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Estimation of Chlorophyll Using SPAD

Article in International Journal of Environment and Climate Change · October 2023

DOI: 10.9734/IJECC/2023/v13i113348

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 International Journal of Environment and Climate Change

Volume 13, Issue 11, Page 1901-1912, 2023; Article no.IJECC.108018

ISSN: 2581-8627
(Past name: British Journal of Environment & Climate Change, Past ISSN: 2231–4784)

Estimation of Chlorophyll
Using SPAD meter
Megha Vishwakarma a*, P. S. Kulhare b and G. S. Tagore b
a Shri
Vaishnav Vidyapeeth Vishwavidyalaya, Indore (M.P.), India.
b Department of Soil Science and Agricultural Chemistry, JNKVV, Jabalpur (M.P.), India.

Authors’ contributions

This work was carried out in collaboration among all authors. All authors read and approved the final
manuscript.

Article Information
DOI: 10.9734/IJECC/2023/v13i113348

Open Peer Review History:

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Received: 16/08/2023
Accepted: 23/10/2023
Original Research Article
Published: 31/10/2023

ABSTRACT
A chlorophyll meter measures the greenness of leaves and can indicate the chlorophyll content and
N status. Therefore, by using a chlorophyll meter, proxy information related to the leaf N content
could be obtained and this would avoid the need of laborious laboratory analysis. Keeping in this
view a field experiment was conducted during winter (Rabi) 2019 growing season at the
experimental field of JNKVV, Jabalpur to estimate crop nitrogen (N) and chlorophyll content in
wheat using SPAD. The treatment comprised of 3 sources of nutrient M1- Inorganic sources (NPK
fertilizers), M2- organic sources (FYM, vermicompost, biofertilizers Azotobacter and PSB) and M3-
Integrated sources (50% Inorganic + 50% organic) as main treatments and 5 NPK levels S1-
control, S2- 100% RDF, S3- 150% RDF, S4- 200% RDF, S5- Based on soil test value for target
yield of 6t ha-1 as sub treatments were replicated thrice in a split plot design. The results showed
that the correlation (r = 0.94, 0.90, 0.92 and 0.94, respectively) and coefficient of determination (R 2
= 0.96, 0.94, 0.83 and 0.94, respectively) was significantly and positively correlated with SPAD and
nitrogen concentration in leaf. Similarly significant and positive correlation (r = 0.84, 0.76, 0.78 and
0.71, respectively) found with SPAD and total chlorophyll values. Coefficient of determination (R 2)
SPAD with chlorophyll and leaf N concentration proved to be significant (R 2 = 0.75, 0.76, 0.54 and
0.85, respectively) at different growth stages. It was concluded that SPAD meter are useful for the
determination of proxy chlorophyll and total nitrogen content of wheat plant.
_____________________________________________________________________________________________________

*Corresponding author: E-mail: [email protected];

Int. J. Environ. Clim. Change, vol. 13, no. 11, pp. 1901-1912, 2023
 Vishwakarma et al.; Int. J. Environ. Clim. Change, vol. 13, no. 11, pp. 1901-1912, 2023; Article no.IJECC.108018

Keywords: SPAD; chlorophyll; total N; Inorganic; INM; organic.

1. INTRODUCTION Analysis Development (SPAD) meter has been
introduced as a popular, fast, and cheap
“Nitrogen (N) is a key constituent of all living cells technique to estimate N levels from the
and is essential for the growth and development measurement of leaf transmittance” [15]. Many
of plants” [1]. “The most important role of N in the studies suggest that “a positive correlation
plant is its presence in the structure of protein, between N uptake, leaf N concentration, leaf
the most important building substances from chlorophyll content, and grain yield”
which the living material or protoplasm of every [17,18,19,20]. In this regard, [21] suggested that
cell is made. In addition, nitrogen is also found in “the SPAD chlorophyll meter can be used as an
chlorophyll, the green coloring matter of leaves. alternative to nitrogen nutrition index (NNI) to
Chlorophyll enables the plant to transfer energy measure N status in wheat”. Therefore, SPAD
from sunlight by photosynthesis. Therefore, the values has been successfully used for N fertilizer
nitrogen supply to the plant will influence the management in rice [22,23] wheat [22,24] and
amount of protein, protoplasm and chlorophyll maize [25]. “The device can be used very simply
formed” [2]. Nitrogen is one of the major and effectively for decision making in
nutritional elements that limits crop yields. supplementary application of N fertilizer in wheat
Insufficient N supply results in smaller leaf area field. Wheat is very sensitive to insufficient
[3,4] and lower leaf photosynthesis, chlorophyll nitrogen and very responsive to N fertilization”
content and biomass production [4] leading to the [26]. “The chlorophyll meter is faster than tissue
loss of yield and quality. testing for N and allows “fine tuning” of N
“On the other hand, excessive use of N fertilizer management to field conditions, and
increases not only production cost but also consequently reduces risk of under- or over
environmental pollution” [5]. “Increased N fertilizing the wheat crop” [27]. “There is no doubt
fertilizer application does not always imply an that the nitrogen management practices can be
increase of grain yield; on the contrary, even a adopted by crop growers if the practices are
decrease in crop yields is seen in poorly affordable, accurate in fertilization, easy to use,
managed production systems” [6]. “In the quest and environmentally friendly. The SPAD meter
of achieving high yield of wheat, farmers tend to has been demonstrated and proved to be a
apply nitrogen more than the requirements. It is viable and effective tool in developing and
well known that excess N availability for crops evaluating management recipes across a field”
like wheat and barley can adversely affect plant [28]. Keeping in this view, the objective of this
health, yield, values of the final product and work was to evaluate the potential for Minolta
environmental pollution” [7]. As [8] showed, SPAD 502 chlorophyll meter readings in
“about 67% of total applied N fertilizer in wheat monitoring leaf chlorophyll content and its
production could be lost because of natural relation to N concentration in wheat leaves.
processes like denitrification, leaching, 2. MATERIALS AND METHODS
volatilization (NH3), and greenhouse gas (N2O,
NO or NO2) emissions”. “Nitrogen application at 2.1 Experiment Details
the right amount contributes to higher yields”
[9,10]. Leaf chloroplasts contain 70% of leaf N A field experiment was conducted during winter
concentration [11] thus, the amount of chlorophyll (Rabi) 2019 growing season at the experimental
present in plant leaves is often well correlated field of JNKVV, Jabalpur. (23º 13’ North latitude,
with leaf N [12,13]. “Assessment of crop N 79º 57’ East longitudes; 393 m elevation). The
requirement is critical for optimum N fertilizer soil of the experimental site was Typic
application [14]. The optimum rate and Haplustert, clay in texture neutral in reaction,
application timing of Nitrogen (N) fertilizer are non-calcareous, medium in organic carbon
crucial in achieving a high yield however, content, medium in available nitrogen,
conventional laboratory testing of plant nutrients phosphorus, and potassium and low in DTPA
is time-consuming and expensive” [15]. “Plant extractable Zn. The treatment comprised of 3
tests performed with optical instruments can be a sources of nutrient M1- Inorganic sources (NPK
significant simplification of the methods applied fertilizers), M2- organic sources (FYM,
to attain precise determination of the nutritional vermicompost, biofertilizers Azotobacter and
status of plants during a growing season” [16]. PSB) and M3- Integrated sources (50% Inorganic
“As leaf nitrogen content is strongly correlated + 50% organic) as main treatments and 5 NPK
with chlorophyll content the use of Soil Plant levels S1- control, S2- 100% RDF, S3- 150%

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RDF, S4- 200% RDF, S5- Based on soil test diluted by adding 9 ml of 80% acetone per ml of
value for target yield of 6t ha-1 as sub treatments extract. The extract produced in this way was
were replicated thrice in a split plot design. The subjected to reading on a spectrophotometer at
wheat crop (GW-366) sown on with spacing of 645, and 663 and 470 nm using acetone (80%)
22.5 cm row to row. blank. The amount of chlorophyll ‘a’ and ‘b’ are
determined using the formula given by Arnon
The experimental treatments were arranged in (1949).
split plot design with three replications. Each
experimental plot was 4 m long and 3 m wide, Chl ‘a’= ((12.7 A 663) - (2.69 A 645))
consisting of three rows with one m inter-row
spacing. The plots were kept weed-free and Chl ‘b’= ((22.9 A 645) - (4.68 A 643))
maintained in an optimum soil moisture regime
throughout the experimental period. The specific Total chlorophyll (a+b) = ((20.2 (A 645)
quantity of each fertilizer was calculated based +8.02(A 663)))
on gross plot size as per treatment. Treatment
Where, A = Absorbance, V = Final volume of
wise 1/3 quantity of nitrogen and entire dose of
80% acetone (in ml), W= Weight of plant tissue
phosphorus and potassium were applied as
(in grams) μg g-1 fresh weight (μg/ml final
basal. While, the 1/3 dose of nitrogen was top
volume)/leaf weight (g)
dressed in two equal splits, after CRI and third
irrigation (65 DAS). Urea (46% N), Single super The observations were recorded from each plot
phosphate (16% P2O5) and muriate of potash at crown root initiation (CRI) (21 DAS), tillering
(60% K2O) were used as sources of nitrogen, (45 DAS), flowering (65 DAS) and milking stages
phosphorus and potassium, respectively. FYM, (90 DAS).
VC and biofertilizers were applied as per
treatment 15 days before sowing. The 2.4 Nitrogen Analysis in Plant
observations were recorded from each plot at
crown root initiation (CRI) (21 DAS), tillering (45 The nitrogen content in plant sample was
DAS), flowering (65 DAS) and milking stages (90 estimated by micro Kjeldahl digestion and
DAS) of wheat. distillation method using KEL PLUS system. For
digestion, a known weight of plant sample in the
2.2 Estimation of Chlorophyll Using presence of sulfuric acid with catalyst mixture
SPAD-502 (5:1) under high temperature was digested and
distilled in presence of sodium hydroxide, the
A chlorophyll meter (SPAD-502, Minolta, Japan) released ammonia was condensed and absorbed
was used to obtain readings estimating leaf in boric acid with mixed indicator (Bromocresol
chlorophyll concentration (SPAD value). Five cresol green and methyl red indicator) to form
plants per treatment were selected randomly and ammonium borate, the excess of which was
SPAD values were recorded from the fully titrated with standard sulfuric acid (0.1N) (AOAC,
matured leaves counted from the top of the 1995). The observations were recorded from
plants, the youngest fully expanded leaf. The each plot at crown root initiation (CRI) (21 DAS),
observations were recorded from each plot at tillering (45 DAS), flowering (65 DAS) and milking
crown root initiation (CRI) (21 DAS), tillering (45 stages (90 DAS).
DAS), flowering (65 DAS) and milking stages (90
DAS). 2.5 Statistical Analysis
2.3 Chlorophyll Content Measurement Data were analyzed using SPSS for analysis of
variance and Fisher’s LSD multiple range test
The material was processed in the fresh state was employed for the means comparisons. The
immediately after collection. After fine chopping, correlations between fertilizer application levels
portions weighing 0.5 g were measured off on an and grain yield, SPAD value and grain yield were
analytical balance. The measured-off material tested at 5% level of significance.
was then homogenized in a homogenizer with
the addition of 10 ml of 80 % acetone. A primary 3. RESULTS AND DISCUSSION
acetone extract containing all chloroplast
pigments was obtained in this way. The extract 3.1 SPAD Readings at Different Growth
was then centrifuged at 2500 rpm for 5 min. Stages
Since the concentration of pigments was in most
cases too great for reading to be performed on a It can be seen from the data (Table 2) that the
spectrophotometer, the obtained extract was SPAD reading, was significantly maximum (8.15,

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12.38, 28.58 and 30.65, respectively) with the because nitrogen is a structural element of
application of inorganic sources of nutrient (M1) chlorophyll and protein molecules, and thereby
over integrated (M3) and organic sources of affects formation of chloroplasts and
nutrient (M2) at CRI, tillering, jointing and milking accumulation of chlorophyll in them” [35,36]. Our
stages, respectively. The SPAD readings of 6.36, results supported by Islam et al. 2014 [37]
9.71, 25.54 and 25.00 were registered in M3 and reported that the SPAD values drastically
it was also found significantly superior over M2 reduced in treatments treated with only organic
(4.47, 6.74, 17.26 and 16.65, respectively) at fertilizers. The values were higher in treatment
each stage, respectively. receiving full dose of NPK through inorganic
fertilizers as compared with reduced inorganic
However, the application of S3 (150% RDF) and fertilizers and/or organically supplied fertilizers.
S4 (200% RDF) significantly increased SPAD These results agree with that obtained [37] in
reading over S2 (100% RDF) and S5 (STV potato. “This might be because N supply in
based RDF) at CRI and jointing but at par at organic treatment is generally restricted for slow
tillering. S4 Significant S2 (100% RDF) and S5 N mineralization as compared to crop N demand”
(STV based RDF) but at par with S3. At milking. [38].
The highest SPAD reading was registered in S4
(200% RDF) which was 7.97, 11.07, 27.45 and 3.2 Total Chlorophyll in Leaves (mg g-1
28.02 and 7.29, 11.11, 26.60 and 27.24 with Leaf Tissue) at Different Growth
150% RDF at each stage, respectively. Stages
The interaction effect was found to be significant Table 3 showed that the total chlorophyll of 1.27,
at all growth stages. At CRI stage of wheat, the 1.92, 2.14 and 1.24 mg g-1 leaf tissue was
combination of M1S4 (10.50) was recorded the significantly more where, inorganic sources of
highest SPAD reading, which was significantly nutrient (M1) was applied followed by integrated
superior over all the combination and followed by sources of nutrients (M3) with value of 1.08,
M1S3 (9.23), M1S2 (8.40), M1S5 (8.40), M3S4 1.75, 1.78 and 1.04 mg g-1 leaf tissue over
(7.93) and M3S3 (7.52) all were at par among organic sources of nutrient (M2) which recorded
themselves. At tillering stage, the combination lowest total chlorophyll of 0.93, 1.33, 1.41 and
M1S5 (16.11), M1S4 (15.64), M1S3 (15.28) were 0.87 mg g-1 leaf tissue at CRI, tillering, jointing
found significant over all combination. On and milking stages.
comparing the treatment combination at jointing
stage, applied to wheat, performance of M1S3 However, the application of S3 (150% RDF) and
(33.70), M1S4 (33.48), M1S5 (33.45) were found S4 (200% RDF) significantly increased total
significant over all combinations. However, at chlorophyll over S2 (100% RDF) and S5 (STV
milking stage, the interaction of M1S4 (38.92) based RDF) at each growth stage. The highest
causes significant difference and statistically total chlorophyll was recorded with the
similar with M1S3 (32.44) and M1S5 (32.86), application of S4 at CRI and tillering stage which
M1S2 (31.86). Maximum SPAD reading was was statistically similar with S3 (150% RDF) with
obtained about jointing stage (65 DAS) then the the values of 1.26 and 1.84 mg g-1 leaf tissue at
chlorophyll content gradually decreased till the CRI and tillering stage. However, S3 (150%
end of the vegetation season similar results also RDF) was highest at jointing and milking stage
reported by Juśko et al [29]. Maximum SPAD with the values of 1.35 and 1.86 mg g-1 leaf
reading was found in inorganic fertilizers tissue. Treatment S2 (100% RDF) was found
because there was more nitrogen available to the significant over S5 (STV based RDF) at tillering,
NPK treated plants, there was more chlorophyll jointing and milking stage but at par at CRI stage.
in the leaves, which speeds up photosynthesis
[30]. Our results show that the application of The interaction effect was found to be significant
nitrogen in inorganic form at the right time at CRI stage of wheat. The combination of M1S3
enhances chlorophyll content which resulted in (1.61 mg g-1 leaf tissue) was recorded as the
higher SPAD values [15]. Nitrogen is part of the highest CCI, which was significantly superior
enzymes associated with chlorophyll synthesis over all the combination and at par with M1S4
[31] and the chlorophyll concentration reflects (1.54 mg g-1 leaf tissue). At tillering stage, the
relative crop nitrogen status and yield level [32]. combination M1S4 (2.27) was found maximum
“Very close link between chlorophyll and nitrogen and significant over all combination and at par
content was observed as investigated by many with M1S3 (2.16 mg g-1 leaf tissue). On
investigators” [33,34]. “It is understandable, comparing the treatment combination at jointing

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stage, applied to wheat, performance of M1S4 similar treatment M1S3 (1.45%) and M1S5
(2.58 mg g-1 leaf tissue), was found significant (1.48%).
over all combinations. However, at milking stage,
the interaction of M1S4 (1.54 mg g-1 leaf tissue) “The percentage of total N concentration in dried
causes significant different and statistically leaf increased as larger N doses in mineral
similar treatment M1S3 (1.38 mg g-1 leaf tissue). fertilization were applied. The pattern of nitrogen
accumulation during the vegetation season
The maximum chlorophyll in inorganic sources of deviated from the pattern of chlorophyll
nutrients is usually immediately and fast synthesis. The highest N accumulation was
containing all necessary nutrients that are ready observed during the first measurement.
for plants [39]. Earlier study [40] indicated that Subsequent analyses showed lesser N
abundant nutrient supplementation increases concentration in the leaves. The lowest N
chlorophyll concentration in Sorghum bicolor and concentration was noted later stages, and then it
Zea mays. However, organic sources of nutrients remained roughly at this level till the end of the
perform poorly. This may be due to vegetation season. The fact that the N
disappearance of mineral N in the soil system concentration stayed roughly at the same level
was caused by microbial immobilization under throughout the whole period till the end of the
organic sources [41] which cause lower vegetation season, while chlorophyll content in
availability of N and chlorophyll. “Organic the leaves fell, points to the accumulation of
fertilizer improves physical, biological, and nitrogen compounds other than pigments” [28].
chemical properties of a soil but the nutrients “Maximum N concentration in inorganic fertilizers
may not be as readily available to the plants” may be because inorganic fertilizers releases
[39]. nutrients for the plants instantly and in readily
available forms for the plants during its growth,
3.3 Total N Content in Leaf at Different development and reproductive phases where the
Growth Stages nutrient demand is at its peak. Higher levels of
inorganic fertilizers have increased the activity of
Data presented in Table 4 showed that the total photosynthesis and enzymes which responsible
N content in leaves of 2.39, 2.29, 1.80 and for transformation of energy, carbohydrates, fat
1.38%, respectively was significantly more metabolism and respiration of plant” [42]. “The
where, inorganic sources of nutrient (M1) was nitrogen containing organic compounds in
applied followed by integrated sources of organic manures especially in FYM are more
nutrients (M3) with value of 2.26, 2.15, 1.76 and resistant to decomposition and only about one
1.34%, respectively over organic sources of third of the nitrogen is easily released. The
nutrient (M2) which recorded lowest total remaining amount of nitrogen persisted in the soil
chlorophyll of 2.22, 2.13, 1.60 and 1.19%, for a long period” [43].
respectively at CRI, tillering, jointing and milking
stages. However, M1 is significant over M3 3.4 Correlation and Coefficient of
except milking stage. Determination of SPAD Readings with
Total Chlorophyll and Total N Content
Among the different levels of NPK, the in Leaves at Different Growth Stages
application of S3 (150% RDF) and S4 (200%
RDF) significantly increased N content over S2 Pearson correlation coefficients between total N
(100% RDF) and S5 (STV based RDF) at CRI, concentration in leaf, total chlorophyll content,
jointing and milking stage. S3 (150% RDF) and SPAD values at CRI, tillering, jointing and milking
S4 (200% RDF) were found at par among stages of wheat crop are presented in Table 1
themselves. All levels were found at par among and Fig. 1. Good correlations were found for both
themselves. meters for determination of chlorophyll content
and N content. The SPAD values were positively
The interaction effect was found to be significant correlated with grain yield of wheat at differ good
at jointing and milking stage of wheat. The stages of plants. The SPAD values were also
combination of M1S4 (2.13%) was recorded as significantly correlated (r = 0.94, 0.90, 0.92 and
the highest N content, which was significantly 0.94, respectively) with leaf N concentration at
superior over all the combination and at par with different growth stages in this study. Maximum
M1S3 (2.04%) at jointing stage. However, at correlation was found in CRI and milking stage
milking stage, the interaction of M1S4 (1.56%) followed by jointing and tillering respectively.
causes significantly different and statistically Coefficient of determination R2 between

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Table 1. Correlation and Coefficient of variance of SPAD with total chlorophyll and total N content

Stages Correlation of SPAD with total chlorophyll and total N Coefficient of variance of SPAD with total chlorophyll and total N
content content
Total Total Total Total
Chlorophyll N content chlorophyll N content
CRI 0.94 0.96 0.84 0.75
Tillering 0.90 0.94 0.76 0.76
Jointing 0.92 0.83 0.78 0.54
Milking 0.94 0.94 0.71 0.85

Table 2. Effect of different sources and NPK levels on SPAD readings at different growth stages

M/S SPAD
CRI Tillering Jointing Milking
M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean
S1 4.20 3.83 4.17 4.06 2.70 6.60 4.10 4.47 13.95 17.38 15.82 15.72 17.17 13.47 17.86 16.17
S2 8.40 5.03 5.76 6.40 12.15 7.53 10.58 10.09 28.31 18.14 26.48 24.31 31.86 16.13 25.42 24.47
S3 9.23 5.10 7.52 7.29 15.28 7.15 10.90 11.11 33.70 18.49 27.63 26.60 32.44 20.27 29.01 27.24
S4 10.50 5.47 7.93 7.97 15.64 6.11 11.44 11.07 33.48 19.85 29.01 27.45 38.92 16.48 28.65 28.02
S5 8.40 2.90 6.40 5.90 16.11 6.34 11.53 11.33 33.45 12.43 28.29 24.72 32.86 16.90 24.04 24.60
Mean 8.15 4.47 6.36 6.32 12.38 6.74 9.71 9.61 28.58 17.26 25.45 23.76 30.65 16.65 25.00 24.10
SEm± 0.19 0.48 0.90 1.18
CD(p=0.05) 0.76 1.90 3.56 4.64
SEm± 0.20 0.45 0.93 1.14
CD(p=0.05) 0.60 1.31 2.71 3.34
Int I 0.35 0.77 1.61 1.98
CD(p=0.05) 1.03 2.27 4.70 5.79
Int II 0.50 1.19 2.31 2.95
CD(p=0.05) 1.46 3.48 6.76 8.62
M1- Inorganic sources (NPK fertilizers), M2- organic sources (FYM, vermicompost, biofertilizers Azotobacter and PSB) and M3- Integrated sources (50% Inorganic + 50%
organic) S1- control, S2- 100% RDF, S3- 150% RDF, S4- 200% RDF, S5- Based on soil test value for target yield of 6t ha-1

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Table 3. Effect of different sources and NPK levels on total chlorophyll at different growth stages

M/S Total chlorophyll

CRI Tillering Jointing Milking
M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean
S1 0.74 0.72 0.73 0.73 1.35 1.26 1.26 1.29 1.48 1.19 1.30 1.32 0.92 0.73 0.89 0.85
S2 1.20 0.98 1.06 1.08 2.02 1.36 1.88 1.75 2.14 1.45 1.75 1.78 1.26 0.75 1.14 1.05
S3 1.61 1.05 1.38 1.35 2.16 1.40 2.01 1.86 2.36 1.51 2.01 1.96 1.38 0.90 1.29 1.19
S4 1.54 1.00 1.22 1.26 2.27 1.34 1.92 1.84 2.58 1.72 2.34 2.21 1.54 1.13 0.94 1.20
S5 1.25 0.88 0.98 1.04 1.82 1.27 1.66 1.59 2.16 1.19 1.51 1.62 1.12 0.81 0.92 0.95
Mean 1.27 0.93 1.08 1.09 1.92 1.33 1.75 1.67 2.14 1.41 1.78 1.78 1.24 0.87 1.04 1.05
SEm± 0.029 0.043 0.082 0.028
CD(p=0.05) 0.113 0.169 0.321 0.109
SEm± 0.031 0.044 0.044 0.033
CD(p=0.05) 0.090 0.130 0.129 0.097
Int I 0.053 0.077 0.076 0.057
CD(p=0.05) 0.156 0.224 0.223 0.167
Int II 0.075 0.110 0.177 0.076
CD(p=0.05) 0.218 0.321 0.517 0.221
M1- Inorganic sources (NPK fertilizers), M2- organic sources (FYM, vermicompost, biofertilizers Azotobacter and PSB) and M3- Integrated sources (50% Inorganic + 50%
organic) S1- control, S2- 100% RDF, S3- 150% RDF, S4- 200% RDF, S5- Based on soil test value for target yield of 6t ha-1

Table 4. Effect of different sources and NPK levels on total N content (%) in leaves at different growth stages

M/S Total N content (%)

CRI Tillering Jointing Milking
M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean
S1 2.13 2.13 2.15 2.14 2.09 2.04 1.97 2.03 1.17 1.17 1.23 1.19 1.07 1.10 1.14 1.10
S2 2.33 2.18 2.30 2.27 2.26 2.12 2.18 2.19 1.82 1.32 1.66 1.60 1.36 1.22 1.28 1.29
S3 2.52 2.24 2.35 2.37 2.28 2.15 2.19 2.21 2.04 1.95 2.00 1.99 1.45 1.24 1.41 1.37
S4 2.56 2.31 2.33 2.40 2.41 2.20 2.23 2.28 2.13 2.02 2.11 2.09 1.56 1.23 1.50 1.43
S5 2.40 2.25 2.19 2.28 2.38 2.15 2.20 2.24 1.83 1.52 1.83 1.72 1.48 1.17 1.36 1.34
Mean 2.39 2.22 2.26 2.29 2.29 2.13 2.15 2.19 1.80 1.60 1.76 1.72 1.38 1.19 1.34 1.30
SEm± 0.032 0.029 0.034 0.024
CD(p=0.05) 0.126 0.113 0.136 0.096

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M/S Total N content (%)

CRI Tillering Jointing Milking
M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean M1 M2 M3 Mean
SEm± 0.027 0.032 0.038 0.025
CD(p=0.05) 0.080 0.093 0.111 0.072
Int I 0.047 0.055 0.066 0.043
CD(p=0.05) NS NS 0.192 0.126
Int II 0.077 0.076 0.091 0.062
CD(p=0.05) 2.13 2.13 2.15 2.14
M1- Inorganic sources (NPK fertilizers), M2- organic sources (FYM, vermicompost, biofertilizers Azotobacter and PSB) and M3- Integrated sources (50% Inorganic + 50%
organic) S1- control, S2- 100% RDF, S3- 150% RDF, S4- 200% RDF, S5- Based on soil test value for target yield of 6t ha-1

a b c d
Fig. 1a. Relationship between SPAD and chlorophyll content at different growth stages

a b c d
Fig. 1b. Relationship between SPAD and nitrogen content at different growth stages

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chlorophyll content in SPAD units and nitrogen COMPETING INTERESTS

concentration and leaf N concentration proved to
be significant (R2 = 0.96, 0.94, 0.83 and 0.94, Authors have declared that no competing
respectively). Highest value of Coefficient of interests exist.
determination was found in CRI and followed by
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