American Journal of Plant Sciences, 2012, 3, 1546-1561
http://dx.doi.org/10.4236/ajps.2012.311187 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
Use of the SPAD-502 in Estimating Nitrogen Content in
Leaves and Grape Yield in Grapevines in Soils with
Different Texture
Gustavo Brunetto1, Gustavo Trentin2, Carlos Alberto Ceretta3, Eduardo Girotto3, Felipe Lorensini3,
Alcione Miotto3, Glaucia Regina Zaferi Moser3, George Wellington de Melo4
1Department of Rural Engineering, Program in Agricultural Ecosystems, Universidade Federal de Santa Catarina (UFSC), Flori-
anopolis, Brazil; 2Embrapa Pecuária Sul, Bagé, Brazil; 3Program in Soil Science, Departamento de Solos (DS), Universidade Federal
de Santa Maria (UFSM), Santa Maria, Brazil; 4Embrapa Uva e Vinho, Bento Gonçalves, Brazil.
Received August 9th, 2012; revised September 14th, 2012; accepted October 8th, 2012
ABSTRACT
The SPAD reading may be used in estimating total nitrogen content (N) in leaves and even in estimating grape yield in
grapevines. The objective of this study was to estimate total N content in leaves and grape yield using the SPAD-502 in
grapevines submitted to nitrogen fertilization in soils with clayey and sandy texture. In 2008, two experiments were
installed in the Southern region of Brazil. In Experiment 1, Cabernet Sauvignon grapevines were planted in a soil with
clayey texture and with application of 10, 20, 40 and 80 kg·N·ha−1·year−1. In experiment 2, Cabernet Sauvignon grape-
vines were planted in a soil with sandy texture and with the application of 0, 10, 15, 20, 40, 80 and 120 kg·N·ha−1·year−1.
In the grapevines of the two experiments and during the period from 2008 to 2010, SPAD readings were made on leaves
throughout the flowering period and at change in color of the berries using the portable chlorophyll meter Mi-
nolta-SPAD-502. The leaves were collected, dried, ground and submitted to analysis of the total N content. In addition,
grape yield per hectare was evaluated. The SPAD-502 readings estimated the total N content in flowering and at change
in color of the berries in the Cabernet Sauvignon grapevines grown on soils with clayey texture and sandy texture, es-
pecially in the first year of evaluation. However, the precision of the SPAD-502 readings is low, with there being no
relationship between the SPAD-502 readings and grape yield.
Keywords: SPAD-Readings; Non Destructive Estimation; Foliar Nitrogen; Leaf Analysis; Vitis vinifera
1. Introduction
Grapevines grown on soils with sandy texture and with
low organic matter content are normally submitted to the
application of nitrogen (N) due to supposed low capacity
of the soil to supply this nutrient. On the other hand,
when grapevines are grown on soils with a clayey texture,
with medium to high organic matter content, and thus
with hypothetically good capacity for supplying N, N is
supplied when the natural sources do not meet the de-
mand of the plant for the nutrient [1,2]. Nevertheless, the
organic matter content of the soil provides partial infor-
mation regarding the quantity of N that is potentially
mineralizable in the soil in the medium to long term;
however, it does not accurately predict the values of
available N, such as nitrate (N-NO3−) and ammonium
(N-NH4+), in short periods, as in a growth season or crop
year. For that reason, tissue analysis, like leaf analysis,
which may be collected in different periods, such as
flowering or change in color of the berries, has been
recommended as one of the methods that best represents
the nutritional state of fruit-bearing plants, among them
the grapevine [3-5].
Tissue analysis is a destructive method, which in-
volves collection of the organ, such as leaves, washing,
drying, preparation and laboratory analyses. These ana-
lytical procedures are not very quick and the results gen-
erated most of the time are interpreted and used for defi-
nition of the need and the dose of the nutrient only in the
following year. For that reason, in recent years in some
fruit-bearing species like apple trees [6], peach trees [7],
pear trees [8,9] and also the grapevine [10-12], the total
N content in the leaves has been estimated through the
use of non-destructive methods like those that use port-
able equipment, among them the SPAD-502 (Soil Plant
Analysis Division Value). The SPAD has a flexible shaft
and another rigid shaft and the leaves are held in between
these two shafts through the pressure of the flexible shaft

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1547
in the direction of the rigid shaft. The flexible shaft has
two diodes that emit light beams at 650 nm (red) and 940
nm (near infrared), through the leaf tissue, and two de-
tectors, located on the rigid shaft, measure light trans-
mittance. The light transmitted, dependent on the tone of
green in the leaf, is converted into electric signals and the
ratio transmitted in the two regions of wavelengths cor-
responds to a numeric value, called the SPAD reading
and it is expressed in SPAD units [13]. The SPAD read-
ing is related to the chlorophyll contents of the leaves
determined in laboratory analyses, but also to the total N
content [14,15] and even with yield. Nevertheless, cali-
bration of the SPAD readings with the total N content in
the leaf or with yield is necessary. For that reason, which
organ of the plant, normally the leaf, which part of the
organ, which position of the plant the organ will be lo-
cated, which season, among other aspects, must be pre-
defined for performance of the SPAD readings and, pref-
erentially, they should be obtained in experiments with
plants grown in soils with different textures, organic
matter content and availability of mineral N, which may
be created by the application of increasing doses of min-
eral nitrogen fertilizer in the soil. For that reason, it is
expected that the N content in the soil and, consequently,
inside the plant is variable, which may be diagnosed by
the total N content in the leaf. After that, the organ (leaf)
where the reading was performed must be collected,
dried, ground and prepared for analysis of total N content.
Afterwards, the SPAD readings must be related to the
total N content in the organ, thus obtaining an equation
that will allow estimation of the total N content and even
an estimation of yield. This value, interpreted in tables of
the critical level or ranges of sufficiency, may define the
need for application of N and/or its dose.
The objective of the present study was to estimate,
through use of the SPAD-502, the total N content in
leaves and grape yield in grapevines submitted to nitro-
gen fertilization in soils with clayey and sandy texture.
2. Material and Methods
2.1. Description of the Experiments
2.1.1. Experiment 1—Use of the SPAD-502 in
Estimation of Total Nitrogen Content in Leaves
and of Grape Yield in Grapevines Submitted to
Nitrogen Fertilization and Grown in Soil with a
Clayey Texture
Experiment 1 was conducted in clayey soil in a vineyard
in the experimental area of Embrapa Uva e Vinho in the
municipality of Bento Gonçalves, Rio Grande do Sul
(RS), region of the Serra Gaúcha (Gaucho Highlands),
South of Brazil. The vineyard was of the cultivar Caber-
net Sauvignon, grafted on the root stock SO4 at a density
of 2666 plants per hectare, spacing of 1.5 m between
plants and 2.5 m between rows. The vineyard was im-
planted in 1986 and the plants were trained in a trellis
system. The climate in the region has had temperatures
from 12.8˚C to 21.8˚C throughout the years and precipi-
tation of around 1736 mm, well distributed through the
entire year. The soil was an Udorthent soil [16] and, in
the layer from 0 - 20 cm, before implementing the ex-
periment, it exhibited the following attributes: clay 240
g·kg−1, organic matter 27.0 g·kg−1, pH in water 6.3; ex-
changeable Al 0.0 cmolc·dm−3 (KCl extraction solution 1
mol· L −1); exchangeable Ca 8.8 cmolc·dm−3 (KCl extrac-
tion solution 1 mol·L−1); exchangeable Mg 3.3 cmolc·dm−3
(KCl extraction solution 1 mol·L−1); available P 18.9
mg·dm−3 (Mehlich 1 extraction solution) and available K
188 mg·dm−3 (Mehlich 1 extraction solution).
The grapevines were submitted to the application of 0,
10, 20, 40 and 80 kg·N·ha−1 in August 2008 and also in
2009, totaling throughout the two years 0, 20, 40, 80 and
160 kg·N·ha−1. The doses of N were applied a single time
at the beginning of flowering of the plants, in accordance
with the official recommendation for fertilization and
liming for grapevines established in Brazil for the states
of Rio Grande do Sul and Santa Catarina [17]. The N
was applied manually in the form of urea (45% N) in the
soil surface, without incorporation, in strips of 0.5 m
width under the plant canopy on the same side of the
planting row. Throughout the grapevine cycle, the strip
where the N was applied was submitted to application of
Glyphosate to avoid the occurrence of weeds. In the two
planting seasons, the grapevines were submitted to ap-
plication of phosphorus (P) and potassium (K), following
the official recommendations for the crop [17]. The ex-
perimental design was randomized blocks with three rep-
lications, with each plot formed of five plants, where the
three center plants were assessed.
2.1.2. Experiment 2—Use of the SPAD-502 in
Estimation of the Total Nitroge n Co nten t in
Leaves and of Grape Yield in Grapevines
Submitted to Nitrogen Fertilization and Grown
in Soil with a Sandy Texture
Experiment 2 was conducted in sandy soil in a vineyard
of a property in the municipality of Rosário do Sul, Rio
Grande do Sul, region of the Campanha Gaúcha (Gaucho
Pampa), South of Brazil. The vineyard was of Cabernet
Sauvignon, grafted on the root stock SO4 at a density of
3704 plants per hectare, spacing of 1.0 m between plants
and 2.7 m between rows. The vineyard was implanted in
2004 and the plants were trained in an vertical trellis. The
climate of the region is Cfa, with average temperatures
ranging from 11.9˚C to 23.5˚C and with annual average
rainfall of 1599 mm. The soil was a Sandy Typic

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1548
Hapludalf soil [16] and, in the layer from 0 - 20 cm, be-
fore implementing the experiment, it exhibited the fol-
lowing attributes: clay 70 g·kg−1, organic matter 10.0
g·kg−1, pH in water 5.5, exchangeable Al 0.0 cmolc·dm−3
(KCl extraction solution 1 mol·L−1), exchangeable Ca 0.9
cmolc·dm−3 (KCl extraction solution 1 mol·L−1), ex-
changeable Mg 0.6 cmolc·dm−3 (KCl extraction solution
1 mol·L−1), available P 30.1 mg·dm−3 (Mechlich 1 ex-
traction solution) and available K 48 mg·dm−3 (Mehlich
1 extraction solution).
The Cabernet Sauvignon grapevines were submitted to
the application of 0, 10, 15, 20, 40, 80 and 120 kg·N·ha−1,
in August 2008 and also in 2009, totaling in the two crop
seasons 0, 20, 30, 40, 80, 160 and 240 kg·N·ha−1. The
source of N used was urea (45% N) and its mode of ap-
plication, as well as weed control and fertilization with P
and K, were the same as described in experiment 1. The
experimental design was randomized blocks with three
replications, with each plot formed of five plants, where
assessments were performed on the three center plants.
2.2. SPAD Readings and Assessments of Total N
Content in the Leaves and Grape Yield
In October 2008 and 2009, during flowering of the
grapevines, SPAD readings were performed, using the
portable chlorophyll meter Minolta-SPAD-502. Readings
were performed on mature leaves opposite the first bunch
of the new shoot. Already in January 2009 and 2010,
throughout the change in color of the berries, the SPAD
readings were performed on mature leaves, located in the
middle third of the new shoot. In the two periods, the
readings were taken on three leaves, one on each new
shoot. One leaf was located in the outer part of the plants,
on the right side of the planting row, another leaf was
located in the outer side of the plants, but at the left side
of the planting row, and a third leaf was found in the
center of the plant. Three readings were performed on
each leaf in approximately the center part of the leaf.
Soon after each reading, in the two periods of assessment,
the same leaf submitted to SPAD reading was collected
and reserved. After that, the leaf was dried in a forced air
laboratory oven at a temperature of 65˚C, until constant
weight. Soon afterwards, the leaves were ground in a
Wiley type knife mill, having particles with an average
diameter of approximately 0.1 mm at the end of the
process, which were then prepared for analysis of total N,
following the methodology proposed by [18], which may
thus be described in summary fashion: 0.2 g of dry plant
tissue was added to a 100 mL digestion tube, with the
addition of 0.7 g of digestion mixture (90.9% Na2SO4
and 9.1% CuSO4·5H2O), 2 mL of H2SO4 concentrate and
1 mL of H2O2. After that, the tubes were placed and
heated in a dry block at a temperature of 150˚C. Through-
out digestion, the temperature was gradually raised (50˚C
every 30 minutes), up to 350˚C. After the extracts of the
samples exhibited a clear greenish yellow color, they
remained in the dry block at a temperature of 350˚C for
60 minutes more. Soon afterwards, the digestion tubes
were removed from the block. After that, 20 mL of dis-
tilled water was added to each digestion tube. Then, 10
mL of NaOH 10 mol·L−1 was added to each digestion
tube and it was immediately connected to the semi micro
Kjedahl steam stripping apparatus for distillation of total
N, until collecting 35 mL of distillate in 5 mL of the in-
dicator solution, boric acid. This extract was then titrated
with H2SO4 0.05 mol·L−1, which allowed quantification
of the total N content in each sample.
In March 2009 and 2010, all the grape bunches from
each plant were collected and weighed and then grape
yield per hectare was determined.
2.3. Statistical Analysis
The results of the SPAD readings, of the total nitrogen
content in the leaves, and grape yield obtained in the crop
year of 2008/2009 and 2009/2010 were submitted to
analysis of variance and when the effects were signify-
cant, they were adjusted by the regression equations,
testing the linear model by the F test, with an error prob-
ability less than 5% (P < 0.05).
To validate the equations generated, independent data
were used, derived from the 2009/2010 crop year, which
we considered as observed data. Also to assess perform-
ance of the models, the correlation coefficient (r) [19],
the Willmott dw index [20], the c index [19] and the root
mean square error (RQME) [21] were used.
Quantification of the association between two or more
variables was established by r, which varies from −1 to 1
[22], being calculated by the equation:




0.5
22
111
nnn
iii
rOiOEiEOiO EiE




 
 



 

 


where: Ei = estimated values; Oi = observed values; E
= represents the mean of the estimated values; O =
represents the mean of the observed values.
The agreement index, designated dw, quantifies the
accuracy of the model. Its values vary from zero, for no
agreement, to 1, for perfect agreement [20]. The dw
value is calculated by the formula [19]:


2
2
11
1
nn
ii
dwEi OiEi OOiO





  








The c coefficient, proposed by [19] is obtained by the
product between the r and dw, and the interpretation is
the following: excellent (c > 0.85); very good (c from

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1549
0.76 to 0.85); good (c from 0.66 to 0.75); average (c from
0.61 to 0.65); fair (c from 0.51 to 0.60); poor (c from
0.41 to 0.50) and very poor (c < 0.40).
The RQME expresses the error produced by the model;
the lower the value of this statistic, the better the model
[21]. The RQME value is calculated by the equation:

0.5
2
1
n
i
RQMEEi Oin






where: Ei = estimated values; Oi = observed values; n =
number of observations.
3. Results
3.1. Experiment 1—Use of the SPAD-502 in
Estimation of Total Nitrogen Content in
Leaves and of Grape Yield in Grapevines
Submitted to Nitrogen Fertilization and
Grown in Soil with a Clayey Texture
The application of increasing doses of N (0, 10, 20, 40
and 80 kg·N·ha−1) in the form of urea in year 1 (2008-
2009) and in year 2 (2009-2010) did not affect grape
yield in the Cabernet Sauvignon grapevines grown in soil
with a clayey texture and with 27.0 g·kg−1 of organic
matter (Figure 1). Grape production in the grapevines in
all the treatments was less in year 2 as compared to year
1. In addition, the application of doses of N in the soil
did not affect the total N content in the leaves, when col-
lected at flowering in year 1 (Figure 2(a)), but increased
the N content in the leaves collected at change in color of
the berries in a linear manner (Figure 2(b)), with the
mathematical adjustment being expressed by the equa-
tion: Total N content = 22.44 + 8.53 dose N (R2 = 0.92, P
< 0.05).
It is fitting to comment that the total N content in the
leaves collected at change in color of the berries in all the
treatments and in the two years of assessment were less
than those found in the leaves collected at flowering
(Figures 2 (a) and (b)).
The N applied in year 1 did not affect the SPAD read-
ings performed using the portable chlorophyll meter Mi-
nolta-SPAD-502 in the leaves collected at flowering of
the grapevines (Figure 3(a)), but increased in a linear
manner when they were performed on the leaves during
change in color of the berries (Figure 3(b)), with the
mathematical adjustment being expressed by the equa-
tion SPAD Reading = 37.32 + 0.023 dose N (R2 = 0.98, P
< 0.05). This behavior was the same as observed for the
total N content in the leaves when collected at flowering
and at change in color of the berries of the grapevines
submitted to the application of doses of N (Figures 2(a)
and (b)).
The SPAD readings performed in year 1 on the leaves
Figure 1. Relationship betwee n the dose of nitrogen applied
and grape yield per hectare in Cabernet Sauvignon grape-
vines grown in soil with clayey texture. NS = not significant.
Figure 2. Relationship betwee n the dose of nitrogen applied
and the total N content in the leaf collected at flowering (a)
and at change in color of the berries (b) in Cabernet Sauvi-
gnon grapevines grown in soil with clay ey texture. NS = not
significant.

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1550
Figure 3. Relationship betwee n the dose of nitrogen applied
and the SPAD reading in the leaf collected at flowering (a)
and at change in color of the berries (b), in Cabernet Sau-
vignon grapevines grown in soil with clayey texture. NS =
not significant.
collected at flowering exhibited a linear relationship with
the total N content in the leaves (Total N content =
−95.339 + 3.817 SPAD Reading, R2 = 0.95, P < 0.05)
(Figure 4(a)), which was also observed when the leaves
were collected at change in color of the berries (Total N
content = −8.203 + 0.381 SPAD Reading, R2 = 0.95, P <
0.05) (Figure 4(b)). This linear relationship between the
SPAD readings and the total N content in the leaves was
also found in year 2 when the leaves were collected at
change in color of the berries (Total N content = 0.96
SPAD Reading + 18.9, R2 = 0.69, P < 0.05) (Figure
4(b)). But when the leaves were collected at flowering,
the SPAD readings did not exhibit a relationship with the
total N content (Figure 4(a)).
The SPAD readings performed on the leaves at flow-
ering and at change in color of the berries in year 2 (Fig-
ures 5(a) and (b)) and at change in color of the berries in
year 1 (Figure 5(b)), did not exhibit a relationship with
Figure 4. Relationship between the SPAD reading and the
total nitrogen content in the leaf collected at flowering (a)
and at change in color of the berries (b) in Cabernet Sauvi-
gnon grapevines submitted to nitrogen fertilization and
grown in soil with clayey texture. NS = not significant.
grape yield per hectare. However, when the SPAD read-
ings were performed on leaves during flowering in year 1,
they exhibited a linear relationship with grape production
per hectare (Grape yield = 37581.71 − 822.58 SPAD, R2
= 0.72, P < 0.05) (Figure 5(a )).
In Figures 6(a) and (b) is presented the relationship
between the total N content in the leaf measured, ob-
tained in year 2 (2009/2010), and the total N content es-
timated by the SPAD reading model, obtained in year 1
(2008/2009), when the leaves were collected at flowering
and at change in color of the berries. The values of the
correlation coefficient (r) obtained between the total N
content measured and that estimated in the leaves col-
lected at flowering was 0.19 (Figure 6(a)); and when the
leaves were collected at change in color of the berries,
the r was 0.21 (Figure 6(b)).
The agreement index (dw) [19,20], which represents
the accuracy of the model, was 0.15 when the relation-

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1551
Figure 5. Relationship between the SPAD reading and
grape yield per hectare at flowering (a) an at change in
color of the berries (b) in Cabernet Sauvignon grapevines
submitted to nitrogen fertilization and grown in soil with
clayey texture. NS = not significant.
ship was established between the total N content in the
leaves collected at flowering with the total estimated N
content (Figure 6(a)), and was 0.16 when the leaves
were collected at change in color of the berries (Figure
6(b)). The c coefficient, obtained by the product between
the r and the dw, obtained by the relationship between the
total N content measured in the leaves and the total esti-
mated N content when the leaves were collected at flow-
ering and at change in color of the berries, was 0.03. The
value of the Root Mean Square Error (RMSE) [21], ob-
tained in the relationship between the total N content of
the leaves measured and the estimated total N of the
leaves when the leaves were collected at flowering, was
13.1 g·kg−1 (Figure 6(a)), but when the leaves were col-
lected at change in color of the berries, the RMSE was
3.8 g·kg−1 (Figure 6(b)).
In Figure 7 it is observed that there was no relation-
ship between grape yield per hectare in year 2 (2009/
Figure 6. Relationship between the total N content in the
leaf in year 2 (2009/2010) and the total N content estimated
by the SPAD reading model obtained in year 1 (2008/2009),
which determines the total N content in the leaf, at flower-
ing (a) and at change in color of the berries (b) in Cabernet
Sauvignon grapevines submitted to nitrogen irrigation and
cultivated in soil with clayey texture. The full line is the
straight line 1:1. r = correlation coefficient; dw = Willmott
index; c = c index and RMSE = root mean square error.
2010) and the production estimated by the SPAD reading
model obtained in the leaves collected at change in color
of the berries in year 1 (2008/2009).
3.2. Experiment 2—Use of the SPAD-502 in
Estimation of the Total Nitrogen Content in
Leaves and of Grape Yield in Grapevines
Submitted to Nitrogen Fertilization and
Grown in Soil with a Sandy Texture
The application of growing does of N (0, 10, 15, 20, 40,
80 and 120 kg·N·ha−1) in the form of urea in year 1
(2008-2009) increased grape yield per hectare in a linear
manner (Grape yield = 8271.23 - 21.11 dose N, R2 = 0.60,
P < 0.05) in Cabernet Sauvignon grapevines grown

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1552
Figure 7. Relationship between grape yield in year 2 (2009/
2010) and grape yield estimated by the SPAD reading
model obtained in the leaf at change in color of the berries
in year 1 (2008/2009) in Cabernet Sauvignon grapevines
submitted to nitrogen fertilization and cultivated in soil
with a clayey texture. The full line is the straight line 1:1. r
= correlation coefficient; dw = Willmott index; c = c index
and RMSE = root mean square error. NS = not significant.
in soil with sandy texture and with 10.0 g·kg−1 of organic
matter (Figure 8). In the same way, in year 2, the appli-
cation of increasing doses of N affected grape yield per
hectare; however, the quantity of grapes produced di-
minished with the increase of the dose applied (Grape
yield = 4126.77 − 17.6 dose N, R2 = 0.66, P < 0.05)
(Figure 8). Grape yield calculations per hectare in year 2
in all the treatments with the application of N were less
than those found in year 1. The application of growing
doses of N in the grapevines, for its part, increased the
total N content in the leaves collected at flowering in
year 1 (Total N content = 34.85 + 0.18 dose N, R2 = 0.96,
P < 0.05) and in year 2 (Total N content = 25.54 + 0.09
dose N, R2 = 0.96, P < 0.05) (Figure 9(a)) in a linear
manner. The increase of total N content in the leaves also
occurred when they were collected at change in color of
the berries in year 1 (Total N content = 22.44 + 8.53 dose
N, R2 = 0.92, P < 0.05) and in year 2 (Total N content =
20.6 + 0.02 dose N, R2 = 0.63, P < 0.05) (Figure 9(b)).
Nevertheless, in year 2, the leaves collected at flowering
of the grapevines and at change in color of the berries
exhibited lower N content when compared to leaves col-
lected in year 1.
The application of growing doses of N in the grape-
vines increased the total N content in the leaves collected
at flowering in year 1 (Total N content = 34.85 + 0.18
dose N, R2 = 0.96, P < 0.05) and in year 2 (Total N con-
tent = 25.54 + 0.09 dose N, R2 = 0.96, P < 0.05) in a lin-
ear manner (Figure 9(a)). The increase in the total N
content in the leaves also occurred when they were col-
lected at change in color of the berries in year 1 (Total N
Figure 8. Relationship betwee n the dose of nitrogen applied
and grape yield per hectare in Cabernet Sauvignon grape-
vines cultivated in soil with a sandy texture.
Figure 9. Relationship betwee n the dose of nitrogen applied
and the total N content in the leaf collected at flowering (a)
and at change in color of the berries (b) in Cabernet Sauvi-
gnon grapevines grown in soil with a sandy texture.

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1553
content = 22.44 + 8.53 dose N, R2 = 0.92, P < 0.05) and
in year 2 (Total N content = 20.6 + 0.02 dose N, R2 =
0.63, P < 0.05) (Figure 9(b)). Nevertheless, in year 2, the
leaves collected at flowering of the grapevines and at
change in color of the berries exhibited lower N content
when compared with leaves collected in year 1.
The application of N in the soil in year 1 increased the
SPAD reading in a linear manner in the leaves of the
grapevines collected at flowering (SPAD Reading = 35.1
+ 0.04 dose N, R2 = 0.92, P < 0.05) (Figure 10(a)) and at
change in color of the berries (SPAD Reading = 41.5 +
0.04 dose N, R2 = 0.66, P < 0.05) (Figure 10(b)). Nev-
ertheless, N application did not affect the SPAD reading
in year 2 performed on the leaves during flowering and at
change in color of the berries (Figures 10(a) and (b)),
which in this same year exhibited the lowest contents of
total N (Figures 9(a) and (b)).
SPAD readings in year 1 exhibited linear relationship
Figure 10. Relationship between the dose of nitrogen ap-
plied and the SPAD-502 readings in the leaf collected at
flowering (a) and at change in color of the berries (b) in
Cabernet Sauvignon grapevines grown in soil with sandy
texture. NS = not significant.
with the total N content in the leaves collected at flower-
ing (Total N content = −134.30 + 4.83 SPAD, R2 = 0.94*)
(Figure 11(a)), as well as when collected at change in
color of the berries (Total N content = 11.408 + 0.453
SPAD, R2 = 0.99, P < 0.05) (Figure 11(b)). In year 2,
there was no relationship between the SPAD readings
and the total N content in the leaves collected at flower-
ing and at change in color (Figures 11(a) and (b)).
The SPAD readings, when performed on leaves during
flowering of the grapevines, exhibited a linear relation-
ship with grape yield per hectare (Grape production =
−8786.08 + 488.66 SPAD reading, R2 = 0.59, P < 0.05)
(Figure 12(a)), which was also found when the SPAD
readings were performed on leaves at change in color of
the berries (Grape yield = −21375.81 + 981.97 SPAD
reading, R2 = 0.93, P < 0.05) (Figure 12(b)). However,
the SPAD readings performed in year 2 on the leaves of
the grapevines in flowering or during change in color of
Figure 11. Relationship between the SPAD-502 reading and
the total nitrogen content in the leaf collected at flowering
(a) and at change in color of the berries (b) in Cabernet
Sauvignon grapevines submitted to nitrogen fertilization
and grown in soil with sandy texture. NS = not significant.

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1554
Figure 12. Relationship between the SPAD reading and
grape yield per hectare when performed on leaves at flow-
ering (a) and at change in color of the berries (b), in Cab-
ernet Sauvignon grapevines submitted to nitrogen fertiliza-
tion and cultivated in soil with sandy texture. NS = not sig-
nificant.
the berries did not exhibit a relationship with grape yield
per hectare (Figures 12(a) and (b)).
In Figures 13(a) and (b) is presented the relationship
between the total N content in the measured leaf, ob-
tained in year 2 (2009/2010) and the total N content es-
timated by the SPAD reading model, obtained in year 1
(2008/2009), when the leaves were collected at flowering
and at change in color of the berries. The r obtained in
the relationship between the total N content measured
and that estimated, when the leaves were collected at
flowering, was 0.65 and when the leaves were collected
at change in color of the berries, the r was 0.21. The dw
was 0.62 when the relationship between the total N con-
tent measured in the leaf collected at flowering and the
total N content estimated in the leaf (Figure 13(a)) was
established, but when the leaves were collected at change
in color of the berries, the dw was 0.15 (Figure 13(b)).
Figure 13. Relationship between the total N content in the
leaf in year 2 (2009/2010) and the total N content estimated
by the SPAD reading model obtained in year 1 (2008/2009),
at flowering (a) and at change in color of the berries (b) in
Cabernet Sauvignon grapevines submitted to nitrogen fer-
tilization and grown in soil with sandy texture. The full line
is the straight line 1:1. r = correlation coefficient; dw =
Willmott index; c = c index and RMSE = root mean square
error.
The c coefficient obtained by the relationship between
the total N content measured in the leaves and the total N
estimated when the leaves were collected at flowering
(Figure 13(a)) was 0.40, and when the leaves were col-
lected at change in color of the berries (Figure 13(b)),
the value was 0.03.
The RMSE value obtained between the relationship
between the total N content of the leaves measured and
the total N of the leaves estimated when the leaves were
collected at flowering was 6.3 g·kg−1 (Figure 13(a)), but
when the leaves were collected at change in color of the
berries, the RMSE was 14.6 g·kg−1 (Figure 13(b)). In
Figure 14, it may be observed that there was no relation-
ship between grape yield in year 2 (2009/2010) and the
yield estimated by the SPAD reading model performed

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1555
Figure 14. Relationship between grape yield in year 2 (2009/
2010) and grape yield estimated by the SPAD reading
model obtained in year 1 (2008/2009) when the reading was
performed on leaves collected at flowering (a) and at change
in color of the berries (b) in Cabernet Sauvignon grapevines
submitted to nitrogen fertilization and cultivated in soil
with sandy texture. The full line is the straight line 1:1. r =
correlation coefficient; dw = Willmott index; c = c index and
RMSE = root mean square error. NS = not significant.
on the leaf during flowering and at change in color in
year 1 (2008/2009).
4. Results
The Cabernet Sauvignon grapevines, grown in experi-
ment 1 on a soil with clayey texture and with 27.0 g·kg−1
of organic matter, did not increase grape yield with the
application of up to 80 kg·N·ha−1, in the first and second
year of assessment (Figure 1). This may have occurred
because the moisture conditions in the soil and the mild
temperatures throughout all the months of the years may
have favored the mineralization of the labile organic
matter of the soil, as well as the decomposition of sense-
cent plant residues deposited on the soil surface [23]
which, in the case of Experiment 1, are especially the
residues deposited between the rows of the grapevines.
For that reason, an increase in the availability of N in the
soil is expected, derived from the organic matter and the
residues in decomposition, which, together with the inner
reserves of N accumulated in the grapevines, especially
in the perennial organs like the roots [24-28], may meet
the plant demand for the nutrient, which may be one of
the possible explanations for the lack of response to the
application of N. According to [2], using the same type
of soil and in the same region as Experiment 1, the Cab-
ernet Sauvignon grapevines increase grape production up
to a dose of around 15 kg·N·ha−1, which reinforces the
possibility that the mineralization of the organic matter,
of the cover plant residues, plus the inner reserves of N
provide a large part of the N taken up by the plants. Nev-
ertheless, when the Cabernet Sauvignon grapevines were
grown in soils with a sandy texture, in other words, in
Experiment 2, with 10.0 g·kg−1 of organic matter, the
application of N increased grape production in the first
year up to the dose of 120 kg·N·ha−1 (Figure 8). Never-
theless, in the second year, grape yield diminished with
the increase in the dose of N (Figure 2), which may be
associated in part with the high availability of N in the
soil and the water, since the rainfall in this year, espe-
cially in the months of September, November and De-
cember was frequent and in large quantity (Tables 1 and
2), which stimulates uptake of forms of N in the soil, de-
tected by the increase of total N content in the leaves
collected at flowering and at change in color of the ber-
ries (Figures 9(a) and (b)). For that reason, stimulation
of plant vigor is expected, which may have decreased the
incidence of solar radiation in the inner part of the plant,
favoring the occurrence of fungal diseases, especially in
wetter years, as was the second year of assessment, which,
consequently, may have decreased the number of fertile
flowers, resulting in lower grape production per hectare
[29,30].
The application of N on the Cabernet Sauvignon
grapevines grown in soil with a clayey texture had little
effect on the N content in the leaves during flowering
and at change in color of the berries (Figures 2(a) and
(b)), with an increase in the content of the nutrient being
found only in the leaves collected in the first year during
the change in color of the berries. That may be caused by
the low utilization of the applied N by the grapevine, as
reported by [27] in Riesling Renano and Chardonnay
grapevines grown in the same type of clayey soil as ex-
periment 1 and submitted to the application of N in the
form of urea enriched with the tracer 15 N. That may
also be one of the possible explanations for the lack of
increase in grape production (Figure 1). But when the N
was applied on grapevines grown in soil with a sandy
texture, the total N content in the leaves during flowering

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1556
Table 1. Monthly average of minimum, mean, and maximum air temperature, and accumulated rainfall and sunshine dura-
tion in the agricultural year, and climatological normal minimum, mean, and maximum air temperature, and accumulated
rainfall and sunshine at Bento Gonçalves (RS), Brazil (Experiment 1).
Agricultural Year 1 Agricultural Year 2 Climatological normal
Temperature
Min. Mean Max. Min. Mean Max. Min. Mean Max.
Month
-------------------------------------------------------------------------------˚C-------------------------------------------------------------------------------
July 11.0 14.9 19.6 6.3 10.2 15.1 9.1 12.9 18.2
August 9.8 14.1 19.5 10.3 15.2 20.8 9.3 13.6 19.2
September 8.8 13.2 18.5 10.2 14.6 19.2 10.6 14.9 20.4
October 13.1 16.8 21.2 12.0 16.7 22.6 12.3 17.0 22.8
November 14.7 19.4 25.0 17.6 21.6 26.7 14.2 18.9 24.8
December 15.6 20.3 26.2 17.0 21.2 26.4 16.0 20.7 26.7
January 16.1 20.4 25.7 18.1 22.0 26.8 17.3 21.8 27.8
February 17.8 21.7 26.6 19.1 23.0 28.4 17.3 21.7 27.5
March 17.1 21.0 26.2 16.8 20.7 25.6 16.1 20.3 26.0
April 13.7 18.4 24.1 13.4 17.5 22.4 13.3 17.5 22.9
May 11.1 15.6 20.8 11.1 14.2 17.7 10.4 14.5 20.0
June 7.5 11.2 15.9 9.0 13.1 18.0 8.6 12.8 17.9
Average 13.0 17.3 22.4 13.4 17.5 22.5 12.9 17.2 22.9
Agricultural
Year 1
Agricultural
Year 2
Climatological
normal
Agricultural
Year 1
Agricultural
Year 2
Climatological
normal
Rainfall Sunshine
Month
-----------------------------------mm----------------------------------- ----------------------------------hour----------------------------------
July 73.0 97.8 161.0 175.7 144.9 154.0
August 198.5 257.9 165.0 193.5 183.0 159.0
September 144.1 411.7 185.0 185.8 134.4 162.0
October 309.6 145.1 156.0 154.8 194.1 192.0
November 70.3 359.5 140.0 240.8 141.1 219.0
December 85.8 232.6 144.0 261.7 224.3 239.0
January 269.6 296.4 140.0 235.7 189.2 231.0
February 144.5 167.1 139.0 174.0 205.3 199.0
March 90.6 57.2 128.0 228.1 192.5 208.0
April 24.2 142.1 114.0 243.4 180.1 173.0
May 134.7 154.7 107.0 162.3 98.4 162.0
June 82.9 129.9 157.0 155.4 142.4 142.0
Sum 1627.8 2452.0 1736.0 2411.2 2029.7 2240.0

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1557
Table 2. Monthly average of minimum, mean, and maximum air temperature, and accumulated rainfall and sunshine dura-
tion in the agricultural year, and climatological normal minimum, mean, and maximum air temperature, and accumulated
rainfall and sunshine at Rosario do Sul (RS), Brazil (Experiment 2).
Agricultural Year 1 Agricultural Year 2 Climatological normal
Temperature
Min. Mean Max. Min. Mean Max. Min. Mean Max.
Month
-------------------------------------------------------------------------------˚C-------------------------------------------------------------------------------
July 11.7 14.9 18.2 4.5 10.2 15.8 16.4 23.5 29.6
August 8.6 12.7 18.5 9.4 15.5 20.6 16.8 22.6 28.6
September 8.2 12.1 19.2 9.4 14.4 18.8 15.4 21.2 27.0
October 13.1 16.8 23.2 11.1 18.4 23.9 12.7 17.7 23.1
November 16.0 21.1 29.0 16.7 21.8 26.0 10.1 14.6 19.9
December 16.9 22.3 30.3 16.8 23.1 27.7 8.4 12.3 17.1
January 18.0 23.3 32.1 17.9 25.1 30.1 7.9 11.9 17.2
February 18.4 22.6 30.6 18.9 24.3 28.9 9.1 13.4 19.2
March 17.5 20.8 28.9 17.2 23.2 28.5 9.8 14.6 20.2
April 13.4 17.5 27.0 12.6 18.7 23.9 12.6 17.5 23.1
May 10.3 16.6 22.1 11.2 15.3 19.2 14.3 19.7 25.5
June 5.1 10.8 16.0 8.6 13.0 17.2 16.6 22.2 28.3
Average 13.1 17.6 24.6 12.8 18.6 23.4 12.5 17.6 23.2
Agricultural
Year 1
Agricultural
Year 2
Climatological
normal
Agricultural
Year 1
Agricultural
Year 2
Climatological
normal
Rainfall Sunshine
Month
-----------------------------------mm----------------------------------- -----------------------------------hour-----------------------------------
July 129.9 66.1 120.4 139.8 177.1 155.0
August 145.2 45.3 76.2 200.3 190.5 166.0
September 91.5 269.4 147.3 174.2 167.3 179.0
October 188.1 135.9 155.8 196.8 274.8 222.0
November 29.0 540.8 118.0 278.4 121.9 247.0
December 61.4 219.0 127.8 295.1 236.8 274.0
January 68.6 204.1 138.8 274.5 267.4 264.0
February 256.0 240.7 137.2 219.9 197.5 219.0
March 70.2 58.9 135.9 251.7 247.7 227.0
April 11.8 132.0 183.6 233.2 219.3 192.0
May 123.6 133.4 125.8 167.1 121.8 174.0
June 22.4 33.7 132.1 174.4 130.1 134.0
Sum 1197.7 2079.3 1598.9 2605.4 2352.2 2453.0

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1558
and change in color of the berries increased (Figure 9(a)
and (b)), showing that part of the N applied was taken up
by the plants, moreover, increasing grape production in
the first year (Figure 8).
The addition of doses of N in grapevines grown in
soils with clayey texture did not affect the SPAD read-
ings performed on the leaves during flowering in the first
year and in the second year (Figures 3(a) and (b)) and
that performed on the leaves throughout the change in
color of the berries in the second year (Figure 3(b)). This
may have occurred because the application of N did not
increase the total N content in the leaves (Figures 2(a)
and (b)) which, consequently, may not have increased
the chlorophyll content in the leaves, since the total N
content in the leaves normally and for most crops is
highly related to the chlorophyll content [31-34], and the
chlorophyll content to the SPAD reading [35,36]. This
may be a plausible explanation because in the grapevines
grown in soils with a sandy texture, the total N content in
the leaves collected at flowering and at change in color
increased with the dose of N applied (Figures 9(a) and
(b)), which, moreover, promoted the increase of the
SPAD readings, in this case of the leaves collected at
flowering and at change in color of the berries in the first
year of assessment (Figures 10(a) and (b)). When the
total N content in the leaves was less, for example, in the
leaves collected at flowering and at change in color of
the berries in the second year (Figures 9(a) and (b)), no
relationship was found between the dose applied and the
SPAD reading (Figures 10(a) and (b)). Nevertheless, it
is fitting to report that the SPAD 502 has adequate sensi-
tivity to detect the increase of the chlorophyll content in
leaves that have up to 300 mg·m−2 of chloroplasts [37]
The SPAD readings performed on the grapevines
submitted to application of N and grown in soil with
clayey and sandy texture exhibited a positive linear rela-
tionship with the total N content in the leaves collected at
flowering and at change in color of the berries in year 1.
On the other hand, in year 2, this relationship was only
found when the leaves were collected at change in color
in the grapevines with application of N and grown in soil
with clayey texture (Figures 4(a) and (b), 11(a) and (b)).
The relationship between the two variables allows the
SPAD reading to estimate the total N content in the
leaves which, consequently, may be used to define the
need for and the dose of the nutrient to be added. Thus,
the collection of leaves, drying and chemical analysis in
the laboratory may be unnecessary for the determination
of total N content, which is a slower procedure with a
higher cost.
It is desirable for the SPAD reading to have the capac-
ity of estimating the total N in the leaves in earlier
phenological stages, such as flowering, because this
would allow the nitrogen fertilization, when necessary, to
be applied in the same vegetation and productive cycle of
the plant. The relationship between the SPAD reading
and total N content in the grapevine leaves was also re-
ported by Porro et al. [38] in Chardonnay grapevines
grown in Italy. This author reports that the best determi-
nation coefficients (R2) between the two variables were
found when the leaves were collected in the fruit-set,
similar to that which was obtained by Porro et al. (1995).
Nevertheless, these authors report that the relationship
between the SPAD reading and the total N content is not
found every year, which was also obtained in the present
study because, for example, in year 2, in the grapevines
grown in the soil with sandy texture, no relationship was
found between the SPAD reading and the total N content
in leaves during flowering and at change in color of the
berries (Figures 11(a) and (b)). That may be explained,
in part, by the lower total N contents in leaves in all the
treatments (Figures 9(a) and (b)), probably caused by
the greater leaching of N-NO3− in the soil profile, which
may happen in soils planted to fruit bearing plants, like
grapevines, in traditional producer regions in the world
[39,40], especially because the rainfall in that year was
high (Tables 1 and 2) and the soil has a sandy texture.
Thus, it is observed that the SPAD reading may indeed
have the potential for estimating the total N content in
leaves during flowering and at change in color of the
berries, both in Cabernet Sauvignon grapevines grown in
soils with clayey texture, as well as sandy texture; nev-
ertheless, this is highly dependent on the year of assess-
ment, as has already been reported by [12,39].
The SPAD readings did not exhibit a relationship with
grape yield per hectare when they were performed on the
leaves during the change in color of the berries in the two
years, as well as in flowering in year 2 in the grapevines
submitted to application of N and grown in soil with
clayey texture (Figures 5(a) and (b)). Nevertheless, the
SPAD readings exhibited a positive relationship with
grape yield when they were performed on the leaves of
grapevines grown in soil with sandy texture during flow-
ering and at change in color of the berries in year 1
(Figures 12(a) and (b)). These data obtained throughout
one year of assessment show that it is possible to esti-
mate production using the SPAD reading performed at
flowering or at change in color of the berries. Neverthe-
less, in year 2, there was no relationship between the
SPAD readings and production (Figures 12(a) and (b)),
showing the variability of results from one year to the
next.
When the relationship was established between the to-
tal N content measured in the leaves, which was obtained
in year 2 and the total N content estimated by the SPAD
reading model, generated in year 1, it was observed that

Use of the SPAD-502 in Estimating Nitrogen Content in Leaves and
Grape Yield in Grapevines in Soils with Different Texture
1559
the leaves collected at flowering and at change in color
of the berries in grapevines submitted to application of N
and grown in soils with clayey texture exhibited similar
correlation coefficient values, although low (Figures 6(a)
and (b)). However, when the same model was estab-
lished, but with the leaves of the grapevines submitted to
application of N in soils with sandy texture, it was ob-
served that the greatest correlation coefficient was found
when the leaves were collected at flowering, if compared
to those collected at change in color of the berries (Fig-
ures 13(a) and (b)). The agreement indexes (dw) [19,20],
obtained in the relationship between the total N content
measured and that estimated in the leaves collected at
flowering and at change in color of the berries in the
grapevines grown in soils with clayey texture, were near
to zero (Figures 6(a) and (b)), indicating little agreement
between the two variables. On the other hand, when the
relationship between the total N content measured and
that estimated was obtained in the leaves collected at
flowering of the grapevines grown in soils with sandy
texture, the dw was greater and nearer to one (1) which,
consequently, shows greater agreement if compared to
the value of dw found when the leaves were collected at
change in color of the berries (Figures 13(a) and (b)).
The c coefficient values, obtained by the product be-
tween r and the dw, in the relationship between the total
N content measured in the leaves and the total N content
estimated, in the leaves collected at flowering and at
change in color of the berries, both in the grapevines
submitted to the application of N and those grown in soil
with a clayey and sandy texture, indicated a very poor
performance (c < 0.40) (Figures 6(a), (b), 13(a) and (b)).
On the other hand, the value of the RMSE [21], obtained
in the relationship between the total N content of the
leaves measured and the total N of the leaves estimated
by the SPAD reading model when the leaves were col-
lected at change in color of the berries was less and,
consequently, it is a better model (Figures 6(a) and (b)),
in comparison with the value of RMSE obtained when
the leaves were collected at flowering (Figures 13(a) and
(b)). Nevertheless, in the grapevines grown in soils with
sandy texture, the values of RMSE obtained were con-
trary to those found in the grapevines grown in clayey
soil, with the lowest values of RMSE being verified
when the leaves were collected at flowering. The statis-
tical values, like dw, but especially the c index, shows
that the models generated by the SPAD readings to esti-
mate the total N content in the leaves collected at flow-
ering and at change in color of the berries, both in the
grapevines grown in soil with clayey texture as well as
sandy texture, in a general sense, exhibit low accuracy in
determination of the total N content in the leaves. When
the relationship between the production measured and the
production estimated was established using the SPAD
reading model, performed on the leaves of the grapevines
grown in clayey soils during the change in color of the
berries or in the leaves of the grapevines grown in soil
with sandy texture during the flowering and change in
color of the berries, the SPAD readings also did not ex-
hibit accuracy in estimating grape production (Figures 7,
14(a) and (b)).
5. Conclusion
The SPAD-502 readings estimated the total N content in
the leaves at flowering and at change in color of the ber-
ries in the Cabernet Sauvignon grapevines grown in soils
with clayey and sandy texture, especially in the first year
of assessment. However, the accuracy of the SPAD
readings in estimation of the total N content is low and,
in estimation of grape yield, it is non-existent.
6. Acknowledgements
To the CAPES Foundation (Brazilian Federal Agency for
Support and Evaluation of Graduate Education) for fi-
nancing the project “Prediction of fertilization in grape-
vines with the aim of quality grapes and wines in the
southern region of Brazil” (National Post Doctorate Pro-
gram-PNPD 2007, public notice 034/2007) and for the
Post Doctorate fellowship which was granted to the first,
second and fifth authors. To Fapergs (Research Support
Foundation of Rio Grande do Sul) for the financial assis-
tance (Process no. 0903999). To Embrapa Uva e Vinho
and the Citrosul company for making vineyards available
for performance of the experiments.
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