Chitosan and Laminarin as Alternatives to Copper for Plasmopara

Article pubs.acs.org/JAFC

Chitosan and Laminarin as Alternatives to Copper for Plasmopara viticola Control: Effect on Grape Amino Acid T. Garde-Cerdán,*,† V. Mancini,‡ M. Carrasco-Quiroz,§ A. Servili,‡ G. Gutiérrez-Gamboa,† R. Foglia,‡ E. P. Pérez-Á lvarez,† and G. Romanazzi*,‡ †

Instituto de Ciencias de la Vid y del Vino (CSIC-CAR-UR), Carretera de Burgos Km. 6, 26007 Logroño, Spain Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, 60121 Ancona, Italy § Centro Tecnológico de la Vid y el Vino, Facultad de Ciencias Agrarias, Universidad de Talca, Av. Lircay S/N, Talca, Chile ‡

S Supporting Information *

ABSTRACT: Copper fungicide use is limited by the European regulation; therefore, new strategies have been developed to prevent grapevine downy mildew (GDM). However, there is poor information about their effects on grape amino acid composition. This field trial aimed to evaluate the effect on grape amino acid composition of chitosan and of a mixture of laminarin and Saccharomyces extracts (LamE), applied in different strategies with copper hydroxide. The results showed that all the treatments applied to grapevines decreased the concentration of several amino acids. Moreover, treatments that have mostly decreased these compounds are those with copper hydroxide, especially when applied individually. LamE applied individually or alternately with copper hydroxide had the least negative effect on grape amino acid content. These results provide further information about the negative effects of copper on grape quality, which can be reduced when it is used in strategy with LamE or chitosan in GDM control. KEYWORDS: amino acids, Montepulciano, bordeaux mixture, cooper hydroxide, chitosan, laminarin

■

used in organic farming are limited to 6 kg ha−1 per year in most European countries, including France, Italy, and Spain.12 Therefore, the development and optimization of alternative strategies to reduce the use of classic chemical inputs for protection against diseases in grapevines is becoming a necessity.13 In response to this trend, several techniques have been developed to reduce the damage caused by P. viticola at grapevine plants, including the use of biostimulants, homeopathic products, acid clay-based products (bentotamnio), resistance inducers (chitosan, laminarin, and lignosulfonate), plant extracts (orange extract, propolis, and equisetum), and potassium bicarbonate, applied alone or alternately with copper-based formulation.4,14 Chitosan is one of the most studied elicitors used to control P. viticola and other grapevine pathogens, such as Botrytis cinerea and Erysiphe necator.15−17 This is a polycationic β-1,4linked-D-glucosamine polymer that forms a semipermeable film around plant tissues, inhibiting several pathogens, and induces defense response mechanisms in the host tissues.18,19 It has been shown via in vitro studies that chitosan oligomers of different molecular weight and degree of acetylation triggered an accumulation of phytoalexins, trans- and cis-resveratrol, and their derivatives ε-viniferin and piceid in grapevine leaves.16 Furthermore, chitosan applied with copper sulfate strongly induced an important phytoalexin accumulation, reducing the infections of B. cinerea and P. viticola on grapevine leaves.16

INTRODUCTION Grapevine downy mildew (GDM) is caused by the biotrophic oomycete Plasmopara viticola, and it is one of the most important and devastating diseases of grapevines worldwide.1,2 Most of the grapevine varieties in Europe are highly susceptible to this pathogen because in this part of the world the grapevine growth conditions are characterized by high humidity and abundant rainfall during the spring season.3 Therefore, adequate vineyard control with fungicides is crucial, mainly in rainy climate regions.1 Since more than half the amount of all synthetic fungicides applied in the European Union (EU) is used on grapes, during the last years, the trend has been to reduce their use, especially for the control of downy mildew, that can require more than 10 applications per season.4 In addition, in organic vineyards, GDM is mainly controlled by regular sprays with copper-based products such as copper hydroxide and Bordeaux mixture.5 The long-term use of copper-based fungicides against GDM had led to an accumulation of this heavy metal in vineyard topsoil in many European countries.6,7 The soil copper accumulation may induce phytotoxicity in grapevines. Root and shoot growth reduction as well as leaf number decrease and leaf edge chlorosis were observed in different soil types with additions of ≥400 mg Cu kg−1.8 Besides, heavy metal accumulation in soils negatively affects the agroecosystem sustainability. Copper residues caused decreases of earthworm populations, microbiological and enzymatic alterations, lowered the soil pH, and reduced grapevine growth.9−11 As a consequence of these problems, in organic agriculture, copper fungicide use is currently restricted by European Union Regulation 473/2002. Today, the copper-based formulations © 2017 American Chemical Society

Received: Revised: Accepted: Published: 7379

May 23, 2017 July 27, 2017 July 31, 2017 July 31, 2017 DOI: 10.1021/acs.jafc.7b02352 J. Agric. Food Chem. 2017, 65, 7379−7386

Article

Journal of Agricultural and Food Chemistry

Table 1. Strategies Adopted in the Application of Copper Formulations and Alternative Products (Bordeaux Mixture, Chitosan and Laminarin Applied with Microbial Extract of Saccharomyces spp), and Dates of the Grapevine Foliar Applications strategy A A A A B C D B C D

treatments/formulation application rate

application date (month/day)

Bordeaux mixture (5 kg ha−1) copper hydroxide (2.8 L ha−1) chitosan (5 kg ha−1) laminarin (1 L ha−1) + microbial extract of Saccharomyces spp. (2 L ha−1) copper hydroxide (2.8 L ha−1)/chitosan (5 kg ha−1) copper hydroxide (2.8 L ha−1)/chitosan (5 kg ha−1) chitosan (5 kg ha−1)/copper hydroxide (2.8 L ha−1) copper hydroxide (2.8 L ha−1)/laminarin (1 L ha−1) + microbial extract of Saccharomyces spp. (2 L ha−1) copper hydroxide (2.8 L ha−1)/laminarin (1 L ha−1) + microbial extract of Saccharomyces spp. (2 L ha−1) laminarin (1 L ha−1) + microbial extract of Saccharomyces spp. (2 L ha−1)/ copper hydroxide (2.8 L ha−1)

5/21, 5/21, 5/21, 5/21, 5/27, 5/21, 5/21, 5/27,

5/27, 5/27, 5/27, 5/27, 6/12, 5/27, 5/27, 6/12,

6/4, 6/12, 6/4, 6/12, 6/4, 6/12, 6/4, 6/12, 6/25, 7/8, 6/4, 6/12, 6/4, 6/12, 6/25, 7/8,

6/18, 6/18, 6/18, 6/18, 7/21 6/18 6/18 7/21

6/25, 6/25, 6/25, 6/25,

7/1, 7/1, 7/1, 7/1,

7/8, 7/8, 7/8, 7/8,

7/15, 7/15, 7/15, 7/15,

7/21, 7/21, 7/21, 7/21,

7/28 7/28 7/28 7/28

5/14, 5/21, 5/27, 6/4, 6/12, 6/18 5/14, 5/21, 5/27, 6/4, 6/12, 6/18

central-eastern Italy (latitude: 43°53′84.13″N; longitude: 13°48′15.85″E). The plants were spaced by 0.85 m in the rows, with 2.80 m between the rows, and they were grown according to the Guyot trellis system, leaving 10 buds per grapevine, with grass cover crop between the rows. The height of the fruiting shoot was 60 cm from the ground. The vineyard was not irrigated, the fertilizers were distributed banded under grapevine in the winter, and additional hedging was applied in spring and summer, as common practices for the area. Grapevine Treatments. Ten treatments were tested compared with an untreated control. Treatments were repeated weekly from mid-May until the end of July, with a total of 12 applications per year. Each plot consisted of six/seven grapevines along a row, and the treated rows were separated each from the other by an untreated row. The active ingredients and the application rates of the treatments are listed in Supplementary Table 1. Throughout the experimental period, the distribution of the compounds occurred by adopting 4 different application strategies (Table 1), having the aim to measure GDM control reducing of different magnitude the amount of copper to zero as compared to the standard approach used in organic viticulture. Following the strategy “A”, the plants of each plot were foliar sprayed with the correspond treatment: Bordeaux mixture, copper hydroxide and two natural compounds (chitosan or Laminarin + microbial extract of Saccharomyces spp. (LamE)). Following the strategy “B”, copper hydroxide was applied alternately with a natural compound; following the strategy “C”, copper hydroxide was used for the first 6 applications, while a natural compound was used for the following 6 applications; and with the strategy “D”, the order of the products applied was the inverse respect to the “C” strategy. A randomized block design with four replicates was used, the blocks were oriented perpendicularly regarding the field slope and the treatments were assigned to plots using a random-number generator (Excel; Microsoft Corp., Redmond, WA). Treatments began on 14 May, when the plants started to be sensitive to GDM infection. At the time of the first product application, plants were at the phenological stage of inflorescences swelling (BBCH 55) and the shoots were about 20 cm long. The treatments were distributed by the spraying of a volume equivalent to 1000 L ha−1, using a motorized backpack sprayer (Honda GX 25, 25 cc, 0.81 kW, Tokyo, Japan). Grapes were harvested at their optimum technological maturity, and then were destemmed and crushed. The oenological parameters were determined in the musts obtained. Aliquots of each sample were frozen at −20 °C in order to determine their free amino acids content. Must Oenological Parameters. The quality parameters of the musts, including the total soluble solids (°Brix), titratable acidity (g L−1 of tartaric acid), and pH, were determined through laboratory analysis carried out by Moncaro winery (Ancona, Italy).26,27 Moreover, on harvest day, the grape production per treatment was recorded. Analysis of Must Amino Acid. The must amino acids analysis was performed by the method described by Garde-Cerdán et al.28 Free

There is little information regarding the effect of chitosan application on amino acid composition in must from grapevines.20 In this report, chitosan application to Tempranillo grapevines decreased the concentration of several amino acids in grapes. Therefore, its application to the grapevines could have a significant effect on grape amino acid concentration in relation to GDM control. The β-1,3-glucan laminarin derived from the brown alga Laminaria digitata has been shown to be an efficient elicitor of defense responses in grapevine cells and plants and able to control B. cinerea and P. viticola development on infected plants.21 In addition, laminarin is involved in the induction of genes that encode various pathogenesis-related proteins with antimicrobial properties, acting as biostimulant in plants.22 However, its effect on grape amino acid composition has been little studied and its application to grapevines could provide more information about its effects. Must nitrogen composition plays an important role in final wine quality. Its composition affects yeast metabolism, fermentation kinetics, and the synthesis of fermentative volatiles such as higher alcohols, ethyl and acetate esters which contribute to the desirable wine aroma. From this, certain free amino acids found in the grape must are important precursors of fermentative volatile compounds from the yeast metabolism. Therefore, wine composition is strongly affected by the must amino acid content and composition.23 In fact, a deficient nitrogen concentration, related to a low amino acid concentration in the must, may cause stuck or sluggish fermentations, which is a persistent problem in wine production.24 However, an excessive nitrogen concentration, related to a high amino acid concentration, resulting from excessive nitrogen fertilization, delays fruit maturation, increases berry weight, and decreases considerably the anthocyanin content.25 Therefore, the aim of this study was to evaluate the effect on grape amino acid composition of weekly foliar applications of natural compounds for the control of GDM to Montepulciano grapevines, individually applied or in strategies with copper formulations. The compounds evaluated were Bordeaux mixture, copper hydroxide, laminarin with Saccharomyces spp. extracts (LamE), and chitosan.

■

5/14, 5/14, 5/14, 5/14, 5/14, 5/14, 5/14, 5/14,

MATERIALS AND METHODS

Study Site. The experimental field trials were conducted in 2015 in a 6-year-old commercial vineyard of Vitis vinifera L. cv. Montepulciano, grafted onto 420A rootstock, located at Montesicuro (Ancona), in 7380

DOI: 10.1021/acs.jafc.7b02352 J. Agric. Food Chem. 2017, 65, 7379−7386

Article


Table 2. Grape Production and Oenological Parameters from Grapevines of the Untreated Control and from Those Treated with the Different Formulationsa treatment (strategy)b Bordeaux mixture (A) copper hydroxide (A) chitosan (A) LamE (A) Cu + chitosan (B) Cu + chitosan (B) chitosan + Cu (D) Cu + LamE (B) Cu + LamE (C) LamE + Cu (D) control

production vine (Kg) 6.45 7.65 4.89 6.53 8.15 7.20 6.45 7.79 6.64 6.99 6.17

± ± ± ± ± ± ± ± ± ± ±

sugar content (% Brix)

1.65 2.36 0.66 1.17 3.12 0.93 0.74 1.50 1.94 1.09 1.83

21.50 22.61 22.11 21.70 22.44 22.67 22.47 22.08 21.49 22.20 22.31

± ± ± ± ± ± ± ± ± ± ±

0.76 0.91 1.22 0.53 1.78 0.55 1.24 0.85 1.10 0.30 0.38

total acidity (g L−1)c 3.96 4.32 3.85 3.66 4.23 3.85 4.11 4.35 4.09 4.16 3.80

± ± ± ± ± ± ± ± ± ± ±

0.45 0.59 0.10 0.29 0.43 0.28 0.32 0.70 0.39 0.35 0.57

pH 3.31 3.25 3.26 3.33 3.30 3.28 3.31 3.29 3.27 3.28 3.38

± ± ± ± ± ± ± ± ± ± ±

0.04 0.04 0.04 0.05 0.03 0.03 0.08 0.06 0.03 0.07 0.10

Data are means ± standard deviation (n = 4). b(A) = strategy A; LamE = laminarin applied with microbial extract of Saccharomyces spp.; Cu = copper hydroxide; (B) = strategy B; (C) = strategy C; (D) = strategy D. cAs g L−1 of tartaric acid. The absence of letter indicates that there is no significant differences between treatments using the Tukey test at p ≤ 5. a

Figure 1. Concentration (mg L−1) of the most (a) and least (b) abundant amino acid in must from the untreated control Montepulciano grapevines. amino acids were analyzed by reversal-phase HPLC using an Agilent 1100 Series instrument (Agilent, Palo Alto, CA), equipped with an ALS automatic liquid sampler, a fluorescence detector (FD) and a diode array detector (DAD). Each sample was centrifuged at 3220g for 10 min at 20 °C and then, 5 mL of the sample was mixed with 100 μL of norvaline, internal standard for quantifying all amino acids except proline, and 100 μL of sarcosine, internal standard for quantifying proline. This mixture was filtered through 0.45 μm OlimPeak pore filter (Teknokroma, Barcelona, Spain) and submitted to an automatic precolumn derivatization with o-phthaldialdehyde (OPA Reagent, Agilent) and with 9-fluorenylmethylchloroformate (FMOC Reagent, Agilent). The injected amount from the derivatized sample was 10 μL at 40 °C. All separations were performed on a Hypersil ODS (250 × 4.0 mm, I.D. Five μm) column (Agilent). Two eluents, previously filtered through 0.45 μm Millipore filter, were used as mobile phases: eluent A: 75 mM sodium acetate, 0.018% triethylamine (pH 6.9) + 0.3% tetrahydrofuran; eluent B: water, methanol and acetonitrile (10:45:45, v/v/v). Identification of compounds was performed by comparison of their retention times with their pure reference standards. The pure reference compounds and internal standards were obtained from Sigma-Aldrich. The amino acids analyzed were aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), γaminobutyric acid (GABA), tyrosine (Tyr), cysteine (Cys), valine (Val), methionine (Met), tryptophan (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), and proline (Pro). The treatments were carried out in quadruplicate, so the results for free amino acids correspond to the average of four analyses (n = 4). Statistical Analysis. The oenological parameters statistical analysis was submitted to analysis of variance according to a randomized block design, and the means were separated by Tukey’s HSD tests, at p ≤ 0.05 (Statsoft, Tulsa, OK). The must amino acids were performed

using variance analysis (one-way ANOVA), according to a randomized block design by Statgraphics Centurion XVI.I. Differences between samples were compared using the Duncan’s test at p ≤ 0.05.

■

RESULTS AND DISCUSSION Grape Production and Oenological Parameters. The treatments applied to grapevines, Bordeaux mixture, copper hydroxide, chitosan, LamE, and the application of copper hydroxide alternately with chitosan and LamE, did not significantly affect grape production and oenological parameters (Table 2). Bordeaux mixture is a contact fungicide, containing copper sulfate and slaked lime. In an interesting report that studies the metabolic changes of Vitis vinifera L. cv. Vinhão, it was shown that the content of sucrose, glucose, fructose, malic acid, and tartaric acid was not significantly affected in mature grapes treated with Bordeaux mixture.29 In addition, grapevines cultivated in agar exposed to copper treatment with a dose of 5 μg g−1 had considerably decreased soluble sugar content in leaves without changes in starch or sucrose.30 With regard to the productivity, these authors reported that sap flow and essential elements translocation were reduced after copper application, suggesting that the potassium translocation reduction can be related to limited use of water by the plant and, thus, growth and physiological activity were reduced. In another report, it is shown that the levels of total sugars, reducing and nonreducing, were higher in the grapevine berries sprayed with copper hydroxide compared to control samples.31 Therefore, the scientific evidence shows that the applications based on copper have an important effect on grape oenological 7381


Article


Table 3. Must Amino Acid Concentration (mg L−1) from Grapevines of the Untreated Control and from Those Montepulciano Grapevines Treated Following Strategy A (Copper and the Alternative Compounds Applied Independently)a amino acid Asp Glu Asn Ser Gln His Gly Thr Arg Ala GABA Tyr Cys Val Met Trp Phe Ile Leu Lys Pro total aas total aas-Pro

control 1.38 10.30 4.53 70.51 31.97 17.38 8.82 67.82 174.10 49.66 189.86 11.43 6.17 35.89 17.12 31.12 14.96 39.20 49.27 3.05 643.20 1477.73 834.53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 c 3.50 b 0.40 b 12.85 c 5.51 bc 8.26 bc 2.27 b 15.76 c 48.92 b 14.15 b 17.63 c 2.64 c 2.48 b 3.35 c 0.87 c 8.58 c 2.47 d 6.05 c 8.26 c 0.65 cd 175.12 c 185.24 d 60.38 d

Bordeaux mixture 1.09 2.58 1.34 13.19 22.36 10.45 5.32 6.69 30.29 28.39 120.18 3.03 2.32 8.01 0.49 4.13 4.58 4.34 9.75 2.11 434.07 714.72 280.65

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

copper hydroxide

0.16 b 0.10 a 0.26 a 1.27 a 2.74 ab 1.72 ab 0.24 a 0.89 a 7.23 a 3.67 a 9.81 b 0.21 a 0.24 a 0.85 a 0.16 a 0.93 a 0.69 b 0.69 a 1.32 a 0.23 b 53.96 b 55.60 b 13.39 b

0.79 1.25 1.31 16.30 11.90 4.63 3.53 8.96 11.98 19.29 65.65 1.78 2.43 4.13 0.37 3.97 2.36 2.80 4.79 0.93 224.57 393.73 169.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 a 0.20 a 0.25 a 2.89 a 1.48 a 0.90 a 0.55 a 1.27 a 0.27 a 0.22 a 4.17 a 0.23 a 0.49 a 0.97 a 0.12 a 0.75 a 0.45 a 0.01 a 0.64 a 0.04 a 37.79 a 38.23 a 5.77 a

chitosan 0.82 1.36 1.26 16.84 17.07 2.60 2.84 6.47 27.59 18.20 71.88 1.04 1.49 4.93 0.5 2.61 2.23 3.95 6.36 3.71 336.36 530.13 193.78

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

LamE

0.13 a 0.09 a 0.03 a 1.78 a 3.68 a 0.16 a 0.71 a 1.02 a 3.24 a 1.27 a 8.88 a 0.13 a 0.24 a 0.27 a 0.09 a 0.64 a 0.25 a 0.95 a 1.11 a 0.33 d 31.47 ab 33.21 a 10.59 a

1.29 2.35 1.60 38.37 41.57 19.75 5.77 30.89 220.09 47.64 113.22 6.00 5.09 21.86 5.80 15.69 10.13 20.65 33.37 2.17 618.30 1261.61 643.31

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.12 bc 0.46 a 0.13 a 1.67 b 7.24 c 1.18 c 1.45 a 3.45 b 38.81 b 3.77 b 12.35 b 0.52 b 0.00 b 1.58 b 0.48 b 0.25 b 0.33 c 0.22 b 0.85 b 0.19 bc 55.73 c 69.67 c 41.81 c

a

LamE = laminarin applied with microbial extract of Saccharomyces spp. All the parameters are given with their standard deviation (n = 4). For each parameter, different letters in the same row indicate significant differences between treatments using the Duncan test at p ≤ 0.05.

suitable nitrogen concentrations in musts, so stuck or sluggish fermentations can occur when the yeast assimilable nitrogen (YAN) amount is low.24 Thus, proline to arginine ratio reflects the proportion of not assimilable (proline) to assimilable nitrogen (arginine) that provides a useful indication of the likely nutritional value of the must of a particular cultivar to yeast.35 To our knowledge, there is no evidence of the nutritional behavior of the Montepulciano grape must. For this variety, proline to arginine ratio was around 4, tending to behave as a proline accumulator variety. Grape Amino Acid Content. Table 3 shows the must amino acid concentration from grapevines of the untreated control and from those treated following strategy “A”, where the plants were foliar sprayed with the same compound(s): Bordeaux mixture, copper hydroxide, chitosan and LamE. The copper and chitosan based formulations had a negative effect on must amino acid composition. Bordeaux mixture applied to the grapevines decreased the content of several amino acids, except Gln and His, compared to control samples. Copper hydroxide and chitosan applications to the grapevines decreased the must concentration of all amino acids compared to the control, except for the content of Lys, after application of chitosan alone. Regarding to the amino acids synthesis, LamE had a minor negative effect than Bordeaux mixture and copper hydroxide with chitosan, decreasing only Glu, Asn, Ser, Gly, Thr, GABA, Tyr, Val, Met, Trp, Phe, Ile, and Leu concentration respect to the control. Thus, the formulations based on copper and chitosan showed lower content of total amino acids and total amino acids without proline than control and LamE samples. Table 4 shows the amino acid concentration of must from grapevines of the untreated control and from those treated following strategy “B”, where copper hydroxide was applied

parameters, and productiveness, which was not evidenced in our results (Table 2). The linear β-1,3-glucan laminarin derives from the brown seaweed Laminaria digitata and elicits a variety of defense reactions in several crop plants.21 There is little information regarding to the effect of laminarin on the grape productivity and oenological parameters, however it has been seen that the use of seaweeds as a biostimulant allow to increase the leaf size, chlorophyll content, berry setting, number of bunches per shoot, rachis length, berry weight and size, soluble solid concentration, titratable acidity, must pH, total sugars and reducing sugars.32 In relation to the chitosan, it has been observed that the weekly applications of this compound from May to the end of July had no effects on quantitative and qualitative yield parameters in comparison with the control.33 In another report, Portu et al.34 showed that the chitosan foliar applications to Tempranillo grapevines did not affect grape production and oenological parameters, while the use of elicitors such as methyl jasmonate and yeast extracts to the grapevines had slight effects on grape oenological parameters. Characterization of Must Amino Acids. The most abundant amino acids in musts were Pro, GABA, Arg, Thr, Ser representing around of 78% of total amino acids (Figure 1a). The least abundant amino acids found in musts were Gly, Cys, Asn, Lys, and Asp (Figure 1b). Most of the amino acids are in the normal concentration described for these compounds by Bell and Henschke,35 with the exception of Asp and Glu, whose contents were smaller, and Trp, which concentration was higher than that reviewed by these authors. To our knowledge, this is the first report that characterizes the amino acid composition in must of the Montepulciano variety. Proline was the most abundant amino acid found in must. However, this amino acid is not metabolized by yeast in 7382


7383

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.40 c 0.92 a 0.50 b 6.74 a 2.35 bcd 3.31 b 0.75 a 0.53 b 16.95 c 1.25 d 29.58 b 0.33 b 0.23 a 3.17 b 0.50 b 1.09 a 0.00 c 0.89 a 0.50 b 0.65 b 136.45 bc 140.92 b 35.23 c

Cu + LamE 2.17 3.62 1.81 33.76 31.54 16.76 4.87 30.69 224.79 60.94 133.53 5.51 1.89 12.26 2.50 7.85 8.85 7.10 18.74 2.66 505.19 1117.05 611.86

1.42 1.84 1.42 23.25 26.35 7.84 3.69 15.60 110.17 36.57 90.84 2.73 2.24 8.29 1.61 6.09 4.42 4.75 11.14 1.33 371.83 733.44 361.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.26 b 0.19 a 0.21 ab 5.03 a 5.14 bc 1.53 a 0.68 a 4.35 a 4.76 abc 0.57 abc 7.94 a 0.53 a 0.10 a 1.53 a 0.30 ab 1.69 a 0.41 ab 1.01 a 2.08 a 0.20 a 33.63 ab 36.08 a 13.07 ab

Cu + chitosan ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.35 ab 0.49 a 0.17 a 4.00 a 1.05 a 0.91 a 0.92 a 2.93 a 5.21 a 5.94 ab 14.65 ab 0.15 ab 0.26 a 1.21 a 0.06 a 0.83 a 1.00 b 0.66 a 1.04 a 0.06 a 51.68 a 54.59 a 17.59 a

Cu + LamE 1.28 2.65 1.18 23.79 12.52 6.13 4.29 13.00 33.19 25.64 115.47 2.86 0.88 7.28 0.72 3.31 5.00 4.48 9.43 1.05 291.66 565.80 274.14

strategy C 1.69 2.87 1.07 23.78 20.50 10.69 5.01 21.39 129.42 41.44 119.61 3.66 1.94 5.11 1.17 3.37 3.23 3.76 5.83 1.49 275.38 682.42 407.03

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.30 bc 0.51 a 0.24 a 5.65 a 5.21 abc 1.02 ab 0.72 a 1.30 ab 5.10 abc 1.03 bc 23.59 ab 0.37 ab 0.42 a 0.44 a 0.03 a 0.28 a 0.24 ab 0.95 a 0.10 a 0.14 a 22.15 a 33.74 a 25.45 b

Cu + chitosan

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 a 0.02 a 0.35 ab 7.83 a 13.86 d 0.99 a 0.79 a 5.62 a 145.85 c 9.84 a 17.64 a 0.30 a 0.85 a 0.86 a 0.52 b 0.01 a 0.72 a 0.09 a 1.20 a 0.98 c 46.05 bc 155.22 b 148.23 b

Cu + LamE 0.78 2.03 1.36 22.61 39.85 3.79 3.82 10.09 198.88 21.68 97.71 1.01 1.50 4.15 2.35 6.44 2.74 3.25 5.37 4.99 514.49 948.88 434.39

strategy D

LamE = laminarin applied with microbial extract of Saccharomyces spp.; Cu = copper hydroxide. All the parameters are given with their standard deviation (n = 4). For each parameter, different letters in the same row indicate significant differences between treatments using the Duncan test at p ≤ 0.05.

a

0.41 b 0.52 a 0.17 a 4.21 a 3.83 ab 0.57 a 0.75 a 2.16 a 18.55 ab 4.94 ab 5.10 a 0.45 a 0.51 a 0.60 a 0.13 a 0.37 a 0.57 b 0.58 a 1.04 a 0.23 a 80.34 a 83.00 a 20.87 a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.55 1.96 1.18 21.96 19.74 5.25 3.22 12.69 62.84 24.13 96.94 2.19 2.62 6.42 0.86 5.10 5.00 4.66 9.05 1.04 323.78 612.17 288.39

Cu + chitosan

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 ab 3.50 b 0.40 c 12.85 b 5.51 cd 8.26 b 2.27 b 15.76 c 48.92 bc 14.15 cd 17.63 c 2.64 c 2.48 b 3.35 c 0.87 c 8.58 b 2.47 d 6.05 b 8.26 c 0.65 b 175.12 c 185.24 c 60.38 d

control

1.38 10.30 4.53 70.51 31.97 17.38 8.82 67.82 174.10 49.66 189.86 11.43 6.17 35.89 17.12 31.12 14.96 39.20 49.27 3.05 643.20 1477.73 834.53

amino acid

Asp Glu Asn Ser Gln His Gly Thr Arg Ala GABA Tyr Cys Val Met Trp Phe Ile Leu Lys Pro total aas total aas-Pro

strategy B

Table 4. Must Amino Acid Concentration (mg L−1) from Grapevines of the Untreated Control and from Those Montepulciano Grapevines Treated Following Strategies B, C, and Da

Journal of Agricultural and Food Chemistry Article


Article


with strategy “C”, while following the last strategy the proline concentration was considerably affected. The arginine accumulation began before veraison and continued to full maturity, while proline accumulation occurred late in ripening, around 4 weeks postveraison.38 The grapevine foliar applications shown in Table 1 were carried out before veraison. Therefore, copperbased formulations, applied before veraison following different strategies, affected negatively the grape amino acid composition. Chitosan and laminarin are elicitors that trigger strong defense responses against different pathogens.39 However, it has been observed that the induction resistance through the chemical inducers often results in physiological costs to the plant associated with a decrease in plant growth rate and the consequently synthesis of secondary compounds such as cellulose, hemicellulose, terpenoids, and phenolic compounds.40 Heil et al.41 reported growth and seed set reduction following chemical induction of pathogen defense in spring wheat (Triticum aestivum, cv. ‘Hanno’), suggesting that systemic acquired resistance (SAR) incurs allocation costs. In addition, Hofgaard et al.42 reported that perennial ryegrass pretreated with the elicitors Bion or chitosan and inoculated with pink snow mold (Microdochium nivale) did not display better regrowth after incubation than nontreated, inoculated plants, evidencing physiological costs after use of these elicitors. It is probable that the grapevine chitosan application, individually or following the different application strategies, had important physiological costs related to a considerable decrease on must amino acid concentration. In fact, Romanazzi et al.4 reported that the chitosan application at a dose of 0.8% lowered net photosynthesis, due to reduced stomatal conductance, leaf area, and dry weight, with no negative effects observed on the grapes quantity and their must quality parameters. It is likely that, due to the biostimulant capacity of laminarin, its application to the grapevines following strategy “A” and strategy “B”, alternately with copper hydroxide, had a lower negative effect on the amino acid composition than in the other treatments. In conclusion, the results obtained in this research indicate that weekly foliar applications to Montepulciano grapevines using natural compounds, applied individually or in strategies with copper formulations, can have a negative effect on amino acid composition of must. Copper-based formulations, like Bordeaux mixture and copper hydroxide, significantly decreased the concentration of several free amino acids applied individually. In the same way, copper hydroxide, applied following the different strategies studied, and chitosan considerably decreased several amino acids content. The least negative effect on must amino acid composition was after the application of LamE, individually and following strategy “B”, suggesting as previously reported that laminarin may have a biostimulant action.22 These results provide important information about the effect of the new developed strategies performed against GDM on must quality, according to the current trends of regulation in the use of heavy metals in viticulture. Therefore, different disease management tools can be developed according to the grapevine nitrogen requirements. Thus, in grapevines with low nitrogen requirements and high vegetative expression, it is possible to use copper-base formulation and chitosan to control GDM without affecting grape quality. On the other hand, in grapevines with high nitrogen requirement, that may cause stuck or sluggish fermentation in the wine cellar, the use of copper-based formulations for disease control in the vineyard

alternately in the same plots with chitosan and LamE, respectively, as shown in Table 1. The applications of copper hydroxide and chitosan to grapevines had a negative effect on must amino acid composition, decreasing the content of all amino acids compared to the control, except Asp. The applications with copper hydroxide and LamE to grapevines decreased the concentration of certain amino acids except Asp, Gln, His, Arg, Ala Lys, and Pro compared to control samples. Thus, the must from grapevines treated with copper hydroxide and LamE showed higher Gln, Arg, Ala, Val, Met, Phe, and Leu concentration than that from grapevines treated with copper hydroxide and chitosan. This latter treatment showed lower content of total amino acids and total amino acids without proline than the untreated control and copper hydroxide and LamE samples. Following strategy “C”, where the copper hydroxide was used for the first six applications, while chitosan or LamE was used for the following six applications, both treatments had a negative effect on must amino acid content (Table 4). The foliar grapevine applications with copper hydroxide and chitosan decreased the must concentration of several amino acids, except Asp, Gln, His, Arg, Ala, and Cys, compared to control samples. The foliar grapevine applications with copper hydroxide and LamE decreased several amino acids content, except Asp and His, with respect to control samples. The Gln concentration was higher in must from copper hydroxide plus chitosan treatments than in copper hydroxide plus LamE applications. Total amino acid and total amino acid without proline must concentration decreased with the foliar applications of the treatments compared to control samples. Following strategy “D”, where chitosan or LamE was used for the first six applications, while copper hydroxide was used for the following six applications, the treatments decreased several amino acid concentrationn, except Gln, His, Arg, and Ala, compared to the control. However, LamE and copper hydroxide increased the concentration of Lys, and also did not affect Pro composition compared to control samples. Chitosan and copper hydroxide application did not affect must Asp and Lys concentration compared to control samples. Must total amino acid and total amino acid without proline concentration decreased with the foliar applications compared to control samples. The applications of copper and chitosan had a negative effect on amino acid content, while LamE, following strategies “A” and “B”, had a less negative effect compared to the other treatments. Martins et al.29 showed that the grapevines Bordeaux mixture application decreased the concentration of 19 individual amino acids; this decrease was more severe for asparagine, glutamine, proline, and all the basic amino acids, namely, arginine, histidine, and lysine. In addition, Llorens et al.36 studied the effects of copper treatments on grapevine nitrogen metabolism in a closed system. These authors reported that grapevine copper exposure produces a dramatic change in nitrogen metabolism reducing total nitrogen content, nitrate and free amino acid in root and leaves. Martins et al.37 studied the effect of Bordeaux mixture on regulation of the expression of V. vinifera copper transporters (VvCTr). These authors reported that the VvCTr expression pattern depends on the tissue and the development stage. Bordeaux mixture application caused an increase in the copper concentration in whole grape berries at all stages of their development, due to a greater VvCTrs berries expression mainly occurred before veraison. In our results, it seems that, following strategy “D”, the individual amino acid concentration was less affected than 7384


Article


(9) Paoletti, M. G.; Sommaggio, D.; Favretto, M. R.; Petruzzelli, G.; Pezzarossa, B.; Barbafieri, M. Earthworms as useful bioindicators of agroecosystem sustainability in orchards and vineyards with different inputs. Appl. Soil Ecol. 1998, 10, 137−150. (10) Eijsackers, H.; Beneke, P.; Maboeta, M.; Louw, J. P.; Reinecke, A. J. The implications of copper fungicide usage in vineyards for earthworm activity and resulting sustainable soil quality. Ecotoxicol. Environ. Saf. 2005, 62, 99−111. (11) Pontiroli, R.; Rizzotti, R.; Zerbetto, F. Prove di difesa antiperonosporica in viticoltura biologica. Inf. Fitopatol. 2001, 10, 62−66. (12) European Commission. Commission regulation (EC) No. 473/ 2002. OJEC 2002, 75, 21−24. (13) Delaunois, B.; Farace, G.; Jeandet, P.; Clément, C.; Baillieul, F.; Dorey, S.; Cordelier, S. Elicitors as alternative strategy to pesticides in grapevine? Current knowledge on their mode of action from controlled conditions to vineyard. Environ. Sci. Pollut. Res. 2014, 21, 4837−4846. (14) La Torre, A.; Talocci, S.; Spera, G.; Valori, R. Control of downy mildew on grapes in organic viticulture. Commun. Agric. Appl. Biol. Sci. 2008, 73, 169−178. (15) Romanazzi, G.; Nigro, F.; Ippolito, A.; Di Venere, D.; Salerno, M. Effects of pre- and postharvest chitosan treatments to control storage grey mold of table grapes. J. Food Sci. 2002, 67, 1862−1867. (16) Aziz, A.; Trotel-Aziz, P.; Dhuicq, L.; Jeandet, P.; Couderchet, M.; Vernet, G. Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew. Phytopathology 2006, 96, 1188−1194. (17) Trotel-Aziz, P.; Couderchet, M.; Vernet, G.; Aziz, A. Chitosan stimulates defense reactions in grapevine leaves and inhibits development of Botrytis cinerea. Eur. J. Plant Pathol. 2006, 114, 405−413. (18) El Ghaouth, A.; Arul, J.; Grenier, J.; Benhamou, N.; Asselin, A.; Belanger, R. Effect of chitosan on cucumber plants: suppression of Pythium aphanidermatum and induction of defense reactions. Phytopathology 1994, 84, 313−320. (19) Romanazzi, G.; Feliziani, E.; Bautista Baños, S.; Sivakumar, D. Shelf life extension of fresh fruit and vegetables by chitosan treatment. Crit. Rev. Food Sci. Nutr. 2017, 57, 579−601. (20) Gutiérrez-Gamboa, G.; Portu, J.; Santamaría, P.; López, R.; Garde-Cerdán, T. Effects on grape amino acid concentration through foliar application of three different elicitors. Food Res. Int. 2017, 99, 688. (21) Aziz, A.; Poinssot, B.; Daire, X.; Adrian, M.; Bézier, A.; Lambert, B.; Joubert, J. M.; Pugin, A. Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol. Plant-Microbe Interact. 2003, 16, 1118−1128. (22) Khan, W.; Rayirath, U. P.; Subramanian, S.; Jithesh, M. N.; Rayorath, P.; Hodges, D. M.; Critchley, A. T.; Craigie, J. S.; Norrie, J.; Prithiviraj, B. Seaweed extracts as biostimulants of plant growth and development. J. Plant Growth Regul. 2009, 28, 386−399. (23) Arias-Gil, M.; Garde-Cerdán, T.; Ancín-Azpilicueta, C. Influence of addition of ammonium and different amino acid concentrations on nitrogen metabolism in spontaneous must fermentation. Food Chem. 2007, 103, 1312−1318. (24) Bisson, L. F.; Butzke, C. E. Diagnosis and rectification of stuck and sluggish fermentations. Am. J. Enol. Vitic. 2000, 51, 168−177. (25) Hilbert, G.; Soyer, J. P.; Molot, C.; Giraudon, J.; Milin, S.; Gaudillere, J. P. Effects of nitrogen supply on must quality and anthocyanin accumulation in berries of cv. Merlot. Vitis 2003, 42, 69− 76. (26) Howell, G. S. Sustainable grape productivity and the growthyield relationship: a review. Am. J. Enol. Vitic. 2001, 52, 165−174. (27) Mira de Orduña, R. Climate change associated effects on grape and wine quality and production. Food Res. Int. 2010, 43, 1844−1855. (28) Garde-Cerdán, T.; López, R.; Portu, J.; González-Arenzana, L.; López-Alfaro, I.; Santamaría, P. Study of the effects of proline, phenylalanine, and urea foliar application to Tempranillo vineyards on

could be detrimental to wine production. To control GDM, LamE and chitosan application, individually or alternately with copper hydroxide, would be a suitable alternative that would not affect grape quality.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02352. Copper and alternative (Bordeaux mixture, chitosan and laminarin applied with microbial extract of Saccharomyces spp) formulation applied in the study (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; teresa.garde@icvv. es. *E-mail: [email protected]. ORCID

T. Garde-Cerdán: 0000-0002-2054-9071 Funding

G.G.-G. is thankful for the financial support given by CONICYT through BCH/Doctorado-72170532. T.G.-C. and E.P.P.-A. also thank MINECO for funding their contracts. G.R. thanks Dr. Sandro Nardi, SFR-ASSAM, Marche region, for funding the project “Grapevine downy mildew management with low copper rates and alternative compounds”. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The kind help of Danilo Coppa and Giuliano D’Ignazi, Moncaro winery, in technical assistance and basic must analyses is acknowledged.

■

REFERENCES

(1) Dagostin, S.; Schärer, H. J.; Pertot, I.; Tamm, L. Are there alternatives to copper for controlling grapevine downy mildew in organic viticulture? Crop Prot. 2011, 30, 776−788. (2) Gessler, C.; Pertot, I.; Perazzoli, M. Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathol. Mediterr. 2011, 50, 3−44. (3) Caffi, T.; Rossi, V.; Bugiani, R. Evaluation of a warning system for controlling primary infections of grapevine downy mildew. Plant Dis. 2010, 94, 709−716. (4) Romanazzi, G.; Mancini, V.; Feliziani, E.; Servili, A.; Endeshaw, S.; Neri, D. Impact of alternative fungicides on grape downy mildew control and vine growth and development. Plant Dis. 2016, 100, 739− 748. (5) Speiser, B.; Berner, A.; Häseli, A.; Tamm, L. Control of downy mildew of grapevine with potassium phosphonate: Effectivity and phosphonate residues in wine. Biol. Agric. Hortic. 2000, 17, 305−312. (6) Rusjan, D.; Strlič, M.; Pucko, D.; Korošec-Koruza, Z. Copper accumulation regarding the soil characteristics in sub-Mediterranean vineyards of Slovenia. Geoderma 2007, 141, 111−118. (7) Duca, D.; Toscano, G.; Pizzi, A.; Rossini, G.; Fabrizi, S.; Lucesoli, G.; Servili, A.; Mancini, V.; Romanazzi, G.; Mengarelli, C. Evaluation of the characteristics of vineyard pruning wood residues for energy applications: effects of different copper-based treatments. J. Agric. Eng. 2016, 47, 22−27. (8) Toselli, M.; Baldi, E.; Marcolini, G.; Malaguti, D.; Quartieri, M.; Sorrenti, G.; Marangoni, B. Response of potted grapevines to increasing soil copper concentration. Aust. J. Grape Wine Res. 2009, 15, 85−92. 7385


Article

Journal of Agricultural and Food Chemistry grape amino acid content. Comparison with commercial nitrogen fertilisers. Food Chem. 2014, 163, 136−141. (29) Martins, V.; Teixeira, A.; Bassil, E.; Blumwald, E.; Gerós, H. Metabolic changes of Vitis vinifera berries and leaves exposed to Bordeaux mixture. Plant Physiol. Biochem. 2014, 82, 270−278. (30) Romeu-Moreno, A.; Mas, A. Effects of copper exposure in tissue cultured Vitis vinifera. J. Agric. Food Chem. 1999, 47, 2519−2522. (31) Valarmathi, P.; Kumar-Pareek, S.; Priya, V.; Rabindran, R.; Chandrasekar, G. Studies on the quality of grapevine berries sprayed with copper fungicide. IJSRP 2013, 3, 1−7. (32) Khan, A. S.; Ahmad, B.; Jaskani, M. J.; Ahmad, R.; Malik, A. U. Foliar application of mixture of amino acids and seaweed (Ascophylum nodosum) extract improve growth and physico-chemical properties of grapes. Int. J. Agric. Biol. 2012, 14, 383−388. (33) Romanazzi, G.; Murolo, S.; Feliziani, E. Effects of an innovative strategy to contain grapevine Bois noir: field treatment with resistance inducers. Phytopathology 2013, 103, 785−791. (34) Portu, J.; López, R.; Baroja, E.; Santamaría, P.; Garde-Cerdán, T. Improvement of grape and wine phenolic content by foliar application to grapevine of three different elicitors: Methyl jasmonate, chitosan, and yeast extract. Food Chem. 2016, 201, 213−221. (35) Bell, S. J.; Henschke, P. A. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 2005, 11, 242− 295. (36) Llorens, N.; Arola, L.; Bladé, C.; Mas, A. Effects of copper exposure upon nitrogen metabolism in tissue cultured Vitis vinifera. Plant Sci. 2000, 160, 159−163. (37) Martins, V.; Teixeira, A.; Bassil, E.; Hanana, M.; Blumwald, E.; Gerós, H. Copper-based fungicide Bordeaux mixture regulates the expression of Vitis vinifera copper transporters. Aust. J. Grape Wine Res. 2014, 20, 451−458. (38) Stines, A. P.; Grubb, J.; Gockowiak, H.; Henschke, P. A.; Høj, P. B.; van Heeswijck, R. Proline and arginine accumulation in developing berries of Vitis vinifera L. in Australian vineyards: Influence of vine cultivar, berry maturity and tissue type. Aust. J. Grape Wine Res. 2000, 6, 150−158. (39) Romanazzi, G.; Sanzani, S. M.; Bi, Y.; Tian, S.; GutierrezMartinez, P.; Alkan, N. Induced resistance to control postharvest decay of fruit and vegetables. Postharvest Biol. Technol. 2016, 122, 82−94. (40) Barbosa, M. A. G.; Laranjeira, D.; Coelho, R. S. B. Custo ́ fisiológico da resistência em algodoeiro sob diferentes niveis de nitrogênio. Summa Phytopathol. 2008, 34, 338−342. (41) Heil, M.; Hilpert, A.; Kaiser, W.; Linsenmair, E. Reduced growth and seed set following chemical induction of pathogen defence: does systemic acquired resistance (SAR) incur allocation costs? J. Ecol. 2000, 88, 645−654. (42) Hofgaard, I. S.; Ergon, A.; Wanner, L. A.; Tronsmo, A. M. The effect of chitosan and bion on resistance to pink snow mould in perennial ryegrass and winter wheat. J. Phytopathol. 2005, 153, 108− 119.

7386


Chitosan and Laminarin as Alternatives to Copper for Plasmopara

Recommend Documents