Biological control by the oomycete, Pythium oligandrum, of a pathogenic fungus involved in esca, a grapevine trunk disease

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.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

Protection against a vine trunk attack by Phaeomoniella chlamydospora is concomitant with root colonization by the oomycete Pythium oligandrum

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Bio-suppression of sclerotinia stem rot of tomato and biostimulation of plant growth using tomato-associated rhizobacteria

A collection of 25 rhizobacterial strains, recovered from rhizospheric soils around healthy tomato plants grown in Rhizoctonia-infested fields, belonging to Bacillus amyloliquefaciens, B. thuringiensis, B. megaterium, B. subtilis, Enterobacter cloacae, Chryseobacterium jejuense, and Klebsiella pneumoniae was screened for its suppressive effects of Sclerotinia Stem Rot of tomato caused by Sclerotinia sclerotiorum and plant growth-promoting ability. The inhibitory effects of diffusible and volatile metabolites from these rhizobacteria against pathogen mycelial growth depended significantly upon strains tested. Growth inhibition caused by diffusible and volatile compounds was of about 37-57% and 24-54%, respectively. All strains tested had totally suppressed myceliogenic germination of sclerotia and improved germination of bacterized tomato seeds as compared to the untreated controls. The screening of their disease-suppressive and plant growth-promoting abilities revealed 72-100% decrease in Sclerotinia Stem Rot severity and significant increments in plant height by 52-67%, roots fresh weight by about 66-88% and aerial part weight by 47-75%, compared to S. sclerotiorum-inoculated and untreated control. The most promising strains combining disease-suppressive and growth-promoting abilities were B. subtilis B10 (KT921327) and B14 (KU161090), B. thuringiensis B2 (KU158884), B. amyloliquefaciens B13 (KT951658) and B15 (KT923051), and E. cloacae B16 (KT921429)

Transcriptional analysis of the interaction between the oomycete biocontrol agent, Pythium oligandrum, and the roots of Vitis vinifera L.

The oomycete, Pythium oligandrum, has received considerable attention as a potential biocontrol agent against various pathogenic fungi of plants, including grapevine. However, much remains underexplored on the interaction between grapevine and P. oligandrum. In the present study, following root colonization with P. oligandrum, grape-genome microarray analyses showed significant changes in the grapevine root transcriptome. The main changes concerned the genes involved in the biological processes and cellular functions, more particularly those associated with response to stimuli. Several functional categories were differentially expressed in P. oligandrum-inoculated plants. The highest inductions concerned the functional categories involved in secondary metabolism (terpenoid and flavonoid pathways), abscisic acid metabolism, resistance genes and some RNA regulation transcription factors. Redox state functional category was, however, significantly repressed. Whereas the expression of several transcripts would suggest that the plant sets up defense systems against the oomycete, certain similarities with symbiotic microorganism/root interactions were also observed, the main one being the stimulation of subtilases. These data provide new insights about the pathways involved in the establishment of the complex relationship between P. oligandrum, an oomycete with biocontrol potential, and grapevine

Pythium oligandrum: an example of opportunistic success

International audiencePythium oligandrum, a non-pathogenic soil-inhabiting oomycete, colonizes the root ecosystem of many crop species. Whereas most members in the genus Pythium are plant pathogens, P. oligandrum distinguishes itself from the pathogenic species by its ability to protect plants from biotic stresses in addition to promoting plant growth. The success of P. oligandrum at controlling soilborne pathogens is partly associated with direct antagonism mediated by mycoparasitism and antimicrobial compounds. Interestingly, P. oligandrum has evolved with specific mechanisms to attack its prey even when these belong to closely related species. Of particular relevance is the question of how P. oligandrum distinguishes between self- and non-self cell wall degradation during the mycoparasitic process of pathogenic oomycete species. The ability of P. oligandrum to enter and colonize the root system before rapidly degenerating is one of the most striking features that differentiate it from all other known biocontrol fungal agents. In spite of this atypical behaviour, P. oligandrum sensitizes the plant to defend itself through the production of at least two types of microbe-associated molecular patterns, including oligandrin and cell wall protein fractions, which appear to be closely involved in the early events preceding activation of the jasmonic acid- and ethylene-dependent signalling pathways and subsequent localized and systemic induced resistance. The aim of this review is to highlight the expanding knowledge of the mechanisms by which P. oligandrum provides beneficial effects to plants and to explore the potential use of this oomycete or its metabolites as new disease management strategies

Seaweed and microalgae as major actors of blue biotechnology to achieve plant stimulation and pest and pathogen biocontrol – a review of the latest advances and future prospects

International audienceAbstract Modern agriculture needs proper solutions to face the current trend of pesticides and fertilizers reduction. One of the available leverages to support this transition is the use of bioproducts that are more environmentally friendly and less hazardous for human health. Among them, blue biotechnology and more precisely seaweed and microalgae gain interest every year in the scientific community. In agriculture, seaweeds (Macroalgae) have been used in the production of plant biostimulants while microalgae still remain unexploited. Microalgae are widely described as renewable sources of biofuels, bioingredients and biologically active compounds, such as polyunsaturated fatty acids (PUFAs), carotenoids, phycobiliproteins, sterols, vitamins and polysaccharides, which attract considerable interest in both scientific and industrial communities. They affect agricultural crops for enhancement of plant growth, seedling growth. They can also improve nutrient incorporation, fruit setting, resistance properties against pests and diseases, improving stress management (drought, salinity and temperature). The present review aimed at the interest of blue biotechnology in agronomy, with a specific focus on microalgae, their biological activities and their possible application in agriculture as a potentially sustainable alternative for enhanced crop performance, nutrient uptake and resilience to environmental stress. This review does not only present a comprehensive study of microalgae as plant biostimulants but also as biofertilizers, with a particular emphasis on future challenges these solutions will have to deal with, microalgae being able to synthesize secondary metabolites with potential biopesticidal action

Draft genome sequence of biocontrol agent Pythium oligandrum strain Po37, an oomycota

The oomycota Pythium oligandrum Po37 is used as a biocontrol agent of plant diseases. Here, we present the first draft of the P. oligandrum Po37 genome sequence, which comprises 725 scaffolds with a total length of 35.9 Mb and 11,695 predicted protein-coding genes

Evaluation of the effectiveness of tomato-associated rhizobacteria applied singly or as three-strain consortium for biosuppression of Sclerotinia stem rot in tomato

In the present study, the capacity of three native tomato-associated rhizobacteria (Bacillus subtilis B2, B. thuringiensis B10, and Enterobacter cloacae B16) to suppress Sclerotinia Stem Rot in tomato and to improve growth was investigated in two tomato cultivars. The three bacterial strains were tested against S. sclerotiorum either singly or as consortium and their efficacy was compared to a fungicide control. All bacteria-based treatments were found to be more effective in suppressing disease than chemical fungicide on both cultivars and in both year trials. The disease-suppression and growth-promoting abilities of the treatments tested varied significantly depending on bacterial strains used, tomato cultivars grown, and year trial. Overall, all three strains suppressed the disease more effectively than the chemical fungicide. Indeed, for both year trials and cultivars combined, disease suppression potential, as compared to pathogen-inoculated and untreated control, ranged between 80.79 and 88.01% using the three-strain consortium relative to 70.00-82.07% achieved with single strains and 32.13-58.97% using fungicide. Plants grown in S. sclerotiorum-infected peat and challenged with the three-strain consortium were 38.36 to 80.95% taller than control ones whereas height increment noted using single strains and fungicide was of about 32.35- 79.01 and 29.62-51.85%, respectively. Aerial parts and root fresh weights of pathogen-inoculated and treated plants were enhanced by 51.59-74.69% and 54.00-78.12% using mixed strains and by 39.12-76.83% and 42.02-77.01%, respectively, using single strains compared to 24.04-53.05 and 12.74-67.05% noted on chemically treated plants. The effect of the three biocontrol agents was also examined on the composition of microbial communities inhabiting the rhizosphere of tomato plants. Results of the single strand conformational polymorphism (SSCP)-based profiling revealed that rhizosphere communities differed between cultivars only. However, the introduction of S. sclerotiorum or biocontrol agents did not cause detectable perturbations in the composition of fungal and bacterial communities inhabiting roots of treated tomato plants