Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, ( )-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) ( )-4-chloro-terremutin hydrate (4) and(+)-4- hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-( )-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)( )-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with ( )-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxinsinfo:eu-repo/semantics/publishedVersio

The microbiome of the leaf surface of Arabidopsis protects against a fungal pathogen

We have explored the importance of the phyllosphere microbiome in plant resistance in the cuticle mutants bdg (BODYGUARD) or lacs2.3 (LONG CHAIN FATTY ACID SYNTHASE 2) that are strongly resistant to the fungal pathogen Botrytis cinerea. The study includes infection of plants under sterile conditions, 16S ribosomal DNA sequencing of the phyllosphere microbiome, and isolation and high coverage sequencing of bacteria from the phyllosphere. When inoculated under sterile conditions bdg became as susceptible as wild-type (WT) plants whereas lacs2.3 mutants retained the resistance. Adding washes of its phyllosphere microbiome could restore the resistance of bdg mutants, whereas the resistance of lacs2.3 results from endogenous mechanisms. The phyllosphere microbiome showed distinct populations in WT plants compared to cuticle mutants. One species identified as Pseudomonas sp isolated from the microbiome of bdg provided resistance to B. cinerea on Arabidopsis thaliana as well as on apple fruits. No direct activity was observed against B. cinerea and the action of the bacterium required the plant. Thus, microbes present on the plant surface contribute to the resistance to B. cinerea. These results open new perspectives on the function of the leaf microbiome in the protection of plants

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses

Déterminisme du pouvoir protecteur de <em>Fusarium oxysporum</em> : recherche de gènes impliqués dans l'interaction protectrice avec la tomate

Diplôme : Dr. d'UniversitéFusarium oxysporum is a common soil borne fungus, well represented in every type of soils, throughout the world. This species includes pathogenic strains inducing severe diseases in many crops and strains able to protect a plant against the infection by a pathogenic strain. The protective strains are not only non pathogenic strains isolated from suppressive soils but also pathogenic strains applied to a non host plant. The protective capacity of these strains is mainly based on mechanisms of competition and induced resistance of the plant The main objective of this work was to identify fungal genes involved in the protective capacity of these strains and associated to the elicitation of plant defence mechanisms. The approach consisted in looking for genes differentially expressed during the interaction between tomato cell cultures and either a protective or a non protective strain of F. oxysporum. An experimental model was developed; it is based on a strain of F. oxysporum f. sp. melonis (Fom24) pathogenic on muskmelon (Fom24) and on its transposition mutant (rev157). The wild type strain has the capacity to protect flax and tomato against fusarium wilt, although rev157 has lost the protective capacity. This mutant being neither affected in its capacity to grow saprophytically, nor to colonize the plant roots, the hypothesis that it was affected in its capacity to elicitate the plant defence reactions was proposed. Indeed, when interacting with cell cultures it induced physiological events (H2O2 accumulation, cell death) different from that induced by the wild type strain Fom24. Having no a priori hypothesis regarding the functions of the genes affected by the mutation, a Rapid Subtraction Hybridization (RaSH) technique was used to identify genes differentially expressed during the interaction between tomato cell cultures and either the protective or the non protective strain of F. oxysporum. This technique enabled to identify sequences from both fungal and plant origin. Since data concerning fungal genes are scarce in Genbank, the most informative data concerned plant genes. Regarding fungi, the expression of two genes, a chitinase and a polyphenol oxydase respectively involved in the degradation of cell walls and the oxidative catalysis of phenols was analyzed by Northern blot. Regarding plant genes, no newly expressed sequence was detected, demonstrating that the protective interaction results from a differential regulation of genes already expressed during the non protective interaction between plant cells and conidia of F. oxysporum. The expression profile of 5 genes was analyzed by Northern blot and real time PCR. Two of them are involved in the plant response to pathogen infection (RIN4, chitinase), and the 3 others are involved in important functions related to metabolism and transport (ATPase, ferredoxin-NADP-reductase and porin). The low activity of the ferredoxin-NADP-reductase in the cells interacting with the protective strain can be correlated with the great of amount H2O2 produced by these cells. The most interesting result concerns the demonstration that RIN4, a gene implicated in a guard system, is involved in the interaction between cells of tomato of a susceptible variety and different strains of F. oxysporum. Depending on the genetic environment of the cell (presence or absence of resistance genes) this gene either favors the growth of the pathogen or stimulates the plant defence reactions. The gene RIN4 being down-regulated during the protective interaction, the plant defence reactions would be fully expressed.Au sein de l’espèce Fusarium oxysporum, on distingue des souches pathogènes provoquant des dégâts économiquement importants dans diverses cultures et des souches non pathogènes et protectrices. Ces dernières sont en effet capables de protéger les plantes auxquelles elles sont appliquées contre les formes pathogènes spécifiques de ces plantes. Outre les souches non pathogènes, généralement isolées de sols résistants, des souches pathogènes appliquées à une plante non hôte sont également capables de protéger cette plante contre la fusariose. Cette capacité de protection repose principalement sur des mécanismes de compétition et d’induction de résistance chez la plante. Dans le cadre de l’étude du déterminisme du pouvoir protecteur de certaines souches de F. oxysporum, l’objectif de ce travail est d’identifier des gènes fongiques associés à l’activation des mécanismes de défense de la plante et impliqués dans l’activité protectrice des souches de F. oxysporum. La démarche adoptée consiste à rechercher des gènes différentiellement exprimés lors de l’interaction de cultures cellulaires de tomate avec une souche protectrice et une souche non protectrice. Le modèle d’étude choisi est constitué d’une souche pathogène du melon F. oxysporum f. sp. melonis (Fom24) et de son mutant de transposition rev157. Alors que la souche sauvage Fom24 possède une capacité protectrice vis-à-vis de la fusariose du lin et de la tomate, son mutant rev157 présente une capacité protectrice significativement diminuée. Ce mutant n’étant affecté ni dans ses caractéristiques de croissance in vitro, ni dans sa capacité à coloniser les racines des plantes, l’hypothèse selon laquelle il est affecté dans sa capacité à induire les réactions de défense de la plante a été formulée. En interaction avec des cultures cellulaires, il induit des évènements physiologiques (accumulation de H2O2, mort des cellules) différents de ceux induits par la souche sauvage Fom24. N’ayant aucun a priori quant aux gènes affectés par la mutation, une méthode d’hybridation soustractive rapide a été mise en œuvre pour identifier des gènes différentiellement exprimés lors de l’interaction entre des cultures cellulaires de tomate et les souches de F. oxysporum. Cette méthode a permis d’identifier des séquences d’origine fongique et végétale ; la caractérisation de ces dernières étant plus facile en raison de l’abondante information disponible dans les banques de données. Concernant les champignons, nous avons analysé par Northern blot l’expression de deux gènes de fonction connue impliqués dans la dégradation de la paroi cellulaire (chitinase) ou dans la catalyse oxydative des phénols (polyphénol oxydase). Concernant les gènes de plante, aucune séquence nouvellement exprimée n’a été détectée, démontrant que l’interaction protectrice résulte d’une régulation différentielle de gènes également exprimés lors de l’interaction non protectrice. Le profil d’expression de cinq gènes a été confirmé par Northern blot et RT-PCR en temps réel. Deux d’entre eux sont impliqués dans la réponse de la plante à l’attaque d’un agent pathogène (RIN4, chitinase) et les trois autres correspondent à des fonctions importantes dans le métabolisme ou le transport (sous-unité β de l’ATP synthase, ferredoxine-NADP réductase, porine). La faible activité de la ferredoxine-NADP réductase lors de l’interaction des cellules avec la souche protectrice peut être corrélée à la quantité importante d’H2O2 produite par ces cellules. Le résultat le plus original concerne la mise en évidence du gène RIN4 impliqué dans un système de garde, lors de l’interaction entre des cellules de tomate d’une variété sensible et des souches de F. oxysporum protectrice on non. Selon le contexte génétique (présence ou non de gène de résistance) ce gène contribue en effet à favoriser le développement de l’agent pathogène ou au contraire à stimuler les défenses de la plante. Le gène RIN4 étant sous exprimé lors de l’interaction protectrice, les réactions de défense peuvent s’exprimer pleinement

Déterminisme du pouvoir protecteur de Fusarium oxysporum (recherche de gènes impliqués dans l interaction protectrice avec la tomate)

L objectif de ce travail est d identifier des gènes fongiques impliqués dans l activité protectrice des souches de Fusarium oxysporum. Le modèle d étude choisi est constitué d une souche fongique (Fom24) capable de protéger la tomate et le lin contre la fusariose et de son mutant de transposition (rev157) qui a perdu sa capacité protectrice sur ces espèces végétales. N ayant aucun a priori quant aux gènes affectés par la mutation, une méthode d hybridation soustractive rapide a été mise en oeuvre pour identifier des gènes différentiellement exprimés lors de l interaction entre des cultures cellulaires de tomate et les souches de Fusarium oxysporum. Concernant les gènes fongiques obtenus nous avons approfondi l étude de deux gènes de fonction connue un précurseur de chitinase et une polyphénol oxydase. Concernant les gènes de plantes, le profil d expression de RIN4, une chitinase, une ATP synthase, une ferredoxine-NADP réductase et une porine ont été étudiées.The main objective of this work was to identify fungal genes involved in the protective capacity of Fusarium oxysporum. An experimental model is based on a fungal strain (Fom24) has the capacity to protect tomato and flax against fusarium wilt and on its transposition mutant (rev157) has lost the protective capacity. Having no a priori hypothesis regarding the functions of the genes affected by the mutation, a Rapid Subtraction Hybridization (RaSH) technique was used to identify genes differentially expressed during the interaction between tomato cell cultures and either the protective or the non protective strain of F. oxysporum. Regarding fungi, the expression profile of two genes, a chitinase precursor and a polyphenol oxydase was analyzed by Northern blot. Regarding plant genes, the expression profiles of RIN4, a chitinase, a ATPsynthase, a ferredoxin-NADP-reductase and a porin was analyzed by Northern blot and real time RT-PCR.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Lack of biocontrol capacity in a non-pathogenic mutant of Fusarium oxysporum f.sp. melonis

International audienceThe aim of this study was to assess the biocontrol capacity of rev157, a non-pathogenic mutant of a pathogenic strain of Fusarium oxysporum f. sp. melonis (Fom24). Inoculated in association with the virulent parental strain, the mutant rev157 did not protect the host plant (muskmelon) against infection by Fom24. Applied on flax, a non-host plant, the mutant rev157 was not able to protect it against its specific pathogen F. oxysporum f. sp. lini. On the contrary the parental strain Fom24 did protect flax as well as a soil-borne biocontrol strain (Fo47). Since the mutant rev157 was affected neither in its growth in vitro nor in its capacity to penetrate into the roots, it can be speculated that the mutation has affected traits responsible for interactions within the plant. In F. oxysporum the pair of strains Fom24/rev157 is a good candidate to identify genes involved in the biocontrol capacity of F. oxysporum and to test the hypothesis of a link between capacity to induce the disease and capacity to induce resistance in the plant

Déterminisme du pouvoir protecteur d’une souche de Fusarium oxysporum : recherches de gènes impliqués lors de l’interaction avec la tomate

National audienc

Determinism of the biocontrol capacity of a strain of <em>Fusarium oxysporum</em>: identification of genes expressed during interactions with tomato

International audienc

Newly Isolated Trichoderma spp. Show Multifaceted Biocontrol Strategies to Inhibit Potato Late Blight Causal Agent Phytophthora infestans both In Vitro and In Planta

Potato growers worldwide have been at war for more than 150 years with an enemy whose lifecycle, genome size and architecture, infection rate, and economic impacts are the epitome of a plant pathogen. Phytophthora infestans is an oomycete that causes the notorious late blight infection in potato and tomato fields. This study explored the benefits of the multitalented plant symbiotic fungi Trichoderma spp. and their metabolites as potential biopesticides against P. infestans. Eleven strains of Trichoderma spp. were obtained from soil and tree barks and were identified using DNA sequence analysis of three molecular markers. The antagonistic potential of the strains against P. infestans was first evaluated in vitro. In dual-culture assays, P. infestans growth was significantly inhibited (53 to 95%) by different Trichoderma spp. through direct mycoparasitism, competition for space and nutrients, or antibiosis. The cell-free filtrates (CFFs) of different Trichoderma strains were obtained and characterized for anti-Phytophthora activities as well as biochemical stability. The obtained results indicated that Trichoderma CFFs were chemically stable and strongly decreased P. infestans’ mycelial growth and zoospore motility and viability. Similarly, in leaf-disk assays, Trichoderma CFFs showed significant protection against P. infestans infection. Ultraperformance liquid chromatography analysis revealed the presence of harzianic acid, iso-harzianic acid, and 6-pentyl-2H-pyran-2-one as major compounds in different Trichoderma CFFs. Furthermore, selected Trichoderma strains significantly protected potato plants against soil-mediated late blight infection. Finally, Trichoderma spp. showed high compatibility with a copper-based fungicide, especially at lower concentrations, suggesting that both protective agents could be combined in integrated pest management programs. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Colonization of Tomato Root by Pathogenic and Nonpathogenic Fusarium oxysporum Strains Inoculated Together and Separately into the Soil

In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites