Relationship between discoloration and histological changes in leaves of trees affected by forest decline

171

REMOTE SENSING OF ENVIRONMENT 26:171-184 (1988)

Relationship between Discoloration and Histological Changes in Leaves of Trees Mfected by Forest Decline

ENAMUL HOQUE, PETER

J.

S. HUTZLER, AND HARALD K. SEIDLITZ

Cesellschaft fiir Strahlen- und Umweltforschung, D-8042 Neuherberg, Ingolstaedter Landstr. 1, Federal Republic of Germany

The relationship between discoloration and histological changes induced by forest decline in leaves of Norway spruce (Picea abies) and beech (Fagus sylvatica) is analyzed. Discoloration of leaves by various degrees of damage is quantitatively measured by high resolution reflection spectroscopy. The cell structure of leaves is determined from microscopic images of histological cuts. Major significant changes in spectral reflectance as shown by t-value maxima are observed in the visible, but not in the near infrared region. The related cellular changes are hypertrophy of chloroplasts, reduction of chloroplast number, and formation of large isodiametrical hypodermal cells. These results may be helpful in assessing the forest decline from remote sensing data.

based inspection. The principles of color appearance of plants in general and their Forest decline, suggested to be mainly spectral signature in quantitative access due to air pollution and acid rain (Schuett are analyzed by various authors. In the et al., 1985), has become a tremendous visual range of the electromagnetic specchallenge during the last decade. Na- trum, the spectral signature is mainly tional inventory and monitoring programs characterized by the absorption of the have been started, and yearly status re- chlorophylls and other chromophores, as ports on forest damage are given. Until illustrated by Sauer (1975) and Gausman now, forest decline inventory, as il- et al. (1970). The high reflectance at the lustrated, e.g., by Hildebrandt and Kadro near infrared (IR) region is caused by (1984), is mainly done by ground-based refractive index discontinuities between visual inspection, using criteria like crown hydrated cell walls and intercellular air shape, loss of leaves, and discoloration of spaces. Gausman (1974) demonstrated leaves. It is to be noted that the discolora- that flooding the air spaces with water tion of leaves, especially in Bavarian for- results in a reduction of reflectance at the ests, is considered to be one of the major near IR to one half. The spectral signature of plants is alsymptoms of forest decline in West Germany (Schuett et al., 1984). The color ready analyzed under various aspects. information, which is quantitatively as- Tucker (1979) and Markham et al. (1981) sessed by the spectral reflectance, be- pointed out that the depth of the refleccomes even more important when remote tance minimum at 680 nm can be taken sensing is involved because there is less as a measure of photosynthesis activity, textural information due to reduced spa- and the near IR reflectance of an areal tial resolution, compared with ground- plot can be related to the biomass. They Introduction

©Elsevier Science Publishing Co., Inc., 1988 655 Avenue of the Americas, New York, NY 10010

0034-4257/88/$3.50

17:2

ENAMUL HOQUE ET AL

used a combination of the Landsat compares the spectroscopic data taken Thematic Mapper Bands 3 (0.63-0.69 from individual leaves of various degrees p.m) and 4 (0.76-0.90 p.m) for seasonal of discoloration with the histological revegetation monitoring and harvest grain sult. prediction. Investigations of the spectral signature Materials and Methods of trees were reported by Heller (1969). He pointed out that damage on Pinus Plant materials ponderosa is more obvious in the visible Needle leaves of healthy and affected range than in the near and medium IR. trees of Scots pine (Pinus sylvestris), These results were confirmed by Elling Norway spruce (Picea abies), and broad and Knoppik (1986) by spectrometric leaves of beech (Fagus sylvatica) showmeasurements on a dense layer of spruce ing different degrees of discoloration have branches. Tanner and Eller (1981) analbeen used in this study. Usual visual clasyzed the seasonal variation of the spectral sification methods used by foresters and signature of beech. Recently, Westman us (Hoque, 1985) were employed in order and Price (1988) reported about the to classify the needles in different discolqualitative rise of reflectance of Pine neeoration groups. Table 1 shows the chardles in the range 639-690 nm (Landsat acterization of these plant materials. TM3 Band) when subjected to air-drying, exposures to ozone, and acid rain. Acquisition of histological images However, there is little experience in interpreting spectral reflectance data of Thin cross sections of needles and broad the forest decline which has been expand- leaves were obtained using a sharp razor ing over the past decade. This paper blade and were immediately embedded TABLE 1

SPECIES

Characterization of Plant Materials AGEOF AGEOF SAMPLE SIZE TREES LEAVES (n) (years) (years)

2-3

> 32

SITE OF COLLECTION/ In ABOVE NN

COLOR OF LEAVES/ DISCOLORATION GROUP

MONTH OF COLLECTION/

Munich/670

green yellow brown

d.c.u

YEAR

Scots pine

ca. 100

Norway spruce

ca. 100

2

10

Sauerlach/670

green green-yellow yellowish yellow brown

d.c.O d.c.1 d.c.2 d.c.3 dc.4

August,' 1987

Beech

ca. 100

0.4

10"

Munich/670

green light green yellowish yellow brown

d.c.O d.l'.1 d.l'.2 d.c.3 d.l'.4

August/1987

"Leaf pieces.

Augustj1986

d.(.3 d.c.4

REFLECTANCE AND HISTOLOGY CHANGE DUE TO FOREST DECLINE

in 100% glycerine. A Leitz Orthoplan optical transmission microscope was used for imaging. Images with 25 X magnification were sensed at the first image plane by a high resolution CCD camera with 1/2 in. detector size. Three color extracts were taken from each scene by inserting interference filters at the illumination path of the microscope. The transmission maxima of the filters in use are located at 500 (20), 550 (20), and 680 (10) nm. The value in parentheses indicates the spectral half width of transmission. The images were digitized and stored at a digital video frame store. For color image presentation, the intensity normalized and D/ A converted signals of three color extracts are fed simultaneously to the RGB channels of a color monitor.

173

Particular care was taken to obtain geometrical reproducibility and to avoid any optical gloss effect by the use of a special sample holder. The needles or leaf pieces were pressed gently from below against a slit of 0.5 mm width, and illuminated perpendicularly by the monochromatic light beam of 0.1 mm width. This is illustrated in Fig. Ib). Foam rubber kept the samples wet from beneath. During stage movement the reflected light was recorded continuously by a photomultiplier (Hamamatsu R928) at an angle of 45°, as shown in Fig. 1a). For further evaluation the average reflectance over the scanned object width of 0.5 mm was taken. At each of the selected wavelengths the same measurements were performed with a small strip of Kodak Neutral Grey Measurement of spectral reflectance Chart and a strip coated with Kodak Immediately after cutting of branches White Reflectance Coating positioning in under water in the afternoon, the cut the same measuring place. The latter were edges of the branches were soaked in taken for reference when calculating abwater in order to avoid water stress and solute reflectance values. The grey chart then the branches were transferred to the reflectance with a nominal value of 18% laboratory. The branches with the cut was always verified within an absolute edges in water were stored in darkness at error margin of 2%. In order to be sure that glossy reflec20°C and a relative humidity of 70% tance does not distort the measurements, until measurement the next morning. For measurement of spectral diffuse we used several healthy green needles reflection in the range of 480-850 nm cleaned from waxy layers by acetone wavelengths at intervals of 20 nm, we treatment. Over the entire spectral range used a Zeiss Model KM3 spectropho- the reflectance of these acetone-treated tometer. The optical setup is shown in needles corresponds with the reflectance Fig. Ia). The exit slit of the monochroma- of untreated needles within the instrutor was imaged at unit magnification onto mental relative error range of 10% rethe upper (adaxial) surface of single leaves ferred to the measured value. The intenor leaf pieces. Under these setup condi- sity of the monochromatic illumination tions, the illuminated area was 3.5 X 0.1 was kept below 1 W/ em" in order to mm. A slit width of 0.1 mm corresponds avoid photoinduced changes of the leaf to a spectral bandwidth of approximately surface. The lower limit of the spectral 5nm. interval was chosen such that fluores-

(b)

,.,. •....,00 \..Jf

;q)

MLB

MO

Vo

OOA

e-CJ

0

SH

AI

FIGURE 1. Setup of reflection spectrophotometer: a) main configuration (PMT = photomultiplier, ~U = mirror, L = lens, ES = exit slit of MO. MO = monochromator, LS = light source, W = white reference, S = sample, SH = sample holder); b) schematic exploded view of cut A-A' from Figure Ia) (MLB = monochromatic light beam, S = sample holder, f = wet foam).

ES

LS

....

Z

r-

~

trl tr1 ""':l

c:::

-c

o

~

t""'

c:::

2:

~

tr1

-1 .4

REFLECTANCE AND HISTOLOGY CHANGE DUE TO FOREST DECLINE

cence at 680 nm was not likely to be excited. For statistical interpretation of data, each measurement was performed under identical geometrical conditions with 10 or more leaves or leaf pieces randomly chosen from the same group of discoloration (Table 1). Statistical analysis Statistical analysis of measured reflectance values (%) from each data point (selected wavelength) was performed using SAS Statistics software (Anon., 1985). The arithmetical means, standard deviations (s.d.), and absolute t-values d each data point (subgroup) were calculated, whereby the discoloration groups (d.c.l-d.c.4) were tested vs. d.c.0 group of healthy green needles or broad leaves (Annon., 1985). According to Bonferroni t statistics, the significance level for each component interval can be set at a/k, TABLE 2

175

where k = n u m b e r of t tests (Miller, 1985). Since our p-values (a-values) are very low in most subgroups (wavelengths) of the visible electromagnetic regions of various discoloration groups d.c.0-d.c.4, the fullfilment of required 0.05 significance level--in other words, at p = p ' / k , where p ' = 0 . 0 5 ( p = 0.0045, when k = 11 in a group, Table 2; p = 0.0029, when k = 17 in a group, Tables 3 and 4)--can be easily shown. Further, multivariate tests using Wilk's criterion, Pillai's trace, HoteUing-Lawley trace, and Roy's maximum root criterion were performed in order to find out overall significant differences between the discoloration groups (Anon., 1985; Morrison, 1976). The means and s.d. of reflectance (%) of all sample groups, as well as the t-values and error probability p of the different discoloration groups, vs. the reference group d.c.0 are summarized in a table for each tree species. Additionally, for better visualization the calculated means of reflectance and the t-values of

Reflectance Data and Statistical Analysis of Scots Pine a WAVELENGTH(nm) 500

520

560

600

640

660

680

700

720

760

800

9.00 1.82

7.10 1.35

5.69 1.32

14.24 2.77

33.74 5.10

46.54 4.49

48.06 3.36

d.c.O s.d.

6.80 1.55

10.13 1.47

16.48 2.19

11.57 1.97

d.c.3 Means s.d. t p ~<

8.83 2.54 3.80 .0004

16.19 3.29 9.39 .0001

29.38 2.66 23.78 .0001

26.97 5.63 14.65 .0001

24.01 5.74 15.13 .0001

20.28 7.35 10.09 .0001

12.06 5.28 8.07 .0001

30.24 6.27 13.18 .0001

42.83 7.20 5.80 .0001

45.67 5.60 0.78 .46

47.83 7.16 0.16 .88

d.c.4 Means s.d. t p ~<

8.67 4.92 1.77 .09

10.32 4.99 0.20 .84

15.58 5.02 0.92 .38

22.44 5.36 9.85 .0001

30.20 6.69 14.97 .0001

34.29 6.91 19.66 .0001

38.03 7.10 25.27 .0001

41.64 7.63 17.46 .0001

44.58 8.02 5.93 .0001

49.61 8.23 1.80 .08

53.45 8.46 3.15 .003

a Means, standard deviations (s.d.), absolute t-values, and corresponding p-values of measured reflectance (%) were calculated for various subgroups (wavelengths) using needles of different discoloration groups, whereby the discoloration groups d.c.3 and d.c.4 were tested against d.c.0 (green).

5.91 1.10 6.92 .0001

4.95 1.40 4.12 .0006

2.30 0.45 1.41 .18

2.85 0.78 0.24 .82

2.76 0.93

480

8.69 1.51 3.61 .002

8.20 0.48 3,88 .003

6.26 1.50

520

6.95 1.26 6.89 .0001

8.08 1.49 2.72 .01

11.90 7.10 2.68 1.23 7.30 5.81 .0001 .0001

3.52 0.78 0.27 .79

3.74 0.82 0.79 .44

3.41 1.02

500

10.11 2.00 1.99 .06

15.86 3.18 6.48 .0001

12.42 1.91 4.89 ,0001

11.75 0.51 5.84 .0001

8.46 1.71

540

11.94 2.24 3.68 .0017

18.09 3.11 8.39 .0001

13.69 2.03 5.96 .0001

12.25 0.61 6.20 .0001

8.65 1.73

560

.0001

13.97 2.30 8.54

18,72 2.91 11.76 .0001

11.93 2.08 6.62 .0001

.0001

6.60

10.15 0.88

6.66 1.42

580

9.99 2.25 6.24 .0001

7.84 1.07 .5.65 .0001

4.96 1.21

9.43 2.38 5.63 .0001

7.23 1.10 4.85 .0001

4.59 1.32

640

WAVELENGTH

620

6.50 2.32 3.74 .001.5

4.91 0.90 :3.28 .0004

3 ..52 0.99

660

(nm)

4.0H 1.77 2.21 .04

3.19 0.79 1.31 .21

2.69 0.92

680

16.21 23,01 18.67 21.42 24.58 2,96 2.59 2.79 :3.03 3.23 20.62 11.11 14.24 16.10 19.73 .()O01 .00<)1 .0001 .0001 .0001

1,5.6.5 19.04 19.68 19.00 17.33 2.9.5 3.34 3.00 3.98 3.57 12.78 13.29 11.11 1:3.73 10.65 .0001 .0001 .0001 .0001 .0001

11.19 2.19 6.49 .0001

9.16 0.98 6.12 .0001

5.86 1.40

600

Reflectance Data and Statistical Analysis of Norway Spruce"

27.48 4,29 12.61 .0001

23.10 .5.74 7.53 .0001

16.16 2.64 7.19 .0001

12.79 0.96 5.95 ,()O01

8.64 1.99

700

29.81 4.01 ,').27 .()()O1

30.02 3.96 .5.44 .0001

24.20 3.1.5 2.19 .04

22.3.5 1.45 1.17 .27

20.96 3.47

720

.om>

32.70 4,42 2.92

:31.60 3,42 2.64 .017

27.47 4.82 0.16 .87

26.16 2.2.5 0.67 .51

27,15 4.08

740

34.94 4.87 2.96 .008

32.02 3.64 1.77 .09

27.54 3.82 0.73 .47

27.00 2.21 1.23 .24

28.87 4.28

760

36.71 .'5.48 3.4,5 .0029

33.49 4.41 2.2.5 .01

.,50

27.86 3.83 0.69

27.48 2.19 1.08 .30

29.12 4.28

800

40.S.5 6.18 4.10 .0002

32.92 4,29 2.02 .06

29.0:3 ,'3.90 0.17 .87

27.63 2.22 0.61 .56

28.69 5.06

850

"Means, standard derivations (s.d.), absolute r-values. and corresponding p-values of measured reflectance (%) were calculated for various suhgroups (wavelengths) using needles of different discoloration groups, whereby the discoloration groups d.c.l-o.c.4 were tested against d.c.O (green}

p~

t

d.c.4 Means s.d.

p:(,

t

d.c.3 Means s.d.

p:(,

t

c1.c.2 Means s.d.

p:(,

t

d.c.1 Means s.d.

d.c.O Means s.d.

TABLE 3

r-'

;;..

>--3

~

~

-c C

0

~

F

~ ,-

>

tr'l

Z

~

-I

I-

5.01 1.90 3.46 .01

4.19 1.05 3.91 .003

4.48 1.07 4.56 .002

10.73 .0001

0.30

0.83

2.64 0.36

480

6.26 1.58 5.86 .0005

6.25 0.73 12.03 .0001

.oooi

7.65 1.22 10.72

1.50 0.16 13.12 .0001

2.93 0.26

500

4.63 .002

2.33

8.69

17.23 1.36 25.31 .0001

16.63 1.51 21.79 .0001

5.39 0.50 2.72 .02

4.84 0.25

520

8.19 0.50

560

11.01 3.28 2.73 .03

24.64 1.68 24.93 .0001

22.31 1.70 22.51 .0001

13.08 4.09 3.35 .01

27.09 1.50 31.68 .0001

24.54 1.87 23.72 .0001

10.05 0.64 6.30 .0004 .0001

9.76 0.76 5.53

7.79 0.76

540

15.34 4.80 5.40 .0009

28.14 1.35 41.99 .0001

24.66 2.12 24.57 .0001

7.10 0.40 5.72 .0001

6.03 0.33

580

Reflectance Data and Statistical Analysis of Beech"

17.86 5.57 6.39 .0001

28.86 1.29 49.94 .0001

24.23 2.20 24.11 .0001

6.00 0.39 3.96 .002

5.27 0.31

600

20.75 5.81 7.83 .0001

29.69 1.16 58.41 .0001

23.63 2.59 20.54 .0001

5.19 0.33 3.19 .007

4.66 0.32

620

19.84 1.30 34.04 .0001

29.16 1.41 49.83 .0001

.oooi

21.88 3.02 16.98

3.85 0.29 1.47 .17

3.61 0.33

640

WAVELENGTH

23.60 0.72 61.07 .0001

27.69 1.90 34.42 .0001

17.53 3.78 10.38 .0001

2.70 0.19 3.88 .002

3.55 0.55

660

(nm)

29.20 3.26 21.91 .0001

25.75 1.80 33.64 .0001

14.29 3.68 8.06 .0001

.012 17.95 .0001

2.06

3.76 0.38

680

34.79 2.76 27.22 .0001

34.53 1.43 50.08 .0001

27.83 2.15 25.64 .0001

10.05 0.62 8.04 .0001

7.83 0.45

700

38.95 2.02 18.87 .0001

39.08 1.84 20.54 .0001

33.41 1.23 17.16 .0001

28.31 1.36 6.96 .0001

24.40 0.78

720

43.13 1.41 1.43 .18

40.39 1.89 0.91 .38

37.59 1.21 4.55 .0005

39.69 1.41 2.17 .05

41.76 2.25

740

46.94 1.66 .93 .37

42.96 1.51 4.30 .0009

40.00 1.06 7.24 .0001

46.43 1.72 1.34 .20

48.07 2.96

760

53.78 3.38 1.72 .12

46.29 2.47 3.95 .0017

43.74 1.06 8.11 .0001

47.83 1.50 3.37 .005

51.21 2.36

800

58.54 4.93 2.68 .03

48.56 1.43 6.32 .0001

45.26 1.34 10.81 .0001

50.54 1.28 4.10 .001

53.57 1.64

850

"Means, standard deviations (s.d.), absolute t-values, and corresponding p-values of measured reflectance (%) were calculated for various subgroups (wavelengths) using leaves of different discoloration groups, whereby the discoloration groups d.c.l-d.c.4 were tested against d.c.O (green).

p~

d.c.4 Means s.d. t

p~

d.c.3 Means s.d. t

p~

d.c.2 Means s.d. t

p~

Means s.d. t

d.c.l

d.c.O Means s.d.

TABLE 4

-..J -..J

I--'

tr:l

Z

C

o

tr:l

0

:xl trl V'J '"":l

0

~

0

c::: trl ....,

0

trl

0

Z

>-

o ::r:

-<

0 t"" 0 0

V) '"":l

::r:

0

>Z

t%:l

o ~ Z o

~

~

:xl t%:l

ENAMUL HOQUE ET AL.

17R

rather coarse subdivision into discoloration groups d.c.O, d.c.3, and d.c.4 (Table 1). The arithmetic means and standard deviations of the measured reflectance values are summarized in Table 2, and illustrated by Fig. 2a). The relative standard deviation is about 10% from the mean at the d.c.O group, and increases to 30% at the d.c.3 and d.c.4 group, due to

the discoloration groups are plotted vs. wavelength, and fitted by spline functions. Results Scots pine

Our early experiments in Summer 86 on Scots pine (Pinus sylvestris) used a

REFLECTANCE (%) OF NEEDLES OF VARIOUS COLORS FROM SCOTS PINE 50

40

30

20

10

O _ - - - - - - - - r - - - - -_ _~ - - - - - - - ~ - 460

560

660

760

NM

860

(a) 50,------------------------~

REFLECTANCE (%) OF NEEDLES OF VARIOUS COLORS FROM NORWAY SPRUCE 40

... x· 30

20

10

O\---------r--------~-------~-.----~-____.'

41lO

560

660

760

NM

860

( b)

FIGURE 2. Absolute reflectance of various discoloration (d.c.) groups versus wavelength for: a) Scots 3: (X) 4 pine; b) Norway spruce; c) Beech. d.c.: (+) 0 (green); (L:.) 1 [new sprout in c)]; (D) 2; (brown).

(<»

179

REFLECTANCE AND HISTOLOGY CHANGE DUE TO FOREST DECUNE 60

•• --x

REFLECTANCE (x) OF LEAVES OF VARIOUS COLORS FROM BEECH

50

40

30

20

10

fr······

......

" ·······8··

660

560

760

NM

860

(c)

FIGURE 2 (Continued)

the coarse classification [Fig. 2a), Table 2]. Changing from d.c.O to d.c.3, great differences in reflectance occur between 560 and 660 nm, where the reflectance values increase by a factor of 2-3. Overall significant effects of leaf discoloration on changes of reflectance could be con-

firmed between the tested groups d.c.3 vs. d.c.O, and d.cA vs. d.c.O by multivariate tests (Wilk's criterion, Pillai's trace, Hotelling - Lawley trace, and Roy's maximum root criterion; p ~ 0.00(1), whereas the subgroups, i.e., selected wavelengths, between 500 and 720 nm

30

ABSOLUTE t -VALUES DEPENDING ON WAVELENGTH SCOTS PINE 25

20

15

10

5

..~

~

o, <;o,

01,----......:Y;"'--.::..,.460

560

~-------,____------_;

660

760

NM

660

(a)

FIGURE 3. Student's t-test of discoloration (d.c.) groups paired with the normal green group (d.c.O) plotted vs. wavelength for: a) Scots pine [d.c.: (+) 3 (yellow); (<» 4 (brown)]; b) Norway spruce [d.c.: ( +) 1 (green-yellow); (a) 2; (D) 3; «> 4 (brown)]. c) Beech [d.c.: ( +) 1 (new sprout); (a) 2; (D) 3; (<» 4 (brown)].

IHO

ENAMUL HOQUE ET AL 25.,--------------------------

ABSOLUTE t -VALUES DEPENDING ON WAVELENGTH NORWAY SPRUCE ~

/

20 I

;I'

,/

\

\ \

/

\ \

15

\

:s, \ \

10

460

\

560

660

860

(b I

70

60

------------------~--~----,-,-~_._-~--,-,

ABSOLUTE t -VALUES DEPENDING ON WAVELENGTH BEECH

50

40

30

20

10

O'l-----------,--------,----------=""'-T-- ---------1

460

560

660 (c

760

NM

860

I

FIGURE 3 (Continued)

(d.c.S) and 600 and 720 nm (d.c.4) could be separated by t-tests [Table 2, Fig. 3a)]. The IR shoulder, i.e., the transition from low to high reflectance, for the yellow group d.c.3 is shifted by more than 15 nm towards shorter wavelengths in comparison to the d.c.O group. Reflectance of d.c.4 needles increases monotonically with wavelength. There is no major significant change in

reflectance along the near IR plateau between the discoloration groups (d.c.3 and d.c.4) and d.c.O group [Table 2, Fig. 3a)]. The R760jR680 ratio values of the discoloration groups d.c.3 and d.c.4 given in Table 5 decrease significantly with increasing discoloration (p ~ 0.001-0.0001, Table 5). Needles from the discoloration groups d.c.3 and d.c.4 show t-value maxima of about 13-25 at different regions of

REFLECTANCE AND HISTOLOGY CHANGE DUE TO FOREST DECllNE

181

TABLES Ratio of Reflectance Values (R760, in %) at 760 nm to Reflectance Values (R680, %) at 680 nm (Calculated According to Tucker, 1979t TREE •SPECIES Scots pine Means s.d. t p.., Norway spruce Means s.d, t p.., Beech Means s.d. t p..,

DISCOLORATION GROUP

d.c.O

d.c.I

d.c.2

8.63 2.13

d.c.3

d.c.4

4.41 1.62 9.73 .0001

1.31 0.08 19.46 .0001

12.88 7.07

8.86 3.33 1.61 .13

7.87 3.48 2.01 .07

2.29 0.32 4.73 .001

1.45 0.23 5.10 .0006

12.94 1.82

22.53 1.46 11.34 .0001

2.89 0.90 13.88 .0001

1.67 0.12 16.39 .0001

1.62 0.17 16.42 .0001

"Means, standard deviations (s.d.), absolute z-values and corresponding p-values of ratio data were calculated for the leaves of various discoloration <>TOUps, whereby the discoloration groups d.c.l-d.c.4 were tested versus d.c.O (green).

the visible spectrum. t-Values in the near IR region are below 4 for both groups [Fig. 3a) and Table 2].

spectively, in comparison to the d.c.O reference group. However, a more dramatic change is observed in the histological images of the needles. Whereas healthy d.c.O needles [Fig. 3a)] possess plenty of green chloroNorway spruce plasts of 4-10 f-L m in size, needles of The reflectance spectra of healthy dark discoloration group d.c.2 [Fig. 4b)] congreen (d.c.O) needles and four discolora- tain about 80% hypertrophied chlorotion groups d.c.( n) are summarized in plasts with a size of up to 18 f-Lm. Table 3, and illustrated by Fig. 2b). The The number of chloroplasts is reduced group label n = 0-4 indicates increasing to about 40% of the number for d.c.O discoloration. The reference group d.c.O needles. Green pigmentation is less prevashows a maximum of 10% reflectance at lent, and protective cuticules are de550 nm, and a minimum of 4% reflec- stroyed to a large extent. At the higher tance at 680 nm. Reflectance rises to discoloration group d.c.3, the spectrum is about 30% at the near infrared plateau characterized by an additional increase beyond 750 nm. The discoloration groups of reflectance at 680 nm by a factor d.c.I and d.c.2 are characterized by a of 5 (Table 3). Finally, the reflectance nearly synchronous increase of reflec- spectrum of d.cA needles shows a curvatance (over the range from 560 to 640 ture monotonically increasing with wavenm) by approximately 50 and 85%, re- length, comparable to the reflectance of

11')2

non-vegatative surfaces [Huck et al., 1984; Fig. 2b) and Table 3]. The histological image of d.cA needles [Fig. 3c)] exhibits loss of chloroplasts with a reduction of 86%, as well as a loss of compartmentalization in the cells. Swelling of cell walls and overall browning of cellular structures in d.cA needles could be observed. Remaining chloroplasts in about 70% of parenchymatic cells were hypertrophied with sizes up to ca. 17 p.m. The overall cellular skeleton is retained. In all the groups of needle discoloration (d.c.l-d.cA) examined, relatively lower t-value maxima occur in the near infrared region than those in the visible range [Table 3, Fig. 3b)]. Overall significant effects of leaf discoloration on changes of reflectance could be confirmed between the tested groups d.c.2 vs. d.c.O (p ~ 0.0004), and d.cA vs. d.c.O (p ~ 0.0047) by multivariate tests, whereas the subgroups, i.e., selected wavelengths, between 540 and 660 nm and 700 nm of the discoloration group d.c.I, between 520 and 660 nm and 700 nm of the discoloration group d.c.2, between 480 and 720 nm of the discoloration group d.c.S, and between 480 and 500 nm, 580 and 700 nm, and 800 and 850 nm of the discoloration group d.cA could be separated by Student's t-tests (Bonferroni t = statistics, p ~ 0.0029 at 0.05 significance level; Table 3). The ratio values R760/R68O shown in Table 5 decrease from 13 for d.c.O to 1.5 for d.c.4 needles. Significant changes of ratio values could be detected between d.c.3 and d.c.O (p ~ 0.(01), and d.c.4 and d.c.O (p ~ 0.0006) (Table 5). Beech

The results of reflection spectroscopic measurements of five discoloration groups

ENAMUL HOQUE ET AL.

(d.c.O-d.c.4) are summarized in Table 4 and Fig. 2c). Light green leaves (d.c.l) of affected beech trees were taken from newly emerged shoots in August, and therefore, require special precautions in interpreting results from these leaves. The reference group d.c.O showed a maximum of 8% reflectance at 560 nm, and a minimum of 4% reflectance at 660 nm in the visible region. Reflectance increased to about 50% at the IR plateau above 750 nm. Light green leaves (d.c.l) from newly emerged shoots showed a strong minimum of 2% reflectance at 680 nm, and an increment of only 2% reflectance at 560 nm. The discoloration groups d.c.2 and d.c.3 are characterized by increased reflectance in the range from 560 to 640 nm by factors of 3 and 4, respectively, in comparison to healthy d.c.O leaves. The spectrum of the discoloration group d.c.3 is characterized by a sixfold increase in reflectance at 680 nm. Dramatic changes as in Norway spruce could be observed in histological images of Beech leaves, too. Leaves of d.c.O [Fig. 5a)] and d.c.I groups contained plentiful chloroplasts with their full integrity, whereas d.c.2 and d.c.3 leaves [Fig. 5b)] lost chlorophyll to a whole extent and compartmentalization of cell organelles in the cells was nearly 100% lost. About 70-100% reduction of chloroplast number in palisade parenchymatic cells could be observed. The palisade parenchymatic and spongy parenchymatic cells were observed to be partly separated. Dark deposits could be observed in vascular bundles. Finally, the reflectance spectrum of d.c.d leaves shows a monotonically increasing curvature similar to the respective spectra of Scots pine and Norway spruce. Heavy deposits of brown substances could be observed in d.c.4 leaves. The spongy parenchymatic cells could not

(a)

(h)

(c)

FIGURE 4. Cross section through needles of Norway spruce from three discoloration (d.c.) groups: (left) green (d.c.O); (center) yellowish (d.c.2); (right) brown (d.c.4).

"

• F I

(a)

(h)

, ~ 9

.~

~/.

\ .. ,

* .~ , , . .v . .,

.

(c)

FIGURE 5. Cross section through leaves of beech from three discoloration (d.c.) groups: (left) green (d.c.O); (center) yellow (d.c.3); (right) brown (d.c.4).

REFLECTANCE AND HISTOLOGY CHANGE DUE TO FOREST DECUNE

be differentiated [Fig. 5c)]. Although some significant changes in reflectance in the near IR could be observed in most of the discoloration groups (d.c.1-d.c.3) in comparison to the needle group d.c.O, it should be borne in mind that relatively lower t-value maxima (discoloration groups d.c.1-d.cA vs. d.c.O) occur mainly in the infrared region than those in the visible range [Table 4, Fig. 3c)]. Multivariate tests were not performed due to lack of constant sample size in all the groups while evaluating the measured data. The ratio R760/R680 takes a value of 22 for newly emerged d.c.1 leaves and 12 for d.c.O leaves. With increasing discoloration of leaves, it decreases to 1.62 for d.cA leaves. This significant change is quite similar to Scots pine and Norway spruce (Table 5).

183

sociated with considerable intracellular modification. The density of normal chloroplasts is reduced, and hypertrophic chloroplasts appear in an increasing number. The analysis shows that the spectroscopic characterization of leaf discoloration induced by the forest decline is fully assessable at the visible range of the electromagnetic spectrum. Reflection spectroscopic data in the near infrared give no information on discoloration of the leaves. In order to assess this weak discoloration, using remote sensing techniques, an additional highly sensitive channel between 540 and 680 nm is recommended. Given the background of the given results, the changes in infrared reflectance obtained by integral measuring techniques are obviously worth a more detailed analysis.

The authors acknowledge the skillful technical assistance provided by M. Reflection spectroscopical and histo- Gorgel and the statistical evaluation of logical aspects of the discoloration of our manuscript provided by Dr. W. leaves, a major symptom of the forest Lehmacher, Statistician, GSF. decline in West Germany (Schuett et al., 1984; 1985), have been analyzed. For several degrees of discoloration of Norway spruce and beech the reflecReferences tion spectra of individual leaves and the histological status were compared. A Annonymous (1985), SAS User's Guide: Staweak discoloration (d.c.1-d.c.2) is spectistics, SAS, North Carolina. troscopically assessable first at the visible Elling, W., and Knoppik, D. (1986), Spektrales spectral range, about 540-660 nm, Reflexionsverhalten von Fichtenzweigen whereas the reflectance at 680 nm, being mit Chlorosen und Nekrosen der Nadeln, a measure of chlorophyll content, shows AUg. Forstz. 18:431-432. no significant change in Norway spruce, Gausman, H. W. (1974), Leaf reflectance of but a significant change in Beech, and at near-infrared. Photogramm. Eng. the near infrared region no change in 1974:183-191. Norway spruce, but significant changes Gausman, H. W., Allen, W. A., Cardenas, R., in Beech. Major t-value maxima occur in and Richardson, A. J. (1970), Relation of the visible range in comparison to the light reflectance to histological and physical infrared range (discoloration groups evaluations of cotton leaf maturity, Appl. d.c.1-d.cA). Moderate discoloration is asOpt. 9:545-552. Conclusion

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Heller, R. C. (1969), Large-scale photo assessment of smog damaged pines, in New Horizon in Color Aerial Photography, American Society of Photogrammetry, Falls Church, VA. Hildebrandt, G., and Kadro, A. (1984), Aspects of countrywide inventory and monitoring of actual forest damages in Germany, Bildmessung Luftbildwesen 52:201-216. Hoque, E. (1985), Norway spruce dieback: occurrence, isolation and biological activity of p-hydroxyacetophenone and p-hydroxyacetophenone-O-glucoside and their possible roles during stress phenomena, Eur. ]. For Pathol. 15:129-145. Huck, F. 0., Davis, R. E., Fales, C. L., Aherron, R. M., Arduini, R. F., and Samms, R. W. (1984), Study of remote sensor spectral responses and data processing algorithms for feature classification, Opt. Eng. 23:650-666. Kadro, A. (1981), Untersuchungen der spektralen Reflexionseigenschaften verschiedener Bestande, dissertation, AlbertLudwigs-Universitat, Freiburg. Markham, B. L., Kimes, D. S., Tucker, C. J., McMurtrey, J. E., III (1981), Temporal spectral response of a com canopy, Photogramm. Eng. Remote Sens. 48:1599-1605. Mauser, W., and Stibig, J.-J. (1983), Neue Sensoren fiir die Vegetationsbeobachtung mit Satelliten, AUg. Forstz. 46/47:12421243.

Miller, R. G., Jr., (1985), Simultaneous Statistical Inference, Springer-Verlag, New York. Morrison, D. F. (1976), Multivariate Statistical Methods, 2nd ed., McGraw-Hill, New York. Sauer, K. (1975), in Bioenergetics of Photosynthesis (Govindjee, Ed.), Academic, New York, pp. 115-181. Schuett, P., Blaschke, H., Holdenrieder, 0., Koch, W., Lang, K. J., Schuck, H. J., Stimm, B., and Summerer, H. (1984), Der Wald stirbt an Stress, C. Bertelsmann Verlag, Munich. Schuett, P., Koch, W., Blaschke, H., Lang, K. J., Reigber, E., Schuck, H. J., and Summerer, H. (1985), So stirbt der Wald, BLV Verlagsgesellschaft, Munich. Tanner, V., and Eller, B. M. (1981), Veraenderungen der spektralen Eigenschaften der Blaetter der Buche (Fagus sylvatica L.) von Laubaustrieb bis Laubfall, AUg. Forst Jagdztg. 157:108-117. Tucker, C. J. (1979), Red and photographic infrared linear combinations for monitoring vegetation, Remote Sens. Environ. 8:127-

ISO. Westman, W. E., and Price, C. V. (1988), Spectral changes in conifers subjected to air pollution and water stress: experimental studies, IEEE Trans. Geosci. Remote Sens. 26:11-21. Received 4 January 1988; revised 7 June 1988.