Mixtures of Macro and Micronutrients Control Grape Powdery Mildew and Alter Berry Metabolites

Abstract

Powdery mildew caused by the fungus Erysiphe necator is a major grape disease worldwide. It attacks foliage and berries and reduces yield and wine quality. Fungicides are mainly used for combating the disease. Fungicide resistance and the global requisite to reduce pesticide deployment encourage the use of environment-friendly alternatives for disease management. Our field experiments showed that the foliar application of the potassium phosphate fertilizer Top-KP+ (1-50-33 NPK) reduced disease incidence on leaves and clusters by 15-65% and severity by 75-90%, compared to untreated vines. Top-KP+ mixed with Nanovatz (containing the micronutrients boron (B) and zinc (Zn)) or with TruPhos Platinum (a mixture containing N, P₂O₅, K₂O, Zn, B, Mg, Fe, Mn, Cu, Mo, and CO) further reduced disease incidence by 30-90% and disease severity by 85-95%. These fertilizers were as effective as the fungicide tebuconazole. Tank mixtures of fertilizers and tebuconazole further enhanced control efficacy in the vineyards. The modes of action of fertilizers in disease control were elucidated via tests with grape seedlings, microscopy, and berry metabolomics. Fertilizers applied preventively to the foliage of grape seedlings inhibited powdery mildew development. Application onto existing mildew colonies plasmolyzed mycelia and conidia and arrested the development of the disease. Berries treated with fertilizers or with a fungicide showed a significant increase in anti-fungal and antioxidant metabolites. Twenty-two metabolites, including non-protein amino acids and carbohydrates, known for their anti-fungal and bioactive effects, were significantly upregulated in grapes treated with fertilizers as compared to grapes treated with a fungicide, suggesting possible indirect activity against the pathogen. Esters and organic acids that contribute to wine quality were also upregulated. We conclude that integrating macro and micronutrients in spray programs in commercial vineyards shall control powdery mildew, reduce fungicide deployment, delay the buildup of fungicide resistance, and may improve wine quality.

Keywords: Erysiphe necator; Vitis vinifera; antioxidants; bio-stimulants; fertilizers therapy; integrated pest management; metabolomics; secondary metabolites.

Figure 1

PCA analysis for 840 compounds identified by LC-MS/MS in cv. Riesling grape skin tissues treated by the fungicide Folicur (tebuconazole) 0.02%, Top KP+ 1% plus Nanovatz 0.1%, or Top KP+ 1% plus TruPhos 0.25% and untreated grapes.

Figure 2

Relative amounts (peak area) of metabolites that were up-produced in grape skins of cv. Riesling vines treated with five foliar spays (starting at 14 April until 3 June) of the fungicide Folicur (tebuconazole) 0.02%, or the fertilizers Top KP+ 1% plus Nanovatz 0.1%, Top KP+ 1% plus TruPhos 0.25%, compared to untreated control vines. Analysis was conducted using LC–MS/MS. (A) the peptide Leu-Arg, (B) the peptide dihydroxy-phenylalanine, (C) the peptide Boc-Ser-OH, (D) the peptide Boc-Gln-OH, (E) the peptide L-gamma-Glutamyl-L-leucine, (F) Pheophorbide A, (G) Tuliposide B, (H) Nocardicin E. Different letters represent significant differences (p <  0.05, Tukey-Kramer HSD test).

Figure 3

Relative amounts (peak area) of metabolites that were up-produced in grape skins of cv. Riesling untreated control vines, compared to vines treated with five foliar spays (starting at 14 April until 3 June) of the fungicide Folicur (tebuconazole) 0.02%, or the fertilizers Top Kp+ 1% plus Nanovatz 0.1%, Top KP+ 1% plus TruPhos 0.25%. Analysis was conducted using LC–MS/MS. (A) Cis-Resveratrol, (B) Catechin 3-O-gallate. Different letters represent significant differences (p <  0.05, Tukey-Kramer HSD test).

Figure 4

Heatmap and Hierarchical Cluster Analysis graph for all metabolites significantly (p < 0.05, Tukey-Kramer HSD test) up-produced or down-produced in grape skins of cv. Riesling in the fungicide Folicur (tebuconazole) 0.02% treatment samples and the fertilizers Top KP+ 1% plus Nanovatz 0.1%, or Top KP+ 1% plus TruPhos 0.25% treatments samples. The x-axis represents three repetitions of each treatment.

Figure 5

Relative amounts (peak area) of metabolites detected in grape skins of cv. Riesling vines treated with five foliar spays (starting at April 14 until June 3) of the fungicide Folicur (tebuconazole) 0.02%, Top KP+ 1% plus Nanovatz 0.1%, or Top KP+ 1% plus TruPhos 0.25%. Analysis was conducted using LC–MS/MS. (A) THP(A), (B) N~2~-(4-Aminobenzoyl) arginine, (C) L-Homoarginine, (D) 2-Deoxy-2-(methacryloylamino)-D-glucopyranose, (E) 6-O-Acetyl-D-glucose, (F) L-2-Hydroxyglutaric acid, (G) (2S)-2-(beta-D-Glucopyranosyloxy) succinic acid, (H) 4-O-Acetyl-D-galacturonic acid. Different letters represent significant differences (p <  0.05, Tukey-Kramer HSD test).

Figure 6

PCA analysis for 205 compounds identified by LC-MS/MS in cv. Carignan grape skin tissues treated with six foliar spays (starting at 6 April until 8 June) of the fungicide Folicur (tebuconazole) 0.02%, Top KP+ 1% plus Nanovatz 0.1%, or Top KP+ 1% plus TruPhos 0.25% and untreated grapes.

Figure 7

Heatmap and Hierarchical Cluster Analysis graph for all metabolites significantly (p < 0.05, TukeyKramer HSD test) up-produced or down-produced in grape skins of cv. Carignan in the fungicide Folicur (tebuconazole) 0.02%, the fertilizers Top KP+ 1% plus Nanovatz 0.1%, or Top KP+ 1% plus TruPhos 0.25% and the control untreated treatments samples. The X-axis represents three repetitions of each treatment.

Figure 8

Relative amounts (peak area) of metabolites that were up-produced in grape skins of cv. Carignan vines treated with six foliar spays (starting at April 6 until June 8) of the fungicide Folicur (tebuconazole) 0.02%, or the fertilizers Top KP+ 1% plus Nanovatz 0.1%, Top KP+ 1% plus TruPhos 0.25%, compared to control untreated vines. Analysis was conducted using LC–MS/MS. (A) Trans-3-Indoleacrylic acid, (B) Indole, (C) Phytosphingosine, (D) 6-hydroxysphing-4E-enine, (E) Limocitrin, (F) (±)-(2E)-Abscisic acid. Different letters represent significant differences (p < 0.05, Tukey-Kramer HSD test).

Figure 9

(A) The effect of Top KP+ 1% plus TruPhos 0.25% or Nanovatz 0.1% on conidial germination of E. necator in vitro. Bars represent the standard error of the mean. (B) Prophylactic activity of Top KP+ 1% plus TruPhos 0.25% or Nanovatz 0.1% in comparison to Vivando (metrafenone) 0.03% fungicide against grape powdery mildew on young plants. The percentage of infected leaf area on five leaves of each treated plant was recorded at various time intervals after foliar spray. Different letters indicate significant differences (p < 0.05) according to the Fisher’s LSD K-ratio t-test. (C) Germinating conidia on water agar. (D) Inhibition of conidial germination, and disrupted conidia with shrunken intra-cellular content on water agar embedded with Top KP+ 1% plus TruPhos 0.25%. (E) Inhibition of conidial germination, and disrupted conidia with shrunken intra-cellular content on water agar embedded with Top KP+ 1% plus Nanovatz 0.1%. Bar = 200 µm.

Figure 10

(A) Curative effect on existing powdery mildew colonies on young grape plants by a single application of Top KP+ 1%, and the mixture Top KP+ 1% plus TruPhos 0.25%. Different letters indicate significant differences (p < 0.05) according to the Fisher’s LSD K-ratio t-test; (B–G) Epifluorescent micrographs of hyphae and conidia of E. necator from curative experiment: (B–C) Normal conidiophores and hyphae with smooth hyphal walls from control water treated colony. (D–E) Disrupted conidiophores (marked by arrows), and shrinkage and disruption of hyphae (marked by arrows) on infected leaves exposed to a single application of Top KP+ 1% plus Nanovatz 0.1%. (F–G) Disrupted conidia and conidiophores, and deformed hyphal cells (marked by arrows) on infected leaves exposed to a single application of Top KP+ 1% plus TruPhos 0.25%. Samples for microscopy were taken 24 h post-application. Bar = 20 µm. (H–J) Light microscopy of E. necator conidia: (H) Normal conidia taken from colonies sprayed with water. (I) Deformed and shrunken conidia taken from colonies exposed to a single application of Top KP+ 1% plus Nanovatz 0.1%. (J) Deformed and shrunken conidia taken from colonies exposed to a single application of Top KP+ 1% plus TruPhos 0.25%. Samples for microscopy were taken 24 h post-application. Bar = 20 µm (×100 magnification), or 10 µm (×400 magnification).