Influence of Nitrogen on Grapevine Susceptibility to Downy Mildew

Abstract

Downy mildew, caused by the obligate parasite Plasmopara viticola, is one of the most important threats to viticulture. The exploitation of resistant and susceptibility traits of grapevine is one of the most promising ways to increase the sustainability of disease management. Nitrogen (N) fertilization is known for influencing disease severity in the open field, but no information is available on its effect on plant-pathogen interaction. A previous RNAseq study showed that several genes of N metabolism are differentially regulated in grapevine upon P. viticola inoculation, and could be involved in susceptibility or resistance to the pathogen. The aim of this study was to evaluate if N fertilization influences: (i) the foliar leaf content and photosynthetic activity of the plant, (ii) P. viticola infectivity, and (iii) the expression of the candidate susceptibility/resistance genes. Results showed that N level positively correlated with P. viticola infectivity, confirming that particular attention should be taken in vineyard to the fertilization, but did not influence the expression of the candidate genes. Therefore, these genes are manipulated by the pathogen and can be exploited for developing new, environmentally friendly disease management tools, such as dsRNAs, to silence the susceptibility genes or breeding for resistance.

Keywords: Vitis vinifera; nitrogen fertilization; oomycete; plant pathogen interaction; resistance genes; susceptibility genes.

Figure 1

(A) Box-plot distribution of nitrogen (N) content (%) at different Ca(NO₃)₂ concentrations. Different letters indicate significant differences (p < 0.05) in N (%) among treatments within timepoint. (B) Scatter plot showing the positive linear correlation existing between N content (%) and sporulated area (SA; mm²).

Figure 2

Violin plot distribution of (A) maximum quantum yield (MQY) without acclimatization and (B) disease severity (PSA) recorded at T3 in the leaf discs sampled from plants treated with different Ca(NO₃)₂ concentrations (0, 0.05, 1, 2 and 5 mM). The width of the plot is proportional to the estimated density.

Figure 3

Scatter plot showing the positive linear correlation existing between sporangia production (sporangia mm^–2) and sporulated area (mm²). Grey shade represents confidence interval.

Figure 4

P. viticola structures inside leaves sampled at T3 from plants that were fertilized with 2 mM nitrogen (A) or not fertilized (B–D) and observed at 7 dpi (day post inoculation). (A) Mycelium with haustoria regularly elongating and branching in the mesophyll cells. (B) Short portions of mycelium departing from infected stomata with a sporangiophore. (C,D) Short hypha with a clearly visible haustorium. S = stoma; M = mycelium; HA = haustorium; SP = sporangiophore. Scale bar = 50 µm.

Figure 5

Relative expression level (2^−ΔΔCt method) of genes involved in the N metabolism detected in leaves of Pinot noir plants grown under different N concentrations (0, 0.05, 1, 2 and 5 mM Ca(NO₃)₂) at different timepoints (T1, T2 and T3). (A) Nitrate transporter (VvNRT3.1). (B) Glutamine synthetase (VvGS). (C) Asparagine synthetase (VvAS1). Bars represent the standard deviation. Different letters indicate statistically significant differences.

Figure 6

Scheme of the utilization of the leaves. (A) Five leaves (A, B, C, D, and E) were sampled for each treatment. Two leaf discs (A1-2, B1-2 and C1-2) were cut from leaves A–C, while three leaf discs (D1-3 and E1-3) were sampled from leaves D and E. (B) The leaf discs were placed, lower surface upwards, in a Petri dish for experimental inoculation with P. viticola. The leaf tissues remaining from leaves A–C were used for N quantification (C) while those remaining from leaves D and E were used for gene expression analysis (D).