Scientia Horticulturae 245 (2019) 82–89

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

The application of copper nanoparticles and potassium silicate stimulate the T

tolerance to Clavibacter michiganensis in tomato plants
Claudia Francely Cumplido-Nájeraa, Susana González-Moralesb, Hortensia Ortega-Ortízc,
⁎
Gregorio Cadenas-Pliegoc, Adalberto Benavides-Mendozaa, Antonio Juárez-Maldonadod,
a
Departamento de Horticultura, Universidad Autónoma Agraria Antonio Narro, 25315, Saltillo, Coahuila, Mexico
b
Cátedras CONACyT, Departamento de Horticultura, Universidad Autónoma Agraria Antonio Narro, 25315, Saltillo, Coahuila, Mexico
c
Centro de Investigación en Química Aplicada, 25294, Saltillo, Coahuila, Mexico
d
Departamento de Botánica, Universidad Autónoma Agraria Antonio Narro, 25315, Saltillo, Coahuila, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: The damage by Clavibacter michiganensis in the tomato plants is of great economic importance worldwide due to
Nanotechnology the significant losses it generates in the crop. The advances in nanotechnology provide alternatives that can be
Defense compounds applied in the control of pathogens. Nano-copper and silicon currently have a widespread use for the control of
Antioxidants plant pathogens. The combined use of both is expected to have a synergistic effect on the tolerance of plants
Carotenoids
against pathogens. The objective of the present research was to determine the effect of the application of copper
Biotic stress
nanoparticles and potassium silicate on tolerance to C. michiganensis in tomato. The presence, severity, and the
Plant productivity
impact of the bacteria on the yield of the tomato crop were determined. Levels of activity of defense enzymes
were determined as well as antioxidant compounds in the leaves and fruits of tomato to understand the changes
at the biochemical level caused by the bacteria and by the treatments. The results showed that the application of
copper nanoparticles and potassium silicate was effective in reducing the severity of C. michiganensis. Also, the
loss of yield due to the bacteria was reduced in 16.1%. The biochemical analyzes showed that the application of
copper nanoparticles and potassium silicate positively modified the activity of the enzymes SOD, PAL, GPX, and
APX, as well as the concentration of reduced glutathione and total phenols in the leaves. The activity of the PAL
and GPX enzymes, as well as the contents of lycopene and β-carotene, were elevated in the fruits. The joint
application of copper nanoparticles and potassium silicate changed the levels of enzymatic and non-enzymatic
compounds that are key in defense of tomato plants, increasing the tolerance to C. michiganensis.

1. Introduction
The tomato is one of the most important crops worldwide (Martí
et al., 2018), however, the production of this is affected by external
biotic factors among which are different pathogens. Clavibacter michi-
ganensis is a bacterium that penetrates through the stomata and hy-
dathodes causing severe damage in the crop, among which marginal
necrosis in the leaves is observed, causing great losses (Chalupowicz
et al., 2017). The control of this bacterium is of great importance, and
although there are chemical products for the control of this pathogen,
the efficiency is low, in addition to the risks they present for health
(Nandi et al., 2018), so it becomes necessary propose new alternatives
that are efficient.
An alternative is the induction of tolerance to pathogens from
biostimulation, that is, the application of substances or microorganisms
that do not directly provide essential elements for plants (du Jardin,
2015). Biostimulation in plants with non-essential elements includes
the application of Al, Co, Na, Se and Si, elements that provide different
benefits in crops under stress conditions (du Jardin, 2015). In addition,
it has recently been shown that the application of nanoparticles (NPs)
also generates positive effects in crops (Zuverza-Mena et al., 2017).
The effects of silicon have been well documented, this element is not
considered essential and numerous beneficial effects have been re-
ported for plants (Luyckx et al., 2017). Under conditions of abiotic
stress, it can regulate the generation of ROS from the antioxidant
system, which reduces oxidative stress (Kim et al., 2017). Or by phy-
siological adjustments such as increased stomatal conductance and
transpiration under conditions of salt stress (Rios et al., 2017). Parti-
cularly, the Si improves the resistance of the plants against both fungal
and bacterial pathogens, from enzymes related to defense, stimulation

⁎
Corresponding author.
E-mail address: antonio.juarez@uaaan.edu.mx (A. Juárez-Maldonado).

https://doi.org/10.1016/j.scienta.2018.10.007
Received 2 August 2018; Received in revised form 1 October 2018; Accepted 4 October 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.
C.F. Cumplido-Nájera et al.
of the production of antimicrobial compounds, regulation of signaling
pathways, and activation of the expression of defense-related genes
(Wang et al., 2017).
Meanwhile, nanotechnology can be used to increase crop pro-
ductivity (De la Rosa et al., 2017; Wang et al., 2015). In recent years,
nanotechnology has been used by means of the application of nano-
materials such as nano-fertilizers, nanoparticles, or nano-pesticides for
nutrient management, genetic improvement, plant disease treatment,
and plant growth promotion (De la Rosa et al., 2017). Nanoparticles
(NPs) are materials with at least one dimension less than 100 nm. This
small size gives rise to properties different from those exhibited by the
bulk of the material of the same composition (Bell et al., 2014). So
much so that for metallic elements such as Cu, Fe, Ce, Ti, and Ag, the
cellular responses are very different when induced by ionic forms
compared to the nanometric forms (Zuverza-Mena et al., 2017). These
new properties provide the material with an added value that has
multiple applications in the agricultural industries, among others
(Siddiqui et al., 2015). Specifically, the application of copper nano-
particles (Cu NPs) increases the yield and fruit quality of the tomato
crop (Hernández-Hernández et al., 2017; Juarez-Maldonado et al.,
2016), as well as the quality of the fruit of jalapeño pepper crop
(Pinedo-Guerrero et al., 2017). Under conditions of salt stress, the ap-
plication of Cu NPs increased the activity of antioxidant enzymes (APX,
SOD, CAT, GPX), in addition to inducing better tomato fruit quality
from the increase in lycopene (Hernández-Hernández et al., 2018).
Considering the biostimulation capacity of both silicon and nano-
particles, the combined use of both is expected to have a synergistic
effect on the tolerance of plants against pathogens. Hence, the objective
of the present research was to determine the effect of the application of
copper nanoparticles and potassium silicate on tolerance to C. michi-
ganensis in tomato.
2. Materials and methods
2.1. Crop development
A “El Cid F1” indeterminate growth tomato variety (Harris Moran,
Davis, CA, USA) of the saladette type was used for this experiment. The
transplant took place in black 10 L capacity polyethylene bags. The crop
was established under greenhouse conditions, with average tempera-
tures of 35 °C/20 °C for day and night, relative humidity of 60%, and
photosynthetically active radiation of 1400 μmol m−2 s−1. The crop
was grown from a single stem and developed for 106 days, and the yield
of the crop considered the total of fruits harvested in that period. A
substrate composed by a mixture of perlite-peat moss (1:1 on volume
base) was used with a directed irrigation system. The Steiner nutrient
solution was used for crop nutrition (Steiner, 1961) using the following
micronutrients in chelated form using EDTA (2,2′,2″,2″’-[Ethane-1,2-
diyldinitrilo] tetraacetic acid), Fe EDTA = 3.75 mg L–1; Mn EDTA =
1.85 mg L–1; B = 0.35 mg L–1; Zn EDTA = 0.30 mg L–1; Cu EDTA =
0.15 mg L–1; Mo = 0.10 mg L–1 and the solution pH was adjusted to 6.5
with sulfuric acid each time it was prepared.
2.2. Application of treatments
The treatments consisted in the combined application of silicon and
copper nanoparticles (Cu NPs) in two doses namely low dose (LD) and
high dose (HD), with a total of six treatments: 1) 50 mg L−1 Cu
NPs + 184 mg L−1 Si (Cu NPs LD + Si LD); 2) 50 mg L−1 Cu
NPs + 460 mg L−1 Si (Cu NPs LD + Si HD); 3) 250 mg L−1 Cu
NPs + 184 mg L−1 Si (Cu NPs HD + Si LD); 4) 250 mg L−1 Cu
NPs + 460 mg L−1 Si (Cu NPs HD + Si HD), these four treatments in-
oculated with Cmm, 5) A treatment inoculated with Cmm and without
application of Si or Cu NPs (Cmm), and 6) A Control (T0). Five appli-
cations of treatments in total were made by foliar way, and at intervals
of 14 days starting from two weeks after the transplant. As source of Si
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Scientia Horticulturae 245 (2019) 82–89
potassium silicate was used (K2SiO3). While the Cu NPs used were of
average size of 42 nm and spherical shape and were synthesized ac-
cording to the methodology of (Cadenas-Pliego et al., 2013). Each
treatment consists of 16 plants, so the full experiment considers 90
plants of tomato.
2.3. Inoculation and severity of Clavibacter michiganensis
The bacterium Clavibacter michiganensis subsp. michiganensis (Cmm)
was obtained from the sap of diseased tomato plants. The sap was
placed in Petri dishes with NYA culture medium and incubated at 29 °C
for 48 h. The colonies obtained were analyzed by morphological and
biochemical tests for their identification. The bacterium was increased
in Petri dishes with NYA culture medium (nutritive broth 0.8%, yeast
extract 0.2%, K2HPO4 0.2%, KH2PO4 0.025%, agar 1.5%). The Petri
dishes were incubated at 29 °C for 48 h, after which the bacterial
growth was collected.
The plants corresponding to the treatments with Cmm were in-
oculated at 28 days after transplanting (DAT). A solution of 1 × 106
colony forming units (CFU) per milliliter of Cmm was prepared.
Cuttings were made on the leaves of the tomato plants and were im-
mersed in 30 mL of bacterial solution for 5 min, and the remainder was
sprinkled on the foliage. The severity of Clavibacter michiganensis in
tomato plants was determined following the method of Baysal et al.
(2003).
2.4. Biochemical variables
To determine proteins, catalase, ascorbate peroxidase, superoxide
dismutase, glutathione peroxidase, phenylalanine ammonium lyase,
reduced glutathione, ABTS, and DPPH antioxidant capacity, samples of
leaf tissue were collected. For this purpose, after 73 days of trans-
plantation, random plants were selected, and the third fully expanded
young leaf was taken for biochemical analysis. Samples were stored at
−80 °C until use. For the enzymatic and non-enzymatic determination,
200 mg of lyophilized leaves of each treatment and 20 mg of poly-
vinylpyrrolidone were weighed. After this, 1.5 mL of phosphate buffer
with a pH of 7–7.2 (0.1 M) were added, and the mixture was then
subjected to micro-centrifugation at 16,128 × g for 10 min at 4 °C. The
supernatant was filtered with a nylon membrane (Ramos et al., 2010).
Dilutions of the extract were prepared in a ratio of 1:20 with the
phosphate buffer.
The quantification of total proteins was determined using Bradford’s
colorimetric technique (Bradford, 1976), and were expressed in mg g–1
of dry weight (DW). Catalase (CAT) (QE 1.11.1.6) enzymatic activity
was quantified by the spectrophotometric method used by Dhindsa
et al. (1981), and the values were expressed in U per total proteins (mg
g–1), where U is equal to mM equivalent of H2O2 consumed per milli-
liter per minute. Determination of Superoxide dismutase (SOD) (QE
1.15.1.1) enzymatic activity was carried out using the SOD Cayman
706002® kit, one unit of SOD is defined as the amount of enzyme
needed to exhibit 50% dismutation of the superoxide radical. The re-
sults are expressed in U mL–1 per total proteins (mg g–1). The Glu-
tathione Peroxidase (GPX) (QE 1.11.1.9) enzyme was determined with
the method adapted by Xue et al. (2001) using H2O2 as the substrate,
the results are expressed in U per total proteins (mg g–1), where U is
equal to mM equivalent of GSH per milliliter per minute. The mea-
surement of the enzymatic activity of ascorbate peroxidase (APX) (QE
1.11.1.1) was carried out according to what was established by Nakano
and Asada (1987), the enzymatic activity was expressed as U per total
proteins (mg g–1), where U is equal to μmol QE of oxidized ascorbate
per milliliter per minute. The measurement of the enzymatic activity of
ascorbate peroxidase (APX) (QE 1.11.1.1) was carried out according to
what was established by Nakano and Asada (1987), the enzymatic ac-
tivity was expressed as U per total proteins (mg g–1), where U is equal to
μmol QE of oxidized ascorbate per milliliter per minute. Phenylalanine
C.F. Cumplido-Nájera et al. Scientia Horticulturae 245 (2019) 82–89

Ammonium Lyase (PAL) (QE 4.3.1.5) was determined according to

Sykłowska-Baranek et al. (2012) with modifications, and the results
were expressed as U (production of 1 μM cinnamic acid per min) per
total proteins (mg g−1).
Glutathione quantification was performed using the spectro-
photometric technique by Xue et al. (2001), by means of a 5,5-dithio-
bis-2 nitrobenzoic acid (DTNB) reaction, and the results were expressed
in mM equivalent of GSH ml–1 min–1 total protein–1. The total phenols
were determined according to the methodology of Singleton et al.
(1999), the sample (0.2 g) was extracted with 1 mL of a water:acetone
solution (1:1), and the results are expressed in equivalent milligrams of
gallic acid per 100 g dry weight (mg EGA 100 g–1 DW). The flavonoids
quantification was carried out using Dowd's method adapted by
Arvouet-Grand et al. (1994), and the content was expressed in
equivalent milligrams of Quercetin per 100 g of dry weight (mg EQ
100 g–1 DW). The vitamin C content was determined by the titration
method with 2,6-dichlorophenolindophenol, using 1 g of fresh tissue
and HCl (2%) (Padayatt et al., 2001). Vitamin C is expressed as mg
100 g−1 Fresh Weight (mg 100 g−1 FW).
The content of chlorophylls, lycopene and β-carotene was de-
termined according to Nagata and Yamashita (1992). The absorbances Fig. 1. Severity of Clavibacter michiganensis supsp. michiganensis on tomato
at 453, 505, 645 y 663 nm were determined, and were used in the Eqs. plants. Vertical bars represent the standard deviation. Bars with same letter are
(1)–(4) to determine the content of lycopene, β-carotene, and chlor- statistically equal (LSD Fisher, p ≤ 0.05). T0: Control, LD and HD represent the
ophylls as follow: low or high dose respectively of Cu NPs or silicon, Cmm: Clavibacter michiga-
nensis supsp. Michiganensis.
Lycopene = −0.0458 × Abs663 + 0.204 × Abs645 + 0.372 × Abs505
− 0.0806 × Abs453 (1)

β − carotene = 0.216 × Abs663 − 1.22 × Abs645 − 0.304 × Abs505

+ 0.452 × Abs453, (2)

Chl a= 0.999 × Abs663 − 0.0989 × Abs645, (3)

Chl b= −0.328 × Abs663 + 1.77 × Abs645, (4)

Total chlorophyll is the sum of Chl a and Chl b. All data are ex-
pressed as mg 100 g−1 dry weight (mg 100 g−1 DW).

2.5. Statistical analysis

Six replicates per treatment were considered for each of the eval-
uated variables in a completely random design. Each replicate was
obtained from one different plant. Only for the severity of C. michiga-
nensis and yield of tomato crop 15 replicates were considered. The
analysis of variance and Fisher LSD mean test (p ≤ 0.05) were per-
formed using the software Infostat 2018.

Fig. 2. Yield of tomato crop. Vertical bars represent the standard deviation.
3. Results
Bars with same letter are statistically equal (LSD Fisher, p ≤ 0.05). T0: Control,
LD and HD represent the low or high dose respectively of Cu NPs or silicon,
3.1. C. michiganensis severity and crop growth Cmm: Clavibacter michiganensis supsp. Michiganensis.

The severity of C. michiganensis decreased with all the treatments

3.2. Enzymatic activity and antioxidant compounds
applied in relation to the Treatment with only Cmm that presented a
severity of 68.5% (Fig. 1). The treatment Cu NPs LD + Si HD was the
The enzymatic activity in leaves of the tomato plants was modified
best, since it generated the greatest decrease in the severity of Cmm
by the application of the treatments, only CAT did not present differ-
with 28% less. The rest of the treatments generated a decrease in se-
ences between treatments (Table 1). In SOD activity the two treatments
verity around of 16–20% compared to the treatment of only Cmm.
with Cu NPs LD plus Si LD or Si HD generated a significant decrease
The yield was negatively affected by Cmm, generating a significant
compared to the Control. The rest of the treatments were statistically
decrease in all the treatments compared to the control (Fig. 2). The
equal to each other. Regarding the activity of the GPX enzyme, the two
treatment with only Cmm generated the greatest decrease in yield with
treatments with Cu NPs HD plus Si LD or Si HD generated an increase
43% less than the Control. The treatments Cu NPs LD + Si LD and Cu
compared to the Control, being 161% and 191% respectively. This same
NPs LD + Si HD generated a higher yield than the treatment with only
trend was observed in the activity of APX, where treatment Cu NPs
Cmm, exceeding it by approximately 28%. The treatments Cu NPs
HD + Si LD generated an increase of 279% compared to the Control,
HD + Si LD and Cu NPs HD + Si HD, although they showed a slight
while the treatment Cu NPs HD + Si HD induced an increase of 140%.
increase compared to the treatment with only Cmm, they were not
Regarding the PAL enzyme, again the treatment Cu NPs HD + Si LD
statistically different.

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C.F. Cumplido-Nájera et al. Scientia Horticulturae 245 (2019) 82–89

Table 1 45–74%. Regarding the content of flavonoids, no treatment exceeded

Enzymatic activity of PAL and antioxidant enzymes in leaves of tomato plants. the control, only it was observed that the treatments Cu NPs HD plus
Treatments CAT SOD GPX APX PAL both doses of Si, and Cu NPs LD + Si HD decreased this compound in a
range of 26–28%. In the content of β-carotene although differences
T0 131.49a 13.90a 0.23b 9.07b 0.10b between treatments were observed, no treatment was different from the
Cu NPs HD + Si LD 149.67a 13.18ab 0.60a 34.34a 0.30a
control.
Cu NPs HD + Si HD 70.89a 11.96ab 0.67a 21.79ab 0.17b
Cu NPs LD + Si LD 88.12a 9.32c 0.17b 14.47b 0.13b
Regarding antioxidants in tomato fruits, differences were observed
Cu NPs LD + Si HD 95.72a 11.04bc 0.23b 19.08b 0.18ab between treatments in glutathione, flavonoids, lycopene and β-car-
Cmm 111.24a 12.04ab 0.31b 13.46b 0.13b otene, while in total phenols and vitamin C all treatments were equal to
each other (Table 4). In Glutathione all treatments increased the con-
T0: Control, LD and HD represent the low or high dose respectively of Cu NPs or
tent of this compound, however, only the treatment of only Cmm and
silicon, Cmm: Clavibacter michiganensis supsp. michiganensis. Averages with
the two treatments with Si HD statistically beat the Control. The
same letter per column are statistically equal (LSD Fisher, p ≤ 0.05). CAT:
treatment with only Cmm presented 35% more glutathione content
enzymatic activity of catalase (U total protein−1), SOD: superoxide dismutase
(U ml−1 total proteins−1), GPX: glutathione peroxidase (U total proteins−1), than the Control, while the treatments with Cu NPs LD + Si HD and Cu
APX: ascorbate peroxidase (U total proteins−1), PAL: phenylalanine ammonia NPs HD + Si HD were 31% and 28% higher respectively. In the flavo-
lyase (U total proteins−1). noid content, the two treatments with Cu NPs LD regardless of the dose
of silicon were the best, however, they were not statistically different
Table 2 from the Control. In the lycopene content the treatment Cu NPs LD + Si
Enzymatic activity of PAL and antioxidant enzymes in fruits of tomato plants. HD was the best, surpassing the Control by 78%. The treatments Cu NPs
LD + Si LD and Cu NPs HD + Si LD were also better to the Control, by
Treatments CAT SOD GPX APX PAL
61% and 39% respectively. In the content of β-carotene the treatments
T0 108.31a 15.46a 0.26cd 23.24a 0.19b Cu NPs LD Si HD and Cu NPs LD + Si LD were those that generated the
Cu NPs HD + Si LD 291.57a 15.54a 0.97a 30.20a 0.48a highest content of this compound, surpassing the Control by 83% and
Cu NPs HD + Si HD 138.46a 14.94a 0.59b 15.92a 0.24b
67% respectively. Consistently treatment Cu NPs LD + Si HD generated
Cu NPs LD + Si LD 178.36a 15.64a 0.27cd 20.69a 0.27b
Cu NPs LD + Si HD 214.36a 15.66a 0.21d 20.37a 0.19b positive effects in antioxidants of tomato fruits, especially in glu-
Cmm 114.12a 15.08a 0.48bc 20.53a 0.30b tathione, flavonoids, lycopene and β-carotene. While the treatment
with only Cmm generated negative effects, being the one that presented
T0: Control, LD and HD represent the low or high dose respectively of Cu NPs or the lowest values in flavonoids, lycopene and β-carotene.
silicon, Cmm: Clavibacter michiganensis supsp. michiganensis. Averages with
same letter per column are statistically equal (LSD Fisher, p ≤ 0.05). CAT:
enzymatic activity of catalase (U total proteins−1), SOD: superoxide dismutase 4. Discussion
(U ml−1 total proteins−1), GPX: glutathione peroxidase (U total proteins−1),
APX: ascorbate peroxidase (U total proteins−1), PAL: phenylalanine ammonia 4.1. C. michiganensis severity and crop growth
lyase (U total proteins−1).
Clavibacter michiganensis ssp. michiganensis, is one of the most de-
generated the best result, surpassing the Control by 200%. structive bacterial diseases of tomato worldwide (Chalupowicz et al.,
In enzymatic activity evaluated in the fruits, the results showed that 2017; Nandi et al., 2018). This vascular pathogen generally invades and
the treatments affected only the GPX and PAL enzymes, while in CAT, proliferates in the xylem through natural openings or wounds, causing
SOD and APX there were no differences between treatments (Table 2). wilt and canker symptoms (Nandi et al., 2018). In addition, attack by
In the activity of GPX the best treatment was Cu NPs HD + Si LD, fol- pathogens induces the production of reactive oxygen species (ROS)
lowed by treatment Cu NPs HD + Si HD, generating increases of 273% which in turn generates oxidative stress in plants causing damage at the
and 127% respectively in comparison to the Control. On the other hand, molecular and genetic level, protein synthesis, and ultimately culmi-
the activity of PAL was favored only by the treatment with Cu NPs nates in programmed cell death (Aybeke, 2017).
HD + Si LD generating an increase of 153% compared to the Control. The application of silicon to tomato plants has induced benefits on
The rest of the treatments were statistically equal to each other. the incidence and severity of pathogens such as Ralstonia and Fusarium
The content of chlorophyll A was modified by the application of the (Ghareeb et al., 2011; Huang et al., 2011). This is by different me-
treatments, being the treatment with only Cmm which presented the chanisms that ultimately hinder the multiplication of the pathogen
highest content, however, the treatments with Cu NPs plus Si were which in turn improves the crop development. Silicon applied as silicic
equal to the Control (Table 3). In the content of glutathione it was acid induces biochemical barriers, and once polymerized in amorphous
observed that the treatment Cu NPs HD + Si HD generated the highest silica, generates physical barriers (Cooke and Leishman, 2011; Dann
amount of this antioxidant, surpassing the Control by 152%. In the case and Le, 2017). In addition, its accumulation in cell walls, intracellular
of total phenols, it was observed that with the exception of treatment spaces and leaf trichomes generates firmer and more rigid structures
Cu NPs LD + Si HD, all treatments exceeded the control in a range of (Carré-Missio et al., 2014; Ferreira et al., 2015). In the cytoplasm, it

Table 3
Content of chlorophylls and non-enzymatic antioxidant compounds in the leaves of tomato plants.
Treatments chl a Chl b Total Chl Glutathione Phenols Flavonoids β-carotene

T0 0.61ab 0.30a 0.91a 2.54b 4570.38b 5050.94a 0.12ab

Cu NPs HD + Si LD 0.74ab 0.36a 1.09a 6.39a 7967.24a 3828.74bc 0.15ab
Cu NPs HD + Si HD 0.59b 0.28a 0.87a 4.04b 6873.88a 3740.54c 0.08b
Cu NPs LD + Si LD 0.75ab 0.34a 1.09a 3.61b 6793.68a 4869.50ab 0.17a
Cu NPs LD + Si HD 0.59b 0.26a 0.85a 4.01b 4365.74b 3716.60c 0.14ab
Cmm 0.81a 0.35a 1.16a 3.43b 6623.52a 4927.46a 0.18a

T0: Control, LD and HD represent the low or high dose respectively of Cu NPs or silicon, Cmm: Clavibacter michiganensis supsp. michiganensis. Averages with the same
letter per column are statistically equal (LSD Fisher, p ≤ 0.05). Chl: chlorophyll, chlorophylls are expressed as mg 100 g−1 dry weight, GSH: Glutathione (mM
equivalent of GSH ml–1 min–1 total protein–1), Total phenols as mg EGA 100 g–1 DW, flavonoids as mg EQ 100 g–1 DW, β-carotene as mg 100 g−1 DW.

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C.F. Cumplido-Nájera et al.
Table 4
Non-enzymatic antioxidant compounds in fruits of tomato plants.
Treatments Glutathione Phenols
T0 2.66b 5283.70a
Cu NPs HD + Si LD 3.03ab 6856.28a
Cu NPs HD + Si HD 3.40a 6465.08a
Cu NPs LD + Si LD 3.30ab 6028.92a
Cu NPs LD + Si HD 3.48a 5424.52a
Cmm 3.58a 6875.84a
T0: Control, LD and HD represent the low or high dose respectively of Cu NPs or silicon, Cmm: Clavibacter michiganensis supsp. michiganensis. Averages with the same
letter per column are statistically equal (LSD Fisher, p ≤ 0.05). Glutathione is expressed as mM equivalent of GSH ml–1 min–1 total protein–1, Total phenols as mg EGA
100 g–1 DW, flavonoids as mg EQ 100 g–1 DW, Vitamin C as mg 100 g−1 FW, Lycopene and β-carotene as mg 100 g−1 DW., lycopene and β-carotene as mg g−1 FW.
induces biochemical barriers such as metabolic pathways activation, as
silicic acid (SA) and jasmonic acid (JA) (Bakhat et al., 2018; Jang et al.,
2018; Ning et al., 2014), SOD and CAT increase (Cao et al., 2017),
flavonoid and PAL expression (Rahman et al., 2015). Silicon also par-
ticipates in the overexpression or repression of pathogenicity related
genes (Brunings et al., 2009).
It has been documented that silicon application can stimulate the
systemic acquired resistance (SAR) by regulating genes involved in the
hypersensitivity response and the defense pathway driven by jasmonic
acid inducing tolerance to pathogens (Manivannan and Ahn, 2017).
On the other hand, the use of Cu NPs has been shown to be efficient
for the control of pathogens in plants, and its form of action is in several
ways. The Cu NPs can activate the antioxidant defense systems of the
plants from ROS stimulation, generating changes in the activity of the
enzymes (Choudhary et al., 2017; Zuverza-Mena et al., 2017). These
ROS can prevent the entry of the pathogen into plants, but they can also
stimulate the pathogenesis-associated protein genes as well as the sti-
mulation of salicylic acid, jasmonic acid and ethylene, hormones that
participate as signaling molecules in defense reactions (Tripathi et al.,
2017). Or through a direct inhibitory effect on the pathogen, a fact that
has been demonstrated in in vitro and in vivo studies (Sathiyabama and
Manikandan, 2018).
Silicon is considered a beneficial mineral since it promotes growth,
development, yield and fruit quality in crops (Malhotra et al., 2016).
While Cu NPs function as growth stimulants in various crops
(Hernández-Hernández et al., 2017; Juarez-Maldonado et al., 2016).
Therefore, it is expected that the application of silicon in conjunction
with Cu NPs will generate beneficial effects on tomato growth, and due
to the decrease in the severity of the observed disease, a beneficial
impact on crop yield is generated.
4.2. Enzymatic activity and antioxidant compounds
The attack by pathogens induces the production of reactive oxygen
species (ROS) generating oxidative stress in plants (Aybeke, 2017) and
activating the antioxidant defense system (Kang et al., 2014). ROS have
a double role that depends on duration, site, and concentration. A low
concentration acts as a signal and generates a stress response in plants,
as the activation of the antioxidant defense system, while a higher
concentration interrupts cellular homeostasis which produces oxidative
damage to proteins, DNA, and lipids (Shaw et al., 2014).
In Glycine max inoculated with Cercospora sojina an increase of
antioxidant compounds was observed (Telles Nascimento et al., 2016).
Also, Fusarium oxysporum colonization in tomato plants secretes effector
proteins that are virulence factors, triggering a series of plant responses
(Houterman et al., 2007) as the increase in CAT enzymatic activity
(Dorneles et al., 2017).
Therefore, an increase in enzymatic and non-enzymatic antioxidant
compounds is essential to reduce the damage caused by oxidative stress.
Hence, it is possible to hope that if a greater antioxidant capacity is
induced in plants, they can tolerate to a greater extent the attack of the
different pathogens.
86
Scientia Horticulturae 245 (2019) 82–89
Flavonoids Vitamin C Lycopene β-carotene
268.59ab 34.47 a 0.18c 0.06bc
265.13ab 40.03 a 0.25ab 0.08ab
278.99ab 39.73 a 0.24bc 0.08ab
294.11a 39.00 a 0.29ab 0.10a
301.67a 38.87 a 0.32a 0.11a
250.01b 34.92 a 0.18c 0.05c
Among the antioxidant enzymes are CAT, SOD, APX and GPX, while
non-enzymatic antioxidant compounds include glutathione, vitamin C,
total phenols, flavonoids, among others. SOD acts as the first line of
defense against ROS (Dos Santos et al., 2017) and CAT is one of the
enzymes that protect cells against ROS since it converts H2O2 into H2O
and O2 (Capaldi et al., 2015; Imahori et al., 2016), and its activity is
dependent on the concentration of H2O2 (Dos Santos et al., 2017). Thus,
there is a correlation between the components of the ROS elimination
systems such as CAT and SOD (Wang et al., 2005). APX is an enzyme
that removes H2O2 using ascorbate as an electron donor to reduce H2O2
to water, and it is extremely sensitive to ascorbate concentrations
(Ahmad et al., 2014). APX plays a central role in the ascorbate-glu-
tathione cycle, that is why the induction of APX is a response to oxi-
dative stress caused in plants by biotic or abiotic stress (Imahori, 2014).
And the GPX that participates in the elimination of H2O2 (Alharby et al.,
2016). The activity of these antioxidant enzymes such as SOD is of great
importance in the defense against pathogens (Hao et al., 2011), since
they regulate the concentration of ROS in the cells, thus avoiding
possible damage (Gill and Tuteja, 2010; Tie et al., 2012).
The enzyme PAL modulates L-phenylalanine for transcinnamic acid
manufacture, which in turn form phenylpropanoids (Hajiboland et al.,
2017a), which cause the production of phenolic metabolites (flavo-
noids, coumarins, phenol esters and lignin) (Weaver and Herrmann,
1997). This enzyme is directly related to tolerance to pathogens, so an
increase in its activity will provide greater tolerance to these (Wang
et al., 2017)
Silicon can also function as elicitor (Van Bockhaven et al., 2013)
and generates changes in different metabolic compounds, since this
element is associated with cell wall components (Babalar et al., 2016).
In addition, it reduces the effects caused by biotic stress, and the sus-
ceptibility to pests, fungi and bacteria (Meharg and Meharg, 2015).
Silicon in plants induces physical, biochemical (Pozza et al., 2015) and
genomic barriers (Pavlovic et al., 2013) against pathogens. In addition,
the formation of deposits of Si in root and leaves could prevent in-
cidence of these pathogens (Ma and Yamaji, 2008). This mineral can
also induce defensive responses similar to acquired systemic resistance,
by increasing the enzymatic antioxidant activity and non-enzymatic
constituents (Hasanuzzaman et al., 2018), phenolic compounds, an-
thocyanins, flavones, lignin (Jafari et al., 2015), phytoalexins and
proteins related to pathogenesis. Moreover, it can activate the expres-
sion of defense related genes and add to the transduction of stress
markers such as salicylic acid, jasmonic acid and ethylene (Cai et al.,
2009).
There is a series of works where the effect of silicon against pa-
thogens in crops has been evaluated. Fortunato et al. (2012) and
Fortunato et al. (2014) showed that the supply of Si to banana plants
reduced the intensity of Fusarium wilt. The application of sodium sili-
cate and potassium silicate in in vitro developed cotton, controlled
Fusarium activity (Yassin et al., 2016). Weerahewa and David (2015)
produced larger fruit size in tomatoes infested by anthracnose when
applying sodium silicate. Ouzounidou et al. (2016) reported higher
antioxidant activity in cucumber when applying potassium silicate.
C.F. Cumplido-Nájera et al.
Dallagnol et al. (2015) reported that the application of Silicon in melon
plants inoculated with Podospaera xanthii generated a high concentra-
tion of H2O2, which increased SOD activity as a defense mechanism. It
has been reported that the application of silicon influences phenolic
compound content (Hajiboland et al., 2017b) since Si improves the
pathway of phenylpropanoids (Fortunato et al., 2014), that in turn in-
creases defenses against pathogens.
On the other hand, the application of Cu NPs also generates bios-
timulation, specifically of the antioxidant capacity of the plants
(Hernández-Hernández et al., 2018; Pinedo-Guerrero et al., 2017). Cu
NPs can trigger ROS production resulting in the activation of anti-
oxidant enzymes as a detoxification mechanism (Zuverza-Mena et al.,
2017). This biostimulation response may be related to the phenomena
of surface interaction of the NPs (Bell et al., 2014) caused by the par-
ticular characteristics of these materials and ultimately, the effects of
Cu NPs will be different depending on the characteristics (size, shape,
etc.), amount and plant species (Zuverza-Mena et al., 2017).
The production of antioxidant enzymes in tomato plants by the
application of Cu NPs (Hernández-Hernández et al., 2018) can induce
greater tolerance to the attack of pathogens such as C. michiganensis,
since they will regulate ROS, thus decreasing oxidative stress (Gill and
Tuteja, 2010).
As observed in the present study, apparently the application of
potassium silicate in conjunction with Cu NPs stimulated the activity of
antioxidant enzymes, which surely generated greater capacity of to-
mato plants to tolerate oxidative stress caused by C. michiganensis. This
result in a decrease in the severity of the pathogen, which in turn had a
lower yield loss.
Non-enzymatic antioxidant compounds as well as antioxidant en-
zymes are very important in controlling oxidative stress caused by pa-
thogens such as C. michiganensis. Among the main compounds of this
type are vitamin C, glutathione, total phenols, flavonoids and car-
otenoids, among others.
In plants, vitamin C has many cellular functions mostly linked to its
ability to give up electrons (Truffault et al., 2014), and may act directly
as an antioxidant by trapping ROS, preventing or minimizing oxidative
damage (Gill and Tuteja, 2010). In addition, it can also regenerate the
glycoprotein and tocopherol radicals, and act as a cofactor for many
enzymes such as APX (Muzolf-Panek et al., 2017). It plays an important
role in photosynthesis as an enzymatic cofactor (including the synthesis
of ethylene, gibberellins, flavonoids, and anthocyanins) (Gest et al.,
2013).
Carotenoids are important by the antioxidant actions based on their
ability to quench singlet oxygen and to trap peroxyl radicals (Havaux,
2013; Latowski et al., 2014; Yuan et al., 2015). Lycopene is one of the
main carotenoids in tomato fruits and it is also one of the main anti-
oxidants (Klunklin and Savage, 2017), and one of the most efficient
singlet oxygen neutralizers among the natural carotenoids (Latowski
et al., 2014). In plants, lycopene synthesis is usually derived from the
mevalonic acid (MVA) and methylerythritol phosphate (MEP) path-
ways. These pathways synthesize isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP), which function as precursors to
the synthesis of carotenoids (Barrios et al., 2017).
Phenols are antioxidant compounds that trigger the synthesis of a
series of secondary metabolites from the shikimic acid pathway or
through phenylpropanoids under conditions of abiotic stress (Ren and
Sun, 2014; Swieca, 2016). Flavonoids act as antioxidants, protecting
plants from oxidative stress by the elimination of H2O2 and singlet
oxygen generated under biotic or abiotic stress (Hernández et al., 2009;
Hollman and Arts, 2000).
Silicon can also function as elicitor (Van Bockhaven et al., 2013)
and generates changes in different metabolic compounds, since this
element is associated with cell wall components (Babalar et al., 2016).
It has been reported that the application of this element influences
phenolic compound content (Hajiboland et al., 2017b). Silicon is as-
sociated with cell wall components and with polyphenol metabolism
87
Scientia Horticulturae 245 (2019) 82–89
alterations (Babalar et al., 2016), which improves water permeability
(Liang et al., 2015), and increases defenses against pathogens.
Fortunato et al. (2014) reported that Si improves the pathway of phe-
nylpropanoids in banana plants under Fusarium sp. stress so there is a
direct relationship with phenol production. The enzyme PAL modulates
L-phenylalanine for transcinnamic acid manufacture, which in turn
form phenylpropanoids (Hajiboland et al., 2017a), which cause the
production of phenolic metabolites (flavonoids, coumarins, phenol es-
ters and lignin) (Weaver and Herrmann, 1997).
The application of Cu NPs stimulates the production of antioxidant
compounds (Pinedo-Guerrero et al., 2017) as a response to the oxida-
tive stress that it generates (Shobha et al., 2014). This has been docu-
mented in different crops, in tomato the lycopene was increased by the
application of Cu NPs + Chitosan, reporting 12% more than the Control
(Juarez-Maldonado et al., 2016). The foliar application of NPs of TiO2
and ZnO (0–1000 mg kg–1) increased the lycopene content (80–113%)
in tomato fruits (Raliya et al., 2015).
In the case of glutathione it has been observed that this compound
increases in response to conditions of biotic or abiotic stress (Kumar and
Trivedi, 2016). Regarding the total phenols content of fruits, Jalapeno
pepper fruits showed an increase in the content of the total phenols
(5.9%) when 2.0 mg of Cu NPs + Chitosan-PVA were applied (Pinedo-
Guerrero et al., 2017). Additionally, foliar application of nano-fertili-
zers of Zn and B increased the phenols content in pomegranate fruits
(Davarpanah et al., 2016). In addition, in A. Thaliana, the concentration
of anthocyanins increased when CuO nanoparticles were applied (Saito
et al., 2013).
García-Gómez et al. (2017) reported that the application of ZnO NPs
to the soil (3, 20, and 225 mg kg–1) in bean and tomato crops stimulated
the antioxidant capacity considerably. The Cu NPs application can
generate certain stress in tomato plants, since this produced an increase
in ROS which activates a defense mechanism (Rizwan et al., 2017;
Shobha et al., 2014), increasing the production of antioxidant com-
pounds such as ascorbate, glutathione, carotenoids, and flavonoids,
among others (Seminario et al., 2017). The same response is observed
under conditions of mild stress caused by various NPs, however, it
changes when high doses are applied due to an overproduction of ROS,
resulting in the decrease of antioxidants (Rizwan et al., 2017).
5. Conclusions
The joint application of copper nanoparticles and potassium silicate
was effective in reducing the severity of C. michiganensis in all doses
probed. Also, the loss of yield due to the bacteria was reduced, espe-
cially the treatments that included the low dose of Cu NPs.
The application of copper nanoparticles and potassium silicate sti-
mulated the activity of the enzymes SOD, PAL, GPX, and APX, as well as
the concentration of reduced glutathione and total phenols in the
leaves, which probably favored tolerance to oxidative stress caused by
C. michiganensis. The joint application of copper nanoparticles and po-
tassium silicate stimulated the levels of enzymatic and non-enzymatic
compounds that are key in defense of tomato plants, increasing the
tolerance to C. michiganensis.
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