Maryline Magnin-Robert

Biological control of Botrytis cinerea by selected grapevine-associated bacteria and stimulation of chitinase and β-1,3 glucanase activities under field conditions

In this study, the biocontrol ability of seven grapevine-associated bacteria, previously reported as efficient against Botrytis cinerea under in vitro conditions, was evaluated in two vineyard orchards with the susceptible cv. Chardonnay during four consecutive years (2002–2005). It was shown that the severity of disease on grapevine leaves and berries was reduced to different levels, depending on the bacterial strain and inoculation method. Drenching the plant soil with these bacteria revealed a systemic resistance to B. cinerea, even without renewal of treatment. Accordingly, this resistance was associated with a stimulation of some plant defense responses such as chitinase and β-1,3-glucanase activities in both leaves and berries. In leaves, chitinase activity increased before veraison (end-July) while β-1,3-glucanase reached its maximum activity at ripening (September). Reverse patterns were observed in berries, with β-1,3-glucanase peaking at full veraison (end-August) and chitinase at a later development stage. Highest activities were observed with Acinetobacter lwoffii PTA-113 and Pseudomonas fluorescens PTA-CT2 in leaves, and with A. lwoffii PTA-113 and Pantoea agglomerans PTA-AF1 in berries. These results have demonstrated an induced protection of grapevine against B. cinerea by selected bacteria under field conditions, and suggest that induced resistance could be related to a stimulation of plant defense reactions in a successive manner.

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 phytotoxins.

Physiological changes in green stems of Vitis vinifera L. cv. Chardonnay in response to esca proper and apoplexy revealed by proteomic and transcriptomic analyses.

Among grapevine trunk diseases, esca proper and apoplexy commonly represent a threat for viticulture worldwide. To retrieve further information about the mechanisms activated in apoplectic and esca proper-affected plants, a two-dimensional gel electrophoresis (2-DE) based analysis was conducted on green stems from 26-year-old standing vines. Symptomatic and asymptomatic stems from both apoplectic (A) and esca proper-affected (E) plants compared to control (without visual symptom since 10 years) stems were studied. Thirty-three differentially expressed proteins were identified by nanoLC-MS/MS and included into three groups conceptually defined as proteins involved in (i) metabolism and energy, (ii) stress tolerance, and (iii) defense response. For nine of them, expression of the relative mRNAs was also monitored by qRT-PCR. Proteome variations were specifically related to apoplexy and esca proper but were more similar in asymptomatic stems than in the symptomatic ones. Remarkable quantitative differences were noted for several proteins in symptomatic stems according to the expressed form, A and E. Results further indicate that similar responses are likely activated in asymptomatic stems but a various quantitative expression is triggered upon onset of apoplexy or esca proper symptoms while both kind of plants are infected by the same pathogenic fungi.

Flowering as the Most Highly Sensitive Period of Grapevine (Vitis vinifera L. cv Mourvèdre) to the Botryosphaeria Dieback Agents Neofusicoccum parvum and Diplodia seriata Infection

Botryosphaeria dieback is a fungal grapevine trunk disease that currently represents a threat for viticulture worldwide because of the important economical losses due to reduced yield of affected plants and their premature death. Neofusicoccum parvum and Diplodia seriata are among the causal agents. Vine green stems were artificially infected with N. parvum or D. seriata at the onset of three different phenological stages (G stage (separated clusters), flowering and veraison). Highest mean lesion lengths were recorded at flowering. Major proteome changes associated to artificial infections during the three different phenological stages were also reported using two dimensional gel electrophoresis (2D)-based analysis. Twenty (G stage), 15 (flowering) and 13 (veraison) differentially expressed protein spots were subjected to nanoLC-MS/MS and a total of 247, 54 and 25 proteins were respectively identified. At flowering, a weaker response to the infection was likely activated as compared to the other stages, and some defense-related proteins were even down regulated (e.g., superoxide dismutase, major latex-like protein, and pathogenesis related protein 10). Globally, the flowering period seemed to represent the period of highest sensitivity of grapevine to Botryosphaeria dieback agent infection, possibly being related to the high metabolic activity in the inflorescences.

Effectiveness of beneficial bacteria to promote systemic resistance of grapevine to gray mold as related to phytoalexin production in vineyards

Background and aimsThe rhizospheric Bacillus subtilis PTA-271 (271) and endophytic Pseudomonas fluorescens PTA-CT2 (CT2) and Pantoea agglomerans PTA-AF2 (AF2) bacteria are able to induce systemic resistance (ISR) in grapevine against B. cinerea, but ISR markers and their costs remained unknown in vineyards. In this study, we investigated the relationship between the effectiveness of single and binary combinations of selected bacteria to induce ISR and their ability to trigger phytoalexin accumulation, as a potential marker for disease resistance, in leaves and berries, as well as their impact on grape yield in vineyards.MethodsGrapevine plants were treated during 2006 in two vineyards by drenching soil with single or binary mixtures of bacteria. Induced resistance against B. cinerea was evaluated and stilbenic phytoalexins were analyzed by HPLC in both leaves and berries. Grape yield was also assessed as number and weight of clusters at ripening.ResultsBoth single and mixtures of bacteria were effective in reducing gray mold severity in the leaves and berries in vineyards. Disease control was accompanied by a significant accumulation of stilbenic phytoalexins, trans-resveratrol and ε-viniferin, in both leaves and berries in the bacterized plants. δ-Viniferin also accumulated, but only in berries of the treated plants. Reduction of disease symptoms and accumulation of resveratrol and viniferins were higher in the plants treated with single CT2 compared to AF2 and 271. Treatment of grapevine plants with binary mixtures of these isolates resulted in a significant performance of CT2+AF2 in leaves and CT2+271 in berries. On the other hand, bacterial treatments did not show any negative effect on grape yield.ConclusionsThese results revealed the efficacy of CT2 alone or in combination with AF2 or with 271 in triggering grapevine resistance against B. cinerea and enhancing systemic accumulation of resveratrol and viniferins, without compromising grape yield.

Changes in plant metabolism and accumulation of fungal metabolites in response to Esca proper and apoplexy expression in the whole grapevine

Trunk diseases have become among the most important grapevine diseases worldwide. They are caused by fungal pathogens that attack the permanent woody structure of the vines and cause various symptoms in woody and annual organs. This study examined modifications of plant responses in green stem, cordon, and trunk of grapevines expressing Esca proper (E) or apoplexy (A) event, which are the most frequent grapevine trunk disease symptoms observed in Europe. Transcript expression of a set of plant defense- and stress-related genes was monitored by quantitative reverse-transcription polymerase chain reaction while plant phytoalexins and fungal metabolites were quantified by high-performance liquid chromatography-mass spectrometry in order to characterize the interaction between the grapevine and trunk disease agents. Expression of genes encoding enzymes of the phenylpropanoid pathway and trans-resveratrol content were altered in the three organs of diseased plants, especially in the young tissues of A plants. Pathogenesis-related proteins and the antioxidant system were severely modulated in A plants, which indicates a drastic stress effect. In the meantime, fungal polyketides 6-MSA, (R)-mellein, and (3R,4R)-4-hydroxymellein, were accumulated in A plants, which suggests their potential effect on plant metabolism during the appearance of foliar symptoms.

Alterations in Grapevine Leaf Metabolism Occur Prior to Esca Apoplexy Appearance

Esca disease is one of the major grapevine trunk diseases in Europe and the etiology is complex, since several inhabiting fungi are identified to be associated with this disease. Among the foliar symptom expressions, the apoplectic form may be distinguished and characterized by sudden dieback of shoots, leaf drop, and shriveling of grape clusters in a few days that can ultimately induce the plant death. To further understand this drastic event, we conducted transcriptomic and metabolomic analyses to characterize responses of leaves during the period preceding symptom appearance (20 and 7 days before foliar symptom expression) and at the day of apoplexy expression. Transcriptomic and metabolomic analyses provide signatures for the apoplectic leaves and most changes concerning the metabolism of carbohydrates, amino acids, and phenylpropanoids. In deciphering glutathione-S-transferase (GST), its preferential location in phloem, correlated with the upregulation of GST genes and a decrease of the glutathione level, offers further support to the putative role of glutathione during apoplexy expression.