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Not Sci Biol, 2011, 3(1):91-94
Print ISSN 2067-3205; Electronic 2067-3264
Notulae Scientia Biologicae
Estimate of Leaf Chlorophyll and Nitrogen Content in Asian
Pear (Pyrus serotina Rehd.) by CCM-200
Mostafa GHASEMI, Kazem ARZANI, Abbas YADOLLAHI, Shiva
GHASEMI, Saadat SARIKHANI KHORRAMI
Tarbiat Modares University, Department of Horticultural Science, Faculty of Agriculture, Tehran, Iran; firstname.lastname@example.org
In many cases evaluation of chlorophyll and nitrogen content in plants need to destructive methods, more time and organic solvents.
Application of chlorophyll meters save time and resources. The aim of this study was estimating of chlorophyll and nitrogen content
in Asian pear leaves using non-destructive method and rapid quantification of chlorophyll by chlorophyll content meter (CCM-
200). This study was conducted on 8 years old Asian pear trees during June 2008 in Tehran, Iran. To develop our regression model,
the chlorophyll meter data were correlated with extracted chlorophyll and nitrogen content data obtained from DMSO and Kejeldal
methods, respectively. The results showed that, there was positive and linear correlation between CCM-200 data and chlorophyll a
(R²=0.7183), chlorophyll b (R²=0.8523), total chlorophyll (R²=0.90), and total nitrogen content (R²=0.76) in Asian pear leaves. Thus, it
can be concluded that, CCM-200 can be used in order to predict both chlorophyll and nitrogen content in Asian pear leaves.
Keywords: Asian pear, chlorophyll, chlorophyll meter, nitrogen
Nitrogen is one of the most important macro nutri-
ents and plays critical role in vegetative growth, flowering
and fruit development (Shaahan et al., 1999). Changes in
chlorophyll content occur as a result of nutrient deficiency,
especially nitrogen with consider to this point that nitro-
gen is leached from soil quickly. Nitrogen deficiency de-
creases chlorophyll content due to chlorophyll decompo-
sition in plant leaves (Kowalczyk-Jusko and Koscik, 2002;
Shaahan et al., 1999). Low concentrations of chlorophyll
limit photosynthetic potential directly (Richardson et al.,
2002). Leaf chlorophyll content is a good indicator of
photosynthesis activity, mutations, stress condition (Nau-
mann et al., 2008), and nutritional status of plants (Wu et
al., 2008). Wright et al. (2009) examined the possibility
of using chlorophyll a fluorescence to measure postharvest
water loss of grape berries non-destructively. Chlorophyll
content estimation by laboratory methods is destructive
and need to time and also chemical materials (Chang and
Robinson, 2003). Since chlorophyll concentration is cor-
related with leaf nitrogen concentration, the instruments
which are used to evaluate chlorophyll concentration such
as SPAD-502 (Minolta, Japan) and Chlorophyll content
meter CCM-200, have been designed to determine nitro-
gen content (Pavan et al., 2004). Such devices provide a
quick, simple and non-destructive method to in situ chlo-
rophyll content estimation (Kowalczyk-Jusko and Koscik,
2002; Richardson et al., 2002). Regarding the relationship
between chlorophyll content and leaf nitrogen content,
chlorophyll meter can be used to improve fertilizer man-
agement (Richardson et al., 2002). A close correlations be-
tween chlorophyll meter with foliar nitrogen and total ex-
tracted chlorophyll has been reported in several fruit trees
such as mango, mandarin, guava and grapevine (Shaahan
et al., 1999), also in other crops such tobacco leaves (Kow-
alczyk-Jusko and Koscik, 2002) and sugar maple (Van den
Berg and Perkins, 2004). Change and Robinson (2003)
also reported significant correlations between CCM-200
data and nitrogen content of four woody species. There-
fore a correlation between chlorophyll content readings
and N content in a plant will be helpful for growers to
monitor nitrogen status of plants.
CCM-200 calculates the chlorophyll content index
(CCI), which is defined as the ratio of percentage of trans-
mission a 935 nm to 635 nm through leaf tissues (Richard-
son et al., 2002). Despite Asian pears need a very precise
nutrient schedule during growth season there is no non-
distractive method to evaluate nutritional status of this
crop. Hence the objective of this study was to isolate the
possible correlations between chlorophyll meter readings
and total chlorophyll measured via DMSO and nitrogen
content of Asian pear leaves to improve nutrient schedul-
ing in order to orchard management.
Materials and methods
This study was conducted on eighteen Asian pear
(Pyrus serotina Rehd) trees in a experimental orchard in
Tehran province (35º45´N, 51º8´E), Iran, during June
2008. Chlorophyll meter (Opti-sciences CCM 200,
USA) which calculates chlorophyll content index (CCI)
Received 29 November 2010; accepted 18 January 2011
Ghasemi, M. et al. / Not Sci Biol, 2011, 3(1):91-94
The leaf samples (each sample containing 4 leaves)
were washed with tap water, 0.01N HCl and then distilled
water. Leaf samples were placed in oven at 60°C until
dry weight was stable. Then the dried leaf materials were
ground. The Kjeldahl method was used to measure nitro-
gen content of leaf samples.
All data were analyzed by SPSS v 12.0 for windows
software and Excel 2002 programs.
Results and discussion
Statistical analysis showed linear correlation between
CCM readings and leaf chlorophyll content and also leaf
Results of chlorophyll a (mg/cm-²), chlorophyll b (mg/
cm-²), total chlorophyll content (mg/cm-²) and CCM
readings of the Asian pears leaves are presented in Tab. 1.
Obtained data by CCM were ranged from 12.8 to 57.1
with a mean of 38.56. Chlorophyll a range was from 0.013
to 0.022 with a mean of 0.0195 mg/cm-² and chlorophyll b
range was 0.001 to 0.0242 mg/cm-² with a mean of 0.0163
mg/cm-². Total extractable chlorophyll content was ranged
from 0.014 to 0.044 mg/cm-² with a mean of 0.0357. Fig.
1 (I, II and III) shows correlations between chlorophyll
meter readings and chlorophyll a, b and total chlorophyll
content in Asian pear leaves. It’s mentionable that, each
reading (each point) by chlorophyll meter is mean of five
readings taken on one leaf. Data analysis indicated that,
based on the ratio of transmittance measurement at 660
and 940 nm was used. Twelve measurements with CCM-
200 were made on four healthy mature leaves of Asian pear
trees from the middle of non fruiting branches and then
nitrogen content of these leaves were analyzed via Kejeldal
method. At the same time five readings also were taken for
chlorophyll analysis. Then all leaves brought to laboratory
for further experiments.
Dimethyl sulfoxide solvent (DMSO) was used for
chlorophyll extraction from five leaf discs in dark accord-
ing to described method by Hiscox and Israelstam (1979).
Absorbance of extracts was read by Beckman grating spec-
trophotometer (model U-1100, ltitach, Ctd, tokyo, Japan)
at 645 and 663 nm. Chlorophyll a (mg/cm-2), Chlorophyll
b (mg/cm-2) and total chlorophyll content (mg/cm-2) were
calculated from absorbance at 663 nm and 645 nm ac-
cording to Arnonʹs (1949) equations:
Chlorophyll a = (ml solvent)[(0.0127 ×Absorbance
663) - (0.00269 Absorbance 645)]/Leaf area (cm²)
Chlorophyll b = (ml solvent) [(0.0229 ×Absorbance
645) - (0.00468 Absorbance 663)]/Leaf area(cm²)
Total chlorophyll content = (ml solvent)(0.0202
×Absorbance 645) + (0.00802 Absorbance 663)]/ Leaf
Fig.1. The linear correlation between chlorophyll meter readings and chlorophyll a, b, total chlorophyll content and total nitrogen
(I,II, III and IV) in Asian pear leaves. Each point is average 12 measurements
Tab. 1. Chlorophyll a (mg/cm-²), Chlorophyll b (mg/cm-2), total chlorophyll content (mg/cm-2) and CCM readings in leaves of Asian pear trees
Tab. 2. Nitrogen content and CCM readings in leaf of the Asian pear trees
Ghasemi, M. et al. / Not Sci Biol, 2011, 3(1):91-94
there was linear correlation between chlorophyll a, b, and
total chlorophyll content with obtained data by CCM.
(R²= 0.718, 0.852 and 0.90, respectively). The data sug-
gests the CCM-200 may be able to estimate of chlorophyll
content in studied trees.
Leaf nitrogen percentage and CCM readings are
shown in Tab. 2. The CCM readings were ranged from 10
to 63 (mean of 38.65) and nitrogen percentage was ranged
from 1.29 to 3.00% (Fig 1) and with a mean of 2.39. The
results of nitrogen analysis in the leaves were correlated
with chlorophyll meter readings as shown in Fig. 1 (IV).
Each reading (each point) by chlorophyll meter is mean
of twelve measurements taken on four leaves. Correlation
analysis showed a linear relationship between CCM read-
ings and nitrogen in the leaves (R² = 0.766) and nitrogen
contents were found may be estimated via the following
A decrease in nitrogen percentage was obtained when
the readings were less than 20. CCM-200 measurements
were found to have positive correlations with total chloro-
phyll content and nitrogen content in Asian pear trees.
Relationship between obtained data from chlorophyll
meter and nitrogen concentrations which obtained in this
study was also reported by other researchers. Kowalczyk-
Jusko and Koscik (2002), reported R² = 0.6936 between
SPAD-502 readings and nitrogen in tobacco leaves. Van
den Berg ad Perkins (2004) Observed linear correlation
between CCI and nitrogen concentration in sugar maple
leaves (R²=0.64). Biber (2007) had reported a significant
correlation between CCI and chlorophyll a content with
an R²=0.95 in mangrove leaves. Chang and Robinson
(2003) reported R²=0.73 for SPAD-502 readings and
% nitrogen in Green ash. Also Richardson et al. (2002)
found an R²=0.958 between chlorophyll content and
CCI in paper birch (Betula papyrifera) leaves. Shaahan
et al. (1999) had reported R²=0.99 between chlorophyll
content and CCI in mandarin and grapevine. Wright et
al. (2009) had reported chlorophyll fluorescence also can
be used for non-destructively measure changes in water
status of grape in storage. From the present work, it can
be concluded chlorophyll content meter CCM-200 can
be used for predicting both chlorophyll and nitrogen in
Asian pear trees under field condition.
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