Tree Physiology 23, 1077–1079
© 2003 Heron Publishing—Victoria, Canada
Chlorophyll content monitoring in sugar maple (Acer saccharum)†
THOMAS M. CATE1,2 and T. D. PERKINS1
1
Proctor Maple Research Center, The University of Vermont, P.O. Box 233, Underhill Center, VT 05490, USA
2
Author to whom correspondence should be addressed ([email protected])
Received October 17, 2002; accepted January 17, 2003; published online September 15, 2003
Summary We conducted two experiments to determine the
usefulness of a chlorophyll content meter (CCM) for the measurement of foliar chlorophyll concentration in sugar maple
(Acer saccharum Marsh.) in the fall color period. In Experiment 1, four sugar maple trees were visually assigned to each of
four fall foliage color categories in October 1998. On four
dates in the fall of 1999, leaves were taken from the trees and
analyzed for chlorophyll concentration by absorbance of pigment extracts and by determination of the chlorophyll content
index (CCI) with a CCM. The two measures of chlorophyll
concentration were strongly correlated (P < 0.001, r 2 = 0.72).
In Experiment 2, the CCI of leaves from sugar maple trees
subjected to one of four fertilization treatments (lime, lime +
manure, lime + 10:10:10 N,P,K fertilizer and an untreated control) were determined with a CCM. Treatment effects were
distinguishable between all pairwise comparisons (P < 0.001),
except for the lime versus lime + NPK fertilizer treatments.
Keywords: CCI, CCM, nondestructive testing, spectrophotometry.
Introduction
Chlorophyll content meters (CCMs) have been used for several years by agronomists studying maize (Tenga et al. 1989,
Dwyer et al. 1994, Chapman and Barretto 1997). They have
the advantages of being pocket-portable, inexpensive, nondestructive to leaf tissue, rapid and easy to use, and do not require
hazardous compounds or specially trained personnel. Data acquired can be rapidly downloaded for computer-based analysis.
Typical CCMs operate by differential absorption of light at
two wavelengths, one in the near infrared that passes through
leaf pigments relatively unimpeded and serves as a reference
beam, and one tuned to the peak absorbance of chlorophyll.
The transmission of beam energy, expressed as the ratio of
absorbance beam to reference beam, yields a unitless value
called the chlorophyll content index (CCI), usually in the
range of 1.0 (no pigment) to about 70.0 (very high pigment
content), depending on instrument make and model.
Values of CCI are strongly correlated to extractable chloro-
phyll in annual crops like cotton, wheat fescue, rice, soy and
maize (Marquand and Tipton 1987, Dwyer et al. 1994, Chapman and Barretto 1997). But there are few reports on the use of
CCMs with broad-leaved trees (Campbell et al. 1990, Sibley et
al. 1996). Our objective was to determine the utility of a CCM
for the nondestructive determination of chlorophyll concentration in sugar maple (Acer saccharum Marsh.) leaves expressing a range of colors, and for the detection of changes in
chlorophyll concentration in trees subjected to different soil
amendments.
Materials and methods
Chlorophyll content meter
Leaf greenness was determined with a prototype of the OptiSciences CCM-200 chlorophyll content meter (Opti-Sciences,
Tyngsboro, MA). This instrument uses differential transmission at two wavelengths, 940 and 665 nm, to determine the
absorbance of chlorophyll pigments (whereas the current production model uses a 655 nm absorbance beam). Beam energy
is sampled pre- and post-transmission, and the 940 nm reference beam is standardized according to the manufacturer’s
instructions. The ratio of the red absorbance beam to the standardized infrared reference beam gives the chlorophyll
content index (CCI).
Beams are generated by LEDs, and the leaf tissues and pigments are unharmed by sampling; the region measured by the
sensing head is a circle 0.71 cm 2 in area (9.5 mm diameter).
Experiment 1
In Experiment 1, sugar maple trees growing at the USDA
Forest Service Northeastern Research Station in South Burlington, VT, USA, were assigned to one of four fall foliage
color categories based on their color in October 1998 (Schaberg et al. 2003). The categories were established on the basis
of a two-person visual assessment of whole-tree color character and number of leaves remaining.
Whole, lower-canopy branch segments were taken from
four trees in each category weekly from September 5 to October 13, 1999. Branch segments were transported to the labora-
† This paper was among those presented at the 17th North American Forest Biology Workshop “Rocky Mountain ecosystems: Diversity, complexity and interactions,” sponsored by the Tree Physiology and Forest Genetics working groups of the Society of American Foresters and held
at Washington State University, Pullman, WA.
1078
CATE AND PERKINS
tory in a cooler in sealed plastic bags.
The CCI was sampled on five leaves from each branch segment with the CCM sensing head held as close as possible to
the junction of the central vein and the next adjacent major
vein without including major vein tissue under the device. Five
non-overlapping measurements were taken on each leaf over
homogeneous, healthy leaf tissue, and a mean value calculated
from the measurements for each tree. In the course of the experiment, 16 trees were tested on four dates.
After the CCI had been sampled, five 6.4-mm diameter
disks were punched from each leaf in the approximate locations of the CCI measurements. The disks were extracted in
80% (v/v) acetone at 4 °C in the dark. Transmittance of the
extract was measured with a Spectronic-Unicam Genesis/8
spectrophotometer. Total chlorophyll was calculated according to Lichtenthaler and Wellburn (1983).
Experiment 2
In Experiment 2, we tested the ability of the CCM-200 to detect the effects of four fertilizer treatments applied to sugar
maple trees the previous summer. The trees were located in a
progeny plantation at the Proctor Maple Research Center
(PMRC) in Underhill Center, VT, USA. The plantation was established in 1960 from seedlings of several known mother
trees chosen for their high sugar content. The fertilizer treatments were dolomitic lime (3360 kg ha –1), dolomitic lime and
10:10:10 N,P,K fertilizer (3360 kg ha –1 and 280 kg ha –1,
respectively), and lime and manure (3360 kg ha –1 and 51 m 3
ha –1, respectively). A fourth group of trees that received no
amendment served as a control.
In September 1999, 10 dominant trees were randomly selected from each of the treatment and control plots. Branches
less than 2 cm in diameter were taken from the top third of the
south-facing crown with a pole pruner. Five visually healthy,
green leaves were randomly chosen from the outermost leaves
on each branch. Leaves from trees in different treatments were
visually indistinguishable. The CCI was measured as de-
scribed for Experiment 1.
Statistical analysis
Data were subjected to regression analysis and ANOVA using
the JMP-IN and StatView software applications (SAS Institute, Cary, NC). For Experiment 2, pairwise comparisons were
made by Bonferroni compensated tests.
Results and discussion
In Experiment 1, extractable chlorophyll was strongly correlated with CCI (Figure 1, P < 0.001, r 2 = 0.72, n = 64) across
all tree categories and sampling dates. The same level of significance and correlation was found in previous studies using
CCMs with annual crops (Campbell et al. 1990, Reeves et al.
1993, Dwyer et al. 1994, Ma and Dwyer 1997). Mean leaf
chlorophyll concentration was 0.258 µg mm –2 for all fall color
categories, consistent with the value found by Liu et al. (1997).
In Experiment 2, all treatments yielded higher CCI values
than the control. Significant differences in CCI were found for
all pairwise comparisons (Figure 2, P < 0.0001, n = 40), except
for the lime versus lime + NPK fertilizer treatment (P < 0.48).
Several studies have shown that the CCI exhibits a nonlinear
plateau response at the upper extreme of the measurement
range (Marquand and Tipton 1987, Dwyer et al. 1994, Chapman and Barretto 1997). However the CCI values measured in
the present study did not reach this plateau level.
Angle of incidence and PAR irradiance have been shown to
affect chloroplast distribution and angle (Haupt 1982), and
CCI values are significantly affected by the incident irradiance, typically giving lower values at higher irradiances (Hoel
and Solhaug 1998). For these reasons, CCM devices provide
the most reliable and reproducible measurements when actinic
PAR is low. Differences in leaf optical density, leaf anatomy
and other factors necessitate species-specific regression models for the CCI–chlorophyll content relationship (Schaper and
Chacko 1991).
Figure 1. Relationship between chlorophyll content index (CCI) as measured
by a hand-held chlorophyll content meter and chlorophyll a + b concentration
as determined by absorbance of extracted pigments (P < 0.001, r 2 = 0.72,
n = 64). Mean chlorophyll a + b concentration for all trees was 0.258 µg
mm–2.
TREE PHYSIOLOGY VOLUME 23, 2003
CHLOROPHYLL CONTENT MONITORING IN SUGAR MAPLE
1079
References
Figure 2. Effects of soil treatments on the chlorophyll content index
(CCI) of sugar maple foliage. Different letters indicate significant
differences at a = 0.05 as determined by Bonferroni-compensated
ANOVA (n = 40). Abbreviations: C = control, L = dolomitic lime,
LF = dolomitic lime + 10:10:10 N,P,K fertilizer, LM = dolomitic lime
+ manure. Each bar represents the mean CCI of 10 trees; error bars are
± 1 SE.
In conclusion, CCI values were strongly correlated with
chlorophyll concentration as determined by absorbance of extracted pigments, indicating that the instrument can be used in
the field to monitor leaf chlorophyll concentration in sugar
maple.
Acknowledgments
This work was partly supported by a grant from the U.S. Environmental Protection Agency. We thank the North American Maple Syrup
Council for assistance in purchasing the spectrophotometer used in
this study. Additional thanks to Dan Harkins of Opti-Sciences for the
loan of the CCM-200 prototype and to Dr. William Currier and Tim
Wilmot of the University of Vermont for their helpful comments and
access to unpublished work. Special thanks to Dr. Paul Schaberg and
Paula Murakami of the USDA Forest Service Northeastern Research
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