8/7/2019 Foreco_200_p113

1/5

8/7/2019 Foreco_200_p113

2/5

(Campbell et al., 1990). Cate and Perkins (2003)

reported significant correlations between CCI and

total chlorophyll in sugar maple (Acer saccharum

Marsh.) leaves collected during the fall colorationperiod. Chang and Robison (2003) observed strong

relationships between CCI and foliar nitrogen in four

hardwood species. Neilsen et al. (1995) observed

significant correlations between CCI and foliar

nitrogen concentration in four apple cultivars early,

but not late, in the growing season. However, in a

study that examined 12 red maple cultivars as a

group, Sibley et al. (1996) found no significant

correlations between CCI and foliar nitrogen. These

latter two studies illustrate some limitations of chlor-

ophyll meters. The mathematical relationships

observed between CCI and total chlorophyll and/

or nitrogen content vary between species (Marquard

and Tipton, 1987; Schaper and Chacko, 1991; Shaa-

han et al., 1999; Yamamoto et al., 2002) and within

species during the growing season (Dwyer et al.,

1995; Bullock and Anderson, 1998), with growth

stage (Chapman and Barreto, 1997), with growing

condition (Campbell et al., 1990; Simorte et al.,

2001), and genotype (Peng et al., 1993). Thus,

relationships must be quantified for each species

of interest, and once determined, the relationship

cannot be generally applied even within thatspecies.

These limitations notwithstanding, hand-held chlor-

ophyll meters can still be effective tools for evaluating

forest tree species. After establishing a general corre-

lative relationship for a particular species, it is possible

to use a chlorophyll meter in applications for which

precise values are not necessary. For example, rapid

assessment of relative chlorophyll and/or nitrogen

content in sugar maple canopies would be of particular

utility for managers or researchers investigating sugar

maple decline, the symptoms of which include foliarnutrient deficiencies and reduced chlorophyll content

(Liu et al., 1997). The accuracy of a handheld chlor-

ophyll meter in predicting chlorophyll and nitrogen

content in growing-season sugar maple leaves has not

been previously reported. Thus, the objective of this

study was to establish the ability of a portable

chlorophyll meter to estimate total chlorophyll and

nitrogen content in large, heterogeneous samples of

sugar maple leaves collected during the growing

season.

2. Materials and methods

2.1. CCI versus nitrogen

Samples were collected on August 17, 2001, within

0.25 miles of the Proctor Maple Research Center

(PMRC) in Underhill Center, VT (elevation 400 m).

About 100 individual, visually-healthy sugar maple

leaves were taken randomly from both understory and

open-grown saplings and seedlings. In the case of

seedlings, two small leaves were used to comprise

one sample.

Following collection, samples were placed in plas-

tic bags and stored in a refrigerator until further

analyses could be completed. Five chlorophyll content

measurements were collected from each leaf with the

CCM-200 portable chlorophyll meter (Opti-Sciences,

Tyngsboro, MA), which calculates a unitless chloro-

phyll content index (CCI) value from the ratio of

optical absorbance at 655 nm to that at 940 nm. Major

veins and areas of obvious visual damage or disease

were avoided and all measurements were completed

within 2 h of collection. These data yielded an average

CCI for each leaf sample. Immediately following

collection of CCI values, leaf samples were dried in

an oven at 70 8C.

Nitrogen analysis of individual leaf samples wasconducted at the Agricultural and Environmental

Testing Laboratory of the University of Vermont

(Burlington, VT). Dried leaf samples were ground

in a mill to pass through a no. 20 sieve (840 mm).

One leaf sample contained insufficient mass for reli-

able nitrogen analysis, thereby reducing the sample

size of the study to 99. Nitrogen content, as a percen-

tage of dry weight (Ndw), was determined by combus-

tion analysis with a CHN elemental analyzer (CEC

Elemental Analyzer Model 440 with PC Compatible/

CEC-490 Interface Unit, Leeman Labs. Inc., Lowell,MA). Simple linear regression was used to determine

the relationship between Ndw and CCI.

2.2. CCI versus total chlorophyll

One-hundred healthy-appearing sugar maple leaves

were collected from individual small trees, saplings,

and first-year seedlings. Collections were made over

three dates in July 2001 from sites representing a wide

range of growing conditions, from dry to moist and

114 A.K. van den Berg, T.D. Perkins / Forest Ecology and Management 200 (2004) 113117

8/7/2019 Foreco_200_p113

3/5

shaded to open-grown from approximately 90400 m

in elevation in the Champlain Valley and Green

Mountains of Vermont. Leaf samples were placed

in plastic bags and transported to PMRC where theywere stored in a refrigerator until the time of CCI

measurement.

CCI was measured using procedures described in

the first portion of the study. Following chlorophyll

index measurements, five 6.45 mm-diameter discs

were collected from each leaf in the approximate

locations of CCI measurements. Chlorophyll was

extracted from the discs at 4 8C in the dark in a

solution of 80% acetone (v/v). Total chlorophyll

(chlorophyll a b) content on a leaf area basis was

estimated with a Spectronic Genesys 8 spectrophot-

ometer with a 10 mm light path using equations

derived by Lichtenthaler and Wellburn (1983).

Two obvious outliers resulting from likely transpo-

sition errors were removed from the analysis. Simple

linear regression was used to determine the relation-

ship between total extractable chlorophyll and CCI.

3. Results and discussion

3.1. CCI versus nitrogen

Nitrogen (Ndw) values ranged from 1.4 to 2.6%

(Fig. 1), which are within published ranges for sugar

maple leaf tissue from trees (Ellsworth and Liu, 1994)

and seedlings (Ellsworth and Reich, 1992). Chloro-

phyll content index (CCI) in the 99 samples ranged

from 6.2 to 25.4 with a mean of 13.9 (0.43).

The relationship between CCI and Ndw was sig-

nificantly linear (P < 0.001). Correlation analysis

indicates 64% of the variation in N was predicted

by CCI (Fig. 1). Peng et al. (1993) found that most

within-species variation in relationships between CCIand Ndw could be explained by differences in leaf

thickness. Indeed, relationships between CCI and N

were found to be improved by calculating specific leaf

weight (Peng et al., 1993) or considering N on an area

(Na), rather than dry weight, basis (Peng et al., 1995).

Chang and Robison (2003) also found that examining

N on an area basis improved correlation coefficients

both within and across dates in sweetgum (Liquidam-

bar styraciflua L.) leaves. Further study is necessary to

examine whether the linear relationship between CCI

and N in sugar maple leaves is improved by consider-

ing Na, and if the relationship remains linear for maple

leaves pooled from a wider range of collection dates

and growing conditions. The data from this study do

represent a relatively wide range of N values for sugar

maple leaves (1.42.6%) collected from different light

environments. Thus, it is probably appropriate to

conclude that for most sugar maple leaves collectedduring the growing season, the CCM provides a

reasonable, linear approximation of total N.

3.2. CCI versus total chlorophyll

Total extractable chlorophyll values from 98 sam-

ples ranged from 0.08 to 0.47 mg/mm2 with a mean of

0.26 (0.01). These values are within the published

range for sugar maple leaves (Cate and Perkins, 2003).

CCI ranged from 2.4 to 23.7 with a mean of 11.7

(0.52).The relationship between total extractable chloro-

phyll and CCI was significantly linear, with r2 indicat-

ing that 76% (P < 0.001) of the variation was

explained by a linear model (Fig. 2a). A logarithmic

model described the relationship marginally better,

explaining 81% of the variation (Fig. 2b). That the

relationship had a slightly better fit following natural

log transformations of CCI and total chlorophyll

values is likely indicative of observations made by

other researchers that the accuracy of portable

Chlorophyll Content Index

5 10 15 20 25 30

PercentNitrogen

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

y = 0.044x + 1.403

r2 = 0.641

Fig. 1. Linear regression of chlorophyll content index (CCI) vs.

total percent nitrogen (by dry weight) in 99 sugar maple leaves.

A.K. van den Berg, T.D. Perkins / Forest Ecology and Management 200 (2004) 113117 115

8/7/2019 Foreco_200_p113

4/5

chlorophyll meters decreases at high CCI values

(Monje and Bugbee, 1992; Richardson et al., 2002).

However, at least for moderate CCI values, it appears

that the CCM is able to provide a relative estimate of

total chlorophyll in most sugar maple leaves based on

a linear model.

4. Conclusions

The data in this study indicate that the CCM is an

effective tool for rapid and nondestructive estimation

of relative chlorophyll and nitrogen content in sugar

maple leaves during the growing season. Once general

relationships are established for a particular species, it

should be possible to use the CCM as a tool for a

variety of management and research applications for

which precise chlorophyll or nitrogen values are not

required, including the assessment of relative healthstatus, the assessment of physiological changes over

time and delineating the effects of management prac-

tices such as fertilization.

Acknowledgements

This research was funded by grants from the Envir-

onmental Protection Agency and the North American

Maple Syrup Council.

References

Azia, F., Stewart, K., 2001. Relationships between extractable

chlorophyll and SPAD values in muskmelon leaves. J. Plant

Nutr. 24, 961966.

Bullock, D.G., Anderson, D.S., 1998. Evaluation of the Minolta

SPAD-502 chlorophyll meter for nitrogen management in corn.

J. Plant Nutr. 21 (4), 741755.

Campbell, R.J., Mobley, K.N., Marini, R.P., Pfeiffer, D.G., 1990.Growing conditions alter the relationship between SPAD-501

values and apple leaf chlorophyll. Hortscience 25 (3), 330331.

Cate, T.M., Perkins, T.D., 2003. Chlorophyll content monitoring

in sugar maple ( Acer saccharum). Tree Physiol. 23 (15),

10771079.

Chang, S.X., Robison, D.S., 2003. Nondestructive and rapid

estimation of hardwood foliar nitrogen status using the

SPAD-502 chlorophyll meter. For. Ecol. Manage. 181,

331338.

Chapman, S.C., Barreto, H.J., 1997. Using a chlorophyll meter to

estimate specific leaf nitrogen of tropical maize during

vegetative growth. Agron. J. 89, 557562.

Dwyer, L.M., Tollenaar, M., Houwing, L., 1991. A non-destructive

method to monitor leaf greenness in corn. Can. J. Plant Sci. 71,

505509.

Dwyer, L.M., Anderson, A.M., Ma, B.L., Stewart, D.W., Tollenar,

M., Gregorich, E., 1995. Quantifying the nonlinearity in

chlorophyll content meter response to corn leaf nitrogen

concentration. Can. J. Plant Sci. 75, 179182.

Ellsworth, D.S., Liu, X., 1994. Photosynthesis and canopy nutrition

of four sugar maple forests on acid soils in northern Vermont.

Can. J. For. Res. 24, 21182127.

Ellsworth, D.S., Reich, P.B., 1992. Leaf mass area, nitrogen content

and photosynthetic carbon gain in Acer saccharum seedlings in

contrasting forest light environments. Func. Ecol. 6, 423435.

Ln (Chlorophyll Content Index)

1.0 1.5 2.0 2.5 3.0

Ln(TotalChlorophyll(g/m

m2))

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

y = 0.6141x - 2.8326

r2 = 0.809

Chlorophyll Content Index

5 10 15 20

TotalChlorophyll(g/mm

2)

0.0

0.1

0.2

0.3

0.4

0.5

y = 0.0135x + 0.1046

r2 = 0.756

(a) (b)

Fig. 2. Linear regression of chlorophyll content index (CCI) vs. (a) total extractable chlorophyll (mg/mm2

) in 98 sugar maple leaves and (b)

linear regression of the natural log of CCI values vs. the natural log of total extractable chlorophyll (mg/mm2) in 98 sugar maple leaves.

116 A.K. van den Berg, T.D. Perkins / Forest Ecology and Management 200 (2004) 113117

8/7/2019 Foreco_200_p113

5/5

Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations

of total carotenoids and chlorophylls a and b of leaf

extracts in different solvents. Biochem. Soc. Trans. 603, 591

592.

Liu, X., Ellsworth, D.S., Tyree, M.T., 1997. Leaf nutrition andphotosynthetic performance of sugar maple ( Acer saccharum)

in stands with contrasting health conditions. Tree Physiol. 17,

169178.

Marquard, R.D., Tipton, J.L., 1987. Relationship between extrac-

table chlorophyll and in situ method to estimate leaf greenness.

HortScience 22, 1327.

Monje, O.A., Bugbee, B., 1992. Inherent limitations of nondes-

tructive chlorophyll meters: a comparison of two types of

meters. HortScience 27, 6971.

Neilsen, D., Hogue, E.J., Neilsen, G.H., Parchomchuk, P., 1995.

Using SPAD-502 values to assess the nitrogen status of apple

trees. HortScience 30, 508512.

Peng, S., Garcia, F.V., Laza, R.C., Cassman, K.G., 1993.

Adjustment for specific leaf weight improves chlorophyll

meters estimate of rice leaf nitrogen concentration. Agron. J.

85, 987990.

Peng, S., Laza, R.C., Garcia, F.V., Cassman, K.G., 1995.

Chlorophyll meter estimates leaf-area based nitrogen content

in rice. Commun. Soil Sci. Plant Anal. 26, 927935.

Peoples, M.B., Dalling, M.J., 1988. The interplay between

proteolysis and amino acid metabolism during senescence

and nitrogen reallocation. In: Nooden, L.D., Leopold, A.C.

(Eds.), Senescence and Aging in Plants. Academic Press, San

Diego, CA.

Richardson, A.D., Duigan, S.P., Berlyn, G.P., 2002. An evaluation

of noninvasive methods to estimate foliar chlorophyll content.

New Phytol. 153, 185194.

Rodriguez, I.R., Miller, G.L., 2000. Using a chlorophyll meter to

determine the chlorophyll concentration, nitrogen concentra-tion, and visual quality of St. Augustinegrass. HortScience 35

(4), 751754.

Schaper, H., Chacko, E.K., 1991. Relation between extractable

chlorophyll and portable chlorophyll meter readings in leaves

of eight tropical and subtropical fruit-tree species. J. Plant

Physiol. 138, 674677.

Shaahan, M.M., El-Sayed, A.A., Abou El-Nour, E.A.A., 1999.

Predicting nitrogen, magnesium and iron nutritional status in

some perennial crops using a portable chlorophyll meter. Sci.

Hortic. 82, 339348.

Sibley, J.L., Eakes, D.J., Gilliam, C.H., Keever, G.J., Dozier Jr.,

W.A., Himelrick, D.G., 1996. Foliar SPAD-502 meter values,

nitrogen levels, and extractable chlorophyll for red maple

selections. HortScience 31, 468470.

Simorte, V., Bertoni, G., Dupraz, C., Masson, P., 2001. Assessment

of nitrogen nutrition of walnut trees using foliar analysis and

chlorophyll measurements. J. Plant Nutr. 24, 16451660.

Wood, C.W., Tracy, P.W., Reeves, D.W., Edmisten, K.L., 1992.

Determination of cotton nitrogen status with a hand-held

chlorophyll meter. J. Plant Nutr. 15, 14351448.

Yamamoto, A., Nakamura, T., Adu-Gyamfi, J.J., Saigusa, M., 2002.

Relationship between chlorophyll content in leaves of sorghum

and pigeonpea determined by extraction method and by

chlorophyll meter (SPAD-502). J. Plant Nutr. 25, 22952301.

A.K. van den Berg, T.D. Perkins / Forest Ecology and Management 200 (2004) 113117 117