soybean seedling colonization by Fusarium oxysporum and F. solani and disease severity studies were conducted using soil collected from areas in a field (P. Minor field, King and Queen County), which exhibited poor stands and growth of Essex soybean in 1986. Tests were conducted also with soil collected from a field (Holland field, Suffolk), which had no history of poor soybean emergence or growth. Population densities of F. oxysporum and F. solani in the P. Minor soil were significantly higher (P≤0.05) than that in the Holland soil. In P. Minor soil maintained at -0.01 MPa water potential, no significant differences were found in disease severity for 15, 20 and 25 C after 6 days. However, disease severity was significantly (P≤0.05) higher at 20 C than at 15 C or 25 C, 13 days after planting. F. oxysporum and F. so/ani were isolated from lesions on Essex cotyledons and hypocotyls at all three temperatures. Rhizoctonia so/ani was isolated with highest frequency from hypocotyl lesions at 25 C. Colonization of soybean plant parts by F. oxysporum and F. solani in P. Minor soil was studied by plating asymptomatic, sequential, 2-mm long tissue segments on Komada's selective medium. Tissue segments from cotyledons, yielded significantly (P≤0.05) more F. oxysporum than F. solani, 3 and 4 days after planting. Both fungi were recovered at high frequencies from hypocotyl segments although F. solani recovery was significantly (P≤0.05) higher than F. oxysporum after 4 days of planting. From root segments, F. solani isolation frequency was significantly higher (P≤0.05) than F. oxysporum after 4 days. In Holland soil, the colonization patterns of F. oxysporum and F. solani were similar but the frequencies were lower than for P. Minor soil. Of 102 representative F. oxysporum, F. solani and R. solani isolates tested, 40% gave disease severity ratings on Essex soybean that were significantly (P≤0.05) higher than for the control, using artificially infested, pasteurized P. Minor field soil. In greenhouse soil- temperature tanks, all Fusarium and Rhizoctonia isolates tested caused significant (P≤0.05) reductions in stem length and plant fresh weight after 13 days at 20 C in soil maintained at -0.01 MPa water potential. Disease severity ratings for all F. oxysporum and F. solani isolates were significantly (P≤0.05) higher than that for the control. All F. oxysporum, F. solani and R. solani isolates delayed emergence of seedlings, compared to the control, but final stands were affected only slightly. / M.S.

Sixty-six isolates of <I>F. oxysporum</I> and <I>F. solani</I> were recovered from healthy and necrotic Essex soybean seedlings grown in naturally infested soil. These were tested for pathogenicity at 20 C and -0.01 MPa water potential in artificially infested, autoclaved field soil. Highly pathogenic, moderately pathogenic, and hypovirulent isolates of both species were identified. Fifty-seven <I>F. oxysporum</I> and nine <I>F. solani</I> isolates were tested for the presence of dsRNA. The presence of dsRNA was not associated with hypovirulence in <I>F. oxysporum</I> since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing <I>F. oxysporum</I> isolates, three were hypovirulent, two were moderately pathogenic, and one isolate was highly pathogenic. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7, and 2.2 kb, were detected in extracts of all six <I>F. oxysporum</I> isolates. No morphological differences were found between dsRNA-containing and dsRNA-free <I>F. oxysporum</I> isolates. Attempts to cure dsRNA-containing hypovirulent <I>F. oxysporum</I> isolates, either by single-sporing of isolates or by using a range of concentrations of cycloheximide, were not successful. No dsRNA was found in any of the F. solani isolates tested. Pythium ultimum, an associate in Essex seedling disease, was isolated from water-soaked lesions and interfered with evaluations of disease caused by the Fusarium spp. Metalaxyl was used to control P. ultimum and had no apparent effect on symptoms associated with <I>F. oxysporum</I> and <I>F. solani</I> in field soil. Prior inoculation of Essex soybean seeds with conidia of dsRNA-free hypovirulent <I>F. oxysporum</I> isolates, plus metalaxyl seed treatment, significantly (p<0.05) reduced disease severity on both cotyledons and hypocotyls and increased the rate of seedling emergence in field soil, compared to the control plants treated with metalaxyl alone or not treated with metalaxyl. No significant (p>0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent <I>F. oxysporum</I> isolates in their effects on the reduction of disease severity. A mixture of two hypovirulent <I>F. oxysporum</I> isolates was significantly (p<0.05) more effective than single hypovirulent <I>F. oxysporum</I> isolates in increasing the rate of seedling emergence. Symptoms associated with P. ultimum were not affected by the prior inoculation of seeds with individual hypovirulent <I>F. oxysporum</I> isolates. / Master of Science

Cette étude visait à suivre l’abondance relative d’une combinaison de quatre souches de Fusarium graminearum au cours du processus d’inoculation artificielle de parcelles d’orge. Trois amplicons présentant des polymorphismes (SSR, SNP) spécifiques ont été examinés par pyroséquençage 454 sur des échantillons récoltés à tous les stades (souches pures, mélange des souches, inoculum frais, inoculum au champ, grains d’orges infectés). Seuls les marqueurs SNP ont permis d’obtenir une signature unique à chaque souche et ont permis d’estimer l'abondance des souches dans chaque échantillon. Cette analyse a révélé qu’il était difficile d’obtenir des mélanges parfaitement égaux des souches et que les souches se développaient de manière inégale sur les grains de maïs servant de support à l’inoculum. Au champ, ces différences se sont largement maintenues, tandis que des profils d’abondance distinctifs sont apparus sur les trois sites d’essai chez les grains d’orge infectés, mais sans différences significatives entre les cultivars d’orge. / The aim of this study was to track the evolution of a combination of four Fusarium graminearum strains within a mixed inoculum and the ensuing stages of artificial inoculation on barley cultivars. Three amplicons containing specific polymorphisms (SSR, SNP) were examined by 454 pyrosequencing on samples collected at all stages of the process (pure strains, strain mixture, primary inoculum, inoculum in the field and infected barley kernels). Only SNP markers provided unique and accurate signatures for each strain. This analysis revealed that it was difficult to obtain perfectly equal mixtures of strains and that these strains differ in their development on corn kernels used as a growth medium for the inoculum. In the field, these differences remained, while distinctive abundance profiles were detected in infected barley kernels samples at the three nurseries; however, no significant differences between barley cultivars were observed.

En France, la récente apparition et implantation de l’espèce Fusarium langsethiae sur les orges de brasserie est devenue une source d’inquiétude. Ce champignon étant connu comme producteur de toxines T-2 et HT-2 cela pose le problème de la présence éventuelle de ces mycotoxines dans la bière. Des études ont proposé la levure Geotrichum candidum comme possible outil de contrôle du niveau de contamination par ces molécules. Par contre, le mécanisme d’action est encore inconnu. L’objectif de ce travail était de comprendre l’interaction entre F. langsethiae et G. candidum pour évaluer l’effet sur le développement de F. langsethiae et sur sa production de la toxine T-2. Nous avons évalué l’interaction entre ces micro-organismes en utilisant des cultures séquentielles et des co-cultures. Dans ce dernier cas la différentiation des deux micro-organismes a été faite par DOPE-FISH et par quantification de l’ADN. Les résultats ont montré que la croissance de F. langsethiae n’est pas inhibée dans un milieu pré-fermenté par G. candidum. Par contre, la présence d’un agent actif thermorésistant produit lors du développement de G. candidum affecte la concentration de la toxine T-2. Uneréduction supérieure à 90% est observée en comparaison au témoin. Dans les co-cultures on obtient le même pourcentage de réduction de la concentration de la toxine par rapport au témoin. Dans ce cas les deux micro-organismes se développent mais en moindre quantité que dans leur culture pure, F. langsethiae semblant plus inhibé que G. candidum. / F. langsethiae has been recently installed and detected in French barley used for malting. This species is known as a T-2 and HT-2 mycotoxin producer and for this reason, the possible presence of these mycotoxins in beer is a concern. G. candidum has been suggested to have a control activity in this issue, however the mechanisms is not yet described. The aim of this work was to understand the interaction between F. langsethiae and G. candidum to identify any effect in F. langsethiae growth and the T-2 toxin production. In this work, the interaction was evaluated using sequential cultures and co-cultures. Differentiation of these microorganisms in co-culture was achieved using DOPE-FISH and DNA quantification. Results showed that in the medium prefermented by G. candidum F. langsethiae growth was not inhibited but a thermoresistant active agent affected the T-2 toxin concentration during F. langsethiae development. A reduction over 90% of the T-2 toxin was observed in comparison to the concentration detected in the control. For mixed cultures the same toxin concentration reduction was observed while both microorganisms grew but at a lesser extent compared to pure cultures. In this case the growth of F. langsethiae was more affected than the one of G. candidum.

Propomos avaliar a influência da rotação de culturas na infecção de milho transgênico (Bt) e cobertura morta por F. verticillioides e na presença de fumonisinas em amostras provenientes de Palmital e Capão Bonito. O isolamento fúngico da cobertura morta e do milho foi realizado em DG18 e DRBC, respectivamente. Os fungos isolados pertencentes ao gênero Fusarium foram identificados até espécie. Utilizamos, para análise de fumonisinas, Cromatografia Líquida de Alta Eficiência. Micobiota da cobertura morta revelou maior frequência de Cladosporium spp. Nas amostras de milho, F. verticillioides foi o fungo mais isolado. Dentro do gênero Fusarium, a espécie F. verticillioides foi a mais frequente. Análise micotoxicológica do milho revelou a presença de fumonisinas em 88,9% das amostras provenientes de Palmital e em 86,1% de Capão Bonito. Dos isolados de F. verticillioides 84,6% foram produtores de FB1 + FB2. A presença de F. verticillioides e fumonisinas nos grãos de milho estabelece um problema econômico e de saúde pública. / The objective of this study was to evaluate the influence of crop rotation systems on the infection of maize grains and mulch by F. verticillioides and on the presence of fumonisins in maize samples collected in Palmital and Capão Bonito. Surface seeding (DG18) and direct seeding (DRBC) were employed for fungal isolations from mulch and maize, respectively. Identified isolates belonging to the genus Fusarium were further classified at the species level. Analysis of the mulch mycobiota revealed a higher frequency of Cladosporium spp., whereas in maize samples, F. verticillioides was the most frequently isolated fungus. High-performance liquid chromatography (HPLC) was used for mycotoxicological analysis of the grain samples. Maize from Palmital and Capão Bonito presented fumonisins in 88.9% and 86.1% of the samples, respectively. From the F. verticillioides isolates, 84.6% were producing FB1 + FB2. The high incidence of F. verticillioides in maize grains represents a serious problem, due to its potential in causing diseases in humans and animals.

Na agricultura moderna, formas alternativas de alcançar maior produtividade de modo sustentável são prioridades. Entretanto, o sucesso agrícola é ainda excessivamente dependente do emprego de fertilizantes químicos e de defensivos agrícolas. A severidade de algumas doenças é um fator preocupante na produção de várias culturas. Dentre alguns patógenos de planta, destaca-se o gênero Fusarium, o qual apresenta uma expressiva importância na agricultura por ser patógeno em várias culturas de interesse econômico.. Por outro lado, a utilização de microrganismos endofíticos como agentes de biocontrole vem se tornando cada vez mais atrativa, pois, surge como uma alternativa capaz de amenizar gastos excessivos com controle químico e danos ao meio ambiente. Dentre esses microrganismos, destaca-se o gênero Burkholderia, conhecidamente capaz de produzir uma vasta gama de antimicrobianos, com diferentes níveis de especificidade. Essas bactérias vivem em interações com diversos microrganismos e plantas, em um ambiente altamente competitivo, resultando em uma fonte de metabólitos secundários, bacteriocinas dentre outros pepitideos A linhagem TC3.4.2R3 Burkholderia seminalis, isolada endofiticamente de raízes de cana-de-açúcar, é capaz de controlar vários fungos e bactérias fitopatogênicas. Apartir dessa linhagem foi construída uma biblioteca de mutantes randômicos por meio de transposon. Assim, por meio dessa biblioteca, o objetivo do trabalho foi selecionar mutantes defectivos no controle de diferentes espécies de Fusarium, identificar e caracterizar os genes nocauteados, visando o melhor entendimento do controle da fusariose. Os resultados obtidos por meio da caracterização do teste de antagonismo demonstrou que a linhagem TC3.4.2R3 selvagem produz metabólitos capazes de inibir in vitro o crescimento de Fusarium spp. A partir da biblioteca de mutantes, foram obtidos 8 mutantes defectivos para o controle de Fusarium verticilioides FV-01-CTC que não apresentaram alta especificidade quando avaliados contra outros Fusarium spp. Dentre os genes nocauteados foram identificadas sequências codificadoras para a proteína glutamato sintase, TolB e FAD. Até o momento não foi encontrado relatos sobre o glutamato como um biocontrolador de patógenos, entretanto o uso de mutantes sítio dirigido para esse gene confirmou o papel do mesmo no controle de FV-01-CTC. TolB é uma proteína relacionada ao transporte de substâncais e pode ter seu papel de controle relacioanado à secreção de metabolítos secundários, visto que in silico, no genoma da TC3.4.2R3, foram encontrados 18 clusters resposáveis pela produção de metabólitos secundários. Assim, o presente trabalho abre novas perspectivas de estudo, visto que, os mecanismos de controle da fusariose por B. seminalis TC3.4.2R3 são amplos e sinergisticos. Há uma enorme perspectiva em desvendar o papel do glutamato e TolB no controle de fitopatógenos. Os mutantes caracterizados, pode ser fonte de novos estudos para o complexo entendimento da interação microrganismo-planta-patógeno, visto que há a possibilidade de verificar por exemplo o papel do glutamato proveniente da linhagem TC3.4.2R3 na promoção de crescimento vegetal. / In modern agriculture, alternative ways of achieving high productivity with sustainability are priority. However, agricultural success is still excessively dependent on the use of chemical fertilizers and pesticides. The severity of some diseases are worrying factor in the production of differents crops. Among plant pathogens, the genus Fusarium stands out, which has an important role in agriculture being disease causer in several crops such as: sugarcane, corn, passion fruit, tomato, banana, rice, and others. Fusarium can be found inhabiting soil in the most diverse geographical regions of the world, especially in tropical and subtropical climates. On the other hand, studies using endophytic microorganisms as biocontrol agents has become increasingly attractive, since they appear as an alternative to chemical control. Among these microorganisms, the genus Burkholderia stands out. This genis is able to produce a wide range of antimicrobials, with different levels of specificity. Moreover, Burkholderia has been received special attention because its potential plant growth promoter, bioremediation agents and recent studies aimed at biocontrol of diseases. These bacteria live in interactions with diverse microorganisms and plants in a highly competitive environment representing significant source of new bioactive secondary metabolites, bacteriocins among other pepitides. The B. seminalis strain TC3.4.2R3, endofitically isolated from sugarcane roots, is able to control diverse phytopathogenic fungi and bacteria. By Tn5 randomic mutations, it was obtained a library of TC3.4.2R3 mutantes. Thus, using this library, the objective of this work was select defective mutants to control of Fusarium spp., with the aimed the better understand the fusariose control by identificayion and characteriazation of knockouted genes. The results obtained through the characterization of the antagonism assays allowed us to conclude that wild-type TC3.4.2R3 produces many metabolites with antifungal activity under in vitro conditions, being able to control the growth Fusarium spp. From the mutants library we found 8 defectives mutants to control of F. verticilioides FV-01-CTC. Among the knockouted genes we found sequences enconding glutamato syntase, TolB and FAD. To date no reports have been found on glutamate as a pathogen biocontroller, however the use of mutant site directed to this gene confirmed the role of the same in the control of FV-01-CTC. TolB is a protein related to the transport of substances and may have its biocontrol role related to the secretion of secondary metabolites, since in silico analisys in the genome of TC3.4.2R3, 18 clusters were found as responsible for the production of secondary metabolites. Thus, the present work opens new perspectives of study, confirming that the mechanisms of control of fusariosis by B. seminalis TC3.4.2R3 are ample and synergistic. There is a huge prospect in unraveling the role of glutamate and TolB in controlling of plant pathogens. The characterized mutants may be the source of new studies for understanding the complex interaction among microorganism-plant-pathogen, since it is possible to verify, for example, the role of glutamate from the TC3.4.2R3 strain in the plant growth-promotion.

Le complexe d'espèces Fusarium solani regroupe des champignons phytopathogènes également impliqués en pathologie humaine dans des infections parfois profondes et souvent de mauvais pronostic. Dans un premier temps, une méthode de MLST, s'appuyant sur 5 gènes de ménage a donc été développée. Validée sur 51 isolats épidémiologiquement distincts, cette méthode stable et reproductible présente un pouvoir discriminant de 99,1 %. Après comparaison à la technique de référence utilisée en phylogénie, un schéma consensus à 8 loci a été proposé. Dans un second temps, une étude de la sensibilité de ce pathogène à l'amphotéricine B et au voriconazole a été menée par deux techniques d'évaluation des CMI : microdilution CLSI M38-A2 et bandelettes E-test. Devant le paradoxe entre une sensibilité diminuée in vitro au voriconazole et la recommandation de cette molécule pour le traitement curatif de la fusariose humaine, des mécanismes de résistance ont été exploré. L'hypothèse d'un phénomène d'efflux n'a pas été retenue alors que celle d'une modification de la cible, la 14 alpha stérol déméthylase, peut être envisagée après la description de différentes mutations pour les isoformes CYP51A, B et C / Fusarium solani species complex includes phytopathogenic fungi also involved in human infections with poor prognosis. Firstly, MLST method, based on five housekeeping genes has been developed. This method has been validated on 51 isolates epidemiologically distinct, and has been shown to be stable and reproducible and provides a discriminating power of 99.1%. After comparison with the reference technique used in phylogeny, a consensus method with 8 loci has been proposed. Secondly, a study of the susceptibility to amphotericin B and voriconazole has been conducted with two MIC determination methods : CLSI M38-A2 microdilution and E-test. The paradox between decreased susceptibility to voriconazole in vitro and recommendation of this molecule for the curative treatment of Fusarium infections has lead to the exploration of resistance mechanisms. The hypothesis of an efflux phenomenon has not been retained whereas a change in the target, the sterol 14 alpha demethylase may be considered following the description of different mutations on proteins CYP51A, B and C