DataSheet_1_Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.pdf

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.</p

Priming of ROS and HR-like cell death are key defenses during PS3-IR to <i>P. viticola</i>.

<p><b>A, B.</b> Primed transcript accumulation of genes encoding a NADPH-oxidase (<i>RbohD</i>) and a HR-related lipase (<i>HSR203J</i>) revealed by qPCR in plants treated with PS3 (5 g l⁻¹) or Adj (0.05%) at 0, 1 and 2 dpi. Means of relative expression after the two treatments derived from four independent biological experiments were compared within a timepoint. Asterisks indicate significant differences using unpaired heteroscedastic Student's <i>t</i>-test (<i>P</i><0.05). The 2^nd and 3^rd youngest full-sized leaves of four plants were sampled and combined per treatment and per timepoint. The relative expression in Adj at 0 dpi was set as 1 and the others were adjusted accordingly. The error bars represent the standard error of the mean. <b>C.</b> Diphenylene iodonium (DPI) partly abolishes the PS3-IR to <i>P. viticola</i> in grapevine. Leaf disks were treated during 1 day with 10 µM DPI, washed and then treated with 2.5 g l⁻¹ PS3 during 1 day, washed and, finally inoculated with <i>P. viticola</i>. Leaf sporulating area evaluated at 8 dpi. Different letters indicate statistically significant differences (<i>P</i><0.05; ANOVA followed by LSD test). Data are representative of three independent experiments (n = 3). <b>D.</b> Microscopic analyses on the same grapevine leaf discs show that DPI inhibits the primed H₂O₂ production (black arrows and inset) and callose deposition (white arrows and inset) during PS3-IR, leading to <i>P. viticola</i> spreading. Aniline blue and DAB staining were realized to detect callose and H₂O_2, respectively. Pictures are representative of three independent experiments. Bar = 100 µm.</p

PS3- and Lam-treated plants share a common stress transcriptome that partly overlaps the SA- and JA-dependent ones.

<p><b>A.</b> Hierarchical clustering analysis of genes expressed in plants sprayed with PS3 (5 g l⁻¹), Lam (5 g l⁻¹), salicylic acid (SA, 1 mM), or jasmonic acid (JA, 40 µM), 12 h post-treatment (hpt). Three independent biological experiments were performed (n = 3) and hierarchical clustering was realized with TIGR MeV software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088145#pone.0088145-Saeed1" target="_blank">[42]</a> after normalization with the corresponding controls: adjuvant (0.05%) for PS3 and Lam and DMSO (0.1%) for SA and JA. <b>B.</b> Venn diagram showing specific and common genes regulated by PS3, Lam, SA or JA at 12 hpt (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088145#pone.0088145.s005" target="_blank">Table S2</a> for gene identifiers). <b>C.</b> Gene Ontology enrichment analysis of the PS3-up-regulated genes at 12 hpt realized with the AgriGO software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088145#pone.0088145-Du1" target="_blank">[36]</a>. An asterisk (*) implies statistical significance between PS3-induced transcripts <i>vs</i> reference transcripts (Fisher's exact test with Benjamini-Yekutieli false detection rate correction, <i>Q</i><0.05; Fold-change cut-off 2 used). A hash mark (#) indicates a significant difference between PS3-induced transcripts <i>vs</i> reference transcripts when a more stringent fold-change cut-off of 4 was used.</p

The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine's Induced Resistance against <i>Plasmopara viticola</i>

<div><p>Grapevine (<i>Vitis vinifera</i>) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The β-glucan laminarin (Lam) and its sulfated derivative (PS3) have been previously demonstrated to induce resistance in grapevine against downy mildew (<i>Plasmopara viticola</i>). However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR)-proteins and phytoalexin production, PS3 triggered grapevine resistance <i>via</i> a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i) the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii) grapevine immune responses primed by PS3 during <i>P. viticola</i> infection. Our results showed that i) PS3 was unable to elicit reactive oxygen species (ROS) production, cytosolic Ca²⁺ concentration variations, mitogen-activated protein kinase (MAPK) activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii) PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA) and jasmonate-(JA)-dependent ones. After <i>P. viticola</i> inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance.</p></div

Plasma membrane depolarization mediates the primed ROS production during the PS3-IR to <i>P. viticola</i>.

<p><b>A.</b> The anionic channels inhibitor glibenclamide (Gli, 200 µM) blocked the plasma membrane depolarization triggered by PS3 in grapevine cell suspensions revealed by the DIBAC₄ probe fluorescence. <b>B.</b> Sporulating areas indicate that Gli blocked the PS3-IR to <i>P. viticola</i> in grapevine leaf discs. Leaf discs were treated during 24 h with Gli (200 µM), washed and then treated with 2.5 g l⁻¹ PS3 during 24 h, washed and, finally inoculated with <i>P. viticola.</i> Leaf sporulating area evaluated at 8 dpi. Different letters indicate statistically significant differences (<i>P</i><0.05; ANOVA followed by LSD test). Data are representative of three independent experiments (n = 3). <b>C.</b> Microscopic analyses on the same grapevine leaf discs show that Gli inhibits the primed H₂O₂ production (black arrows) and callose deposition (white arrows) during PS3-IR, leading to <i>P. viticola</i> spreading. Aniline blue and DAB staining were realized to detect callose and H₂O_2, respectively. Pictures are representative of three independent experiments. Bar = 100 µm.</p

Effects of β-1,3 glucans on early signaling events in grapevine cells.

<p><i>V. vinifera</i> cell suspensions were treated with PS3 (2 g l⁻¹), Lam (2 g l⁻¹) or water (control). <b>A.</b> Cytosolic [Ca²⁺] variations in aequorin expressing grapevine cells. <b>B.</b> H₂O₂ production measured by luminol chemiluminescence assays. FWC: fresh weight of cells. <b>C.</b> MAPK activation revealed by western blotting using an antibody raised against active phosphorylated MAPK or total MAPK. <b>D.</b> Plasma membrane depolarization revealed by fluorescence of the DIBAC₄ probe in grapevine cells. RFU: relative fluorescence unit. Results are from one of three representative experiments (n = 3).</p