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Field Crops Research 209 (2017) 4757

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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


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
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).

The hyperspectral signature from green leaves carries a vast amount
Sweet sorghum (Sorghum bicolor [L.] Moench) has garnered much of information that has been related to various plant characteristics,
attention as a source for biofuel due to its high stem sugar content including N status, thereby oering a non-destructive and quick
(Putnam et al., 1991; Rooney et al., 2007). Sweet sorghum is an annual assessment under eld conditions (Peuelas and Filella, 1998;
C4 crop with high photosynthetic capacity, uses water and nutrients Richardson et al., 2002; Sims and Gamon, 2002). Although selected
more eciently than C3 crops, and can be grown on marginal lands spectral features and their sensitivity to specic stresses can signi-
(Anten et al., 1995; Young and Long, 2000). Although sorghum is cantly dier between crops species (Gausman et al., 1984; Sims and
considered a soil nutrient scavenger and has shown excellent nitrogen Gamon, 2002), assessments of the relationships between canopy
(N) use eciency, application of N fertilizer is critical for optimum spectral reectance and sweet sorghum characteristics have been
production (Maw et al., 2016; Serro et al., 2012). However, excess N limited. Selection of the most sensitive wavebands to construct the
application not only increases production costs but also leads to reectance model is a crucial step towards developing predictive
environmental concerns (Snyder et al., 2009). Ecient monitoring of models for crop and leaf characteristics (Carter and Spiering, 2002;
plant N status oers an opportunity for appropriate timing of N Gausman, 1974; Singh et al., 2013; Zhao et al., 2005). Spectral models
application and site-specic N management, which could reduce the comprised of simple waveband and multiple waveband indices derived
use of N fertilizers (Blackmer et al., 1994; Ma et al., 1996). Remote from visible and near-infrared (NIR) regions indicate that these models
estimation of leaf/canopy reectance has been widely used to non- can be used to estimate leaf N and pigment concentrations (Huang
destructively assess plant N status and health in agricultural and et al., 2015; Sims and Gamon, 2002; Singh et al., 2013; Yu et al., 2014).
forestry ecosystems (Gausman et al., 1984; Peuelas and Filella, 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: (S.K. Singh), (F.B. Fritschi).
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. Field Crops Research 209 (2017) 4757

growth and yield (Gray et al., 2010; Ma et al., 2001; Peuelas and numbers, and (c) to determine eects of N availability on sweet
Filella, 1998; Ryu et al., 2011; Yi et al., 2007; Zhao et al., 2005; Zhao sorghum growth and development, leaf N concentration and total
et al., 2007). The N reectance index developed by Bausch and Duke chlorophyll (TChl) content.
(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 2. Materials and methods
subsequent study, Bausch and Diker (2001) used the N reectance
index to detect in-season corn N deciency and make N fertilizer 2.1. Crop management and N treatments
application decisions. Using this approach, they were able to reduce
fertilizer application by 39.2 kg N ha1 in a farmers eld without The experiment was conducted at the Bradford Research Center
reducing the corn grain yield. These results indicated that a canopy near Columbia, MO, USA (38 53N, 92 12 W) in 2009 and 2010 on a
reectance index derived from one experimental setting can be Mexico Silt Loam (ne, smectitic, mesic, Vertic Epiaqualf) soil. The eld
suciently robust to be applied successfully on a dierent site and was tilled with a disk to a depth of approximately 0.15 m before
year for N-management decisions, thus illustrating the potential for planting. On 7 June 2009 and 18 June 2010, sweet sorghum cultivars
broader applicability of indices developed on a single or small numbers Top 76-6 and Dale were sown 0.02 m deep, in rows 0.76 m apart to
of eld environments. achieve an approximate population of 208,000 plant ha1. The main
Spectral reectance models may be comprised of one or more plots consisted of N treatments of 0, 56, 112, 168, and 224 kg N ha1
wavebands/spectral features as single-waveband, simple-ratios or in- (hereafter referred to as 0N, 56N, 112N, 168N and 224N) and were
dices to determine plant N status or chlorophyll concentration arranged in a randomized complete block design with four replications,
(Chappelle et al., 1992; Gray et al., 2010; Zhao et al., 2007; Zhao and subplots consisted of sweet sorghum cultivars that were arranged as
et al., 2005). Often these spectral models are composed of distinct strips within each replication. Main plots were 12 rows wide and 15 m
wavebands (e.g. in the visible and/or NIR regions) of the electromag- long, and subplots were six rows wide and 15 m long. SuperU urea
netic spectrum. Daughtry et al. (2000) found that spectral indices that (Koch Agronomic Services, Wichita, KS) was broadcast as applications
combined reectance at visible and NIR regions of the spectrum are of 56 kg ha1 (56N) and 112 kg ha1 (112N, 168N, and 224N) at
sensitive to both leaf spectral characteristics and background reec- planting, and supplemented with 56 kg ha1 or 112 kg ha1 for 168N
tance. Thus, an integrated approach to develop hyperspectral reec- and 224N approximately two weeks after emergence. The same eld
tance models using features from two distinct wavebands oers better and plot design were used in both study years.
precision and accuracy for the prediction of plant growth, leaf N, or For each growing season, rainfall and temperature data from
pigment concentration. Due to the high degree of collinearity among planting to the end of measurements were obtained from a weather
waveband reectance from the plant canopy, use of fewer wavebands station located at the Bradford Research Center. Total in-season rainfall
to construct an empirical model is encouraged (Gardner et al., 1985). for 2009 and 2010 was 288 mm and 514 mm, respectively. The average
Models with fewer wavebands usually oer the advantage of being growing season temperature ( standard deviation) in 2009 was
straightforward and rapid compared to the more complex models with 28.8 3.5 C and in 2010 was 22.7 4.6 C. Long-term (between
many spectral features. Once the sensitive wavebands are identied, a 1971 and 2000) records of rainfall (395 mm) and temperature
relatively simple spectroradiometer that measures those particular (22.3 3.3 C) in this region for the same period were obtained from
wavebands can instantaneously calculate the desired plant trait by the High Plains Regional Climate Center (HPRCC) website (http://
using appropriate coecients derived from the reectance model; Columbia WSO AP, MO, USA, Station # 231791).
(Peuelas and Filella, 1998).
Sweet sorghum has gained much interest as a potential biofuel crop 2.2. Measurements
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 Plant samplings for determination of N concentration and chlor-
et al., 2012). Although, N fertilizer application and management is ophyll content and canopy reectance measurements were conducted
crucial in both crops, studies describing the impact of N deciency and twice each year. The rst measurements were conducted between 50
its relation with canopy reectance under eld condition in sweet and 55 days after planting (DAP) in both years. The second measure-
sorghum are scarce compared to maize (Blackmer et al., 1994; ments were conducted 70 DAP in 2009 and 78 DAP in 2010. In 2010,
Daughtry et al., 2000; Ma et al., 1996; Zhao et al., 2005). Zhao et al. plant height (PH) and main stem node numbers (MSN) for four
(2005) and Foster et al. (2016) found that plant tissue N or chlorophyll randomly selected plants, and canopy reectance were also measured
correlated with spectral reectance measured at leaf or canopy levels in ten times in each subplot between 40 and 120 DAP. The average
grain and high-biomass sorghum, respectively. Mani et al. (1991) number of leaves during the rst measurement was approx. 56 in 0N
reported that canopy reectance of grain sorghum was highly sensitive and 78 in the N-treated plants. At the second measurement, there were
to growth and nutrient application around 55 and 65 days after 89 leaves in the 0N and 1013 leaves in the N-treated plants. Plant
planting. Further, Serro et al. (2012) found that leaf N concentration height was measured from ground to the apex of the plant. The stem
was the best indicator of sugar content in sweet sorghum. Foster et al. elongation rate (SER) and node addition rate (NAR) were calculated as
(2016), reported that a normalized N index constituting reectance at maximum plant height or node number divided by the number of days
wavebands in the blue region of the spectrum (400 and 510 nm) was required to reach the maximum.
strongly correlated with the total N concentration. These studies
indicate the presence of empirical relationships between sweet sorghum 2.3. Determination of leaf N concentration
N status and canopy reectance properties that can be utilized to
identify sensitive wavebands to construct spectral models. In association with the canopy spectral reectance measurements,
A crops N fertilizer requirement is often determined based on the plants were harvested from one square meter area in each subplot, and
laboratory analyses of soil and/or plant N status, but sample collection leaves and stems were dried separately in a forced air-drier at 55 C
and analyses can be time-consuming and expensive. Therefore, an until weights stabilized. Dried leaves were ground using a Wiley Mill
empirical model developed using canopy spectral reectance would be (Thomas Scientic, NJ, USA) to pass a 2.0 mm screen, and subsequently
useful for a quick and non-destructive assessment of sweet sorghum N ground a second time with a UDY Cyclone Mill (UDY Corp., Fort
status. Thus, the objectives of this study were (a) to identify spectral Collins, CO) to pass a 1.0 mm screen. Ground materials were homo-
features most sensitive to N deciency, (b) to develop simple spectral genized, and total N concentration was determined by dry combustion
models to quantify crop N status, TChl content, plant height and node with a LECO TruSpec N Elemental Determinator (LECO Corp., MI, USA)

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

according to AOAC (2006). 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
2.4. Determination of leaf total chlorophyll content spectral reectance (independent variable) to N concentration or TChl
content (dependent variables). The reectance data from two sampling
Total chlorophyll (TChl) content was determined from ve 0.68 cm2 dates in each year were pooled to establish the relationships between
leaf disks excised from the uppermost, fully expanded leaf from ve reectance and N concentration or TChl content. To determine the
plants of each subplot. Leaf disks were placed into vials containing 5 mL simple ratios, the reectance values of the most sensitive wavebands
of ethanol (95%, v/v) and incubated at room temperature in the dark with the greatest r2 were used as the numerator and the reectance at
for 24 h. The absorbance of the supernatant was measured at 664 and each of all other wavebands throughout the whole spectrum as the
648 nm using a Genesys 10uv spectrophotometer (Thermo Electron denominator. The resulting ratio values of reectance were then
Corporation, Madison, WI). Total chlorophyll was estimated using the regressed with the N concentration or TChl content in a similar manner
equations of Lichtenthaler (1987): Total chlorophyll (g ml1) = as for the single-waveband to obtain the simple-ratio. Three simple
(5.24 A664) + (22.24 A648), where A664 and A648 are the absor- ratios with the greatest r2 were selected for N concentration as well as
bance values of the supernatant at 664 and 648 nm, respectively. The for TChl content. Similarly, a simple ratio with the highest r2 was also
values were expressed in g cm2 by using the equation: TChl content developed by linear regression of the reectance with plant height and
(g cm2) = (Total chlorophyll, g ml1 5)/3.40, where ve is the node number.
amount of ethanol (solvent, ml) and 3.40 is the leaf area (cm2) of the Additionally, a pre-existing chlorophyll index and a simple-ratio
ve leaf disks. model showing good predictive capabilities in previous studies were
selected for evaluation in the present study (Gray et al., 2010; Zhao
2.5. Acquisition of canopy spectral reectance et al., 2005). The total canopy chlorophyll content index (CHLindex)
calculated as the ratio of the area under the curve in the 840870 nm
Canopy spectral reectance was measured on sunny days between regions and 720730 nm regions [ R840-870/ R720-730] (Gitelson et al.,
1000 and 1400 h in each subplot using an ASD FieldSpec, FR spectro- 2005; Gray et al., 2010) was determined. Since Zhao et al. (2005) found
radiometer (Analytical Spectral Devices Inc., Boulder, CO, USA) with a a good correlation with N concentration or chlorophyll content in
25 eld of view. Three measurements were taken within the middle sorghum, the simple ratio of the reectance between 1075 nm and
rows near the center of each subplot and averaged. Reectance was 735 nm (R1075/R735) was also used.
measured at wavelengths ranging from 350 to 1800 nm with a 1-nm To determine models with consistent performance across year and
interval. However, the reectance between 1350 nm and 1450 nm was cultivar, the reectance models for N concentration and TChl content
excluded from the analysis due to the strong interference of water derived using single-waveband or simple ratios from the data set of
absorption bands in this region (Gausman et al., 1984), and the spectral 2009 were validated on the data set of 2010 and vice-versa. The r2, root
domains of 4001350 and 14511800 nm were used for analyses. To means squared error (RMSE) and the percent relative error (RE) were
measure the canopy reectance, the optical head was held approxi- determined and used as measures of model performance. The RMSE and
mately 1 m above the canopy at the nadir-viewing (nadir position) RE evaluate the accuracy (the relative closeness of the predictions to
geometry at each sampling interval. After optimization of the ASD the actual values) of the models and model validation as used in other
instrument, reectance measurement of the device was set to 100% by studies (Blackburn, 2007; Ryu et al., 2011; Yi et al., 2007) and
measuring the reectance of a Spectralon reference panel (white computed as:
reference panel 25.4 25.4 cm calibrated as 99% reective). The
white reference was measured approximately every 510 min to check i =1 (yi yi)2 100 RMSE
RMSE = RE = y
the instrument stability for 100% reectance. The white reference n

measurements were conducted by bringing the sensor close (approx.

where y and yi are the predicted and actual pigment concentrations,
0.4 m) to the Spectralon to ensure that only the reference panel
respectively; y is the average of the actual pigment concentration, and n
reectance was measured. The rst measurements of spectral reec-
is the number of observations. Although RMSE measures the closeness
tance (5055 DAP) were made from the ground as plant height did not
of the predicted to the actual value, comparisons of RMSE between
exceed the reach for proper measurement by a person standing between
models derived from dierent sets of data are not meaningful. How-
the rows. Due to the tall plants at the time of the second spectral
ever, RE can be compared between models; the model with the smaller
reectance measurements (7078 DAP), the spectroradiometer was
RE has predicted values that are closer to the actual values.
operated by an individual positioned on a stage attached to a tractor-
Analysis of variance (ANOVA) was performed using PROC MIXED
mounted forklift. Spectral readings were acquired after adjusting the
procedure of SAS (SAS Enterprise Guide, 4.2, SAS Institute Inc., NC,
height and location of the forklift. The canopy reectance was auto-
USA). Cultivar, DAP, and N treatments were considered as xed eects
matically adjusted for the white reference and processed instantly on a
and replication as a random eect. Since signicant Cultivar N
laptop computer by RS3 Spectral Acquisition Software (v. 5.6,
interactions were observed for PH and NAR, all growth parameters
Analytical Spectral Devices Inc., Boulder, CO, USA) attached to the
(PH, MSN, SER and NAR) were analyzed by cultivar. Cultivar N and
ASD instrument, and saved for future retrieval using ViewSpec Pro
DAP N interactions were not signicant for leaf N concentration and
software (v. 5.6.8, Analytical Spectral Devices Inc., Boulder, CO, USA).
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
2.6. Selection of sensitive wavebands and data analysis
means presented. Treatment comparisons were conducted by the least
square means (LSMEANS) procedure (at = 0.05) with the letter
The aim was to identify N- and chlorophyll-sensitive single-wave-
grouping obtained using pdmix800 macro (Saxton, 1998).
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- 3. Results
proaches: (a) the sensitivity was calculated as percentage change in
the reectance at each waveband for the 0N, 56N, 112N and 168N 3.1. Plant height and node development
against the 224N treatment (Zhao et al., 2005) and (b) based on the
single-wavebands with the highest coecient of determination (r2) The eects of cultivar and N treatment were signicant for PH,
from the linear regression of spectral reectance with N concentration MSN, SER and NAR (Table 1). The season-end PH ranged from 1.86 m

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

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

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

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

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.













Fig. 3. Results from linear regressions of leaf nitrogen concentration or total chlorophyll


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



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

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



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

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



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


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

thetic enzymes to enhance carbon assimilation and plant growth (Osaki





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

tissue N concentration did not dier signicantly between 168 and


224 kg ha1 N treatments across cultivars, indicating that sweet

sorghum achieved optimum growth at 168 kg ha1 N rate.


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




N, TChl, PH, and MSN for model development



Canopy reectance measurements conducted in this study revealed


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

resulted in the identication of the most sensitive wavebands near the


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

strong sensitivity of canopy reectance near 595 nm and 701 nm



wavebands of the visible spectrum for leaf N concentration and TChl




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


chlorophyll molecules near these wavebands (Carter and Spiering,


2002; Chappelle et al., 1992). Plant height and MSN also showed a


high correlation with spectral reectance in the visible region of the


spectrum across cultivars.

Simple-ratio models introduce an integrative response from two



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


the three wavebands identied as the denominator of simple-ratio

models for leaf N concentration and TChl content was consistently from

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

See Table 3 for additional information.

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

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


Top 76-6




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


Pre-existing index/ratio

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


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





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

Table 4


both visible and NIR regions of the spectrum.

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

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

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

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
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
none have been conducted for sweet sorghum. As part of the present
relationship of the simple reectance ratio with PH (b) or MSN (c) for 2010. Data were study, the most sensitive wavebands for the detection of tissue N
pooled across both cultivars and represent the mean of four replications measured ten concentration and chlorophyll content in sweet sorghum were revealed
times between 40 and 120 days after planting. The equation for linear regression is given. and used to develop the rst spectral model to estimate these traits in
n = 100. R represents the reectance at a given wavelength. RMSE, root means squared sweet sorghum.
error; RE, relative error.

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


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

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

predictions was not surprising, particularly given the variability in the

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
Fig. 7. Actual versus predicted leaf nitrogen (N) concentration (a) and total chlorophyll
than tissue N or chlorophyll in the reectance data, such limitations
(TChl) content (b) during the validation phase. The validation of models selected based cannot be overlooked completely. Nonetheless, as opposed to other
on cultivar Dale models using Top 76-6 reectance data are shown for data pooled across models (Blackburn, 2007; Yu et al., 2014), the models developed in this
2009 and 2010. RE represents the percent (%) relative error. Dotted lines represent the study were validated using an entirely dierent dataset than the one
1:1 lines. 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-
4.3. Validation of single-waveband and simple-ratio spectral reectance world conditions.
models for leaf N concentration and TChl content
4.4. Further evaluation of selected and validated models by cross-validation
Based on lower RE values, the best validated models to predict leaf
for cultivars
N concentration and TChl content were R595/R1240 (RE 21.4%) and
R701/R1348 (RE 17.5%), respectively. The RE value obtained during the
Four models, two from the development phase for leaf N concentra-
validation phase also indicated that models with the highest r2 were not
tion (R595/R1676) and TChl content (R595/R508) and two from the
the same as those with the lowest RE. Since the reectance models were
validation phase for leaf N concentration (R595/R1240) and TChl content
developed and validated on dierent data sets, variation in the
(R701/R1348) were further evaluated by cross-validating between sweet
accuracy (RMSE or RE) of the leaf N concentration or TChl content
sorghum cultivars across years to identify the single best model with the

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

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