Le black-rot en recrudescence dans certaines parcelles de vignes

La saison 2022 a été relativement facile au niveau de la protection de la vigne en bio. Cependant une maladie fongique de la vigne a fait son apparition, le black-rot. L'article de journal s'axent sur la description et fonctionnement de la maladie fongique, les mesures préventives et les diverses méthodes de protection précoces à appliquer

Effect of sulfur deficiency on the resistance of oilseed rape to fungal pathogens and expression profiling of the glutathione S-transferase family of "Arabidopsis thaliana"

La réduction de la pollution atmosphérique due au dioxyde de soufre a provoqué au début des années nonante des problèmes de carence de soufre dans les champs principalement au nord de l’Europe. Le soufre est un macroélément essentiel pour le développement de la plante. Des composés soufrés tels que des protéines riches en soufre, des phytoalexines et des glucosinolates, jouent un rôle important dans les mécanismes de défense. Des plants de colza carencés en soufre, mais ne montrant aucun symptôme visible, présentaient une forte réduction de leur teneur en soufre total et en glutathion. Ces plantes montraient aussi une augmentation de leur susceptibilité au pathogène de la nécrose du collet, Leptosphaeria maculans, au nécrotrophe généraliste Botrytis cinerea ainsi qu’à l’oomycète Phytophthora brassicae. Dans le but d’analyser les causes de cette augmentation de susceptibilité nous avons testé le potentiel antifongique d’un extrait protéique et d’un extrait au méthanol contenant des métabolites secondaires. Ces extraits ont été obtenus à partir de colza cultivé avec et sans soufre, et préalablement induit ou non au moyen d’une inoculation avec Botrytis cinerea. L’extrait méthanolique obtenu à partir de plantes normales avait une activité antifongique qui était presque entièrement perdue dans le même extrait obtenu à partir de colza carencé en soufre. Cette perte d’activité antifongique corrélait avec une forte réduction du contenu en glucosinolates de l’extrait. Ceci suggère que la réduction du contenu en glucosinolates pourrait être la cause de la réduction du potentiel antifongique. Toutefois aucun lien de causalité n’a encore pu être démontré. L’affaiblissement général des plantes carencées pourrait aussi jouer un rôle additionnel dans la diminution de leur résistance aux pathogènes fongiques. Le métabolisme secondaire des végétaux contribue significativement à la défense de la plante contre divers stress environnementaux. Pour étudier les changements transcriptionels des gènes d’Arabidopsis thaliana impliqués dans les mécanismes de détoxification nous avons développé un «DNA array» (MetArray) contenant des sondes spécifiques pour chaque membre des familles de gènes impliquées dans les étapes successives de détoxification. Le MetArray contient des sondes pour la totalité des gènes codant pour des glycosyltransférases, pour 63 enzymes utilisant du glutathion ainsi que 62 cytochrome P450 monooxygénases et 28 ABC transporteurs. Leur expression a été analysée dans les différents organes de la plante ainsi que dans les feuilles suite à divers traitements tel que herbicides, irradiation avec des UV-B, hormones endogènes liées à la réponse au stress et infection avec un pathogène. Une analyse en composante principale basée sur le transcriptome de ces familles de gènes détermine des réponses distinctes. Les traitements avec le méthyljasmonate et l’éthylène forment un groupe. Le traitement avec l’acide salicylique deux herbicides sulfonylurée et l’inoculation avec une souche avirulente de Pseudomonas syringae en forment un autre. Enfin la réponse au bromoxynil un herbicide, et le traitement avec des UV-B forment un troisième groupe clairement séparé. Il faut relever un groupe de gènes qui était induit par le pathogène P. syringae mais qui ne montrait aucune réponse aux trois hormones liées aux voies de signalisation. De plus un petit groupe de gènes qui pourrait jouer un rôle dans les mécanismes de défense était régulé par diverses voies de signalisation convergentes. La régulation différenciée des divers gènes contredit l’hypothèse d’une grande redondance fonctionnelle dans ce quatre grandes familles de gènes. Les glutathion S-transférases (GST) sont des enzymes multifonctionnels qui sont codés par une famille de gènes. Le génome d’Arabidopsis thaliana contient 47 glutathion S-transférases. Une GST de la classe phi, GSTF8 (At2g47730) est induite par divers traitements dont le stress oxydatif et possède une forte activité GSHperoxydase. Nous avons montré que GSTF8 était localisée dans le chloroplaste en fusionnant le peptide signal de GSTF8 avec une protéine fluorescente (GFP). Comme GSTF8 est la seule GST présente dans le chloroplaste et qu’elle possède une activité GSH-peroxidase, elle constitue un candidat idéal pour une analyse fonctionnelle. Pour ce faire, une lignée transgénique surexprimant GSTF8 a été produite et un mutant insertionel (T-DNA) a été isolé. Toutefois aucun changement phénotypique n’a été observé aussi bien en croissance normale qu’après traitements avec du peroxyde d’hydrogène et du paraquat, induisant tout deux un stress oxydatif. Ceci indique que GSTF8 n’est soit pas impliqué dans la protection contre le stress oxydatif, soit qu’en plus de GSTF8 d’autres mécanismes contribuent à cette protection.The reduction of S02 atmospheric pollution in the early nineties caused sulfur-deficiency problems in the agriculture of northern Europe. Sulfur is essential for plant development and sulfur containing compounds such as sulfur rich antifungal proteins, phytoalexins and glucosinolates play an important role in plant defense against pathogens. Sulfur starved Brassica napus with no visible symptoms showed a strong decrease in total sulfur and glutathione content and an increased susceptibility to the blackleg fungus Leptosphaeria maculans, to the generalist necrotroph Botrytis cinerea and to the oomycete Phytophthora brassicae. To test the cause of this increased susceptibility, a methanol extract containing secondary metabolites and a water extract of soluble proteins of plants grown with and without sulfur fertilization were used in fungal growth inhibition tests. MeOH extract of normally grown plants showed strong antifungal activity and this activity was almost totally lost in extracts of S-starved plants. Plants preinoculated with B. cinerea did not contain an increased antifungal potential indicating that phytoalexins do not contribute to this activity. The loss of antifungal activity correlated with a strong reduction of the glucosinolate content of the methanol extract suggesting that the reduced level of glucosinolates might be the cause of the reduction of the antifungal potential. However, no causal link could yet be demonstrated. The general loss of fitness of sulfur-starved plants could play an additional important role in the reduction of resistance. Plant secondary metabolism significantly contributes to defense against adverse environmental cues. To investigate stress-induced alterations at the transcriptional level, a DNA array (MetArray) harboring gene specific probes was established, which combined Arabidopsis thaliana effector gene families encoding enzymes acting consecutively in secondary metabolism and defense reactions. It contained the complete set of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 28 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses. Methyl jasmonate and ethylene treatment was separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae. The responses to the herbicide bromoxynil and UV-B radiation were separate from both groups. A few genes were diagnostic in their specific response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. In addition, small groups of comprehensively induced effector genes may be part of defense mechanisms activated by several converging pathways. The differentiating expression patterns detected by the MetArray provide a framework of information regarding the function of individual genes and argue against widely redundant functions within the large gene families analyzed. Plant glutathione S-transferases are multifunctional enzymes encoded by a large gene family containing 47 members in Arabidopsis thaliana. A member of the Phi class GST, AtGSTF8 (At2g47730), is upregulated by various treatments including oxidative stress and exhibits GSH-peroxidase activity. The chloroplastic localisation of GSTF8 was demonstrated by expressing a fusion protein consisting of the predicted GSTF8 signal pepetide and GFP in transgenic Arabidopsis. Analysis of the GST family indicated that GSTF8 is the only chlorplastic GST in Arabidopsis, making it a promising candidate for functional analysis. To this end, GSTF8 over-expressing transgenic lines were produced and a T-DNA insertion knock out mutant was isolated from the SALK-collection. However, no change in phenotype could be seen under normal growth condition and under conditions of oxidative stresses conditions like treatments with hydrogen peroxide and the herbicide paraquat. This indicates that GSTF8 is either not involved in protection from oxidative stress in chloroplasts or, alternatively, that in addition to GSTF8 other mechanisms contribute to this protection

Sulphur deficiency causes a reduction in antimicrobial potential and leads to increased disease susceptibility of oilseed rape

The reduction of atmospheric sulphur dioxide pollution is causing increasing problems of sulphur deficiency in sulphur-demanding crop plants in northern Europe. Elemental sulphur and many sulphur containing compounds such as cysteine-rich antifungal proteins, glucosinolates (GSL) and phytoalexins play important roles in plant disease resistance. The aim of this work was to analyse the effect of inadequate sulphur supply on disease resistance of oilseed rape (Brassica napus). Compared with fertilized oilseed rape, healthy looking S-deficient plants showed increased susceptibility to the blackleg fungus Leptosphaeria maculans, to the generalist necrotroph Botrytis cinerea and to the oomycete Phytophthora brassicae. To analyse possible causes of the increased disease susceptibility of S-deficient plants, protein extracts and methanolic extracts of secondary metabolites of plants grown with and without adequate sulphur supply were tested for antimicrobial activity. None of the protein extracts showed antimicrobial activity. However, extracts containing secondary metabolites from normally grown plants showed a strong antimicrobial activity in in vitro tests with various fungal and bacterial pathogens. This activity was almost totally lost in extracts derived from S-deficient plants. The antimicrobial activity did not appear to be based on the activity of phytoalexins because it was present in healthy plants and was not increased by a previous inoculation with Botrytis cinerea. The loss of antifungal activity in S-deficient plants correlated with a strong reduction of various GSL, thus suggesting a reduced level of GSL as a possible cause of the reduced antimicrobial potential. However, limited tests of commercially available GSL or their degradation products did not demonstrate a causal link. Our results show that S-deficiency of oilseed rape negatively affects disease resistance and suggest that this effect is at least partially caused by a reduction of sulphur-dependent pytoanticipins

Détection des spores de mildiou et d’oïdium en temps réel à haute résolution spatiale

Réduire l’usage des produits phytosanitaires nécessite de traiter de manière sélective, tant dans l’espace que dans le temps, ce qui se traduit par une connaissance fine du risque d’infection à l’échelle de la parcelle. Nous présentons un réseau de stations de mesure en temps réel de la présence des spores de mildiou et d’oïdium dans l’air, grâce à une méthode de détection par laser. En identifiant la présence de spores avant qu'elles n'infectent les vignes et avec une précision de quelques centaines de mètres, ces instruments permettent de récolter de précieuses informations quantitatives sur le développement de l’infection. Cette quantification de la présence de spores pourrait être intégrée dans les modèles de prévision afin de disposer de nouveaux outils d'aide à la décision pour optimiser les mesures de lutte contre les maladies fongiques

Susceptibility to downy mildew in grape clusters: New microscopical and biochemical insights

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and ο-viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed. © 2011 Elsevier Masson SAS.We gratefully acknowledge the Juana de Vega Foundation (Spain) for its financial support.Peer Reviewe

Vitis vinifera Canes, a New Source of Antifungal Compounds against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea

Methanolic and ethanolic crude extracts of Vitis vinifera canes exhibited significant antifungal activity against the three major fungal pathogens affecting grapevines, Plasmopara viticola, Erysiphe necator and Botrytis cinerea. The active extracts were analyzed by LC-PDA−ESI-MS, and selected compounds were identified. Efficient targeted isolation using medium-pressure liquid chromatography afforded six pure constituents in one step. The structures of the isolated compounds were elucidated by NMR and HRMS. Six identified compounds (ampelopsin A, hopeaphenol, trans-resveratrol, ampelopsin H, ε-viniferin, and Evitisin B) presented antifungal activities against P. viticola. ε-Viniferin also exhibited a low antifungal activity against B. cinerea. None of the identified compounds inhibited the germination of E. necator. The potential to develop a novel natural fungicide against the three major fungal pathogens affecting V. vinifera from viticulture waste material is discussed

Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in <em>Vitis vinifera</em> tissues

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples. Methods and results: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola. Conclusion: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew. Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations

Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in Vitis vinifera tissues

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.Methods and results : Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specific for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola.Conclusion : A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations."Premiers Grands Crus" of BordeauxPeer reviewe

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato. The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions