Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization

<div><p>Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in <i>Botryosphaeriaceae</i> fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of <i>Neofusicoccum parvum</i> and <i>Diplodia seriata</i>, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that <i>Botryosphaeriaceae</i> were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. <i>N</i>. <i>parvum</i> is able to degrade parietal polysaccharides, whereas <i>D</i>. <i>seriata</i> has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on <i>N</i>. <i>parvum</i> Bourgogne S-116. We further show that <i>Botryosphaeriaceae</i> are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for <i>D</i>. <i>seriata</i>. In conclusion, the different <i>Botryosphaeriaceae</i> isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable <i>Botryosphaeriaceae</i> to bypass chemical and physical barriers of the grapevine plant. The specific signature of <i>Botryosphaeriaceae</i> aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.</p></div

Resveratrol metabolization by <i>Botryosphaeriaceae</i> fungi.

<p>Resveratrol (initially added at 50 μM) was quantified by LC-MS in PDA medium extract of <i>N</i>. <i>parvum</i> Bourgogne S-116, <i>N</i>. <i>parvum</i> Bt-67 and <i>D</i>. <i>seriata</i> 98.1. The resveratrol quantity is expressed in relative quantity (% of control) compared to control medium without fungi at different time points (3, 6 and 11 days; A), or when fungi have saturated 95% of the Petri Dish surface (B). Values are means and SD of three biological replicates, each calculated from the mean of three technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0.05 (Tukey Contrasts).</p

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization - Fig 2

<p><b>(A) Laccase activity.</b> Oxidation of ABTS in the radical cation ABTS^.+ was measured with total secreted proteins from <i>N</i>. <i>parvum</i> Bourgogne S-116, <i>N</i>. <i>parvum</i> Bt-67 and <i>D</i>. <i>seriata</i> 98.1. Total proteins were isolated from culture filtrates of fungi grown in malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). <b>(B) Manganese peroxidase activity.</b> Oxidation coupling of MBTH/DMAB was measured in presence of H₂O₂ and Mn²⁺. Total proteins were isolated from culture filtrates of fungi grown in liquid malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). Values are means and SD of three biological replicates, each calculated from the mean of two technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0,05 (Tukey Contrasts).</p

Effect of resveratrol on <i>Botryosphaeriaceae</i>.

<p>The resveratrol was tested at a final concentration of 50 μM (grey lines) and 250 μM (gray dotted lines) on mycelial growth of <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C). Negative control was performed by adding DMSO instead of resveratrol. Values are means and SD of two biological replicates, each calculated from the mean of three technical replicates. Means with a * are significantly different from control at <i>p</i> ≤ 0,05 (Tukey Contrasts).</p

δ-viniferin metabolization by <i>Botryosphaeriaceae</i> fungi.

<p>δ-viniferin (initially added at 50 μM) was quantified by LC-MS in PDA medium extract of <i>N</i>. <i>parvum</i> Bourgogne S-116, <i>N</i>. <i>parvum</i> Bt-67 and <i>D</i>. <i>seriata</i> 98.1. The δ-viniferin quantity is expressed in relative quantity (% of control) compared to control medium without fungi at different time points (3, 6 and 11 days: A), or when fungi have saturated 95% of the Petri Dish surface (B). Values are means and SD of three biological replicates, each calculated from the mean of three technical replicates. Means with a same latter are not significantly different at <i>p</i>≤0,05 (Tukey Contrasts).</p

Effect of δ-viniferin on <i>Botryosphaeriaceae</i>.

<p>The δ-viniferin was tested at a final concentration of 50 μM (grey lines) and 250 μM (gray dotted lines) on <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C) growth. δ-viniferin was dissolved in DMSO and negative control was performed by adding DMSO alone. Values are means and SD of two biological replicates, each calculated from the mean of three technical replicates. Means with a * are significantly different from control at <i>p</i> ≤ 0.05 (Tukey Contrasts).</p

Polysaccharide degrading enzymatic activities.

<p>Glucose liberation resulting from cellulase, hemicellulase and total activities of secreted proteins from <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C), was measured with a colorimetric method using dinitrosalicylic acid (DNS). Total proteins were isolated from culture filtrates of fungi grown in liquid malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). Values are means and SD of three biological replicates, each calculated from the mean of three technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0.05 (Tukey Contrasts).</p

Table_2_Wood degradation by Fomitiporia mediterranea M. Fischer: Physiologic, metabolomic and proteomic approaches.DOCX

Fomitiporia mediterranea (Fmed) is one of the main fungal species found in grapevine wood rot, also called “amadou,” one of the most typical symptoms of grapevine trunk disease Esca. This fungus is functionally classified as a white-rot, able to degrade all wood structure polymers, i.e., hemicelluloses, cellulose, and the most recalcitrant component, lignin. Specific enzymes are secreted by the fungus to degrade those components, namely carbohydrate active enzymes for hemicelluloses and cellulose, which can be highly specific for given polysaccharide, and peroxidases, which enable white-rot to degrade lignin, with specificities relating to lignin composition as well. Furthermore, besides polymers, a highly diverse set of metabolites often associated with antifungal activities is found in wood, this set differing among the various wood species. Wood decayers possess the ability to detoxify these specific extractives and this ability could reflect the adaptation of these fungi to their specific environment. The aim of this study is to better understand the molecular mechanisms used by Fmed to degrade wood structure, and in particular its potential adaptation to grapevine wood. To do so, Fmed was cultivated on sawdust from different origins: grapevine, beech, and spruce. Carbon mineralization rate, mass loss, wood structure polymers contents, targeted metabolites (extractives) and secreted proteins were measured. We used the well-known white-rot model Trametes versicolor for comparison. Whereas no significant degradation was observed with spruce, a higher mass loss was measured on Fmed grapevine culture compared to beech culture. Moreover, on both substrates, a simultaneous degradation pattern was demonstrated, and proteomic analysis identified a relative overproduction of oxidoreductases involved in lignin and extractive degradation on grapevine cultures, and only few differences in carbohydrate active enzymes. These results could explain at least partially the adaptation of Fmed to grapevine wood structural composition compared to other wood species, and suggest that other biotic and abiotic factors should be considered to fully understand the potential adaptation of Fmed to its ecological niche. Proteomics data are available via ProteomeXchange with identifier PXD036889.</p

Changes in the Triterpenoid Content of Cuticular Waxes during Fruit Ripening of Eight Grape (Vitis vinifera) Cultivars Grown in the Upper Rhine Valley

Triterpenoids present in grape cuticular waxes are of interest due to their potential role in protection against biotic stresses, their impact on the mechanical toughness of the fruit surface, and the potential industrial application of these biologically active compounds from grape pomace. The determination of the triterpenoid profile of cuticular waxes reported here supplements existing knowledge of the chemical diversity of grape, with some compounds reported in this species for the first time. Common compounds identified in eight examined cultivars grown in the Upper Rhine Valley include oleanolic acid, oleanolic and ursolic acid methyl esters, oleanolic aldehyde, α-amyrin, α-amyrenone, β-amyrin, cycloartanol, 24-methylenecycloartanol, erythrodiol, germanicol, lupeol accompanied by lupeol acetate, campesterol, cholesterol, sitosterol, stigmasterol, and stigmasta-3,5-dien-7-one, whereas 3,12-oleandione was specific for the Muscat d’Alsace cultivar. Changes in the triterpenoid content of cuticular waxes were determined at three different phenological stages: young grapes, grapes at véraison (the onset of ripening), and mature grapes. The results reveal a characteristic evolution of triterpenoid content during fruit development, with a high level of total triterpenoids in young grapes that gradually decreases with a slight increase in the level of neutral triterpenoids. This phenomenon may partially explain changes in the mechanical properties of the cuticle and possible modulations in the susceptibility to pathogens of mature grapes