Size-mediated foliar response to ozone in black cherry trees

Environmental Pollution, Vol. 91, No. 1, pp. 53~53, 1996

Copyright © 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0269-7491/96 $09.50+ 0.00

~ I ¸~"4 ~ ''711 " ELSEVIER

0269-7491(95)00032-1

SIZE-MEDIATED FOLIAR RESPONSE TO OZONE IN BLACK CHERRY TREES T. S. Fredericksen, a J. M. Skelly, b K. C. Steiner, c T. E. Kolb a & K. B. K o u t e r i c k b "Environmental Resources Research Institute, 220 Forest Resources Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802, USA bDepartment of Plant Pathology, Pennsylvania State University, Pennsylvania, USA CSchool of Forest Resources, Pennsylvania State University, Pennsylvania, USA dSchool of Forestry, Northern Arizona University PO Box 15018, Flagstaff, Arizona 86011-5018, USA

(Received 10 December 1994; accepted 3 April 1995) ponents of cellular membranes and disrupting enzymes important in key physiological processes (Mudd, 1984; Guderian et al., 1985). Palisade cells in the leaf mesophyU are particularly sensitive to ozone injury and the collapse of individual cells or groups of these cells produces visible symptoms on the adaxial leaf surface in many species (US EPA, 1975). Some studies indicate that physiological or 'hidden' injury (e.g. decreased photosynthesis, altered stomatal conductance) may occur before or without the onset of visible symptoms (Stoklasa, 1923; Reich and Amundson, 1985; Reich et al., 1986). Conversely, visible foliar symptoms may not necessarily be indicative of physiological injury if the leaf is able to balance injury to some cells through a compensatory response by uninjured ones (Jacobsen, 1982; Roberts, 1984). Black cherry (Prunus serotina Ehrh.) is a commonlyoccurring tree species in eastern North America of regional ecological and economic importance, especially in the Appalachian mountains. Studies have shown that black cherry is one of the most consistently sensitive eastern forest tree species to tropospheric ozone in terms of visible foliar injury. Correlation of symptom expression with ozone exposure have been reproducible with increasing ozone exposures in continuously-stirred tank reactors (Davis & Skelly, 1992a,b), open-top chambers (Simini et al., 1992; Kouterick et al., 1994; Skelly et al., 1994), and field studies (Fredericksen et al., 1995; Hildebrand et al., 1996). Results from studies examining growth responses to ozone with this species are less clear. Long and Davis (1991) found that black cherry seedlings treated with the antioxidant ethylenediurea (EDU) had 47% greater biomass than untreated trees. However, despite the presence of foliar symptoms and early leaf senescence under high ozone exposure, Simini et al. (1992) found that ozone did not affect above-ground biomass after three growing seasons. Few studies have investigated how ozone uptake (ozone concentration × stomatal conductance) relates to adaxial stipple and early leaf senescence in the species and if this visible injury is related to injury or to impairment of physiological injury. Because of its high apparent sensitivity to ozone, black cherry is considered a potential field bioindicator species of high ozone concentrations and potential plant injury (Davis, 1983; Simini et al., 1992; Brantley et al., 1994), Black cherry

Abstract Local ozone concentration and v&ible foliar injury were measured over the 1994 growing season on open-grown black cherry (Prunus serotina Ehrh.) trees of varying size (age) within forest stands and adjacent openings at a site in north-central Pennsylvania. Relationships were determined between visible ozone injury and ozone exposure, as well as calculated between injury and ozone uptake expressed as the product of stomatal conductance and ozone concentration. In addition, simultaneous measurements of visible symptoms and leaf gas exchange were also conducted to determine the correlation between visible and physiological injury and ozone exposure. By September, the amount of leaf area affected by visible foliar ozone injury was greatest for seedlings (46%), followed by canopy trees (20%) and saplings (15%). A large amount of variability in foliar ozone symptom expression was observed among trees within a size class. Sum40 and Sum60 (ozone concentration > 40 and > 60 nl liter-1) cumulative exposure statistics were the most meaningful indices for interpretation of foliar injury response. Seedlings were apparently more sensitive to ozone injury than larger trees because their higher rates of stomatal conductance resulted in higher rates of ozone uptake. Seedlings also had higher rates of early leaf abscission than larger trees with an average of nearly 30% of the leaves on a shoot abscised by 1 September compared to approximately 5% for larger trees. However, per unit ozone uptake into the leaf, larger trees exhibited larger amounts of foliar injury. The amount of visible foliar injury was negatively correlated (r e = 0.82) with net photosynthetic rates, but was not related to stomatal conductance. Net photosynthesis and stomatal conductance thus became uncoupled at high levels of visible foliar injury.

Keywords: Air pollution, black cherry, foliar injury, ozone, ozone exposure, ozone uptake, scaling.

INTRODUCTION

Ozone is perhaps the most important phytotoxic air pollutant affecting forest trees (Reich, 1987; Hogsett et al., 1988). Absorbed through leaf stomata, ozone causes foliar injury by oxidizing protein and lipid corn53

54

T . S . Fredericksen et al.

would be a more valuable bioindicator of ozone injury for forest health monitoring assessments if a demonstrated linkage existed between visible symptoms and physiological injury parameters, such as net photosynthesis or growth. Most of the present information on how ozone affects forest trees comes from studies conducted on seedlings. In order to predict the response of larger forest trees to ozone, more information is needed on how ozone exposure-response relationships may differ by tree size. Preliminary studies with black cherry indicate a potential for large differences in foliar injury among differentsized trees of this species (Fredericksen et al., 1995) and between individuals within a size class (Skelly et al., 1994). The objectives of this study were to (i) quantify the phenological development of visible injury symptoms (adaxial stipple and early leaf senescence) on black cherry by tree size, (ii) determine relationships between seasonal cumulative ozone exposure, calculated uptake, and visible foliar injury symptoms, and (iii) determine the strength of the relationship, if any exists, between the presence of visible symptoms of ozone injury and physiological injury manifested as impaired gas exchange. We tested the null hypotheses of no difference in the amount of visible foliar symptoms (adaxial stipple, early leaf senescence) among different-sized trees for both ozone exposure and ozone uptake. In addition, we tested the null hypothesis of no relationship between visible injury symptoms and two physiological measures, net photosynthesis and stomatal conductance. The study was conducted in the context of comparing the physiology and ozone injury-response of seedlings under conditions typical of open-top experiments to that of mature trees growing in an actual forest environment.

MATERIALS AND M E T H O D S Study site

The study site is located within the Moshannon State Forest in Clearfield County, Pennsylvania (lat. 41008 '

N, long. 78031' W, elev. 655 m). Study areas are located within a 3-km radius containing 70-80-year-old mixed hardwood forest stands with mature black cherry trees, clearings containing naturally-regenerated black cherry saplings, and a recently-cleared ( < 2 years) mixed forest stand containing planted black cherry seedlings from a central Pennsylvania seed source. Site description statistics are presented in Table 1. The seedling site was chosen based on what would be typical for the open plot of an open-top chamber experiment and not necessarily by an exact matching of physical site characteristics. Phenological development of foliar injury

Four individuals from each of three tree size classes (canopy trees, open-grown naturally-regenerated saplings, and open-grown planted seedlings) were selected for intensive study throughout the 1994 growing season. Canopy trees (21 m tall) consisted of two pairs of closely-spaced dominants selected from a larger population based on their similarity to other trees on the site and the ability to access the crowns of both trees within a pair from a single scaffold tower. Saplings (3-7 m tall) were selected in a similar manner. Seedlings were threeyear-old individuals, planted as 1 ~ nursery stock in the fall of 1992, selected randomly from a group of 250. Before budbreak in May 1994, five randomly selected shoots were permanently tagged in each of the upper and lower crown halves of each canopy and sapling trees. Because seedlings had a larger number of leaves per shoot than canopy trees and saplings, only three representative shoots were tagged on each tree of this size class. From 3 May to 1 September 1994, the percentage of symptomatic area relative to total leaf area for each leaf on all tagged shoots was recorded at approximately two-week intervals. Visible foliar ozone symptoms on this species are evident as a black, brown, or purple adaxial stipple (US EPA, 1975; Sinclair et aL, 1987; Skelly et al., 1989). Percentage foliar injury was estimated visually for each leaf using the Horsfall-Barratt classification scale (Horsfall and Barratt, 1945), which includes injury

Table 1. Soil properties on sites where measurement black cherry trees are located, including seedlings, saplings, and canopy trees. Few quantitative soils data are available for the sapling site. Soil moisture and soil temperature values are growing season means from measurements at depths of 10 and 30 cm

Soil property Soil series pH Nitrogen Phosphorus Potassium Magnesium Calcium CEC Mean soil moisture content (1994) Mean soil temperature (1994)

Units

Seedlings

Saplings

Canopy trees

--

Hazelton channery loam 4.6 0.70 42.6 0.05 0.30 1.00 9.10 20.79 11.20

Hazelton channery loam -------23.72 8.82

Cookport channery loam 4.7 0.65 30.3 0.04 0.12 0.52 9.80 21.44 9.07

-% kg ha -l meq 100g-1 meq 100g-l meq 100g-1 meq 100g-1 % of soil dry weight °C

--Site intermediate between seedlings and saplings.

Size-mediated foliar response to ozone in black cherry trees

percentage classes of 0, 3, 6, 12, 25, 50, 75, 88, 94, 97 and 100. Ratings were made independently by two observers trained to proficiency in symptom rating for black cherry using the ForestHealth expert system (Nash et al., 1992). A 10% resampling of leaves was conducted during each sampling for quality assurance in symptom ratings. Overall, there was an average agreement of 85% between initial ratings and resample ratings, and 92% of remeasurements were within one injury class of initial ratings. On each sampling date, the number of leaves was recorded on each shoot to document the lifespan of leaves. The area of each leaf was also measured non-destructively on each date with a card containing leaf shapes of known area so that the exposure area of foliage could be determined. Variation in visible fofiar injury symptoms Preliminary studies by the authors indicated potentially high variability among trees within a size class (Fredericksen et al., 1994; Skelly et al., 1994). However, restrictions in scaffolding equipment and time did not allow for evaluation of more than four individuals per size class. Sampling of the 12 trees (four per size class) for foliar injury symptom ratings involved making up to 1778 independent observations per sampling date. To verify that intensively-sampled trees for each size class were representative of the larger population of black cherry trees within that size class, visible ozone injury symptoms were evaluated once near the end of the growing season (early September) on a larger sample (20) of individuals for each tree size class. For canopy trees and saplings in that sample, three branch segments were randomly selected and removed by a tree climber from both the upper and lower crown for symptom ratings on the ground. Each branch segment had at least 30 leaves. For seedlings, each tree was rated on a wholecrown basis in the field. During these ratings, percentage of leaves affected on rated trees or tree branches was first estimated. Average percentage injury to leaves was then estimated visually for each tree or tree branch using the Horsfall-Barratt classification scale. Average percentage of leaves affected was then multiplied by percentage leaf injury to determine the percentage leaf injury relative to the total area of foliage rated. Similar to the intensively-monitored trees, ratings were made independently on the larger sample of trees by at least two observers trained to proficiency at rating per cent foliar surface injury for ozone-induced injury using the ForestHealth expert system. Relation of ambient ozone concentration, calculated uptake, and foliar symptoms Measurements of ozone concentrations were made with a TECO model-49 (Thermo Environmental Instruments, Inc., Franklin, MA, USA) ozone analyzers positioned at the mature canopy, sapling, and seedling sites described above. One analyzer was located near trees of each size class. Each monitor was calibrated bi-weekly using a TECO ozone calibrator certified for accuracy by the US Environmental Protection Agency (EPA). In

55

addition, near the end of the growing season (midSeptember), each monitor passed an EPA quality assurance audit. Air samples were drawn through teflon tubing from locations within the canopy boundary layer of an open grown black cherry seedling stand within the upper and lower crowns of a black cherry sapling stand and two mature-canopy black cherry trees. Ozone concentrations were sampled several times per hour at each location and averaged. Sampling was conducted on a 24-h basis from early May through to the end of August. Ozone uptake was calculated as the product of average hourly ozone concentration and stomatal conductance (Reich, 1987; Wieser & Havranek, 1993). Stomatal conductance measurements were made at weekly intervals from 3 May to 1 September on leaves distributed throughout the crown of the same opengrown seedlings, saplings, and canopy trees used in season-long ratings of foliar ozone injury symptoms. Measurements were made at 3-h intervals from 06:0000:00 h using two cross-calibrated Li-Cor 6200 photosynthetic systems (Li-Cor, Inc., Lincoln, Nebraska, USA). Dew formation on leaves often prevented sampling during the early morning hours. Stomatal conductance was assumed to be near zero from 00:0006:00 h. This assumption was verified by two testsampling periods conducted during this time in 1993 and 1994 (data not shown). During each sampling period, measurements were made on 16 leaves of one tree at each crown location for canopy trees, on eight leaves for one sapling, and on eight leaves of one seedling. A different individual was used for sampling each week, so that each study tree was sampled for gas exchange at least once per month. Using these values, a diurnal conductance curve using hourly means was constructed for each sampling day and extrapolated to a weekly basis. Hourly stomatal conductance values for each week were multiplied by matching ozone concentration values averaged for that week to calculate cumulative ozone uptake. Ozone uptake values calculated using stomatal conductance to water vapor were corrected to conductance to ozone by dividing by 1.68, the ratio of diffusivities between ozone and water vapor. Ozone concentrations within leaf intercellular spaces was assumed to be near zero (Laisk et al., 1989). Both exposure and uptake calculations were weighted by percentage leaf expansion relative to the seasonal maximum for a given tree size. This weighting adjusted for reduced whole-crown exposure early in the growing season compared with later in the season due to incomplete leaf area expansion for all sizes and for the free growth habit of seedlings and saplings later in the season compared to the fixed growth of canopy trees. Relation of visible symptoms and physiological foliar injury In late August, an additional eight planted black cherry seedlings and saplings were selected because they displayed a wide range in foliar injury symptoms on leaves within each of their crowns. Randomly selected leaves from these trees were measured for simultaneous

56

T . S . Fredericksen et al.

measures of leaf gas exchange and percentage visible foliar injury. Percentage of visible injury estimated to the nearest 5% was immediately followed by measurement of leaf net photosynthetic rate with a Li-Cor 6200 photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Gas exchange was measured while leaves were attached to the shoot using a 0.25 liter cuvette and a 30 s measurement period at ambient CO2 concentrations. Measurements were made under full sunlight on an 8.4 cm 2 area of leaf representative of the foliar injury level of the entire leaf. At least 15 measurements were made on each seedling or sapling using randomlyselected leaves, but incorporating the full span of injury variability for leaves of each seedling. Leaves selected for measurement were located from the middle portion of terminal or lateral long shoots. Measurements of other black cherry leaves growing in open-top chambers with charcoal filtered air indicated that net photosynthetic rates were fairly constant over the length of terminal or lateral long shoots, with the exception of decreased photosynthetic rates for leaves near the extreme basal and terminal portions of the shoot (Kouterick & Fredericksen, unpublished data). Data analysis

Foliar injury and leaf abscission data were averaged as per cent injury and leaf abscission rates over all leaves on permanently-tagged shoots. For stomatal conductance and ozone uptake rates, data were averaged over all leaf measurements within a sample period on each month for each size class. Mean monthly ozone concentrations were calculated for all average hourly concentrations using standard 24-h, 12 h (07:00-19:00 EST), and 7-h (09:00-16:00 EST) time periods. With the exception of ozone concentration data (which are derived from 1 ozone monitor per size class), tests for differences among size classes were performed with a Kruskal-Wallis one-way analysis of variance. For tests with a significant (a < 0.05) size effect, mean separation tests were conducted using Duncan's Multiple Range Test. Ozone exposure-response and ozone uptakeresponse curves were constructed using percentage adaxial stipple per unit of ozone exposure or uptake. Indices of exposure and uptake included SumO, Sum40, Sum60, and Sum80 statistics, representing the cumulative hourly concentrations greater than 0, 40, 60, and 80 nl liter -l by volume, respectively. Regression analysis was used to determine the significance and strength of the relationship between visible symptom percentage and physiological response variables. All statistical analyses were conducted using the procedures of Statistical Analysis Systems (SAS, Inc., Cary, NC, USA).

trees (Table 2), although higher nocturnal ozone concentrations resulted in higher 24-h average concentrations near canopy trees compared to smaller trees (Fig. 1). Foliar injury varied to a large degree among individuals within a size class (Fig. 2(a)-(c)). Foliar injury symptoms (adaxial stipple, Fig. 2(a)-(c); early leaf senescence, Fig. 3) first appeared in late June for seedlings and early July for larger trees. Thereafter, seedlings had significantly greater amounts of adaxial stipple and early leaf senescence than larger trees. By 1 September, intensively-sampled seedlings had an average of nearly one-half of their photosynthetic foliar surface area covered by adaxial stipple, compared to approximately 20% for canopy trees and 15% for saplings (Fig. 2(a)-(c)). The percentage of injury per Table 2. Monthly ambient ozone concentrations (nl liter-1) recorded from May to August within the canopy of seedling, sapling, and canopy black cherry trees in the Moshannon State Forest, Clearfield County, Pennsylvania. Concentrations are presented as 7-h (09:00-16:00 EST), 12-h (07:00-19:00 EST), and 24-h averages with one standard error in parentheses

Tree size

May

June

July

August

46.77 (1.03) 44.49 (0.88) 44.71 (0.87)

52.51 (1.06) 51.91 (0.97) 50.26 (0.98)

52.12 (1.01) 52.08 (1.02) 48.79 (1.05)

43.86 (1.06) 43.68 (1.03) 47.88 (1.11)

44.87 (0.76) 42.44 (0.66) 42.91 (0.66)

48.27 (0.82) 47.95 (0.77) 47.96 (0.76)

46.55

(0.83) 46.96 (0.83) 46.22 (0.84)

38.93 (0.79) 39.27 (0.78) 44.66 (0.81)

38.26 (0.60) 36.56 (0.54) 40.50 (0.47)

39.47 (0.66) 37.46 (0.69) 45.92 (0.52)

37.97 (0.62) 38.00 (0.64) 44.60 (0.55)

32.63 (0.57) 33.11 (0.59) 42.44 (0.53)

7 h average

Seedlings Saplings Canopy trees 12 h average

Seedlings Saplings Canopy trees 24 h average

Seedlings Saplings Canopy trees

% Symptomatic Leaf Area SO 40 30 20 10

0±~ ~~ 0

:

.

. . 10

.

20

30

40

50

Ozone Uptake (retool/m2)

RESULTS Phenologicai development of foliar injury

Diurnal ozone concentrations were similar within the canopy boundary layer of different-sized black cherry

Seedlings

---4--

Saplings

--x(--Canopy

Fig. 1. Hourly ambient ozone concentrations averaged over the growing season within the crown of seedling, sapling, and canopy black cherry trees from May to September 1994 at Moshannon State Forest, Pennsylvania.

Size-mediated foliar response to ozone in black cherry trees

57

% Absciaaed Leavea

% Symptomatic Leaf Area

30 26

6o

20

/

40

/

15

/

3O 10 / 5

10 = 5/16

0 6103

e 6/01

6/14

6/29

7/16

a/o3

e/19

9/01

j J

5103

5116

6101

--

Seedling 1

-~

Seedling 2

~

711661036119

61146129

Date

s

9101

Date Seedling 3

~ ; ~ eeedllng 4 --

Seedlings

Saplings

~

~

Canopy Trees

(a)

80

Fig. 3. Average percentage leaf abscission on permanentlytagged shoots of seedling, sapling, and canopy black cherry trees from 3 May to l September 1994 at Moshannon State Forest, Pennsylvania. Kruskal-Wallis ANOVA tests for overall differences among tree sizes by date and Duncan's multiple range test for mean separation between size classes: (Seedlings = SE, S a p l i n g s = SA, Canopy Trees = CT). 6/29: X 2 =

% Symptomatic Leaf Area

70 60 50 40

0.002, SE > C T = SA; 7/15:.y2 = 0.0002, SE > SA = CT; 8/03: X ~ = 0.0001, SE > S A = CT; 8/19:.t,2 = 0.0004, SE > SA = CT; 9/01: X 2 = 0.0001, SE > SA = CT.

jJ

30 20 10 0

- -

5103

i¢ 6•29

i

5/16

67Ol

* -6/14

7118

8103

8/19

9/01

Date Sapling 1

~

Sapling 2

~

Sapling 3

-g~ Sapling 4

(b) 80

% Symptomatic Leaf Area

70 ~

6° I

50

4o i ao}

10 0 5103

6116 '

6 ;o 1

6/14 :

6•29

7116

8108

8119

9101

Date --

Tree 1

~

Tree 2

~

Tree 3

' - ~ - Tree 4

(c) Fig. 2. Average percentage leaf area affected by ozone injury symptoms (dark adaxial stipple) on permanently-tagged shoots of (a) seedling, (b) sapling, and (c) canopy black cherry trees from 3 May to 1 September 1994 at Moshannon State Forest, Pennsylvania. Kruskal-Wallis AOV tests for overall mean differences among tree sizes by date and Duncan's multiple range test for mean separation between size classes: (Seedlings = SE, S a p l i n g s -- SA, Canopy Trees = CT). 6/29: X 2 = 0.006, SE > S A = CT; 7/15: X 2 = 0.001, SE > C T = SA; 8/03: X 2 = 0.01, SE > S A = CT; 8/19: X ~ = 0.06, S E > C T = SA; 9/01: X 2 = 0.05, SE > C T = SA.

1 September, nearly one-third of intensively-sampled seedling leaves had abscised compared with less than 5% for saplings and canopy trees (Fig. 3). The relative expansion in leaf area through the growing season on leaves of long (nonspur) shoots followed a similar pattern for each tree size class with the maximum leaf area reached around mid-July (Fig. 4). Seedlings had a greater number of leaves on long shoots compared to larger trees, averaging 23.1 (3.7 SE) leaves per shoot upon full leaf area expansion compared to 12.7 (1.5 SE) leaves per shoot for saplings and 11.3 (1.2 SE) leaves per shoot for canopy trees. Seedlings and saplings (to a lesser extent) displayed free growth (new development) on some shoots throughout the growing season, a trait not observed for canopy trees. Seedlings had the greatest amount of foliar injury (adaxial stipple) per unit ozone exposure of all tree size classes for SumO, Sum40, and Sum60 indices (Fig. 5(a)-(c)). Saplings appeared to have greater injury per unit exposure than seedlings for the Sum80 index,

1oo% of Maximum Leaf AreaJ~:~--~ . ~ : ~ 60

4O

2O 0 6103

individual tree was nearly identical for intensivelysampled seedlings (4) and seedlings in the larger sample (20). Intensively-sampled saplings showed less injury than the larger sapling sample by 12.5% while intensively sampled canopy trees appeared to be more sensitive than the larger canopy sample by 15%. Also by

6116

6101

6114

6129

7116

L 6103

i 6119

9101

Date --

Seedlings

--+-- Saplings

~

Canopy Trees

Fig. 4. Leaf area as a percentage of the season maximum on permanently-tagged shoots of seedling, sapling, and canopy black cherry trees from 3 May-1 September 1994 at Moshannon State Forest, Pennsylvania.

58

T . S . Fredericksen et al. % Symptomatic Leaf Area

6o

60

40

40

30

30

20

20

10

10

6: 0

20

40

60

80

100

120

140

% Symptomatic Leaf Area

13 0

6

Ozone Exposure > 0 nl/I (ppm.hrs) Seedlings

Saplings

~

~

10

15

Canopy Trees

Seedlings

----- Saplings

(a) 6o

% 60

40

40

30

30

20

20

10

10 .

.

.

.

20

.

.

I 40

Seedlings

60

----- Saplings

30

-~-- Canopy Tree'*

80

/ ,

0 100

Symptomatic Leaf Area

0

~

4"

I

0.5

~

J

1

1.5

2

2.5

3

3.5

Ozone Exposure > 80 nl/I (ppm.hrs)

Ozone Exposure > 4 0 nl/I (ppm.hrs) --

28

(b)

% Symptomatic Leaf Area

0

20

Ozone Exposure > 60 nl/I (ppm.hrs)

Canopy Trees

Seedlings

(c)

--4-- Saplings

- - ~

Canopy Trees

(d)

Fig. 5. Average percentage leaf area affected by ozone injury symptoms (dark adaxial stipple) on permanently-tagged shoots of seedling, sapling, and canopy black cherry trees as a function of ozone exposure calculated as: (a) the sum of all average hourly ozone concentrations greater than 0 nl liter -I (Sum0); (b) the sum of all average hourly ozone concentrations greater than 40 nl liter-l (Sum40); (c) the sum of all average hourly ozone concentrations greater than 60 nl 1-1 (Sum60); (d) the sum of all average hourly ozone concentrations greater than 80 nl liter-1 (Sum80).

but exposure-response curves became vertical when using this statistic (Fig. 5(d)). Saplings had a similar ozone-response curve to canopy trees using the SumO statistic (Fig. 5(a)), but appeared to have greater injury per unit exposure than canopy trees when the Sum40 or Sum60 statistic was used (Fig. 5(b)-(c)). With the exception of the Sum80 statistic, threshold exposures for injury were similar by size class: approximately 60 ppm h for SumO, 40 ppm h for Sum40, and 10 ppm h for Sum60. Seedlings had significantly higher rates of stomatal conductance (Table 3) and ozone uptake (Table 4) than larger trees during May and June, although rates were more similar among tree-size classes in July and August. Canopy trees had greater amounts of adaxial stipple per unit ozone uptake than smaller trees for SumO, Sum40, and Sum60 statistics (Fig. 6(a)-(c)). Again, response curves using the Sum80 statistic were difficult to interpret (Fig. 6(d)). Seedlings and saplings had similar uptake-response curves using the SumO and Sum40 (Fig. 6(a)-(b)) statistics, but saplings had greater amounts o f foliar injury than seedlings using the Sum60 statistic (Fig. 6(c)) As with exposure-response curves, threshold levels for visible injury appeared to be con-

Table 3. Monthly mean rates of stomatal conductance (mol m -2 s -1) recorded from 09:00-16:00 EST during May and August on leaves of seedling, sapling, and canopy black cherry trees (n = 4 for each size) in the Moshannon State Forest, Cleadleld County, PA. One standard error of the mean is presented in parentheses. The same letters following means indicate no statistical difference at ~ = 0.05 using Duncan's multiple range test. Results of the Kruskal-Wallis one-way A N O V A are presented below

Tree size

May

June

July

August

0.24a (0.02) Saplings 0.1 lb (0.01) Canopy trees 0.1 lb (0.01) ANOVA for size (df = 2)

0.47a (0.03)

0.54a (0.04)

0.58a (0.04)

(0.03) 0.18c (0.01)

(0.05) 0.43b (0.03)

(0.09)

X2= P > X2

17.20 0.0002

1.29 0.525

1.78 0.41

Seedlings

7.58 0.02

0.28b

0.50a

0.62a

(0.02) 0.50a

sistent among tree size classes for SumO (approximately 10-20 mmol m -2) and Sum 40 (approximately 8-15 mmol m-2), and for Sum60 (3-8 mmol m-2).

Size-mediated foliar response to ozone in black cherry trees Table 4. Monthly mean rates of ozone uptake (pmol m - z h -1) computed from simultaneous hourly (from 07:00-19:00 EST) measurements of stomatal conductance and local ozone concentration. Measurements were made from May to Angust on leaves of seedling, sapling, and canopy black cherry trees (n = 4 for each size) in the Moshaunon State Forest, Clearlield County, PA. One standard error of the mean is presented in parentheses. The same letters following means indicate no statistical difference at ~ = 0.05 using Duncan's multiple range test. Results of the KruskaI-Wams one-way analysis of variance (ANOVA) are presented below

Tree size

May

Seedlings

44.83a (2.97) Saplings 19.45b (2.06) Canopy trees 13.78c (1.17) ANOVA for size (df = 2) .t/2 = 25.91 P > X2 0.0001

June

July

August

41.79a (4.01) 27.73b (2.34) 26.05b (2.47)

45.10a (3.81) 37.31a (3.47) 26.28b (2.77)

31.08a (3.43) 28.56a (3.13) 24.31a (3.02)

10.46 0.005

12.61 0.002

4.41 0.1104

Percentage visible foliar ozone injury observed as adaxial stipple on individual leaves o f black cherry

seedlings and saplings was negatively related (r 2 = 0.82, P < 0.0001) to the net photosynthetic rate of those leaves (Fig. 7(a)). However, there was no significant relationship between per cent visible foliar injury and stomatal conductance (Fig. 7(b)). As a result, stomatal conductance and net photosynthetic rates became uncoupled on leaves with large amounts of visible foliar injury as indicated by increasing stomatal conductance to net photosynthesis ratios (Fig. 7(c)).

DISCUSSION

The greater foliar ozone injury levels with decreasing tree size in this study suggest that seedlings may be more sensitive to ozone than larger trees. These results are consistent with those from a previous survey with these species (Fredericksen et al., 1995). The few other studies c o m p a r i n g response to ozone o f different-sized trees have yielded conflicting results. Grulke and Miller (1994) found that seedling giant sequoia trees (Sequoiadendron giganteum Bucholz.) were m o r e sensitive than m a t u r e trees. However, studies with n o r t h e r n red o a k (Quercus rubra L.) (Edwards et al., 1993; H a n s o n et al.,

% Symptomatic Leaf Area

% Symptomatic Leaf Area

50

50

40

4O

30

30

2O

2O

10

10

/ /

/

0

10

20

30

•

--~

Sapling=

,

10

20

Ozone Uptake --

--~- Canopy

Seedlings

~

(a)

40

40

3O

30

0

40

(retool/m2)

Saplings

~

Canopy

% Symptomatic Leaf Area

50

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60

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Percent Leaf Area Affected (c) Fig. 7. Relation of instantaneous measurements of.' (a) net photosynthetic rate, (b) stomatal conductance, and (c) the ratio of stomatal conductance to water vapor (g) to net photosynthesis (A) of individual leaves and the percentage of area of the same leaf affected by dark adaxial stipple. Measurements were conducted on eight seedling/sapling black cherry trees. 1994; Samuelson & Edwards, 1994) found that the net photosynthesis, apparent quantum yield, and carboxylation efficiency of mature trees was more negatively affected by ozone than seedlings. It is apparent that more such scaling studies incorporating a wider range of species will be required to determine if there are any consistent patterns among tree species ofi.size-related differences in sensitivity to ozone.

Increases in foliar injury with decreasing tree size in this study were not uniform or linear. Amounts of adaxial stipple and early leaf senescence decreased dramatically after the seedling stage and injury amounts were often similar on sampling dates for canopy trees and saplings. Canopy trees had a greater amount of injury on average than saplings (Fig. 2), perhaps due to a reduced whole-crown exposure for some sapling shoots that grew indeterminately (i.e. produced new leaves). Canopy trees selected for intensive study also appeared to be slightly more symptomatic than a larger sample of local canopy trees, while saplings were slightly less sensitive. Although not tested statistically, canopy trees appeared to also have higher ozone exposure than saplings or seedlings during morning and evening hours and also late in the growing season (Fig. 1, Table 2). Ozone concentrations were much higher at night for canopy trees than saplings or seedlings (Fig. 1), reflected in greater differences in ozone exposure among tree sizes in 24-h averages than in 7- or 12-h diurnal averages. Dew formation on leaves and lowlying fog was often observed near seedlings and saplings during the evening and early morning hours, which was not observed near canopy trees. Scavenging of ozone by ground level moisture may have accounted for lower ambient concentrations near saplings and seedlings. Such differences in nocturnal ozone concentrations probably had little effect on ozone uptake, since no significant nocturnal stomatal conductance was observed in this study (data not shown). Some dilution of percentage foliar stipple occurred for seedlings, and for larger trees to a lesser extent, because of early leaf abscission. Similar to Davis and Skelly (1992a) leaves that abscised early tended to be older and had relatively large amounts of adaxial stipple. Early leaf senescence is a commonly observed response to ozone in black cherry and other species (Jensen, 1981; Reich & Lassoie, 1984; Wang et al., 1986; Keller, 1988; Simini et al., 1992; Stow et al., 1992). Early leaf abscission may have been enhanced in seedlings because of their rapid growth rates and indeterminate shoot growth habit and may be indicative of a greater ability to compensate for foliar injury compared to larger trees. Seedlings produced a large number of leaves on long apical and lateral shoots, a pattern that is consistent for trees with rapid growth rates. Woodbury et al. (1994) found that hybrid poplar (Populus sp.) leaves exposed to ozone compensated for early leaf abscission by an increase in new leaf production. Trees with free growth, like seedlings in this study, may be able to remobilize nutrients from older leaves injured from ozone exposure and translocate them to newly developing leaves (Jensen, 1982; Pye, 1988; Laurence et al., 1994; Pell et al., 1994). In contrast, the fixed growth pattern of canopy trees may have limited the ability for compensation of damage to older leaves. For most exposure indices, threshold levels of ozone exposure for adaxial stipple were surprisingly similar for trees within a size class. However, despite a similarlyshaped response curve, threshold exposures were not

Size-mediated foliar response to ozone in black cherry trees

similar to another field study conducted with black cherry using identical methods. Hildebrand et al. (in press) found that threshold levels for symptom response of leaves on canopy black cherry trees in the Shenandoah National Park in Virginia did not occur until after 140 ppm h for SumO and 35 ppm h for Sum60. Comparative injury threshold levels in this study were much lower (70 ppm h for Sum0 and 15 ppm h for Sum60). Exposure-response studies may not show concurrence because exposure to ozone does not necessarily indicate entry into the leaf mesophyll, the principle action site for ozone injury. Other studies have advocated the use of measured or calculated ozone uptake rather than ambient exposure for use in dose-response assessments because of its greater physiological relevance (Reich, 1987; Wieser & Havranek, 1993). The decreasing foliar injury symptoms with increasing tree size in this study is consistent with observations that ozone sensitivity increases with increasing growth rates of plants (Reich & Amundson, 1985; Reich, 1987; Adams et al., 1988), perhaps because of a general correlation of growth rates and high stomatal conductance with ozone uptake. When injury is compared on the basis of uptake rather than exposure, ozone injury sensitivity of different-sized trees appears to be more similar. Interestingly, for Sum60 uptake, the order of apparent sensitivity is reversed, with canopy trees more sensitive per unit uptake than saplings and seedlings. Differences in injury might occur because of a greater cumulative ozone exposure to leaves with increasing tree size associated with fixed shoot growth. However, exposure differences due to shoot growth phenology should have been removed by the weighting of exposure and uptake by relative leaf area expansion. The greater sensitivity of canopy trees per unit uptake might also be partially explained by the chance selection of a group of measurement trees that is somewhat more sensitive than the larger population on the site. However, this reasoning does not explain the greater sensitivity per unit uptake of saplings than seedlings. Stomatal conductance is the principal regulator of ozone uptake (Heath, 1980; Guderian et al., 1985). Uptake of ozone into the leaf through the stomata is thought to be four orders of magnitude greater than uptake through fractures in the leaf cuticle (Kerstiens & Lendzian, 1989). Ozone uptake is thus largely related to stomatal conductance because stomatal guard cells control diffusion of ozone into the leaf. Because of this important regulatory role in uptake, it has been proposed that differences in ozone sensitivity within a species could be explained to a large degree by differences in stomatal conductance (Harkov & Brennan, 1979; Tingey & Taylor, 1982; Reich & Amundson, 1985; Runeckles, 1992). Seedlings in this study had greater ozone uptake rates, and apparently greater foliar ozone injury, because of their higher rates of conductance. However, Fredericksen et al. (1995) found greater injury in lower compared to upper crown leaves of black cherry trees despite greater uptake for upper crown

61

leaves. These results, as well as those from other studies (Tjoelker et al., 1993; Volin et al., 1993) indicate that other factors, besides stomatal conductance and ozone uptake, may be important in determining ozone injury. Tjoelker et al. (1993) proposed that the ratio of stomatal conductance, or uptake, to net photosynthesis could possibly be used as an index of sensitivity to ozone because more photosynthate is necessary with increasing ozone uptake for anti-oxidant defense and repair of damaged tissue. However, such an index was not useful in predicting ozone sensitivity in this study. Bennett et al. (1992) and Fredericksen et al. (1995) found a close correlation between ozone sensitivity and leaf thickness in black cherry. The pathway of ozone from the leaf surface to target sites within the cell is less than a millimeter, but processes occurring at this level may be important in determining the effects of ozone on the plant (Taylor & Hanson, 1992). More studies are needed to determine the physiological or biochemical mechanisms of genotypic sensitivity in forest trees. For both exposure and uptake response curves, Sum40 and Sum60 indices appeared to be the most meaningful in interpreting foliar injury response. The Sum0 index shows an identical exposure-response curve for saplings and canopy trees, but a separation between saplings and canopy trees is indicated by Sum40 and Sum60 indices. Sum60 uptake-response curves also show this separation, while Sum0 and Sum40 curves do not. The aberrant (vertical) portions of Sum80 curves suggest that significant responses to ozone occur at concentrations lower than 80 nl liter -1. Other studies on many types of plants have shown that sum statistics that emphasize higher ozone concentrations, such as Sum60, and functions that give greater weighting to high concentrations, perform better than cumulative measures or mean values that use all concentrations greater than zero (Lefohn & Runeckles, 1987; Hogsett et al., 1988; Krupa et al., 1994a,b; Hildebrand et al., 1996). However, recent studies suggest that moderately high ozone concentrations may be more important than extreme concentrations, often because high ozone concentrations are often out of phase with periods of high ozone flux into canopies or high stomatal uptake (Grunhage et al., 1993; Krupa et al., 1994a,b; Legge et al., 1994). No evidence of compensation for visibly injured leaf mesophyll cells by uninjured ones in the same leaf was observed in this study. Visible foliar injury percentage was negatively correlated with net photosynthesis for individual leaves of this species. Samuelson (1994) also observed that reduction in leaf gas-exchange and growth occurred along with visible foliar injury for black cherry seedlings. A preliminary study with this species (Fredericksen & Skelly, 1994) also suggests that there is little detrimental effect of ozone on net photosynthesis for leaves prior to symptom development. These results suggest that black cherry may be a useful indicator species, not only for the presence of high ozone levels, but also for the occurrence of detrimental physiological effects due to exposure to the pollutant. The relation of visible injury to other negative effects of

62

T . S . Fredericksen et al.

ozone, such as reduced growth, altered carbon allocation, or increased respiration needs to be further investigated for this species. One limitation to the usefulness of black cherry as a bioindicator is the large apparent intra-species variation in sensitivity (Fig. 2). Sensitive and tolerant genetic lines have been identified for this species (Simini et al., 1992; Skelly et al., 1994), which will be essential for controlling variability if the species is to be used as a bioindicator of ozone.

SUMMARY Seedlings had greater absolute amounts of foliar stipple and early leaf senescence than saplings or canopy trees. However, both seedlings and saplings had some ability to compensate for injury because of their indeterminate shoot growth which lowered overall exposure of leaves to ozone and afforded some ability to compensate for injury on older leaves by production of new undamaged ones. Greater foliar injury sensitivity of younger trees may be related to greater rates of stomatal conductance and ozone uptake. After the greater ozone uptake rates o f seedlings were accounted for, seedlings showed less ozone injury at a given uptake level than larger trees, suggesting that factors not related to uptake may also be important in determining injury. Adaxial leaf stipple was negatively related to net photosynthetic rates in this species. In addition, threshold levels for exposure and uptake were similar for different-sized trees. These results, along with consistent visible sensitivity to ozone when exposed to high concentrations, indicates that black cherry may be a valuable ozone indicator species. In this study, ozone, environmental and plant genetic effects cannot be separated. However, large amounts of phenotypic variation in foliar injury need to be controlled when this species is used as an ozone bioindicator. ACKNOWLEDGEMENTS This study was funded by the United States Environmental Protection Agency cooperative agreement CR820417q)l-0 with additional cooperation from the Pennsylvania Department of Environmental Resources, Bureau of Forestry Region No. 9; Penelec Corporation, GPU; and the Pennsylvania Conservation Corps. Although the research described in this article has been funded wholly or in part by the US Environmental Protection Agency agreement CR820417-01-0, it has not been subjected to the Agency's review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. The authors thank Jim Savage, Ken Snyder, Brian Joyce and Jon Ferdinand for their assistance in this study.

REFERENCES Adams, M. B., Kelly, J. M. & Edwards, N. T. (1988). Growth of Pinus faeda L. seedlings varies with family and ozone exposure level. Water, Air Soil Pollut., 38, 137-50.

Bennett, J. P., Rassat, P., Berrang, P. & Karnosky, D. F. (1992). Relationships between leaf anatomy and ozone sensitivity of Fraxinus pennsylvanica Marsh. and Prunus serotina Ehrh. J. Exp. Bot., 32, 33-41. Brantley, E. A., Anderson, R. L. & Smith, G. (1994). How to Identify Ozone Injury on Eastern Forest Bioindicator Plants. USDA Forest Service Protection Report R8-PR 25, Southeast Forest Experiment Stafon, Asheville, NC, USA, 6 pp. Davis, D. D. (1983). Prumos serotina--a perennial bio-indicator plant to detect ozone. Aquilo. Ser. Bot., 19, 197. Davis, D. D. & Skelly, J. M. (1992a). Foliar sensitivity of eight eastern hardwood species to ozone. J. Air, Water Soil Pollut., 62, 269-77. Davis, D. D. & Skelly, J. M. (1992b). Growth response of four species of eastern hardwood tree seedlings exposed to ozone, acidic precipitation, and sulfur dioxide. J. Air. Waste Manage. Assoc., 42, 309-11. Edwards, G. S., Wullschleger, S. D. & Kelly, J. M. (1993). Growth and physiology of northern red oak: Preliminary comparisons of mature tree and seedling response to ozone. Environ. Pollut., 83, 215-21. US EPA (1975). Diagnosing Vegetation Injury Caused by Air Pollution. US Environmental Protection Agency, Washington, DC. Fredericksen, T. S. & Skelly, J. M. (1994). Relation of visible and physiological ozone injury in two eastern hardwood tree species. Phytopathol., 84(11), 1371. Fredericksen, T. S., Skelly, J. M., Kouterick, K. B., Joyce, B. J., Kolb, T. E., Steiner, K. C., Savage, J. E. & Snyder, K. R. (1994). Levels of variation in foliar ozone injury among black cherry trees. Phytopathol., 84, 543. Fredericksen, T. S., Joyce, B. J., Skelly, J. M., Steiner, K. C., Kolb, T. E., Kouterick, K. B., Savage, J. E. & Snyder, K. R. (1995). Physiology, morphology, and ozone uptake of seedlings, saplings, and canopy black cherry trees. Environ. Pollut., 89(3) 273-83. Grulke, N. E. & Miller, P. R. (1994). Changes in gas exchange characteristics during the life span of giant sequoia: Implications for response to current and future concentrations of atmospheric ozone. Tree Physiol., 14, 659-68. Grunhage, L., Dammgen, U., Haenel, H.-D., Jager, H.-J., Holl, A., Schmitt, J. & Hanewald, K. (1993). A new potential air quality criterion derived from vertical flux densities of ozone and from plant response. Angew. Bot., 67, 9-13. Guderian, R., Tingey, D. T. & Rabe, R. (1985). Effects of photochemical oxidants on plants. In Air Pollution by Photochemical Oxidants, ed. R. Guderian. SpringerVerlag, NY, USA, pp. 129-46. Hanson, P. J., Samuelson, L. J., Wullschleger, S. D., Tabberer, T. A. & Edwards, G. S. (1994). Seasonal patterns of light-saturated photosynthesis and leaf conductance for mature and seedling Ouercus rubra L. foliage: differential sensitivity to ozone exposure. Tree Physiol., 14, 1351-66. Harkov, R. & Brennan, E. (1979). An ecophysiological analysis of the responses oftrees to air pollution. J. Air Pollut. Contr. Assoc., 29, 157-61. Heath, R. L. (1980). Initial events in injury to plants by air pollutants. Ann. Rev. Plant. Physiol., 31,395-430. Hildebrand, E. S., Skelly, J. M. & Fredericksen, T. S. (1996). Foliar response of ozonesensitive hardwood tree species in the Shenandoah National Park, Virginia. Can. J. For. Res. Hogsett, W. E., Tingey, D. T. & Lee, E. H. (1988). Ozone exposure indices: Concepts for development and evaluation of their use. In Assessment of Crop Loss from Air Pollutants, ed. W. W. Heck, O. C. Taylor, & D. T. Tingey, Proc. Int. Conf. Elsevier Applied Science, London, pp. 107-38.

Size-mediated foliar response to ozone in black cherry trees Horsfall, J. G. & Barratt, R. W. (1945). An improved grading system for measuring plant disease. Phytopathol., 35, 655. Jacobsen, J. (1982). Ozone and the growth and productivity of agricultural crops. In Effects of Gaseous Air Pollutants in Agriculture and Horticulture, ed. M. H. Unsworth & D. P. Ormrod. Butterworth, London, pp. 293-304. Jensen, K. F. (1981). Growth analysis of hybrid poplar cuttings fumigated with ozone and sulphur dioxide. Environ. Pollut., 26, 243-50. Jensen, K. F. (1982). An analysis of the growth of silver maple and eastern cottonwood seedlings exposed to ozone. Can. J. For. Res., 12, 420-4. Keller, T. (1988). Growth and premature leaf fall in American aspen as bioindicators of ozone. Environ. Pollut., 52, 183-92. Kerstiens, J. & Lendzian, K. J. (1989). Interactions between ozone and plant cuticles. I. Deposition and ozone permeability. New Phytol., 112, 13-19. Kouterick, K. K., Skelly, J. M. & Fredericksen, T. S. (1994). Relationships between foliar ozone injury and physiology among black cherry genotypes. Phytopathol., 84, 544. Krupa, S. V., Nosal, M. & Legge, A. H. (1994a). Ambient ozone and crop loss: Establishing a cause-effect relationship. Environ. Pollut., 83, 269-74. Krupa, S. V., Grunhage, H.-J., Nosal, M. M., Manning, W. J., Legge, A. H. & Hanewald, K. (1994b). Ambient ozone (03) and adverse crop response: A unified view of cause and effect. Environ. Pollut., 87, 119-26. Laisk, A. O., Kull, O. & Moldau, H. (1989). Ozone concentration in leaf intercelluar air spaces is close to zero. Plant Physiol., 90, 1163-7. Laurence, J. A., Amundson, R. G., Friend, A. L., Pell, E. J. & Temple, P. J. (1994). Allocation of carbon in plants under stress: An analysis of the ROPIS experiments. J. Environ. Qual., 23, 412-7. Lefohn, A. S. & Runeckles, V. C. (1987). Establishing a standard to protect vegetation ozone/dose considerations. Atmos. Environ., 21, 561-8. Legge, A. H., Krupa, S. V. & Percy, K. E. (1994). Tropospheric ozone and the protection of forests. In Proc. IUFRO Meet. on Air Pollut. & Multiple Stresses, Fredericton, New Brunswick, Canada, 7-9 September 1994, p. 35. Long, R. P. & Davis, D. D. (1991). Black cherry growth response to ambient ozone and EDU. Environ. Pollut., 70, 241-54. Mudd, J. B. (1984). Effects of oxidants on metabolic function. In Gaseous Air Pollutants and Plant Metabolism, ed. M. J. Koziol & F. R. Whatley. Butterworth, Boston, USA, pp. 189-203. Nash, B. L., Saunders, M. C., Miller, B. J., Bloom, C. A., Davis, D. D. & Skelly, J. M. (1992). ForestHealth, an expert system for assessing foliar and crown health of selected northern hardwoods. Can. J. For. Res., 22, 1770-5. Pell, E. J., Temple, P. J., Friend, A. L., Mooney, H. A. & Winner, W. E. (1994). Compensation as a plant response to ozone and associated stresses: An analysis of ROPIS experiments. J. Environ. Qual., 23, 429-36. Pye, J. M. (1988). Impact of ozone on the growth and yield of trees: A review. J. Environ. Qual., 17, 347-60. Reich, P. B. (1987). Quantifying plant response to ozone: A unifying theory. Tree Physiol., 3, 63-91. Reich, P. B. & Amundson, R. G. (1985). Ambient levels of 03 reduce net photosynthesis in tree and crop species. Science, 230, 566-70. Reich, P. B. & Lassoie, J. P. (1984). Effect of low level ozone exposure on leaf diffusive conductance and water use efficiency in hybrid poplar. Plant Cell Eviron., 7, 661-8.

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Reich, P. B., Schoettle, A. W., & Amundson, R. G. (1986). Effects of 03 and acidic rain on photosynthesis and growth in sugar maple and northern red oak seedlings. Environ. Pollut., 40, 1-15. Roberts, T. M. (1984). Effects of air pollutants in agriculture and forestry. Atmos. Environ., 18, 629-52. Runeckles, V. C. (1992). Uptake of ozone by vegetation. In Surface Level Ozone Exposures and their Effects on Vegetation, ed. A. S. Lefohn. Lewis Publishers Inc., Chelsea, MI, USA, pp. 157-88. Samuelson, L. J. (1994). Ozone exposure-responses of black cherry and red maple seedlings. Environ. Exp. Bot., 34, 355-62. Samuelson, L. J. & Edwards, G. S. (1994). A comparison of sensitivity to ozone in seedlings and trees of Quercus rubra L. New Phytol., 125, 373-9. Simini, M. J., Skelly, J. M., Davis, D. D., Savage, J. E. & Comrie, A. C. (1992). Sensitivity of four hardwood species to ambient ozone in northcentral Pennsylvania. Can. J. For. Res., 22, 1789-99. Sinclair, W. A., Lyon, H. H. & Johnson, W. T. (1987). Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY. Skelly, J. M., Davis, D. D., Merrill, W., Cameron, E. A., Brown, H. D., Drummond, D. B. & Dochinger, L. S. (ed.) (1989). Diagnosing Injury to Eastern Forest Trees. Agricultural Information Services, College of Agriculture, Pennsylvania State University, University Park, PA, USA, 122 pp. Skelly, J. M., Savage, J. E., Snyder, K. R., Fredericksen, T. S. & Kouterick, K. B. (1994). Response of open-pollinated black cherry families to ambient ozone under open-top chamber conditions. Phytopathol., 23, 547. Stoklasa, J. (1923). Die Beschadigung der Vegetation durch Rauchgase und Fabriksexhalationen. Urban and Schwartzenberg Verlag, Berlin und Wien, Germany, 487 pp. Stow, T. K., Allen, H. L. & Kress, L. W. (1992). Ozone impacts on seasonal foliage dynamics of young Ioblolly pine. For. Sci., 38, 102-19. Taylor, G. E. Jr & Hanson, P. J. (1992). Forest trees and tropospheric ozone: Role of canopy deposition and leaf uptake in developing exposure-response relationships. Agric. Ecosys. Environ., 42, 255-73. Tingey, D. T. & Taylor, G. E. Jr (1982). Variation in plant response to ozone: a conceptual model of phsyiological events. In Effects of Gaseous Air Pollutants on Agriculture and Horticulture, ed. M. H. Unsworth & D. P. Ormrod. Butterworth, London, pp. 113-38. Tjoelker, M. G., Volin, J. C., Oleksyn, J. & Reich, P. B. (1993). Light environment alters response to ozone stress in seedlings of Acer saccharum Marsh. and hybrid Populus L. I. In-situ net photosynthesis, dark respiration, and growth. New Phytol., 124, 627-36. Volin, J. C., Tjoelker, M. G., Oleksyn, J. & Reich, P. B. (1993). Light environment alters response to ozone stress in seedlings of Acer saccharum Marsh. and hybrid Populus L. II. Diagnostic gas exchange and leaf chemistry. New Phytol., 124, 637--46. Wang, D., Karnosky, D. F. & Bormann F. H. (1986). Effects of ambient ozone on the productivity of Populus tremuloides Michx. grown under field conditions. Can. J. For. Res., 16, 47-55. Wieser, G. & Havranek, W. M. (1993). Ozone uptake in the sun and shade crown of spruce: Quantifying the physiological effects of ozone exposures. Trees, 7, 227-32. Woodbury, P. B., Laurence, J. A. & Hudler, G. W. (1994). Chronic ozone exposure alters the growth of leaves, stems, and roots of hybrid Populus. Environ. Pollut., 85, 103-8.