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Soil and foliar nickel application in coffee seedlings alters leaf
nutrient balance
L.O. MACEDO1, J.L. FAVARIN1, T. TEZOTTO1, A.P. NETO1, S.A.L. ANDRADE2,
P. MAZZAFERA1,2,*
1Departamento de Produção Vegetal, Escola Superior de Agricultura Luiz de Queiroz, Universidade de
São Paulo, Piracicaba, SP, Brasil
2Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas,
SP, Brasil
Keywords: Coffea arabica, mineral nutrition, Ni recovery efficiency, urea,
urease
S. – Nickel (Ni) is a micro-nutrient demanded in very low amounts by
plants and poorly studied in coffee. Coffee seedlings received Ni via soil and foliar
spray, and were evaluated for growth, biochemical parameters and nutrient contents in
stems and roots both in leaves developed before and after Ni application. Both forms
of Ni application caused several changes in the nutrient contents in pre-existing and
new developed leaves. Ni recovery efficiency (RENi = total plant Ni/applied Ni) was
higher with foliar application. Foliar urease increased with both forms of Ni application.
Growth parameters did not change but Ni increased dry mass and specific mass of new
leaves in both application forms. It is concluded that foliar application is the best way
to supply Ni to coffee plants and it is recommended that further experiments under field
conditions are carried out to establish the benefits of Ni on coffee productivity.
I. – Coffee is a nitrogen-demanding plant, and doses
between 250 and 450 kg ha-1 are usually used for fertilization depend-
ing on the age of the trees (V-R et al., 1997). N is fundamental
for plant growth as it is constituent of nucleic acids, amino acids and
proteins, chlorophyll and several others no less important compounds
involved in photosynthetic, and other primary and specialized metabo-
lisms (M, 2011). Because urea has the lowest price per kg
of nitrogen it is the most used N-fertilizer in coffee cultivation. In the
soil, microorganisms use the enzyme urease to transform urea into NH4+
which is subsequently converted by nitrifying bacteria into NO3-, the
main form of inorganic N absorbed by plants (P et al., 2013).
The essentiality of Ni is plants has been mainly related with the
enzyme urease (P et al., 2013). In leaves, a small fraction of the
organic N is in the form of urea [(NH2)2CO], which is produced from the
Agrochimica, Vol. LXIV - N. 2 April-June 2020
Received May 12, 2019 – Received in revised form May 30, 2019 – Accepted June 5, 2019
DOI 10.12871/00021857201917
* Corresponding author: pmazza@unicamp.br
L.O. MACEDO ET AL.
168
degradation of arginine by the enzyme arginase. Accumulation of urea
in leaves can lead to necrotic lesions and its degradation to NH3 and CO2
by plant urease prevents this damage (P et al., 2013). Urease is
a metalloenzyme that has Ni as part of its active site, and this allows Ni
to be classified as an essential nutrient for plants (M et al.,
2013). This enzyme is found in every plant, whose gene expression var-
ies widely depending on the species. In woody plants, like coffee, urease
gene expression is lower than in herbaceous plants, such as potato, lotus,
tomato and soybean (M et al., 2013).
Besides urease, new roles have been suggested for Ni in plants,
supporting its essentiality. Ni also appears to act in the regeneration
of reduced glutathione in the methylglyoxal pathway (F et
al., 2015). It has been also suggested that by substituting Fe2+ in the
aminocyclopropane carboxylate oxidase (ACC-oxidase) and forming
an inactive enzyme-metal complex Ni can control ethylene biosynthe-
sis (T et al., 2015). Additionally, Ni seems to be involved in
plant disease resistance (E et al., 2020) and seed germination
(B et al., 1987) but without a proof of its exact metabolic function.
A comprehensive review on the essentiality of Ni in plants and meta-
bolic functions can be found in P et al. (2013).
A major problem to study Ni essentiality is because it is requested in
very low amount by plants and even small contaminations may mask the
results. B et al. (1987) grew three consecutive barley generations
in nutrient solution without Ni and observed that the Ni content in the
seeds of the first generation was sufficient to grow all generations without
display of any symptom of deficiency. K et al. (2014) observed
Ni symptom deficiency in soybean plants grown in nutrient solution only
when the Ni content in the seeds was low, but no detrimental effect was
observed in the grain yield. Thus, if not supplied as a fertilizer, the Ni
content in the seeds has a direct relationship with Ni soil content.
The total Ni content in the earth’s crust is similar to that of cobalt
(Co) and iron (Fe), ranging from 0.2 to 450 mg dm-3 Ni, although the
exchangeable content in most world soils is less than 1.0 mg dm-3
(K-P and P, 2001). A few reports showed that
in Brazilian soils Ni is found in a wide range of concentrations, from
< 0.0014 to 1,167 mg kg-1 (R et al., 2014).
Little is known about the effect of Ni in coffee plants. Increasing
Ni content in coffee leaves led to an increase of nitrate reductase activ-
ity (R et al., 2009), suggesting an induction of N metabolism. Field
NI APPLICATION IN COFFEE
169
experiments showed that Ni supply via soil increased leaf protein
contents and prolonged leaf life span what suggests an interference in
ethylene metabolism (T et al., 2012). On the other hand, at toxic
levels Ni impairs growth, interferes with coffee metabolism (T
et al., 2012).
Aiming to increase our knowledge on the beneficial effects of Ni in
coffee, seedlings were supplied with this micronutrient either via soil or
foliar application and growth, urease activity, chlorophyll, and nutrient
contents were evaluated.
M M. – Plant material and growth condi-
tions. – Coffee seedlings (Coffea arabica L. cv. Catuaí Vermelho IAC
99) were obtained from a commercial coffee nursery and transferred to
pots containing as substrate 3 dm3 of a mixture of a loam-textured soil,
sand and organic commercial substrate (1:1:1 by vol.), and kept under
greenhouse conditions, without control of temperature and receiving
daily watering. The plants were fertilized with macro- and micronu-
trients, as recommended by V-R et al. (1997). Plants presenting
similar development (height and number of lateral branches) were cho-
sen for the Ni treatments. Plants were subjected to two Ni treatments,
soil and foliar application, following a completely randomized design
with four plants as replicates. The source of Ni used was nickel chloride
(NiCl2·6H2O). For soil application, two doses of Ni (3 and 30 mg per
plant, what corresponded to 1 and 10 mg Ni dm-3 soil) were used. The
Ni salt was diluted in 250 mL of distilled water and evenly distributed
on the soil surface. For foliar application, the soil surface in each pot was
previously protected with a plastic film to avoid contamination during
the foliar spraying of the plants. Ni aqueous solutions (0.65 g and 6.5 g
of Ni diluted in 250 mL water) were applied with a hand sprayer until
the whole surface of most of the coffee leaves was wetted but without
dripping. We recorded the initial and final volume of the solution in the
sprayer and found that approximately 15 mL were sprayed per plant,
corresponding to approximately 0.038 and 0.38 mg of Ni per plant. Five
to ten times higher Ni amounts (soil) or concentrations (foliar) started to
induce toxicity in the plants, observed by leaf growth inhibition. Control
plants did not receive the nutrient. After Ni application the irrigation
in the greenhouse was suspended for 2 days and afterwards water was
daily supplied with a sprinkler irrigation system. At the beginning of the
experiment, the last internodes of the stem and plagiotropic branches
L.O. MACEDO ET AL.
170
of each plant were marked with plastic tape to differentiate the leaves
developed previously and after Ni treatment.
Plant growth and nutrients content. – After 90 days of the Ni appli-
cation, the stem diameter was determined with a digital calliper at 2 cm
from the base of the stem. The height was measured taking as references
the cotyledon leaf scars and the stem apex. The number of leaves devel-
oped before and after Ni application was also determined.
After 100 days of Ni application, plants were removed from the
pots and separated into roots, woody parts (stem and branches) and
leaves, separating those already developed before Ni application, and
those developed after Ni application. Roots were washed in running
tap water to remove soil. Leaf area was determined using an area meter
(LiCor 3100, Nebraska, USA). Leaves, stem + branches and roots were
oven-dried at 70°C for 72 h for dry mass determination. For each plant,
specific leaf mass (SLM) was obtained by calculating the ratio between
leaf dry mass (mg) and leaf area (cm2). Substrate was sampled from the
pots for Ni chemical analysis.
For nutrients determination, dried plant material was ground in a ball
mill. Total N-concentration was determined after sulfuric acid digestion,
using a semi-micro Kjeldahl analytical method (B, 1965). Part
of the ground material was digested with nitric acid and perchloric acid
in an open system for Ni determination by inductively coupled plasma
optical emission spectroscopy (ICP-OES). Other nutrients, such as
phosphorus (P), calcium, (Ca), potassium (K), sulphur (S), magnesium
(Mg), iron (Fe) and zinc (Zn), were determined by energy-dispersive-ray
fluorescence (EDXRF) (T et al., 2012). Ni recovery efficiency
(RENi) was calculated using the equation: RENi (%) = (Ni accumulated
in plant/Ni applied) × 100.
Chlorophyll content and urease activity determinations. – As we
expected that biochemical changes take place earlier than measurable
growth changes, we determined chlorophyll content and urease activity
after 50 days of Ni application. Chlorophyll was indirectly determined
using a Minolta SPAD on the adaxial side of fully expanded leaves. Leaf
urease activity was determined in vivo, based on the evaluation of NH4+
derived from urea hydrolysis (H et al., 1983). Briefly, the disks (1
cm diameter) were obtained with a cork-borer from the first expanded
leaf pair of lateral (plagiotropic) branches and infiltrated under vacuum
(2 × 2 min) with a reaction buffer containing urea. Then after 3 h of
incubation at 30°C, the released NH4+ was determined.
NI APPLICATION IN COFFEE
171
Fig. 1. – Stem diameter and plant height (A, B), number of leaves (C, D) and leaf area (E, F) of plants
that received Ni via soil (A, C, E) and foliar spray (B, D, F). “Leaves after Ni” and “leaves before Ni”
indicate leaves developed after/before Ni treatment. Bars indicate the standard error of the mean (n = 4).
Fig. 2. – Dry mass of leaves, branches and stem, and roots (A, B) and specific leaf mass (C, D) of
plants that received Ni via soil (A, C) and foliar spray (B, D). “Leaves after Ni” and “leaves before
Ni” indicate leaves developed after/before Ni treatment. Bars indicate the standard error of the mean
(n = 4). Letters indicate statistical difference in the same tissue by the Tukey test (p < 0.05).
L.O. MACEDO ET AL.
172
Statistical analysis. – Four plants were used as replicates in all
measurements. We used Statistica Software for assessing data normal-
ity (Shapiro-Wilk test) and data were submitted to analysis of variance
(ANOVA), and means were compared by Tukey test with a confidence
level of 95% (p < 0.05).
Fig. 3. – Nickel concentrations in leaves, stem and branches, and roots of plants that received Ni
via soil (A) and foliar spray (B). “Leaves after Ni” and “leaves before Ni” indicate leaves developed
after/before Ni treatment. Ni was determined in all tissues but in those figures in which histograms
are not shown Ni was detected at trace levels, and below the confidence limit of the Ni standard
curve. Bars indicate standard error of the mean (n = 4).
Fig. 4. – Nitrogen (A, B) and potassium concentrations (C, D) in leaves, stems and branches, and
roots of plants that received Ni via soil (A, C) and foliar spray (B, D). “Leaves after Ni” and “leaves
before Ni” indicate leaves developed after/before Ni treatment. Bars indicate the standard error of the
mean (n = 4); Letters indicate statistical difference in the same tissue by the Tukey test (p < 0.05).
NI APPLICATION IN COFFEE
173
R. – Plant growth. – Ni application also did not change
significantly stem diameter and plant height (Figs 1A and 1B), num-
ber of leaves (Figs 1C and 1D) and leaf area (Figs 1E and 1F). On the
other hand, leaves developed after Ni application, regardless of the way
of application, had higher dry mass than the leaves of control plants
(Figs 2A and 2B) and also showed an increase in leaf specific mass (Figs
2C and 2D), despite the leaf area remained unchanged.
Ni in the soil and plant. – The substrate used to growth coffee
seedlings had originally 0.1 mg Ni dm-3, which increased to 0.5 mg and
3.2 mg Ni dm-3 (DTPA-extractable) after soil application of 3 mg and
30 mg Ni per pot, respectively. Ni was detected in all coffee plant tis-
sues, but some plants presented very low levels, which were below the
confidence limit of the regression curve produced with pure standards to
estimate Ni concentration in the acid digested extracts (Fig. 3). Ni was
detected in tissues of plants receiving the highest dose in the soil and in
the roots of plants receiving the lowest dose. When Ni was applied via
leaf, it was detected only at the highest dose, but not in leaves developed
after Ni application (Fig. 3). The lack of Ni detection at the lowest con-
centrations in both Ni treatments may also be related to the fact that after
Ni application the plants remained in the green house under daily sprin-
kler irrigation for a hundred days, with a possible removal by drainage
from substrate. The calculated RENi was 57.5% for foliar application of
0.38 mg Ni per plant and only 3.9 and 1.9% for the 3 and 30 mg Ni soil
doses, respectively.
Nutrient contents and biochemical evaluations. – Both, soil and
leaf applications, decreased N concentration in leaves already present
before Ni application (Figs 4A and 4B). On the contrary, K concentra-
tion increased in the new-formed leaves developed after soil or foliar
Ni applications (Figs 4C and 4D). Ni application also led to an increase
of Ca concentration in new leaves and roots (Figs 5A and 5B). S con-
centration was reduced in leaves developed before and after foliar and
soil Ni applications (Figs 5C and 5D). There was an increase of P only
in plants with the foliar Ni application treatment and in both analysed
leaf types (Figs 6A and 6B). There were not significant changes in
plant Mg concentrations (Figs 6C and 6D). There was an increase in
Zn concentration only in the leaves developed after Ni foliar applica-
tion (Figs 7A and 7B). Fe decreased in pre-existing leaves and stem
and branches in plants receiving both, foliar and soil Ni applications
(Figs 7C and 7D).
L.O. MACEDO ET AL.
174
Fig. 5. – Calcium (A, B) and sulfur concentrations (C, D) in leaves, stems and branches, and roots of
plants that received Ni via soil (A, C) and foliar spray (B, D). “Leaves after Ni” and “leaves before
Ni” indicate leaves developed after/before Ni treatment. Bars indicate the standard error of the mean
(n = 4); Letters indicate statistical difference in the same tissue by the Tukey test (p < 0.05).
Fig. 6. – Phosphorus (A, B) and magnesium concentrations (C, D) in leaves, stems and branches,
and roots of plants that received Ni via soil (A, C) and foliar spray (B, D). “Leaves after Ni” and
“leaves before Ni” indicate leaves developed after/before Ni treatment. Bars indicate the standard
error of the mean (n = 4); Letters indicate statistical difference in the same tissue by the Tukey
test (p < 0.05).
Chlorophyll was not significantly changed by Ni application
(Fig. 8A). Leaves of plants treated with Ni had higher urease activity
than leaves of control plants and the highest activities were found at the
highest Ni doses (Fig. 8B).
NI APPLICATION IN COFFEE
175
D. – Since the pioneering works, which proved the essenti-
ality of Ni for plants (P et al., 2013), compared with other micro-
nutrients, little progress has been made with this element in relation to
its influence in plant physiology. Most of the recent research on Ni in
plants has dealt with its toxicity. Because the intense anthropogenic
activities, Ni may increase in the environment and reach toxic levels in
soils, which has stimulated studies on its toxicity and tolerance in plants
(S et al., 2018). In highly polluted soils, Ni concentration can
reach 26.000 mg kg-1 (S et al., 2018) while regular Ni concen-
Fig. 7. – Zinc (A, B) and iron concentrations (C, D) in leaves, stems and branches, and roots of plants
that received Ni via soil (A, C) and foliar spray (B, D). “Leaves after Ni” and “leaves before Ni”
indicate leaves developed after/before Ni treatment. Bars indicate the standard error of the mean (n =
4); Letters indicate statistical difference in the same tissue by the Tukey test (p < 0.05).
Fig. 8. – SPAD-chlorophyll (A) and urease
activity (B) in leaves of plants that received
Ni via soil and foliar spray. Bars indicate the
standard error of the mean (n = 4); Letters
indicate statistical difference in the same
tissue by the Tukey test (p < 0.05).
L.O. MACEDO ET AL.
176
tration in soils is around 100 mg dm-3 (S et al., 2018). However,
the exchangeable amount in most soils is less than 1.0 mg dm-3, which
varies depending on soil type and parent material (K-P and
P, 2001).
Here, application of Ni to the soil increased its concentration from
0.1 mg dm-3 to 0.5 and 3.2 mg dm-3, when 3 mg and 30 mg Ni were
applied per plant, respectively. For soils of São Paulo State, Brazil,
A et al. (2005) determined that Ni phytotoxicity ranged between
30 mg and 65 mg dm-3. Thus, the Ni amounts applied to the soil in this
research were far below these toxicity limit.
As a micronutrient, at non-toxic levels Ni can be beneficial. Ni rang-
ing between 0.1 and 10 mg kg-1 soil increased the N content of soybean
shoots by 42 and 50% (M et al., 2016). The benefits of Ni seem
to be mainly related to the increased efficiency of N metabolism, its
involvement in the activation of various enzymes and by acting on plant
antioxidant metabolism (P et al., 2013; F et al., 2015;
S et al., 2018).
In agreement with several previous studies (P et al., 2013),
here we observed an increase of urease activity in leaves of plants receiv-
ing Ni by both soil and foliar application. Urease activity increased even
in tissues in which we were not able to precisely quantify Ni concentra-
tion, detected at trace levels. Such higher activity may be supported by
previous studies that showed that the amount of Ni in barley seeds was
sufficient to support three new generation of plants, without any appli-
cation of Ni and visible symptom of deficiency (B et al., 1987).
Thus, the requirement of Ni for urease activity is very low.
In coffee plants that received Ni soil application, the highest Ni con-
centration was detected in the roots, as observed for other plant species
(S et al., 2016), which were fertilized with Ni. Ni accumulation
in plant roots has been shown to be related with membrane transporters
of the family iron-regulated transporter, which are specifically induced
by Ni exposure (N et al., 2012). Part of the Ni in the roots may
be retained in the cell wall, and probably by the root associated micro-
organisms, especially by the chitin of the cell wall of mycorrhizal fungi
(A et al., 2009). It is well known that coffee roots may be heav-
ily colonized by mycorrhizal fungi (A et al., 2009). In addition,
root cells may store essential and non-essential elements into vacuoles,
controlling the transport to the shoots (R et al., 2018),
thus contributing to accumulation of Ni in the roots.
NI APPLICATION IN COFFEE
177
Although the amount of Ni applied per plant by foliar spraying
(0.038 and 0.38 mg) was about eight times lower than the amount
applied via soil (3 and 30 mg), plant Ni concentrations were somewhat
similar for both application types (Fig. 3), what is a clear indication
of its low recovery efficiency from soils. Ni has high affinity for soil
organic matter or Mn and Fe oxides (K-P and P,
2001). When we calculated Ni recovery efficiency (RENi), it was only
3.92% and 1.92% for plants that received 3 mg and 30 mg Ni per plant
via soil, while RENi was 57,5% when applied via foliar spray of 0.38
mg Ni per plant. L et al. (2019) tested different doses of Ni applied
to the soils of soybean plantations at different soil types and concluded
that the responses to Ni, including plant growth and urease activity, were
strongly influenced by soil clay amount. Thus, it seems that the best way
to supply Ni to coffee plants is via foliar application.
Ni was found in the roots of plants that received 0.38 mg via foliar
application, indicating the mobility of this nutrient in the phloem (P
et al., 2006). Supportively, in wheat, the application of 63Ni to the leaves
showed high phloem mobility, and radioactivity was found in the roots
(R and F, 2005).
Our results showed that soil Ni application reduced Fe concentration
in leaves developed before Ni application and in stems and branches.
In general, the Fe concentration in older leaves were higher than in
the new developed leaves. It is known that Fe, Zn and Ni compete for
the same membrane protein transporters in the epidermal root cells
(K et al., 1999). However, here this may not be true for Zn,
which contents remained unaltered despite the way Ni was supplied.
Reduced Fe translocation to the shoots has been already observed in
other Ni exposed plant species (F et al., 2019) and related to the
Ni suppression of root-to-shoot Fe signalling, suggested to be linked to
ethylene signalling (N et al.,2012).
Calcium has been studied for its ability to relieve the effect of heavy
metal stress on plants. Regarding the interaction between Ni and Ca,
the application of Ca ameliorates Ni toxicity improving plant growth
and photosynthetic parameters of rice plants (A et al., 2015). Ca
application also decreased Ni in the shoot and roots of the rice plants,
indicating that it interfered with metal absorption (A et al., 2015). Ni
have similar ionic radius to Ca, competing by soil binding sites (C
et al., 2009). Our results showed that Ca concentrations increased in new
leaves developed when Ni was supplied via soil. Because their chemi-
L.O. MACEDO ET AL.
178
cal similarity, Ni supplied via soil may have displaced Ca in the soil,
increasing absorption and explaining its higher content in young leaves.
But foliar application also increased Ca in the same leaves. Since Ca is
poorly remobilized in plants (M, 2011), it is not possible to
find a reasonable explanation for such observation.
Both foliar and soil Ni application influenced N and K contents, but
differently. While there was a decrease in N content in leaves present
before Ni application, K content increased in leaves formed after Ni
application. The variation of these two nutrients, as well as Ca, allows
speculation about their involvement in the dry mass of leaves devel-
oped after Ni application. The drop in N content in the leaves developed
before Ni application may indicate, at first, remobilization to sink tissues.
The higher urease activity in Ni-exposed plants may also be related to
N-remobilization processes (P et al., 2013). But, the only sig-
nificant variation in growth parameter was the dry mass of these young
leaves and, consequently, of the leaf specific mass, probably because of
an increase in leaf thickness. Since the greatest amount of Ca in plants is
in the cell wall and the middle lamella (M, 2011), its increase in
leaves might be related to the increase in leaf thickness and consequently
the higher leaf mass. Higher specific leaf mass was accompanied by lower
concentrations of N and S, which could be a consequence of a dilution
effect caused by an increase in structural carbon compounds, such as
cellulose, lignin, cutin and waxes, in young leaves. Higher leaf specific
mass has been associated to greater metabolic activities to attend carbon
skeletons demand (B et al., 2000). On the other hand, since the role
of K in plants is closely associated with the control of the water potential
(M, 2011), a high K concentration in new leaves would indicate
increased turgor pressure in response to an increased cell volume.
Although the amount of Ni supplied to the coffee plants was far
from toxic levels, Ni might have induced cell wall thickening. Cell wall
thickening formation is considered a detoxification strategy to reduce
metal toxicity and can contribute to increases in specific leaf mass
in Ni-exposed plants, as reported by M et al. (2016). In response
to Ni stress, these authors observed changes in the ultrastructure of
tomato mesophyll cells, that included increased cell wall thickness and
Ni-vacuolization (M et al., 2016).
A pronounced effect of Ni on coffee was the reduction of S con-
tent in both leaf types of plants of both Ni treatments. S participates
in many important metabolic pathways in plants and is part of several
NI APPLICATION IN COFFEE
179
compounds such as the amino acids cysteine and methionine, and mem-
brane sulpholipids. S assimilation into organic compounds occurs in
chloroplast and has a close link to N assimilation, as N limitation affects
the S metabolism (H et al., 2010). As for N, Ni-treated coffee plants
showed reduced S content in the leaves present before Ni application.
However, while there was only a tendency of N reduction in the young
leaves, the S concentration was significantly reduced in the leaves devel-
oped before Ni application. Although the close relationship between the
metabolism of N and S, the amount of S required for plant metabolism is
much smaller than that of N, in a ratio of one to ten. Therefore, a slight
variation in N content could have a greater effect on S content.
C. – Foliar application was a more efficient way of
supplying Ni to coffee than soil application, as indicated by RENi cal-
culation. While RENi values were greater than 50% for foliar applica-
tion, just 4% of recovery was found when Ni was applied via soil. Ni
was readily mobile in coffee plants, being transported from the leaves
to the roots. Ni supply increased leaf thickness and increased the leaf
concentrations of K, Ca, Zn and P, while decreased those of N, S and
Fe. Ni increased foliar urease activity when Ni was supplied either via
soil or foliar spraying. Our results indicated that coffee seedlings were
metabolically and nutritionally affected by Ni application. We recom-
mend long-term field experiments to unravel the beneficial Ni effects on
coffee productivity as well as on beverage quality.
A. – PM and JLF thank the Brazilian Research
Council (CNPq) for a research fellowship. This study was partially
funded by São Paulo Foundation (FAPESP – grant 12/01607-3).
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