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

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

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

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