Field Crops Research 209 (2017) 4757

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Field Crops Research

journal homepage: www.elsevier.com/locate/fcr

Assessment of growth, leaf N concentration and chlorophyll content of sweet MARK

sorghum using canopy reectance

Shardendu Kumar Singh , James H. Houx III, Michael J.W. Maw, Felix B. Fritschi
University of Missouri, Division of Plant Sciences,1-33 Agriculture Building, Columbia, MO 65211, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Remote estimation of leaf nitrogen (N) or pigments through hyperspectral reectance oers an opportunity to
General purpose model non-destructively diagnose plant N status. Two sweet sorghum (Sorghum bicolor [L.] Moench) cultivars (Top 76-6
Near-infrared region and Dale) were grown with 0, 56, 112, 168, and 224 kg N ha1 in 2009 and 2010. Reectance measurements
Simple-ratio model were coupled with plant height, main-stem node number, leaf N concentration, and total chlorophyll content to
Validation
establish the relationship of these traits with canopy reectance. Canopy reectance was most sensitive to N
Visible region
status in the visible region, specically near green (595 nm) and red (701 nm) wavebands. Simple-ratio spectral
models comprised of visible wavebands or wavebands from the visible and near infrared region outperformed
models developed using only the most sensitive single-waveband. Based on the cross-validation of spectral
models between data from two years and two cultivars, the simple-ratio models comprising the reectance (R)
ratios of 595 nm vs. 1676 nm and 595 nm vs. 508 nm predicted leaf N concentration and chlorophyll content
with the greatest accuracy (highest r2 and lowest relative error, RE). These simple-ratio models were used to
develop general-purpose spectral models to derive coecients to estimate leaf N concentration (-66.63 R595/
R1676 + 34.14; r2 0.52; RE 16.8%) and chlorophyll content (-49.12 R595/R508 + 107.47; R2 0.64; RE 17%).
The identied spectral models can be used to assess growth, diagnose sweet sorghum N status and may be useful
to make N management decisions for site-specic fertilizer applications.

1. Introduction 1998; Richardson et al., 2002; Zhao et al., 2005).

Sweet sorghum (Sorghum bicolor [L.] Moench) has garnered much
attention as a source for biofuel due to its high stem sugar content
(Putnam et al., 1991; Rooney et al., 2007). Sweet sorghum is an annual
C4 crop with high photosynthetic capacity, uses water and nutrients
more eciently than C3 crops, and can be grown on marginal lands
(Anten et al., 1995; Young and Long, 2000). Although sorghum is
considered a soil nutrient scavenger and has shown excellent nitrogen
(N) use eciency, application of N fertilizer is critical for optimum
production (Maw et al., 2016; Serro et al., 2012). However, excess N
application not only increases production costs but also leads to
environmental concerns (Snyder et al., 2009). Ecient monitoring of
plant N status oers an opportunity for appropriate timing of N
application and site-specic N management, which could reduce the
use of N fertilizers (Blackmer et al., 1994; Ma et al., 1996). Remote
estimation of leaf/canopy reectance has been widely used to non-
destructively assess plant N status and health in agricultural and
forestry ecosystems (Gausman et al., 1984; Peuelas and Filella,
The hyperspectral signature from green leaves carries a vast amount
of information that has been related to various plant characteristics,
including N status, thereby oering a non-destructive and quick
assessment under eld conditions (Peuelas and Filella, 1998;
Richardson et al., 2002; Sims and Gamon, 2002). Although selected
spectral features and their sensitivity to specic stresses can signi-
cantly dier between crops species (Gausman et al., 1984; Sims and
Gamon, 2002), assessments of the relationships between canopy
spectral reectance and sweet sorghum characteristics have been
limited. Selection of the most sensitive wavebands to construct the
reectance model is a crucial step towards developing predictive
models for crop and leaf characteristics (Carter and Spiering, 2002;
Gausman, 1974; Singh et al., 2013; Zhao et al., 2005). Spectral models
comprised of simple waveband and multiple waveband indices derived
from visible and near-infrared (NIR) regions indicate that these models
can be used to estimate leaf N and pigment concentrations (Huang
et al., 2015; Sims and Gamon, 2002; Singh et al., 2013; Yu et al., 2014).
Further, the models can be used to assess plant nutritional status,

Corresponding author.

Corresponding author at: Adaptive Cropping Systems Laboratory, United States Department of Agriculture Agricultural Research Service, Beltsville, MD 20705, USA.
E-mail addresses: singh.shardendu@gmail.com (S.K. Singh), fritschif@missouri.edu (F.B. Fritschi).

http://dx.doi.org/10.1016/j.fcr.2017.04.009
Received 16 December 2016; Received in revised form 14 April 2017; Accepted 15 April 2017
Available online 26 April 2017
0378-4290/ 2017 Elsevier B.V. All rights reserved.
S.K. Singh et al.
growth and yield (Gray et al., 2010; Ma et al., 2001; Peuelas and
Filella, 1998; Ryu et al., 2011; Yi et al., 2007; Zhao et al., 2005; Zhao
et al., 2007). The N reectance index developed by Bausch and Duke
(1996) to monitor corn N status also is based on canopy reectance in
the visible (green, 520600 nm) and NIR (760900 nm) regions. In a
subsequent study, Bausch and Diker (2001) used the N reectance
index to detect in-season corn N deciency and make N fertilizer
application decisions. Using this approach, they were able to reduce
fertilizer application by 39.2 kg N ha1 in a farmers eld without
reducing the corn grain yield. These results indicated that a canopy
reectance index derived from one experimental setting can be
suciently robust to be applied successfully on a dierent site and
year for N-management decisions, thus illustrating the potential for
broader applicability of indices developed on a single or small numbers
of eld environments.
Spectral reectance models may be comprised of one or more
wavebands/spectral features as single-waveband, simple-ratios or in-
dices to determine plant N status or chlorophyll concentration
(Chappelle et al., 1992; Gray et al., 2010; Zhao et al., 2007; Zhao
et al., 2005). Often these spectral models are composed of distinct
wavebands (e.g. in the visible and/or NIR regions) of the electromag-
netic spectrum. Daughtry et al. (2000) found that spectral indices that
combined reectance at visible and NIR regions of the spectrum are
sensitive to both leaf spectral characteristics and background reec-
tance. Thus, an integrated approach to develop hyperspectral reec-
tance models using features from two distinct wavebands oers better
precision and accuracy for the prediction of plant growth, leaf N, or
pigment concentration. Due to the high degree of collinearity among
waveband reectance from the plant canopy, use of fewer wavebands
to construct an empirical model is encouraged (Gardner et al., 1985).
Models with fewer wavebands usually oer the advantage of being
straightforward and rapid compared to the more complex models with
many spectral features. Once the sensitive wavebands are identied, a
relatively simple spectroradiometer that measures those particular
wavebands can instantaneously calculate the desired plant trait by
using appropriate coecients derived from the reectance model
(Peuelas and Filella, 1998).
Sweet sorghum has gained much interest as a potential biofuel crop
to minimize the dependence on maize (Zea mays L.) to produce ethanol
(Maw et al., 2016; Putnam et al., 1991; Rooney et al., 2007; Serro
et al., 2012). Although, N fertilizer application and management is
crucial in both crops, studies describing the impact of N deciency and
its relation with canopy reectance under eld condition in sweet
sorghum are scarce compared to maize (Blackmer et al., 1994;
Daughtry et al., 2000; Ma et al., 1996; Zhao et al., 2005). Zhao et al.
(2005) and Foster et al. (2016) found that plant tissue N or chlorophyll
correlated with spectral reectance measured at leaf or canopy levels in
grain and high-biomass sorghum, respectively. Mani et al. (1991)
reported that canopy reectance of grain sorghum was highly sensitive
to growth and nutrient application around 55 and 65 days after
planting. Further, Serro et al. (2012) found that leaf N concentration
was the best indicator of sugar content in sweet sorghum. Foster et al.
(2016), reported that a normalized N index constituting reectance at
wavebands in the blue region of the spectrum (400 and 510 nm) was
strongly correlated with the total N concentration. These studies
indicate the presence of empirical relationships between sweet sorghum
N status and canopy reectance properties that can be utilized to
identify sensitive wavebands to construct spectral models.
A crops N fertilizer requirement is often determined based on the
laboratory analyses of soil and/or plant N status, but sample collection
and analyses can be time-consuming and expensive. Therefore, an
empirical model developed using canopy spectral reectance would be
useful for a quick and non-destructive assessment of sweet sorghum N
status. Thus, the objectives of this study were (a) to identify spectral
features most sensitive to N deciency, (b) to develop simple spectral
models to quantify crop N status, TChl content, plant height and node
48
Field Crops Research 209 (2017) 4757
numbers, and (c) to determine eects of N availability on sweet
sorghum growth and development, leaf N concentration and total
chlorophyll (TChl) content.
2. Materials and methods
2.1. Crop management and N treatments
The experiment was conducted at the Bradford Research Center
near Columbia, MO, USA (38 53N, 92 12 W) in 2009 and 2010 on a
Mexico Silt Loam (ne, smectitic, mesic, Vertic Epiaqualf) soil. The eld
was tilled with a disk to a depth of approximately 0.15 m before
planting. On 7 June 2009 and 18 June 2010, sweet sorghum cultivars
Top 76-6 and Dale were sown 0.02 m deep, in rows 0.76 m apart to
achieve an approximate population of 208,000 plant ha1. The main
plots consisted of N treatments of 0, 56, 112, 168, and 224 kg N ha1
(hereafter referred to as 0N, 56N, 112N, 168N and 224N) and were
arranged in a randomized complete block design with four replications,
and subplots consisted of sweet sorghum cultivars that were arranged as
strips within each replication. Main plots were 12 rows wide and 15 m
long, and subplots were six rows wide and 15 m long. SuperU urea
(Koch Agronomic Services, Wichita, KS) was broadcast as applications
of 56 kg ha1 (56N) and 112 kg ha1 (112N, 168N, and 224N) at
planting, and supplemented with 56 kg ha1 or 112 kg ha1 for 168N
and 224N approximately two weeks after emergence. The same eld
and plot design were used in both study years.
For each growing season, rainfall and temperature data from
planting to the end of measurements were obtained from a weather
station located at the Bradford Research Center. Total in-season rainfall
for 2009 and 2010 was 288 mm and 514 mm, respectively. The average
growing season temperature ( standard deviation) in 2009 was
28.8 3.5 C and in 2010 was 22.7 4.6 C. Long-term (between
1971 and 2000) records of rainfall (395 mm) and temperature
(22.3 3.3 C) in this region for the same period were obtained from
the High Plains Regional Climate Center (HPRCC) website (http://
www.hprcc.unl.edu; Columbia WSO AP, MO, USA, Station # 231791).
2.2. Measurements
Plant samplings for determination of N concentration and chlor-
ophyll content and canopy reectance measurements were conducted
twice each year. The rst measurements were conducted between 50
and 55 days after planting (DAP) in both years. The second measure-
ments were conducted 70 DAP in 2009 and 78 DAP in 2010. In 2010,
plant height (PH) and main stem node numbers (MSN) for four
randomly selected plants, and canopy reectance were also measured
ten times in each subplot between 40 and 120 DAP. The average
number of leaves during the rst measurement was approx. 56 in 0N
and 78 in the N-treated plants. At the second measurement, there were
89 leaves in the 0N and 1013 leaves in the N-treated plants. Plant
height was measured from ground to the apex of the plant. The stem
elongation rate (SER) and node addition rate (NAR) were calculated as
maximum plant height or node number divided by the number of days
required to reach the maximum.
2.3. Determination of leaf N concentration
In association with the canopy spectral reectance measurements,
plants were harvested from one square meter area in each subplot, and
leaves and stems were dried separately in a forced air-drier at 55 C
until weights stabilized. Dried leaves were ground using a Wiley Mill
(Thomas Scientic, NJ, USA) to pass a 2.0 mm screen, and subsequently
ground a second time with a UDY Cyclone Mill (UDY Corp., Fort
Collins, CO) to pass a 1.0 mm screen. Ground materials were homo-
genized, and total N concentration was determined by dry combustion
with a LECO TruSpec N Elemental Determinator (LECO Corp., MI, USA)
S.K. Singh et al.
according to AOAC (2006).
2.4. Determination of leaf total chlorophyll content
Total chlorophyll (TChl) content was determined from ve 0.68 cm2
leaf disks excised from the uppermost, fully expanded leaf from ve
plants of each subplot. Leaf disks were placed into vials containing 5 mL
of ethanol (95%, v/v) and incubated at room temperature in the dark
for 24 h. The absorbance of the supernatant was measured at 664 and
648 nm using a Genesys 10uv spectrophotometer (Thermo Electron
Corporation, Madison, WI). Total chlorophyll was estimated using the
equations of Lichtenthaler (1987): Total chlorophyll (g ml1) =
(5.24 A664) + (22.24 A648), where A664 and A648 are the absor-
bance values of the supernatant at 664 and 648 nm, respectively. The
values were expressed in g cm2 by using the equation: TChl content
(g cm2) = (Total chlorophyll, g ml1 5)/3.40, where ve is the
amount of ethanol (solvent, ml) and 3.40 is the leaf area (cm2) of the
ve leaf disks.
2.5. Acquisition of canopy spectral reectance
Canopy spectral reectance was measured on sunny days between
1000 and 1400 h in each subplot using an ASD FieldSpec, FR spectro-
radiometer (Analytical Spectral Devices Inc., Boulder, CO, USA) with a
25 eld of view. Three measurements were taken within the middle
rows near the center of each subplot and averaged. Reectance was
measured at wavelengths ranging from 350 to 1800 nm with a 1-nm
interval. However, the reectance between 1350 nm and 1450 nm was
excluded from the analysis due to the strong interference of water
absorption bands in this region (Gausman et al., 1984), and the spectral
domains of 4001350 and 14511800 nm were used for analyses. To
measure the canopy reectance, the optical head was held approxi-
mately 1 m above the canopy at the nadir-viewing (nadir position)
geometry at each sampling interval. After optimization of the ASD
instrument, reectance measurement of the device was set to 100% by
measuring the reectance of a Spectralon reference panel (white
reference panel 25.4 25.4 cm calibrated as 99% reective). The
white reference was measured approximately every 510 min to check
the instrument stability for 100% reectance. The white reference
measurements were conducted by bringing the sensor close (approx.
0.4 m) to the Spectralon to ensure that only the reference panel
reectance was measured. The rst measurements of spectral reec-
tance (5055 DAP) were made from the ground as plant height did not
exceed the reach for proper measurement by a person standing between
the rows. Due to the tall plants at the time of the second spectral
reectance measurements (7078 DAP), the spectroradiometer was
operated by an individual positioned on a stage attached to a tractor-
mounted forklift. Spectral readings were acquired after adjusting the
height and location of the forklift. The canopy reectance was auto-
matically adjusted for the white reference and processed instantly on a
laptop computer by RS3 Spectral Acquisition Software (v. 5.6,
Analytical Spectral Devices Inc., Boulder, CO, USA) attached to the
ASD instrument, and saved for future retrieval using ViewSpec Pro
software (v. 5.6.8, Analytical Spectral Devices Inc., Boulder, CO, USA).
2.6. Selection of sensitive wavebands and data analysis
The aim was to identify N- and chlorophyll-sensitive single-wave-
bands or simple-ratios (ratio between two wavebands) from the whole
domain of the spectrum with the best predictive capability. The N and
chlorophyll sensitive wavebands were determined using two ap-
proaches: (a) the sensitivity was calculated as percentage change in
the reectance at each waveband for the 0N, 56N, 112N and 168N
against the 224N treatment (Zhao et al., 2005) and (b) based on the
single-wavebands with the highest coecient of determination (r2)
from the linear regression of spectral reectance with N concentration
Field Crops Research 209 (2017) 4757
or TChl content. The PROC REG procedure of SAS (v. 9.2, SAS Institute
Inc., Cary, NC USA) was used for linear regression to relate the canopy
spectral reectance (independent variable) to N concentration or TChl
content (dependent variables). The reectance data from two sampling
dates in each year were pooled to establish the relationships between
reectance and N concentration or TChl content. To determine the
simple ratios, the reectance values of the most sensitive wavebands
with the greatest r2 were used as the numerator and the reectance at
each of all other wavebands throughout the whole spectrum as the
denominator. The resulting ratio values of reectance were then
regressed with the N concentration or TChl content in a similar manner
as for the single-waveband to obtain the simple-ratio. Three simple
ratios with the greatest r2 were selected for N concentration as well as
for TChl content. Similarly, a simple ratio with the highest r2 was also
developed by linear regression of the reectance with plant height and
node number.
Additionally, a pre-existing chlorophyll index and a simple-ratio
model showing good predictive capabilities in previous studies were
selected for evaluation in the present study (Gray et al., 2010; Zhao
et al., 2005). The total canopy chlorophyll content index (CHLindex)
calculated as the ratio of the area under the curve in the 840870 nm
regions and 720730 nm regions [ R840-870/ R720-730] (Gitelson et al.,
2005; Gray et al., 2010) was determined. Since Zhao et al. (2005) found
a good correlation with N concentration or chlorophyll content in
sorghum, the simple ratio of the reectance between 1075 nm and
735 nm (R1075/R735) was also used.
To determine models with consistent performance across year and
cultivar, the reectance models for N concentration and TChl content
derived using single-waveband or simple ratios from the data set of
2009 were validated on the data set of 2010 and vice-versa. The r2, root
means squared error (RMSE) and the percent relative error (RE) were
determined and used as measures of model performance. The RMSE and
RE evaluate the accuracy (the relative closeness of the predictions to
the actual values) of the models and model validation as used in other
studies (Blackburn, 2007; Ryu et al., 2011; Yi et al., 2007) and
computed as:
n
i =1 (yi yi)2 100 RMSE
RMSE = RE = y
n
where y and yi are the predicted and actual pigment concentrations,
respectively; y is the average of the actual pigment concentration, and n
is the number of observations. Although RMSE measures the closeness
of the predicted to the actual value, comparisons of RMSE between
models derived from dierent sets of data are not meaningful. How-
ever, RE can be compared between models; the model with the smaller
RE has predicted values that are closer to the actual values.
Analysis of variance (ANOVA) was performed using PROC MIXED
procedure of SAS (SAS Enterprise Guide, 4.2, SAS Institute Inc., NC,
USA). Cultivar, DAP, and N treatments were considered as xed eects
and replication as a random eect. Since signicant Cultivar N
interactions were observed for PH and NAR, all growth parameters
(PH, MSN, SER and NAR) were analyzed by cultivar. Cultivar N and
DAP N interactions were not signicant for leaf N concentration and
TChl content in a given year. Therefore, Leaf N and TChl data of the two
cultivars and two samplings (DAP) were pooled within each year and
means presented. Treatment comparisons were conducted by the least
square means (LSMEANS) procedure (at = 0.05) with the letter
grouping obtained using pdmix800 macro (Saxton, 1998).
3. Results
3.1. Plant height and node development
The eects of cultivar and N treatment were signicant for PH,
MSN, SER and NAR (Table 1). The season-end PH ranged from 1.86 m
49
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Table 1
Inuence of nitrogen (N) treatment on sweet sorghum cultivar (Top 76-6 and Dale) plant height (PH) and number of main stem nodes (MSN) 118 days after planting in 2010. Stem
elongation rate (SER) and node addition rate (NAR) were determined based on days after planting when maximum plant height and node number were reached. Data points represent
means of four replications. Analysis of variance (ANOVA) results are presented as P-values.

N treatment (Kg ha1) PH (m) MSN (no.) SER (cm d1 plant1) NAR (d1 plant1)

Top 76-6 Dale Mean Top 76-6 Dale Mean Top 76-6 Dale Mean Top 76-6 Dale Mean

c c b b b d c c c c
0 1.86c 2.53 2.19 15.3 12.5 13.9 1.578 2.140 1.859 0.129 0.124 0.127d
56 2.65b 2.84b 2.75b 16.3a 13.3a 14.8a 2.246c 2.682b 2.464b 0.138b 0.142bc 0.140c
112 2.91a 2.98ab 2.94a 16.6a 13.1ab 14.9a 2.468bc 2.795b 2.631b 0.141ab 0.147ab 0.144bc
168 3.06a 3.11a 3.08a 16.6a 13.5a 15.1a 2.590ab 3.225a 2.907a 0.141ab 0.161ab 0.151ab
224 3.04a 3.01ab 3.03a 16.6a 13.0ab 14.8a 2.739a 3.476a 3.107a 0.145a 0.167a 0.156a
Mean 2.71B 2.89A 16.3A 13.1B 2.324B 2.863A 0.139A 0.148A

ANOVA P-values
Cultivar 0.0340 0.0003 0.0026 0.0398
N < 0.0001 0.0042 < 0.0001 0.0032
Cultivar N < 0.0001 0.4278 0.2446 0.0351

Means in a column followed by the same lowercase letter and means in a row followed by the same uppercase letter are not signicantly dierent at P = 0.05.

concentration was 34.8%, 25.1%, and 13.7% lower in the 0N, 56N, and
112N treatments, respectively. The mean TChl content across cultivars
and years ranged between 19.0 and 34.8 g cm2 at 0N and 224N
treatments, respectively. Compared to the 224N treatment, across
cultivar and year mean TChl content was 45.4%, 31.9%, and 21.8%
lower at 0N, 56N, and 112N, respectively.

3.3. Canopy reectance and N sensitivity

Canopy reectance across all N treatments showed a similar pattern

Fig. 1. Inuence of nitrogen (N) treatments on the seasonal patterns of plant height (PH)
(a,b) and main stem node numbers (MSN) (c,d) of sweet sorghum cultivars Top 76-6 (a, b)
and Dale (c, d) in 2010. Data represent means SD (n = 4).
(0N) to 3.06 m (168N) in Top 76-6 and 2.53 m (0N) to 3.11 m (168N) in
Dale, and in the two high N treatments (164 and 224 kg ha1) PH of
both cultivars exceeded three meters (Fig. 1a, b and Table 1). Relative
to the 224N treatment, plants of Top 76-6 were 39 and 13% shorter in
0N and 56N treatments, respectively. For Dale, plants were 17 and 6%
shorter in 0N and 56N treatments, respectively. At the end of the
season, an average of 16 main stem nodes was observed for Top 76-6
and 13 for Dale (Table 1). Final node number was similar among the N-
fertilized treatments and approximately one node lower in the 0N
treatments, with bigger dierences observed earlier in the season
(Fig. 1 c,d). Compared to Top 76-6, Dale had a lower number of MSN
in all N treatments and greater PH at 0N and 56N. Rates of stem
elongation (SER) and node addition (NAR) increased with N treatment
in both cultivars (Table 1). The SER was greater in Dale versus Top 76-
6, and the NAR increased with N in both cultivars but was more
responsive to N treatment in Dale than in Top 76-6.
3.2. Eect of N treatments on leaf N concentration and TChl content
Averaged across cultivars and DAP, leaf N concentration and TChl
content increased with N treatment in a given year (Table 2). The mean
leaf N concentration was signicantly greater (13%) in 2009 compared
to 2010. However, the mean TChl content did not dier signicantly
between the years. The mean leaf N concentration across cultivars and
years ranged between 14.8 and 22.7 g kg1 for the 0N and 224N
treatments, respectively. Compared to the 224N treatment, the leaf N
of minimum reectance in the visible portion (400750 nm) of the
measured electromagnetic spectrum (wavelength) resulting from the
absorption by the leaf pigments (Fig. 2a). Based on the percentage
sensitivity, the eect of N treatment was most distinguishable in the
visible portions of the spectrum, especially those in green (centered
near 595 nm) and red (centered near 701 nm) regions (Fig. 2b, c).
These spectral regions also exhibited the greatest coecient of deter-
mination (r2) for the linear relationship of reectance with the leaf N
concentration or TChl content in both cultivars (Fig. 2d, e). The
averaged reectance at 595 and 701 nm wavebands for 0N compared
to 224N was 89 and 50% greater in Top 76-6 and Dale, respectively.
The canopy reectance at 595 and 701 nm wavebands also increased
430% in other treatments (56N, 112N, and 168N), particularly in Top
76-6 relative to the 224N treatment (Fig. 2b).
3.4. Model development
Since the greatest sensitivity of the canopy reectance to N supply
was found in the spectral domains centered on 595 and 701 nm, the
single-waveband spectral models from these two wavebands were
developed for each cultivar in both years. The r2 of the linear regression
analysis of the reectance at single wavebands (595 and 701 nm) with
leaf N concentration ranged from 0.34 to 0.74 (Table 3) and with TChl
content from 0.19 to 0.50 (Table 4). To determine the simple-ratio
spectral models, the reectance values of the previously selected two
single-wavebands (595 and 701 nm) were used as the numerator and
the reectance at each of all other wavebands as the denominator
(Fig. 3). Three wavebands as the denominator for simple-ratio spectral
models with the best performance (in reference to r2, RMSE, and RE)
were identied for single-wavebands (numerator) R595 and R701 (Tables
34). These simple-ratio models diered between N concentration and
TChl content (Tables 3 and 4). The denominator wavebands in the
simple-ratio spectral models for N concentration were 518, 1240 and
1676 nm (Table 3) and for TChl content were 452 or 508, and 1348 and
1790 nm (Table 4).
The performance of each spectral model based on either single-
waveband or simple-ratio also diered between the cultivars and years.

50
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Table 2
Inuence of nitrogen (N) treatment on sweet sorghum leaf N concentration (Leaf N) and total chlorophyll (TChl) content in 2009 and 2010. Analysis of variance (ANOVA) results are
presented as P-values.

N treatment (Kg ha1) Leaf N (g kg1) TChl (g cm2)

2009 2010 Mean 2009 2010 Mean

c c c d
0 16.0c 13.6 14.8 20.5 17.6 19.0d
56 18.7bc 15.2c 17.0c 25.2b 22.2c 23.7c
112 20.8ab 18.5b 19.6b 25.6b 28.8b 27.2b
168 22.1ab 20.9a 21.5ab 31.6a 33.6a 32.6a
224 23.7a 21.7a 22.7a 35.5a 34.2a 34.8a
Mean 20.3A 18.0B 27.7A 27.3A

ANOVA, Eect P-values

Cultivar 0.1357 0.1176 0.1448 0.0189
DAP 0.0029 0.0033 0.0214 0.2326
N < 0.0001 < 0.0001 < 0.0001 < 0.0001
Cultivar DAP 0.8814 0.5415 0.0441 0.0172
DAP N 0.3807 0.5429 0.9927 0.1804
Cultivar N 0.9155 0.4889 0.4866 0.5673
Cultivar DAP N 0.4805 0.5796 0.9404 0.0014

Values are means of 16 measurements averaged across two sweet sorghum cultivars Top 76-6 and Dale and two samplings obtained at separate days after planting (DAP) in a given
year of study.

Means in a column followed by the same lowercase letter and means in a row followed by the same uppercase letter are not signicantly dierent at P = 0.05.

However, compared to the models developed from single-wavebands,
the simple-ratio models showed improved precision and accuracy as
measured by r2 and RE (Tables 3 and 4, Fig. 4). The best simple-ratio
spectral model exhibiting the highest r2 and the lowest RMSE and RE
was selected for leaf N concentration, R595/R1676 (r2 0.79, RE 10.5%,
Table 3) and TChl content, R595/R508 (r2 0.73, RE 15.1%, Table 4).
Examination of the pre-existing chlorophyll index (CHLindex) or ratio
(R1075/R735) revealed that they did not perform better than the best
simple-ratio models identied in the current study (Tables 3 and 4).
The relationships of the canopy spectral reectance with PH and
MSN also showed the greatest r2 in the visible spectrum near the blue
region and both centered at 440 nm (Fig. 5a). The reectance at 440 nm
was used as the numerator to develop simple-ratio spectral models for
PH and MSN across cultivars (Fig. 5bc). The wavebands associated
with the simple-ratio models diered between PH and MSN. The
simple-ratios R440/R525 and R440/R1525 showed the best predictive
capabilities (r2 0.49) for PH or MSN, respectively.
3.5. Validation of the single-waveband and simple-ratio models for leaf N
and TChl between years
The spectral models, as shown in Tables 3 and 4 for N concentration
and TChl content, respectively, developed from 2009 data sets were
validated using 2010 reectance data sets, and vice versa (Table 5).
Validation of a spectral model developed in one year for the respective
data set in the other year resulted in the same, r2 values as for the
model-development phase, but RMSE and RE values diered. For
instance, r2, RMSE and RE values for the reectance model R595 for
leaf N concentration of Top 76-6 in 2009 were 0.74, 2.29 and 11.7%,
respectively (Table 3). When this data set was validated using the R595
model from 2010, the r2, RMSE, and RE values were 0.74, 4.24 and
21.7, respectively (Table 5). The RE values resulting from model
validation were greater (Table 5) than those from the development
phase for a given data set (Tables 3, 4). However, since RE can be used
to compare the accuracy (lower is better) of dierent models, results
from the validation phase were used to select the best model based on
the RE (lowest) for leaf N concentration (R595/R1240) and TChl content
(R701/R1348) (Table 5, Fig. 6). Examination of the selected models
revealed that the model for leaf N concentration tended to under-
estimate higher values while the model for TChl content tended to
overestimate lower values (Fig. 6).
3.6. Evaluation of selected and validated models by cross-validation for
cultivars
The two best spectral models (the model with the highest r2 and
lowest RE values from the development phase and the model with the
lowest RE value from the validation phase) each for leaf N concentra-
tion and TChl content were further evaluated to determine their
accuracies by cross-validating between cultivars across years
(Table 6). For the prediction of leaf N concentration, the r2 and RE
values ranged from 0.42 to 0.63 and from 14.9 to 18.6%, respectively.
For the prediction of TChl content, the r2 and RE values ranged from
0.52 to 0.67 and from 16.9 to 20.1%, respectively. The cross-validation
results showed that the models selected based on the development
phase R595/R1676 and R595/R508 had the best accuracies (the highest r2
and the lowest RE) for the prediction of leaf N concentrations and TChl
contents (Table 6, Fig. 7). Thus, these simple-ratio models were chosen
as nal models for the development of general purpose models (GPMs)
for leaf N concentration and TChl content.
For the development of GPMs, all data were pooled (not averaged)
across years and cultivars and the simple-ratios R595/R1676 for leaf N
concentration and R595/R508 for TChl content were derived from the
reectance data. The resulting simple-ratio values were then regressed
with N concentration or TChl content to obtain the coecients for each
trait. These coecients were used to develop the universal equations
for the GPMs to estimate leaf N concentrations or TChl contents of
sweet sorghum cultivars across years (Fig. 8). The actual and predicted
leaf N concentrations and TChl contents from the GPMs closely aligned
with the 1:1 line (Fig. 8). The equation derived to estimate sweet
sorghum leaf N concentration was 66.63 R595/R1676 + 34.14 (r2
0.52; RE 16.8%) and the equation to estimate TChl content was
49.12 R595/R508 + 107.47 (R2 0.64; RE 17%).
4. Discussion
4.1. Eect of N deciency on plant growth and leaf N concentration and
TChl content
The observed decrease in plant height and leaf number under N
deciency was accompanied by lower tissue N concentration and TChl
content, and is likely a consequence of reduced carbon assimilation
(Singh et al., 2014; Zhao et al., 2005). While both node number and
internode length were inuenced by N treatment, the impact of low N
availability on the internode length was more pronounced than on node

51
 S.K. Singh et al.

harvests and years (a) or harvests and years (b-e).

Dale allowed this cultivar to reach maximum plant height approxi-

previously been reported and partly attributed to a strategy aimed at

concentration and total chlorophyll content. The sensitivity of reectance was calculated

number (Table 1, Fig. 1). Relatively greater growth rates observed in

reectance, (b, c) sensitivity of reectance to N status in reference to 224 kg N ha1, and

reducing the overall nutrient demand and to optimize plant growth

ments. The observed slower crop growth rates under N deciency have
mately 524 days earlier than Top 76-6, depending on the N treat-
224 kg N ha1) 100. Data are means of sweet sorghum cultivars (Top 76-6 and Dale),
Fig. 2. Inuence of nitrogen (N) treatments on canopy reectance: (a) mean spectral

as ((reectance of N treatment reectance of the 224 kg N ha1)/reectance of the

(d,e) coecient of determination (r2) for the relationship of reectance with the leaf N

52
Table 3
Model development results from linear regression analyses between leaf nitrogen (N) concentration and spectral models with the highest coecient of determination (r2) derived using a single-waveband, a simple ratio or a preexisting index/ratio.
Data from four replications of each of ve N treatments across two sampling dates for each cultivar in a given year were used (i.e. n = 40). The slope (b) and the intercept (c) are provided. A typical model can be constructed using the equation:
spectral model b + c, where spectral model represent the reectance at a single wavelength, reectance ratio between two wavelengths (simple ratio) or the index. CHLindex refers to the chlorophyll index. RMSE, root mean square error (g kg1);
RE, relative error (%).

Spectral model Top 76-6 Dale

2009 2010 2009 2010

2 2 2
b c r RMSE RE b c r RMSE RE b c r RMSE RE b c r2 RMSE RE

R595 251.23 31.33 0.74 2.29 11.7 145.81 25.27 0.37 3.46 19.5 293.93 34.56 0.52 3.67 17.6 155.23 25.87 0.34 3.16 17.0
R701 181.74 31.43 0.72 2.35 12.0 108.74 25.58 0.39 3.40 19.2 214.84 34.87 0.50 3.72 17.8 119.68 26.41 0.37 3.08 16.6
R595/R518 61.90 89.64 0.62 2.74 14.0 51.63 76.61 0.73 2.28 12.8 67.32 96.86 0.44 3.94 18.9 54.77 79.48 0.59 2.49 13.4
R595/R1240 112.65 33.73 0.75 2.24 11.5 89.42 28.82 0.56 2.89 16.3 130.00 36.91 0.60 3.34 16.0 82.18 28.18 0.50 2.76 14.9
R595/R1676 73.18 36.94 0.79 2.06 10.5 73.79 34.20 0.64 2.62 14.7 85.13 40.54 0.60 3.34 16.0 62.92 31.48 0.57 2.54 13.7
R701/R518 31.38 69.15 0.36 3.58 18.3 35.66 74.61 0.72 2.31 13.0 24.53 59.72 0.14 4.90 23.5 36.58 75.47 0.56 2.57 13.8
R701/R1240 80.43 33.67 0.72 2.35 12.1 66.60 29.26 0.59 2.79 15.7 95.11 37.29 0.58 3.41 16.4 62.23 28.72 0.53 2.66 14.4
R701/R1676 53.18 37.17 0.77 2.15 11.0 55.37 34.99 0.68 2.46 13.9 62.09 40.95 0.57 3.46 16.6 47.92 32.30 0.61 2.41 13.0
Pre-existing index/ratio
CHLIndex 9.96 1.67 0.67 2.58 13.2 7.62 1.06 0.53 2.98 16.8 11.06 2.77 0.54 3.59 17.2 6.86 3.02 0.47 2.81 15.2
R1075/R735 27.34 20.80 0.66 2.60 13.3 20.06 12.78 0.50 3.10 17.5 29.13 22.27 0.53 3.61 17.3 19.13 10.87 0.50 2.75 14.8
Field Crops Research 209 (2017) 4757
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Model development results from linear regression analyses between total chlorophyll content (TChl) and spectral models with the highest coecient of determination (r2) derived using a single-waveband, a simple ratio or a preexisting index/ratio.

23.6
24.1
15.5
21.9
21.0
16.9
21.0
19.9

19.2
18.5
RE
RMSE

7.00
7.14
4.58
6.48
6.23
4.99
6.21
5.90

5.67
5.48
0.23
0.19
0.67
0.34
0.39
0.61
0.39
0.45

0.49
0.52
r2

31.17
110.37

2.18
42.12
40.83

48.08
53.46
66.82
50.08
56.08
c

184.45
230.30

122.61
50.91

91.78
11.47
97.47
73.02

14.25
39.82
2010

Fig. 3. Results from linear regressions of leaf nitrogen concentration or total chlorophyll
21.1
21.5
19.1
17.0
16.1
20.4
16.3
15.3

16.4
17.2
RE

content of sweet sorghum cultivars Top 76-6 and Dale with the canopy reectance ratio of
(a-b) R701/R and (c-d) R595/R. Curves reveal the highest coecient of determination
(r2) used to identify the simple-ratios with the greatest r2.The symbol represent any
RMSE

5.95
6.05
5.36
4.81
4.54
5.73
4.59
4.30

4.62
4.84

reectance between 400 and 1800 nm. Data are means of harvests and years.

under limiting resources such as mineral nutrients in the soil (Lenka

0.44
0.42
0.54
0.63
0.67
0.48
0.66
0.71

0.66
0.63
r2

and Lal, 2012; Singh et al., 2015). Leaf N concentrations and TChl
contents observed for 168N and 224N treatments were comparable to
10.93
41.25

those reported for eld-grown sorghum in other studies (Foster et al.,

115.58
47.57
46.42

55.75
61.35
76.04
57.67
63.82

2016; Serro et al., 2012). Plant leaves respond to increasing soil N

c

supply by accumulating leaf N, chlorophyll pigments, and photosyn-

thetic enzymes to enhance carbon assimilation and plant growth (Osaki
299.69
394.79

174.29
112.52

133.06
53.28

13.69

86.22

et al., 1993; Zhao et al., 2005). The PH, MSN, plant growth rates, and
18.19
46.74
2009
Dale

tissue N concentration did not dier signicantly between 168 and

b

224 kg ha1 N treatments across cultivars, indicating that sweet

sorghum achieved optimum growth at 168 kg ha1 N rate.
23.0
23.3
15.1
20.3
20.4
15.6
19.4
19.4

19.0
19.2
RE

4.2. Canopy reectance sensitivity to N availability: relationship with leaf

RMSE

5.70
5.80
3.75
5.03
5.07
3.86
4.83
4.82

4.73
4.78

N, TChl, PH, and MSN for model development

0.38
0.36
0.73
0.52
0.51
0.71
0.55
0.56

0.57
0.56

Canopy reectance measurements conducted in this study revealed

r2

a pattern typical for green vegetation (Fig. 2a). The evaluation of the
reectance spectra based on both percent sensitivity and r2 (Fig. 2b-e)
29.35
4.13

resulted in the identication of the most sensitive wavebands near the

37.35
36.85
95.83
45.83
52.45
63.21
47.03
54.42

edges of the green (595 nm) and red (701 nm) regions. The observed
c

strong sensitivity of canopy reectance near 595 nm and 701 nm

170.37
228.22

130.95

wavebands of the visible spectrum for leaf N concentration and TChl

43.04

96.46
11.15
99.11
73.93

13.42
35.90

content is attributed to the low in vivo light absorption feature of

2010

chlorophyll molecules near these wavebands (Carter and Spiering,

b

2002; Chappelle et al., 1992). Plant height and MSN also showed a
18.8
19.0
15.8
16.5
16.3
16.0
16.2
15.8

15.7
15.3

high correlation with spectral reectance in the visible region of the

RE

spectrum across cultivars.

Simple-ratio models introduce an integrative response from two
RMSE

5.03
5.08
4.24
4.42
4.38
4.29
4.35
4.23

4.22
4.11

wavebands and often improve the precision and accuracy (RMSE or RE)
of the prediction (Chappelle et al., 1992; Singh et al., 2013; Zhao et al.,
2005). For a given best single-waveband (595 nm and 701 nm), one of
0.50
0.49
0.64
0.61
0.62
0.63
0.62
0.64

0.64
0.66
r2

the three wavebands identied as the denominator of simple-ratio

models for leaf N concentration and TChl content was consistently from
37.83
111.67

the visible region (e.g. R452, R508 and R518) while the other two
6.62
42.55
42.11

49.30
52.44
69.58
49.91
53.69
See Table 3 for additional information.

wavebands were selected from the NIR region (e.g. R1240, R1348,
c

R1676, and R1790). Wavebands in the NIR region have been shown to
respond to the changes in leaf structural properties and leaf layers
240.96
326.32

138.66

102.09
Top 76-6

51.53

84.23
12.20

63.35

throughout the depth of canopy (Gardner et al., 1985). Therefore, the

15.68
43.76
2009

Pre-existing index/ratio

wavebands in the simple-ratio models that integrate visible and NIR

b

regions represent two prominent spectral features, optical properties of

Spectral model

chlorophyll and leaf structure, respectively. The simple-ratio models

developed for PH mainly constituted wavebands from the visible
R595/R1348
R595/R1790

R701/R1348
R701/R1790

R1075/R735
R595/R508

R701/R452

CHLIndex

regions. However, the simple-ratio for MSN integrated wavebands from

Table 4

R701
R595

both visible and NIR regions of the spectrum.

The negative coecient (slope) of identied single-waveband and

53
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Fig. 4. Example relationships between sweet sorghum (cultivar Top 76-6) leaf nitrogen (N) concentration or total chlorophyll (TChl) content with canopy reectance for a given single-
waveband or simple-ratio for years 2009 (lled symbol, solid line) and 2010 (unlled symbol, dashed line). Data are pooled over two sampling dates each year (n = 40). Coecients of
determination (r2) of linear regression analyses are shown. The RE represents the percent (%) relative error, and R represents the canopy reectance at the given wavelength.

simple-ratio models in this study indicate a decrease in canopy Filella, 1998).

reectance as leaf N concentration or TChl content increased. Among
the identied models, four out of six simple-ratios, R595/R1240, R595/
R1676, R701/R518, and R701/R1676 consistently showed excellent predic-
tions (r2 0.50) of leaf N concentration with the highest accuracy (r2
0.79, RMSE 2.06, RE 10.5%) observed for the R595/R1676 ratio. For the
prediction of TChl content, the simple-ratio model R595/R508 was the
only model that consistently had an r2 0.50 across years and cultivars
(r2 0.540.73, RE 15.119.1%). Overall, these simple-ratio models for
leaf N concentration (R595/R1676) and for TChl content (R595/R508)
were the most robust to predict these traits across cultivars and
environments. A good performance of simple-ratio models to predict
N or pigment concentrations also has been illustrated in other studies
(Chappelle et al., 1992; Kanemasu, 1974; Zhao et al., 2005). Models
with fewer wavebands such as in a simple-ratio usually have an
advantage of being more straightforward and rapid compared to the
models with many spectral features (Singh et al., 2013). Once a model
is developed and sensitive wavebands are identied, more simple
sensors can be used to measure these specic wavebands and calculate
the desired parameter by using appropriate coecients (Peuelas and
Contactless, non-imaging spectroradiometer (e.g. ASD FieldSpec)
and camera-based imaging instruments are often used to identify and
develop specic spectral models based on simple-ratio or indices to
investigate crop characteristics at the leaf or canopy level. Spectral
models integrating one or more spectral features are highly dependent
upon their relationships with the plant physio-biochemical status and
may be useful for decision-making (Gardner et al., 1985; Jr Perry and
Lautenschlager, 1984). The most sensitive spectral features that relate
to foliar biochemistry (e.g. N or chlorophyll) can vary signicantly
among crops species, such that a particular index developed for one
crop may not be suitable for another crop (Gausman et al., 1984; Sims
and Gamon, 2002; Singh et al., 2013; Yu et al., 2014). Not surprisingly,
Huang et al. (2015) found that predictive relationships between
spectral reectance and pigment content at the canopy or landscape
(mixed species) level generally were not as good as at the leaf level.
Even at the leaf level, equipment commonly used to estimate chlor-
ophyll concentration, such as the SPAD-502 Plus meter (Spectrum
Technologies, Inc., IL USA), does not always perform well (Fritschi and
Ray, 2007; Richardson et al., 2002; Singh et al., 2013). This underscores

54
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Fig. 6. Actual versus predicted leaf nitrogen (N) concentration (a) and total chlorophyll
(TChl) content (b) during the validation phase of sweet sorghum cultivars. The
validations of 2010 model on 2009 reectance data are shown. The RE represents the
percent (%) relative error. Dotted lines represent the 1:1 lines.

the importance of developing a crop specic spectral model comprised

Fig. 5. Coecients of determination (r2) for the regression of sweet sorghum canopy
reectance with (a) plant height (PH) and main stem nodes number (MSN). The
relationship of the simple reectance ratio with PH (b) or MSN (c) for 2010. Data were
pooled across both cultivars and represent the mean of four replications measured ten
times between 40 and 120 days after planting. The equation for linear regression is given.
n = 100. R represents the reectance at a given wavelength. RMSE, root means squared
error; RE, relative error.
of the most sensitive wavebands in response to a given factor (e.g. N
limitations). Only very few studies have evaluated spectral properties of
sorghum (Foster et al., 2016; Mani et al., 1991; Zhao et al., 2005), and
none have been conducted for sweet sorghum. As part of the present
study, the most sensitive wavebands for the detection of tissue N
concentration and chlorophyll content in sweet sorghum were revealed
and used to develop the rst spectral model to estimate these traits in
sweet sorghum.

Table 5
Results of model validation showing the coecient of determination (r2), root means squared error (RMSE, g kg1 or g cm2) and relative error (RE, %). The spectral models, as shown
in Tables 1 and 2 for nitrogen concentration (N) and total chlorophyll content (TChl), respectively, developed in 2009 were validated using 2010 reectance data, and vice versa.

Model (year) Data (year) Model (N) N (g kg1) Model (TChl) TChl (g cm2)

Top 76-6 Dale Top 76-6 Dale

r2 RMSE RE r2 RMSE RE r2 RMSE RE r2 RMSE RE

2009 2010 R595 0.37 6.30 35.5 0.34 4.54 24.5 R595 0.36 7.89 31.8 0.19 9.74 32.9
2010 2009 0.74 4.24 21.7 0.52 5.66 27.1 0.49 5.33 19.9 0.42 6.72 23.9
2009 2010 R701 0.39 6.20 34.9 0.37 4.48 24.2 R701 0.38 7.81 31.4 0.23 9.61 32.5
2010 2009 0.72 4.24 21.7 0.50 5.65 27.1 0.50 5.27 19.7 0.44 6.60 23.4
2009 2010 R595/R518 0.73 5.57 31.4 0.59 4.96 26.8 R595/R508 0.73 6.77 27.2 0.67 7.03 23.7
2010 2009 0.62 4.49 23.0 0.44 6.01 28.8 0.64 4.73 17.6 0.54 5.53 19.6
2009 2010 R595/R1240 0.56 6.39 36.0 0.50 4.91 26.5 R595/R1348 0.52 7.79 31.4 0.34 8.96 30.3
2010 2009 0.75 4.18 21.4 0.60 5.70 27.3 0.61 4.95 18.5 0.63 5.18 18.4
2009 2010 R595/R1676 0.64 6.68 37.6 0.57 5.82 31.4 R595/R1790 0.51 8.28 33.3 0.39 9.06 30.6
2010 2009 0.79 4.80 24.6 0.60 6.18 29.6 0.62 5.80 21.6 0.67 5.02 17.8
2009 2010 R701/R518 0.72 5.43 30.6 0.56 5.08 27.4 R701/R452 0.71 6.96 28.0 0.61 7.36 24.9
2010 2009 0.36 5.01 25.7 0.14 6.57 31.5 0.63 5.09 19.0 0.48 5.98 21.2
2009 2010 R701/R1240 0.59 6.28 35.4 0.53 4.90 26.4 R701/R1348 0.55 7.63 30.7 0.39 8.77 29.6
2010 2009 0.72 4.41 22.6 0.58 5.73 27.5 0.62 4.87 18.2 0.66 4.91 17.5
2009 2010 R701/R1676 0.68 6.56 37.0 0.61 5.86 31.6 R701/R1790 0.56 8.14 32.8 0.45 8.97 30.3
2010 2009 0.77 4.88 25.0 0.57 6.27 30.1 0.64 5.74 21.4 0.71 4.97 17.7

55
S.K. Singh et al. Field Crops Research 209 (2017) 4757

Table 6
Evaluation of models identied in the development and validation phases by cross-
validation between cultivars for leaf nitrogen (N) concentration and total chlorophyll
(TChl) content.

Model Data Model Selection phase r2 RMSE RE (%)

Cultivar Cultivar

Leaf N concentration
Dale Top 76-6 R595/R1676 Development 0.63 2.77 14.9
Top 76-6 Dale 0.42 3.66 18.6
Dale Top 76-6 R595/R1240 Validation 0.61 2.85 15.3
Top 76-6 Dale 0.47 3.48 17.7
TChl content
Dale Top 76-6 R595/R508 Development 0.67 4.38 16.9
Top 76-6 Dale 0.60 5.27 18.2
Dale Top 76-6 R701/R1348 Validation 0.57 5.18 20.1
Top 76-6 Dale 0.52 5.52 19.1

Fig. 8. Actual versus predicted sweet sorghum leaf nitrogen (N) concentration (a) and
total chlorophyll (TChl) content (b) pooled across cultivars (Top 76-6 and Dale) and years
(2009 and 2010) of study (n = 160). Predicted values of leaf N concentration and TChl
content were determined using the general-purpose simple-ratio models R595/R1676 and
R595/R508, respectively. The letters R and RE refer to the canopy reectance at a given
wavelength and the percent relative error, respectively. Dotted lines represent the 1:1
lines.

predictions was not surprising, particularly given the variability in the

Fig. 7. Actual versus predicted leaf nitrogen (N) concentration (a) and total chlorophyll
(TChl) content (b) during the validation phase. The validation of models selected based
on cultivar Dale models using Top 76-6 reectance data are shown for data pooled across
2009 and 2010. RE represents the percent (%) relative error. Dotted lines represent the
1:1 lines.
4.3. Validation of single-waveband and simple-ratio spectral reectance
models for leaf N concentration and TChl content
Based on lower RE values, the best validated models to predict leaf
N concentration and TChl content were R595/R1240 (RE 21.4%) and
R701/R1348 (RE 17.5%), respectively. The RE value obtained during the
validation phase also indicated that models with the highest r2 were not
the same as those with the lowest RE. Since the reectance models were
developed and validated on dierent data sets, variation in the
accuracy (RMSE or RE) of the leaf N concentration or TChl content
data between years (2009 and 2010) (Gardner et al., 1985; Singh et al.,
2013). When tested on dierent data sets, the relatively lower accuracy
of reectance models have been reported previously (Ryu et al., 2011;
Singh et al., 2013). In general, under eld conditions, the spectral
reectance acquired using the ASD FieldSpec, averages the reectance
from canopy consisting of a mixture of leaves, stems, and soil (if the
canopy was not closed) in one spectrum. Although data analysis
methods were used to minimize confounding eects of factors other
than tissue N or chlorophyll in the reectance data, such limitations
cannot be overlooked completely. Nonetheless, as opposed to other
models (Blackburn, 2007; Yu et al., 2014), the models developed in this
study were validated using an entirely dierent dataset than the one
used to develop the model. As such, the models are expected to be more
robust and suitable for further evaluation under a broad range of real-
world conditions.
4.4. Further evaluation of selected and validated models by cross-validation
for cultivars
Four models, two from the development phase for leaf N concentra-
tion (R595/R1676) and TChl content (R595/R508) and two from the
validation phase for leaf N concentration (R595/R1240) and TChl content
(R701/R1348) were further evaluated by cross-validating between sweet
sorghum cultivars across years to identify the single best model with the

56
S.K. Singh et al.
greatest accuracy (the highest r2 and lowest RE). The analysis revealed
that models R595/R1676 and R595/R508 were the best for the prediction
of leaf N concentration and TChl content (Table 6). However, the
respective models showed the tendency of slightly overestimating leaf
N concentration at the lower end and TChl content at the upper end
(Fig. 7). When developed and tested using distinct data sets, such biases
in the spectral models are not surprising particularly during the
validation phase for the prediction of leaf N concentration or TChl
content as also reported previously (Ryu et al., 2011; Singh et al.,
2013).
General purpose models (GPM) which integrate the variability
across cultivars and environments (e.g. years of study) have been found
to be useful for the prediction of leaf N and photosynthetic pigments
(Ryu et al., 2011; Singh et al., 2013). Thus, the identied best simple-
ratio model in the cross-validation process for leaf N concentrations
(R595/R1676) and TChl content (R595/R508) was used to develop a GPM
for each of the two traits by pooling the data across cultivars and years.
The GPM provided the universal coecients that might be used to
estimate leaf N concentration or TChl content in sweet sorghum
cultivars across years (Fig. 8). The regression line between actual and
estimated leaf N concentration or TChl content closely aligned with the
1:1 line, indicating good agreement between measured and estimated
values from the general-purpose simple-ratio spectral models.
5. Conclusion
In general, up to 168 kg ha1 N fertilizer, increasing the availability
of N enhanced stem elongation rate, node addition, and plant height,
and increased leaf N concentration and TChl content. Sweet sorghum
canopy reectance increased in response to reduced N availability and
was most sensitive to N status in the visible region, specically near
green (595 nm) and red (701 nm) wavebands. Results revealed that to
estimate sweet sorghum growth, leaf N concentration, and TChl
content, simple-ratio models exhibit better accuracy than the models
developed using only the most sensitive single-waveband. The devel-
opment and cross-validation of the models between years of study and
cultivars revealed the simple-ratio models R595/R1676 and R595/R508 as
having the best accuracy for predicting leaf N concentration and TChl
content, respectively. Hence, these simple ratios were used to develop
general-purpose models and derive the coecients to estimate sweet
sorghum leaf N concentration (-66.63 R595/R1676 + 34.14; r2 0.52;
RE 16.8%) and TChl content (-49.12 R595/R508 + 107.47; R2 0.64;
RE 17%) across years and cultivars. The identied spectral models can
be used to assess growth, diagnose sweet sorghum N status and may be
useful to make N management decisions for site-specic fertilizer
applications.
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