Academic literature on the topic 'Pythium oligandrum'

Pythium oligandrum is known to display antagonistic activities against several species of pathogenic fungi. It also produces an elicitor of plant defense named oligandrin, which belongs to the elicitin family (10-kDa proteins synthesized by Phytophthora and Pythium species). Here, the potential of P. oligandrum or its purified elicitin to limit the progression of B. cinerea on grapevine leaf and the resulting plant-microorganism interactions are described. P. oligandrum or oligandrin were applied to roots, and changes in the ultrastructure and at the molecular level were examined. When B. cinerea was applied to leaves of pretreated plants, leaf invasion was limited and the protection level reached about 75%. On leaf tissues surrounding B. cinerea inoculation, modifications of cuticle thickness, accumulation of phenolic compounds, and cell wall apposition were observed, indicating that grapevine can be considered reactive to elicitins. No macroscopic hypersensitive reaction associated with the elicitation treatment was observed. At the molecular level, the expression of three defense-related genes (LTP-1, β-1,3-glucanase, and stilbene synthase) was studied. RNAs isolated from B. cinerea-infected leaves of grapevine challenged or not with P. oligandrum or oligandrin were analyzed by real-time reverse transcription-polymerase chain reaction. In grapevine leaves, LTP-1 gene expression was enhanced in response to oligandrin, and RNA transcript levels of β-1,3-glucanase and stilbene synthase increased in response to all treatments with different magnitude. Taken together, these results open new discussion on the concept of plant reactivity to elicitins, which has until now, been mainly based on plant hypersensitive responses.

The associations of Pythium oligandrum with the root cortex, rhizoplane, and rhizosphere were measured with 11 crop species. This work was expedited by the use of a semiselective technique for isolation of P. oligandrum from soil and plant material. Cortical colonization of roots by P. oligandrum was not detected, and the rhizoplanes of the roots of most crops were free of the fungus. However, P. oligandrum was detected in large quantities with every crop tested when roots with adhering soil (rhizosphere soil) were assayed. Different crop species and cultivars of cantaloupe, cauliflower, and tomato varied in rhizosphere densities of P. oligandrum, but rhizosphere population densities of the fungus were consistently higher than in nonrhizosphere soils with plants grown in P. oligandrum-infested sterilized potting mix or an unsterilized mineral soil. After transplanting tomatoes into potting mix infested with P. oligandrum, increases in CFU occurred over time in the rhizosphere but not in the nonrhizosphere soil. In trials on delivery methods of inoculum of P. oligandrum, the rhizosphere populations of tomato plants grown in potting mix were about sixfold higher compared to seed-coat treatments when ground, alginate pelleted oospores were applied to seedlings growing in plug containers prior to transplanting or to pots containing potting mix before direct seeding.

ABSTRACT Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.

The role of Pythium oligandrum as a biocontrol agent against Pythium aphanidermatum was investigated to avoid the harmful impacts of fungicides. Three isolates of P. oligandrum (MS15, MS19, and MS31) were assessed facing the plant pathogenic P. aphanidermatum the causal agent of Glycine max damping-off. The tested Pythium species were recognized according to their cultural and microscopic characterizations. The identification was confirmed through sequencing of rDNA-ITS regions including the 5.8 S rDNA. The biocontrol agent, P. oligandrum, isolates decreased the mycelial growth of the pathogenic P. aphanidermatum with 71.3%, 67.1%, and 68.7% through mycoparasitism on CMA plates. While the half-strength millipore sterilized filtrates of P. oligandrum isolates degrade the pathogenic mycelial linear growth by 34.1%, 32.5%, and 31.7%, and reduce the mycelial dry weight of the pathogenic P. aphanidermatum by 40.1%, 37.4%, and 36.8%, respectively. Scanning electron microscopy (SEM) of the most effective antagonistic P. oligandrum isolate (MS15) interaction showed coiling, haustorial parts of P. oligandrum to P. aphanidermatum hyphae. Furthermore, P. oligandrum isolates were proven to enhance the germination of Glycine max seedling to 93.3% in damping-off infection using agar pots and promote germination of up to 80% during soil pot assay. On the other hand, P. oligandrum isolates increase the shoot, root lengths, and the number of lateral roots.

Pythium oligandrum was a parasite of Verticillium dahliae in dual culture, impeding the latter's ability to grow and form microsclerotia. Variation occurred among isolates of P. oligandrum in their parasitism toward isolates of V. dahliae, and isolates of V. dahliae expressed a range of susceptibilities to parasitism by P. oligandrum. Temperature and matric potential significantly affected interactions between these fungi. Populations of P. oligandrum in the rhizosphere of pepper (Capsicum annuum) were similar in a potting soil infested and noninfested with V. dahliae, but the populations of V. dahliae in combined samples of rhizosphere and nonrhizosphere soil of pepper were significantly lower in the presence of P. oligandrum than in its absence. In greenhouse experiments with pepper grown in soil infested with V. dahliae, shoot and fruit weights were significantly higher in the presence of P. oligandrum than in its absence. However, when soil was infested only with P. oligandrum, fresh weights of shoots and fruits were 40 to 50% higher than when plants were grown in its absence, evidence that this fungus significantly promoted growth of this crop. Therefore, processes responsible for improved plant health associated with soil treatment with P. oligandrum could be the result of complex interactions between pathogen, host, and mycoparasite.

Pythium oligandrum is a unique biological control agent. This soil oomycete not only acts as a mycoparasite, but also interacts with plant roots and stimulates plant defense response via specific elicitors. In addition, P. oligandrum can synthetize auxin precursors and stimulate plant growth. We analyzed the secretomes and biochemical properties of eleven Pythium isolates to find a novel and effective strain with advantageous features for plants. Our results showed that even closely related P. oligandrum isolates significantly differ in the content of compounds secreted into the medium, and that all strains secrete proteins, amino acids, tryptamine, phenolics, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase), exoglycosidases (especially β-glucosidase), proteases, and phosphatases. The most different strain was identified as a not yet described Pythium species. The changes in metabolism of Brassica napus plants grown from seeds coated with the tested Pythium spp. were characterized. Enhanced levels of jasmonates, ethylene precursor, and salicylic acid may indicate better resistance to a wide variety of pathogens. Glucosinolates, as defense compounds against insects and herbivores, were enhanced in young plants. Altogether, P. oligandrum strains varied in their life strategies, and either they could perform equally as plant growth promoters and mycoparasites or they had developed one of these strategies better.

Objectives Dermatophytosis, commonly known as ringworm, is a superficial fungal skin disease and zoonosis. Pythium oligandrum is a micromycete with mycoparasitic properties that is used in agriculture to control fungal infections on plants. Formulations containing P oligandrum were also developed for the treatment of dermatophytoses, but only a small number of case studies have been published. In order to document the process in simplified conditions in vitro, we investigated the effectiveness of P oligandrum against three pathogenic dermatophytes common in domestic animals. Methods Cultures of the pathogens grown on nutrient media and experimentally infected cat hair were treated with P oligandrum preparations in therapeutic concentration and the changes were documented by microscopic videos and scanning electron microscopy. Results There was strong mycoparasitic activity of P oligandrum against Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. Conclusions and relevance P oligandrum was demonstrated to be effective against three common causes of dermatophytosis in vitro.

Pythium oligandrum is a soil born free living oomycete able to parasitize fungi and oomycetes prey, including important plant and animals pathogens. Pythium oligandrum can colonize endophytically the root tissues of diverse plants where it induces plant defenses. Here we report the first long-read genome sequencing of a P. oligandrum strain sequenced by PacBio technology. Sequencing of genomic DNA loaded onto six SMRT cells permitted the acquisition of 913,728 total reads resulting in 112X genome coverage. The assembly and polishing of the genome sequence yielded180 contigs (N50 = 1.3 Mb; L50 = 12). The size of the genome assembly is 41.9 Mb with a longest contig of 2.7 Mb and 15,007 predicted protein-coding genes among which 95.25% were supported by RNAseq data, thus constituting a new Pythium genome reference. This data will facilitate genomic comparisons of Pythium species that are commensal, beneficial or pathogenic on plant, or parasitic on fungi and oomycete to identify key genetic determinants underpinning their diverse lifestyles. In addition comparison with plant pathogenic or zoopathogenic species will illuminate genomic adaptations for pathogenesis toward widely diverse hosts.

Pythium oligandrum Drechs. belongs to the order Oomycetes. It is parasitic on many fungi from the same or other orders. The antagonism of P. oligandrum is a multifaceted process dependent on the target species involved. P. oligandrum is nonpathogenic on 12 species of crops from six families. It does not attack their tissue but occurs on the root surface, predominantly in the regions of hypocotyl – taproot, together with plant pathogenic fungi. It utilises the root exudates and fungus hyphae on the root surface, including those of the plant pathogens, for its own support. A growth stimulation of plants was observed. P. oligandrum can be utilised for biological control on a wide spectrum of crop plants. Different methods of application have been developed. The effect of a mycoparasite preparation is more preventive.

ABSTRACT The ubiquitous oomycete Pythium oligandrum is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability ofP. oligandrum to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between P. oligandrum andPhytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, includingPhytophthora and Pythium spp., which are frequently encountered in field and greenhouse production.

The oomycete Pythium oligandrnm is used as a biocontrol agent because ofthe symbioses it forms with plants, fungi, and other oomycetes. It can parasitize phytopathogenic fungi and oomycetes providing protection to crop plants. P. oligandrnm grows as an endophyte within roots ofplants without causing disease symptoms or defense responses associated with pathogen attack. Growth of P. oligandrnm within plant tissues induces systemic resistance to pathogens and also stimulates plant growth. Little is known ofthe molecular processes that are involved in these proc7sses. Limited molecular tools were currently available to study this organism. The work set out to develop basic tools, and to acquire the fIrst signifIcant DNA sequencing data for this orgalllsm. P. oligandrnm expressed sequence tags derived from vegetative mycelia and a P. oligandrnm-P. infestans interaction were analyzed in an attempt to fmd sequences that may be involved in its biotic interactions. Many sequences with similarity to previously described effectors from fungi, oomycetes, and bacteria were revealed. ' A transformation protocol was adapted for use in P. oligandrnm, which was used to heterologously express green flourescent protein (gfp), producing florescent hyphae that could be used to follow the interaction with a plant and oomycete host. Several genes were identifIed from the EST libraries that were similar to nematode eggshell protein-encoding genes. Using the transformation protocol, it was possible to silence the expression ofthese genes by homology-dependent gene silencing. Oospores .from silenced strains displayed major ultrastructural abnormalities and were sensitive to degradative enzyme treatment. Using immunocytochemistry, these proteins were localized to the oogonial and oospore wall. It was therefore suggested that these proteins are integral components of the oospore/oogonial wall. From the EST libraries three sequences were identifIed that were similar to sea slug pheromones. Similar sequences were found to form a la:.ge gene family in the genomes ofPhytopthora infestans, P. sojae and P. ramorum. One of these genes in P. infestans, was shown to be up-regulated in the motile zoospore stage, leading to the hypothesis that this gene may be acting as a pheromone in zoospore autoaggregation. An initial characterization found no evidence to support this hypothesis.

Pythium oligandrum est un oomycète mycoparasite du sol présentant un large spectre d'hôtes allant des champignons aux oomycètes. Cependant, la base moléculaire de ce mycoparasitisme est encore méconnue. Pour élucider les mécanismes impliqués dans le mycoparasitisme de P. oligandrum, nous avons effectué le premier séquençage de génome long-read de P. oligandrum avec la souche M1 (ATCC 38472). L’assemblage a été optimisé par une approche d’optical mapping conduisant à l’obtention d’un assemblage presque complet de 39Mb. Basé sur une approche de comparative génomique, j'ai détaillé comment ce projet de séquençage du génome m'a permis de découvrir de nouvelles adaptations génomiques liées au mycoparasitisme telles que l'enrichissement de familles de CAZy spécifiques au sein des Pythiales mycoparasites. Pour mieux comprendre les mécanismes moléculaires du parasitisme, une interaction pathogène a été établie entre P. oligandrum et un agent pathogène majeur du blé Fusarium graminearum permettant d'effectuer une analyse transcriptomique au cours de l’interaction. Cela a montré que pendant le développement parasitaire, une reprogrammation transcriptomique majeure se produit chez P. oligandrum, notamment avec l'induction d'un grand nombre de gènes codant pour des protéines sécrétées (glycosyl hydrolases et protéases) et des protéines de fonction inconnue. Plusieurs gènes codant des enzymes impliquées dans la dégradation des champignons fongiques ont été identifiés, comme une chitinase spécifiquement trouvée dans les espèces mycoparasites de Pythium ainsi que des mécanismes de capture des stérols nécessaires pour permettre l'achèvement du cycle de vie sexuel. Pour évaluer l'activité mycoparasite de P. oligandrum sur d'autres proies, des pathosystèmes ont été développés en utilisant un oomycète phytopathogène, Aphanomyces euteiches et un champignon symbiotique Rhizophagus irregularis. Les résultats ont montré que P. oligandrum peut infecter un large éventail d'espèces non apparentées en produisant de nombreuses hydrolases et peut avoir un effet important sur l'interaction des racines avec des micro-organismes pathogènes et également symbiotiques<br>Pythium oligandrum is a major soil-born mycoparasite oomycete with an extensive host range from fungi to oomycetes. However, the molecular basis of Pythium oligandrum pathogenic mechanisms underlying prey colonization is still poorly described. To decipher P. oligandrum mycoparasite mechanisms, we performed the first long-read genome sequencing of Pythium oligandrum with the M1 strain (ATCC 38472). The optical mapping technique improved the assembly leading to a near-complete assembly of the 39 Mb genome. Based on a comparative genomic approach, I detailed how this genome sequencing project allowed me to unravel novel genomic adaptations related to mycoparasitism, such as enrichment in specific CAZy families among mycoparasite Pythiale species. Pythium oligandrum established a pathogenic interaction with a significant wheat pathogen, Fusarium graminearum, allowing transcriptomic analysis. Transcriptomic analysis showed that a considerable transcriptome reprogramming occurred with the induction of many genes-coded secreted proteins (glycosyl hydrolases and proteases) during parasitic development. In addition, we identified several gene coding enzymes involved in fungal wall degradation, such as a chitinase specifically found in mycoparasite Pythium species and prey sterol scavenging mechanisms to enable sexual lifecycle completion. Besides, we evaluate the mycoparasite activity of P. oligandrum on other prey using two different pathosystems: a phytopathogenic oomycete, Aphanomyces euteiches, and a symbiotic fungus of plant roots, Rhizophagus irregularis. Together, these results showed that P. oligandrum could infect an extensive range of unrelated species by producing specific hydrolytic enzymes and can substantially affect root interaction with pathogenic and symbiotic microorganisms

The nutritional and environmental requirements for mycelial growth, oospore production and germination of Pythium oligandrum were examined. Optimum temperatures for growth of several isolates were in the range of 20 - 30 0 , with little growth occurring below 100 or above 350 Oospore germination occurred over the range of 10-35°. Both growth and oospore germination occurred over the range of pH 4.5 - 9.0 and were optimum between pH 6.0 - 7.5. Growth was reduced markedly below -1.0 to -1.5 MPa osmotic potential and ceased at approximately -2.5 to -3.5 MPa; similar results were obtained for oospore germination. Growth and oospore germination were affected more by low matric than by low osmotic potentials. Oospore production required an exogenous supply of sterols; it was also increased by the presence of calcium and affected by the C:N ratio. Semi-solid, static and aerated culture systems were developed for bulk production of P. oligandrum oospores. A liquid cane molasses medium was particularly convenient and efficient. A range of formulations were prepared using oospores produced mainly in this medium. Formulations were evaluated against pathogens causing damping-off in cress and the level of biocontrol in artificially infested sand was not as good as that obtained in naturally infested soil. Alginate pellets and a perlite preparation survived well in laboratory storage at 5-25° for at least 24 wk. Seeds of cress and sugar beet were coated with oospores using commercial seed-pelleting and film-coating procedures. Both types of seed treatment reduced damping-off of cress caused by P. ultimum in artificially infested sand and potting compost, and by Rhizoctonia solani in artificially infested sand. In general, pelleting of P. oligandrum on cress gave better control than film-coating treatments. P. oligandrum also reduced damping-off of sugar beet in soil naturally infested with Aphanomyces cochlioides and Pythium spp.. Control was equivalent to that achieved with hymexazol fungicide seed-coating treatments and was related to the inoculum potential of A. cochlioides in the soil.

Les recherches sur la lutte biologique (ou biocontrôle) par utilisation de micro-organismes connaissent un essor remarquable, les applications au champ étant cependant encore limitées en raison des variations d’efficacité dans la protection des plantes. Celles-ci sont souvent imputées à la non persistance des agents de biocontrôle dans la rhizosphère ou sur le végétal qu’ils sont censés protéger. Afin de réduire ce risque, une solution consiste à utiliser des micro-organismes isolés du végétal que l’on souhaite protéger. Dans le cadre de cette thèse, Pythium oligandrum, un oomycète colonisateur de la rhizosphère de nombreuses plantes dont la vigne, a été étudié pour lutter contre l’esca, une maladie du bois de la vigne pour laquelle il n’existe actuellement aucune méthode de lutte disponible. Des souches de P. oligandrum ont été isolées de la rhizosphère de ceps cultivés dans 3 régions viticoles (12 vignobles) du Bordelais présentant des sols variés : argilo-calcaire, sable-graveleux et graveleux. Les analyses des communautés fongiques et bactériennes obtenues par empreinte moléculaire (Single Strand Conformation Polymorphism) ont montré que, contrairement aux bactéries, les espèces fongiques différaient selon les régions. Des Pythium spp. aux oospores échinulées ont été isolées à partir des racines des ceps échantillonnés, avec une prédominance de P. oligandrum (séquençage de la région ITS). L’analyse des séquences des gènes codant pour le cytochrome oxydase I et une tubuline a permis de constituer 3 groupes de souches. Le séquençage d’autres gènes codant pour des protéines « élicitines-like » a indiqué que chaque souche présentait au moins un gène codant pour chacun des 2 types d’éliciteurs de P. oligandrum : l’oligandrine et les protéines de la paroi cellulaire (CWPs). Il apparaît que le type de sol et la microflore associée à la rhizosphère n’exerceraient pas une influence suffisante pour que la structure génétique des populations de P. oligandrum soient associées à un contexte tellurique particulier. En revanche, le type de porte-greffe et la méthode de désherbage (chimique ou mécanique) pourraient avoir une incidence sur la colonisation racinaire par P. oligandrum. Les relations entre P. oligandrum et les racines de la vigne ont été étudiées par analyse transcriptomique (microarray Vitis vinifera de 29 549 gènes). Les résultats obtenus montrent que de jeunes plants de vigne ont répondu à la colonisation racinaire par P. oligandrum en modifiant l’expression de gènes intervenant dans plusieurs voies métaboliques. Deux aspects a priori opposés ont été observés : P. oligandrum serait perçu comme (1) un agresseur contre lequel la plante a mis en place des réactions de défense mais en même temps, comme (2) un micro-organisme symbiotique car un certain nombre de modifications transcriptionnelles étaient similaires à celles reportées dans les interactions rhyzosphèriques symbiotiques (e.g. forte stimulation de gènes codant pour des subtilases). Un essai visant à induire chez la vigne une protection contre un champignon pathogène impliqué dans l’esca, Phaeomoniella chlamydospora, grâce à P. oligandrum, a été réalisé. La colonisation des racines par P. oligandrum a été associée à une réduction de la longueur des nécroses dues à P. chlamydospora. En adéquation avec ce résultat, l’analyse transcriptomique par RT-PCRq et microarrays a montré une surexpression de la voie de l’éthylène. Plusieurs gènes spécifiquement induits constitueraient des marqueurs de résistance qu’il conviendra de valider lors de prochaines expérimentations<br>Biocontrol research based on the use of microorganisms is expanding very rapidly. However, the use of such bioncontrol agents is still too inconsistent to effectively protect plants in field applications. This phenomenon is often attributed to the non-persistence of biocontrol agents in the rhizosphere or on the plants. In order to reduce the risk of this happening, one solution consists in using microorganisms that are isolated from the plants needing protection. In this thesis, an oomycete called Pythium oligandrum, which colonizes the rhizosphere of many plants, including grapevine, was assessed for the control of esca, a grapevine trunk disease for which no control method is currently available. P. oligandrum strains have been isolated from the rhizosphere of vines cultivated in 3 wine-growing regions (12 grapevines) of Bordeaux with different types of soil: stony-sandy, silty and stony. Analyses of fungal and bacterial communities using a molecular fingerprinting method (Single Strand Conformation Polymorphism) showed that, unlike bacteria, the fungal species varied according to the sampling region. Roots of all the vines sampled were colonized by echinulated-oospore Pythium spp., with P. oligandrum strains predominating. Phylogenetic analyses based on the genes encoding the cytochrome oxidase I and one tubulin allowed these strains to be clustered into three groups. The sequencing of the elicitin-like genes, whose proteins are key components in inducing systemic resistance in plants, showed that each strain held at least one gene encoding for each of the two kinds of P. oligandrum elicitors (i.e. oligandrin and Cell Wall Proteins). Sequencing and molecular fingerprinting analyses showed thus that the type of soil and the rhizosphere microbiota did not shape the population structure of P. oligandrum. However, other factors such as the different kinds of rootstock and weeding management can also have an influence on the root colonization by P. oligandrum. The relationship between P. oligandrum and grapevine was studied using a transcriptomic approach (microarray Vitis vinifera, 29 549 genes). The results highlighted the modifications induced by young vines in response to P. oligandrum root colonization, in the genetic expression of several genes belonging to different metabolic pathways. Two aspects, that are usually opposed, were observed: P. oligandrum was perceived by the plant either (i) as a pathogen because certain defence reactions were triggered (e.g. calcium signalling, resistance genes, abscissic acid metabolism) or as (ii) a symbiotic microorganism since several transcriptional changes were similar to those reported in symbiotic interactions (e.g. induction of subtilase genes). An assay aimed at protecting grapevine against a pathogenic fungus involved in esca, and known to be responsible for wood necrosis, i.e. Phaeomoniella chlamydospora, was carried out. The root colonization by P. oligandrum was associated with a reduction in the length of necroses. In line with this result, transcriptomic analyses by microarrays and RT-qPCR showed overexpression of several genes, particularly those of the ethylene pathway. Some of these induced genes could be thus used as resistance markers, but this needs to be validated in further experiments

Une des principales limites de la lutte biologique réside dans la variabilité des résultats expérimentaux souvent imputée à la non-persistance des micro-organismes introduits sur les végétaux. Dans le cadre de cette thèse, Pythiurn oligandrum, un oomycète connu pour ses propriétés d’agent antagoniste et d’inducteur de résistance chez les plantes, a été utilisé. L’objectif principal a été d’étudier la colonisation et la persistance de P. Oligandrum après son introduction au niveau du système racinaire de plants de tomate en culture hors-sol, et d’apprécier son incidence sur les communautés microbiennes de la rhizosphère et des effluents de serre. Trois souches de P. Oligandrum ont d’abord été sélectionnées pour leur production d’oospores (structure de résistance) ainsi que de molécules ayant des effets positifs sur la croissance (une auxine : la tryptamine) et la résistance des plantes (un éliciteur : l’oligandrine). La colonisation des racines et des solutions circulantes par P. Oligandrum et Pythium dissotocum, un agent pathogène mineur très fréquent en hors-sol, a été estimée par méthodes culturales et moléculaires. L’antagoniste a persisté à des taux élevés tout au long de la saison culturale (avril à septembre), la rhizosphère des plantes ayant été colonisée à 90% par la souche produisant le moins d’oospores (détermination par Inter Simple Sequence Repeat). Malgré son abondance au niveau racinaire, peu ou pas de P. Oligandrum a été détecté dans les différents effluents de la serre. P. Dissotocum a, quant à lui, colonisé les racines et les effluents de drainage pendant les mois d’été. Un léger effet retard et une moindre colonisation au niveau des racines colonisées par l’antagoniste ont été mis en évidence. L’analyse des communautés microbiennes (fongiques et bactériennes) racinaires et circulantes par SSCP (Single-Strand Conformational Polymorphism) a révélé que ces communautés avaient subi des changements structurels importants au cours de la saison culturale. Ces évolutions temporelles sont intervenues indépendamment de l’inoculation et de la persistance de l’agent antagoniste P. Oligandrum. Des archées ont également été détectées durant toute la saison culturale dans les effluents mais uniquement aux mois de septembre et octobre au niveau des racines. Dans ce cas aussi, l’inoculation par P. Oligandrum ne semble pas avoir engendré d’effet sur ces communautés. Les résultats décrits précédemment reposent sur l’extraction et l’amplification d’ADN. Un des inconvénients de cette stratégie est qu’elle ne permet pas d’étudier les communautés microbiennes d’un point de vue fonctionnel. Afin d’évaluer et de comparer la diversité structurelle et fonctionnelle des populations fongiques rhizosphériques, ADN et ARN ont été extraits puis analysés par SSCP en fonction de trois cibles génétiques différentes: la région ITS1, l’ARNr 28S, la grande sous-unité de l’ARNr mitochondrial. Les informations générées par l’analyse de ces trois marqueurs génétiques sont relativement différentes, confirmant ainsi l’intérêt d’utiliser des amorces ciblant des régions de l’ADN très différentes pour obtenir une vision plus exhaustive de la microflore fongique<br>Biocontrol efficacy is mainly limited by the variability of the rhizosphere competence of the inoculated microorganisms. This doctoral thesis focused on Pythium oligandrum, an oomycete acknowledged as an antagonistic organism able to protect plants from pathogenic attacks. The aim of this work was to study the colonisation and the persistence of P. Oligandrum after its introduction in the root system of tomato plants grown in soilless culture; and to assess its impact on microbial communities colonizing the rhizosphere and the greenhouse effluents. Three strains of P. Oligandrum were selected on the basis of their ability to produce oospores (resting structures) and production of tryptamine (an auxin like compound) and of oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter Simple Sequence Repeat (ISSR) analysis showed that, among the three strains inoculated, the one producing the lowest amount of oospores was detected at 90%. Despite its abundance on roots, no traces of P. Oligandrum were detected in the different effluents of the soilless greenhouse. P. Clissotocum (ubiquitous tomato root minor pathogen) colonized the rhizosphere and the effluents only in summer. There was a reduction of P. Dissotocum populations in inoculated root systems. Single-Strand Conformational Polymorphism (SSCP) analysis revealed that the genetic structure of microbial communities (fungi and bacteria) colonizing the rhizosphere and the effluents, evolved throughout the cropping season. This temporal evolution was independent from the inoculation and the persistence of the antagonist P. Oligandrum. Effluents were also colonized by Archaeabacteria but roots, only during the last two months of culture. These populations grew independently from P. Oligandrum. Results previously described, rely on DNA extraction and amplification. This strategy suffers from the inability to investigate active microbial communities. DNA and RNA data obtained by SSCP analysis of three different genetic regions (ITS1, rRNA 28S, mitochondrial RNA large subunit) highlighted the interest of using different primers for having an exhaustive view of the fungal microflora

Horseradish roots, due to the content of many valuable nutrients and substances with healing and pro-health properties, are used more and more in medicine, food industry and cosmetics. In Poland, the cultivation of horseradish is considered minor crops. In addition, its limited size causes horseradish producers to encounter a number of unresolved agrotechnical problems. Infectious diseases developing on the leaves and roots during the long growing season reduce the size and quality of root crops. The small range of protection products intended for use in the cultivation of horseradish generates further serious environmental problems (immunization of pathogens, low effectiveness, deterioration of the quality of raw materials intended for industry, destruction of beneficial organisms and biodiversity). In order to meet the problems encountered by horseradish producers and taking into account the lack of data on: yielding, occurrence of infectious diseases and the possibility of combating them with methods alternative to chemical ones in the years 2012–2015, rigorous experiments have been carried out. The paper compares the impact of chemical protection and its reduced variants with biological protection on: total yield of horseradish roots and its structure. The intensification of infectious diseases on horseradish leaves and roots was analyzed extensively. Correlations were examined between individual disease entities and total yield and separated root fractions. A very important and innovative part of the work was to learn about the microbial communities involved in the epidemiology of Verticillium wilt of horseradish roots. The effect was examined of treatment of horseradish cuttings with a biological preparation (Pythium oligandrum), a chemical preparation (thiophanate-methyl), and the Kelpak SL biostimulator (auxins and cytokinins from the Ecklonia maxima algae) on the quantitative and qualitative changes occurring in the communities of these microorganisms. The affiliation of species to groups of frequencies was arranged hierarchically, and the biodiversity of these communities was expressed by the following indicators: Simpson index, Shannon–Wiener index, Shannon evenness index and species richness index. Correlations were assessed between the number of communities, indicators of their biodiversity and intensification of Verticillium wilt of horseradish roots. It was shown that the total yield of horseradish roots was on average 126 dt · ha–1. Within its structure, the main root was 56%, whereas the fraction of lateral roots (cuttings) with a length of more than 20 cm accounted for 26%, and those shorter than 20 cm for 12%, with unprofitable yield (waste) of 6%. In the years with higher humidity, the total root yield was higher than in the dry seasons by around 51 dt · ha–1 on average. On the other hand, the applied protection treatments significantly increased the total yield of horseradish roots from 4,6 to 45,3 dt · ha–1 and the share of fractions of more than 30 cm therein. Higher yielding effects were obtained in variants with a reduced amount of foliar application of fungicides at the expense of introducing biopreparations and biostimulators (R1, R2, R3) and in chemical protection (Ch) than in biological protection (B1, B2) and with the limitation of treatments only to the treatment of cuttings. The largest increments can be expected after treating the seedlings with Topsin M 500 SC and spraying the leaves: 1 × Amistar Opti 480 SC, 1 × Polyversum WP, 1 × Timorex Gold 24 EC and three times with biostimulators (2 × Kelpak SL + 1 × Tytanit). In the perspective of the increasing water deficit, among the biological protection methods, the (B2) variant with the treatment of seedlings with auxins and cytokinins contained in the E. maxima algae extract is more recommended than (B1) involving the use of P. oligandrum spores. White rust was the biggest threat on horseradish plantations, whereas the following occurred to a lesser extent: Phoma leaf spot, Cylindrosporium disease, Alternaria black spot and Verticillium wilt. In turn, on the surface of the roots it was dry root rot and inside – Verticillium wilt of horseradish roots. The best health of the leaves and roots was ensured by full chemical protection (cuttings treatment + 6 foliar applications). A similar effect of protection against Albugo candida and Pyrenopeziza brassicae was achieved in the case of reduced chemical protection to one foliar treatment with synthetic fungicide, two treatments with biological preparations (Polyversum WP and Timorex Gold 24 EC) and three treatments with biostimulators (2 × Kelpak SL, 1 × Tytanit). On the other hand, the level of limitation of root diseases comparable with chemical protection was ensured by its reduced variants R3 and R2, and in the case of dry root rot, also both variants of biological protection. In the dry years, over 60% of the roots showed symptoms of Verticillium wilt, and its main culprits are Verticillium dahliae (37.4%), Globisporangium irregulare (7.2%), Ilyonectria destructans (7.0%), Fusarium acuminatum (6.7%), Rhizoctonia solani (6.0%), Epicoccum nigrum (5.4%), Alternaria brassicae (5.17%). The Kelpak SL biostimulator and the Polyversum WP biological preparation contributed to the increased biodiversity of microbial communities associated with Verticillium wilt of horseradish roots. In turn, along with its increase, the intensification of the disease symptoms decreased. There was a significant correlation between the richness of species in the communities of microbial isolates and the intensification of Verticillium wilt of horseradish roots. Each additional species of microorganism contributed to the reduction of disease intensification by 1,19%.