AN ABSTRACT OF THE THESIS OF
Peter K. Ziminski for the degree of Master of Science in General Science
presented on June 24 1993.
Title: Ozone Caused Chances in Competition Between Western Wheatgrass and
Sideoats Grama
Abstract Approved:
Redacted for privacy
William E. Winner
In both agricultural and natural ecosystems, plants grow in competition with
other plants for many resources. Competition is just one of the multiple stresses
plants must cope with simultaneously. Few studies have looked at the effects of
tropospheric ozone (03) on plants with the additional stress of competition.
The goals of these experiments were: 1) quantify the role of 03 in modifying
intraspecific and interspecific competition, 2) determine if monocultures and
mixtures of these species are equally sensitive to 03, 3) determine whether intra-
and inter-specific competition are equally sensitive to 03, 4) quantify 03 uptake
for two grasses with differing 03 absorption capacities, so that 5) the role of 03
uptake in modifying intraspecific and interspecific competition could be
determined.
Two experiments were conducted using two commonly associated range
grasses, Western wheatgrass (Agropyron smithii), a C3 species, and Sideoats
grama (Bouteloua curtipendula), a C4, species, planted in a modified
replacements series design. Plants in three chambers (Continuously stirred tank
reactors - CSTR's) received charcoal filtered air while plants in three other
chambers were exposed to 44 ppm-hr of 03 over 28 days in Experiment 1, and
71 ppm-hr of 03 over 42 days in Experiment 2. Environmental conditions for
both experiments were set to 27/20 °C day/night temperatures, 50/50% day/night
relative humidity, and 16 hour photoperiod at 400 moles m-2 sec'.
03 uptake
was calculated for the second experiment using predicted leaf area, whole plant
conductance, and actual 03 concentrations in each chamber.
Results show that 1) A. smithii shoot, root, and total plant biomass is
sensitive to 03 while B. curtipendula is insensitive, 2) in A. smithii with 03, both
intra- and inter-specific competition decreased, 3) monocultures of A. smithii
were more sensitive to 03 than mixtures, 4) the 03 insensitive B. curtipendula
responded to the reduced growth of A. smithii with increased yield only at the
lowest C3 density, 5) while 03 decreased leaf area in A. smithii, whole plant
conductance increased, 6) monocultures of A. smithii showed higher conductance
than mixtures and thus, 7) increased 03 uptake is the reason for monocultures
being more sensitive to 03 than mixtures.
The modified replacement series experimental design provided a good
method of determining the effects of 03 on both intraspecific and interspecific
competition. Changes in intraspecific competition due to 03 is dependent upon
the species sensitivity to 03. Changes in interspecific competition due to 03 is
dependent upon the proportion of 03 sensitive-to-insensitive species.
The relationship between 03 uptake and reductions in dry weight showed a
positive correlation. Thus, knowing 03 uptake can provide some estimation of
potential growth reduction. However, a plant's internal sensitivity and response
to 03 (eg., its ability to repair damage) can vary by species, and therefore makes
the uptake-growth response relationship more complex. In addition, uptake
provided little information about the potential changes in intraspecific or
interspecific competition.
The results suggest that when attempts are made to predict air pollution
effects on plant communities, inferences should be based upon studies which
utilize a mixture of plants and not on those designed to determine individual
plant responses.
Ozone Caused Changes in Competition Between Western Wheatgrass and
Sideoats Grama
by
Peter K. Ziminski
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed June 24, 1993
Commencement June 1994
APPROVED:
Redacted for privacy
Associate Professor of Botany in charge of major
Redacted for privacy
Head of department of General Science
Redacted for privacy
Dean of Graduate $c ool
Date thesis is presented
Type by author for
24 June 1993
Peter K. Ziminski
ACKNOWLEDGEMENTS
This has been a long road traveled. Working full time and taking one
course a term is not the fastest way to accomplish a Master's degree. However,
I could not have done it without being in the position I am in - working for
Man Tech Environmental Technology, Inc. under contract with the U.S.
Environmental Protection Agency.
Man Tech provided tuition support while my supervisor (initially Tom Moser,
and currently Kent Rodecap) worked with the EPA Project Leader Bill Hogsett
in coordinating a work schedule that would allow time for classes. After
completing the required course work, I was allowed to design a thesis that
would be of benefit to current research goals at the EPA. Thus, part of my
responsibilities at Man Tech included conducting research for this thesis. Thanks
to all my cohorts at work who not only endured my ups and downs during this
time period, but provided encouragement and ideas.
I would also like to thank my committee members Drs. William E. Winner,
William E. Hogsett, and Patricia Muir for providing the insight, direction and
focus of the project as a whole. Invaluable statistical help was provided by Dr.
E. Henry Lee. Operation, calibration, and data management of the exposure
system, as well as other tasks, were skillfully done by Blake Farnsworth. Stock
Seed Farms (Murdock, Nebraska) graciously donated the grass seeds for the
experiments.
Finally, a special thanks to my wife Sharon - whose powers of observation
far exceed mine. The road has been long for her too.
TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
THE IMPORTANCE OF OZONE EFFECTS ON COMPETITION
THE ROLE OF CONTROLLED ENVIRONMENTS IN AIR
POLLUTION EFt4ECTS RESEARCH
PLANT COMPETITION EXPERIMENTAL DESIGNS
Additive Experiments
Substitutive Experiments
Systematic Experiments
Neighborhood Experiments
AN 03/ PLANT COMPETITION MODEL
Experimental Design Selection
Combining 03 and Competition Experiments
Species Selection
RESEARCH OBJECTIVES
REFERENCES
1
1
2
4
4
4
7
8
8
8
9
9
10
12
CHAPTER 2: ANALYSIS OF PRODUCTIVITY.
INTRODUCTION
Ozone Effects
Competition Effects
Competition in North American Temperate Grasslands
Research Objectives
MATERIALS AND METHODS
Experimental Design
Plant Culture
Exposure System
Data Analysis
RESULTS
Total Plant Yield
Shoot Yield
Root Yield
Shoot/Root Ratios
DISCUSSION
Extension of Laboratory Results to the Field
General Conclusions
REFERENCES
14
14
14
15
17
CHAPTER 3: ANALYSIS OF OZONE UPTAKE.
INTRODUCTION
Research Objectives
MATERIALS AND METHODS
Experimental Design
48
48
48
50
50
17
19
19
19
21
25
27
27
34
37
40
41
42
43
45
Table of Contents, Continued
Plant Culture
Exposure System
Leaf Area Measurements
Stomatal Conductance Measurements
Ozone Uptake Calculation
RESULTS
Leaf Area
Conductance
Ozone Uptake
DISCUSSION
General Conclusions
REFERENCES
CHAPTER 4: BIBLIOGRAPHY
50
51
55
56
57
58
58
58
60
67
68
69
70
LIST OF FIGURES
Figure 1. Replacement Series Models Proposed by Harper (1977).
6
Figure 2. Planting Density (C3:C4 Plants/Pot). Note: Pots were randomly
arranged within each chamber.
21
Figure 3. 28-day repeating episodic 03 profile.
23
Figure 4. Modified Replacement Series Diagrams for total dry weight
(grams/pot) in control (A) and 03 (B). A. smithii= C3, B. curtipendula =
C4.
31
Figure 5. Intraspecific Competition (Relative monoculture response) for total
dry weight in control and 03. A. smithii = C3, B. curtipendula = C4.
33
Figure 6. Interspecific competition (Relative mixture response) for total dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
33
Figure 7. Modified Replacement Series Diagram for shoot dry weight
(grams/pot) in control (A) and 03 (B). A. smithii = C3, B. curtipendula =
C4.
35
Figure 8. Intraspecific competition (relative monoculture response) for shoot
dry weight in control and 03. A. smithii = C3,13. curtipendula = C4.
36
Figure 9. Interspecific competition (relative mixture response) for shoot dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
36
Figure 10. Modified Replacement Series Diagrams for root dry weight
( grams/pot) in control (A) and 03 (B). A. smithii = C3, B. curtipendula =
C4.
38
Figure 11. Intraspecific competition (relative monoculture response) for root
dry weight in control and 03. A. smithii = C3, B. curtipendula = C4.
39
Figure 12. Interspecific competition (relative mixture response) for root dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
39
Figure 13. 03 caused changes in A. smithii shoot/root ratios in monocultures
and mixtures.
40
Figure 14. 03 caused changes in B. curtipendula shoot/root ratios in
monocultures and mixtures.
40
List of Figures, Continued
Figure 15. Planting Density (C3:C4 Plants/Pot). Pots were randomly arranged
within each chamber.
52
Figure 16. 28-day repeating episodic 03 profile.
54
Figure 17A. 03 caused leaf area changes over time in A. smithii monocultures
and mixtures. Density=2, Error Bars = 1 S.E., n=4.
61
Figure 17B. 03 caused leaf area changes over time in A. smithii
monocultures and mixtures. Density=4, Error Bars = 1 S.E., n=4.
62
Figure 17C. 03 caused leaf area changes over time in A. smithii
monocultures and mixtures. Density=6, Error Bars = 1 S.E., n=4.
62
Figure 17D. 03 caused leaf area changes over time in A. smithii
monocultures. Density=8, Error Bars = 1 S.E., n=4.
62
Figure 18A. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =2, n=4, Error Bars= 1 S.E.
63
Figure 18B. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =4, n=4, Error Bars= 1 S.E.
63
Figure 18C. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =6, n=4, Error Bars= 1 S.E.
64
Figure 18D. 03 caused leaf area changes over time in B. curtipendula
monocultures. Density =8, n=4, Error Bars= 1 S.E.
64
Figure 19. Single leaf diurnal conductance after 14 days of 03 exposure.
Error bars represent 1 standard error. n=8. A. smithii = C3, B.
curtipendula = C4.
65
Figure 20. Whole plant conductance during 6 weeks of 03 exposure. Error
bars represent 1 standard error. n=8. A. smithii = C3, B. curtipendula = C4. 65
Figure 21. 03 caused changes in A. smithii conductance in monocultures and
mixtures. Error bars = 1 standard error. n= 4.
66
Figure 22. 03 uptake in A. smithii (C3) and B. curtipendula (C4) grown in
monocultures and mixtures.
66
LIST OF TABLES
Table 1. Experimental conditions and parameters measured for analysis of
productivity.
22
Table 2. Total plant dry weight means and predicted means for Experiments
1 and 2.
28
Table 3. Shoot dry weight means and predicted means for Experiments 1 and
2.
29
Table 4. Root dry weight means and predicted means for Experiments 1 and
2.
30
Table 5. Mean percent change ± 1 S.E. in growth across all densities due to
03.
37
Table 6. Experimental conditions and parameters measured for analysis of 03
uptake.
53
OZONE CAUSED CHANGES IN COMPETITION BETWEEN WESTERN
WHEATGRASS AND SIDEOATS GRAMA
CHAPTER 1: INTRODUCTION
THE IMPORTANCE OF OZONE EFFECTS ON COMPETITION
Since at least the time of Charles Darwin, competition has been considered
one of the major forces shaping the morphology of plants as well as the structure
of plant communities (Grace and Tilman, 1990). Changes in intraspecific or
interspecific competition can lead to changes in ecosystem structure (eg.,
diversity and abundance) and function (eg., hydrology and nutrient cycling).
Stresses, including those from air pollution, can simplify an ecosystem's
structure (Woodwell, 1970), and cause changes in its functions (Smith, 1974;
Odum, 1985; Smith, 1992). Due to high concentrations of pollutants, point
source air pollution causes ecosystem changes which are rapid, dramatic, and
localized. Regional-scale air pollutants, such as ozone (03), are of moderate to
low pollutant concentrations and cause changes which are gradual, subtle and
extend over large areas (Smith, 1992).
03 affects plants directly and indirectly. Direct effects of 03 include
reduction in photosynthetic rates and changes in carbon allocation.
2
These direct effects are dependent upon:
1) the physical, chemical, and biological environment of the plant;
2) the concentration, duration, and dynamics of the exposure;
3) plant physiology and genetics (Hogsett et. al., 1988).
Indirect effects of 03 include increased susceptibility to diseases, insect attacks
(Mi 111er et. al., 1982), winter injury (Johnson, 1989), and changes in genetic
diversity (Berrang, et. al., 1986).
The most important reason to consider 03 effects on competition is that
these direct and indirect effects of 03 reduce a plants' ability to compete by
reducing its carbon gain and consuming energy reserves for defenses instead of
growth. While these effects occur at the plant level, they eventually affect plant
populations, and ultimately will alter ecosystem structure, and potentially, its
function. Changes in competition between species in a community have been
implicated as a mechanism for some of the observed indirect effects and changes
in community structure (Armentano and Bennett, 1992). Accurately predicting
the changes in competition due to 03 would allow for greater understanding and
prediction of the subsequent changes in ecosystems due to this stress.
THE ROLE OF CONTROLLED ENVIRONMENTS IN AIR POLLUTION
EFFECTS RESEARCH
During the late 1940's photochemical oxidants
primarily 03 were shown to
be a phytotoxic component of southern California's smog (Cowling and Heck,
1988). Since then, research on 03 effects on plants has been focused in several
directions including pollutant exposure/plant response, mechanisms controlling
3
responses, and field observations of changes in plant community structure.
Deciphering either competition or air pollution effects in the field is extremely
difficult because of the numerous variables present and the lack of adequate
experimental controls. Thus, experiments which minimize confounding
influences of environmental factors, and provide for adequate experimental
replication and controls are required to determine which mechanisms are
occurring in the field.
Open-top chambers situated in "field" conditions control the pollutant levels
within the chambers, reduce the confounding environmental factors, and provide
for adequate replication and controls. To further minimize environmental
influences, chambered systems can be situated in a laboratory or a growth
chamber. These indoor systems are capable of controlling temperature,
humidity, and solar radiation as well as the pollutant concentrations within the
chambers. Early experiments in these systems consisted of acute exposures (ie.,
square-wave exposures at greater than ambient doses for short durations). Only
in the past decade, with the use of microcomputers have researchers been able to
mimic the long term episodic nature of ambient 03 in the laboratory.
The relationship between ambient 03 concentrations and its effects on
plants is directly related to 03 absorption. Absorption is defined as the product
of the flux rate of 03 through leaf stomata and the duration of exposure. The
continuous-stiffed tank reactor (CSTR) exposure chamber was developed for
determining pollutant absorption by plants. The CSTR design assures thorough
mixing of pollutants within the chamber and a low leaf boundary layer
resistance. Measurement of pollutant, CO2, and H2O concentrations at the
chamber's inlet and outlet allow for calculation of pollutant uptake, plant
photosynthesis, and transpiration rates (Hogsett et. al., 1987).
4
PLANT COMPETITION EXPERIMENTAL DESIGNS
Density, spatial arrangement, and proportion of species, are important factors
in the study of plant competition. Four major experimental designs have been
developed to study plant competition - each addressing one or more of these
factors in different ways. They have been reviewed by Radosevich (1987) and
are discussed below.
Additive Experiments
In additive experiments, the density of one species is held constant while the
density of another species is varied. This method is commonly used in
agriculture and forestry studies where a fixed density of a "crop" species is
invaded by a "weed" species. Since both the total plant density and the
proportion of the two species vary simultaneously, interpretation of the effects of
these two factors is difficult. Neither can the effects of intraspecific or
interspecific competition be differentiated.
Substitutive Experiments
Two types of substitutive experimental designs are commonly used: the
Dial lel and the replacement series. The diallel design combines an individual of
5
each species in a community into all possible pairs, and then compares the
growth of the mixtures with the monocultures. For example, in a community
with two species, an individual of each species is grown alone (A and B), two
individuals are grown together (AA and BB), and 1 individual of each species in
grown together (AB). Intraspecific competition is measured by comparing A
with AA and B with BB, while interspecific competition is measured by
comparing AB with AA and BB. In the diallel design, the influence of density
on competition between the species cannot be determined.
In the second type of substitutive design, the replacement series experimental
design used by de Wit (1960), the total plant density remains constant while the
proportion of the species varies. Typical species proportion are 100:0 - Species
1 monoculture, 75:25, 50:50, 25:75, and 0:100 - Species 2 monoculture. The
four possible results (Figure 1) from this experimental design have been
modelled (Harper, 1977). Model I shows that neither species has an effect on
the growth of the other, in Model II, one species reduces the growth of the
other, Model III - both species reduce the growth of each other; and for Model
IV, both species increase the growth of each other.
When intraspecific competition equals interspecific competition, yield is
linearly related to species proportion. This linear trend has been called the
"expected yield" and is represented as the straight lines in the four replacement
series models (Figure 1.). Deviations from the "expected yield" are interpreted
as being the outcome of unequal competitive ability of the two species in the
mixture. A "relative crowding coefficient" is calculated as an index of
competition. The relative crowding coefficient, however, is only valid when
species are mixed in equal proportion and does not partition the effects of
competition into intraspecific and interspecific components (Jolliffe et. al., 1984).
6
Thus, a modification of the de Wit replacement series design was proposed by
Jolliffe et. al. (1984) to enable this partitioning regardless of proportions and
densities.
The modified replacement series design differs from the classic replacement
series design in both the design itself and the analysis of its results. The
modified replacement series design includes a monoculture for each species at
each density being used in the mixtures. The analysis of the modified
replacement series designs differs by using a graphical approach to understand
the changes in growth which occur in the monocultures and mixtures.
Model I
Model 11
Y
I
A+B
L
D
0
100
Imlay
100
0
Model II
0
100
kraj
100
0
100
Ilsoef
100
0
Figure 1. Replacement Series Models Proposed by Harper (1977).
The first step in the analysis of the modified replacement series design is
the determination of a "projected yield". The projected yield is derived based on
7
the hyperbolic relationship of the species monoculture yield:density curve. This
projected yield is indicative of the potential yield a species would have in the
absence of any competition. The reduction of the actual monoculture yield is
due to intraspecific competition. Thus, intraspecific competition is calculated as
the difference between the actual monoculture yield and the projected yield. The
yield reduction from the monoculture curve to mixture curve is the effect of
interspecific competition. Thus, interspecific competition is the difference
between the monoculture yield and the mixture yield calculated at each density.
Systematic Experiments
Two approaches also occur in systematic experiments: the Nelder design and
the addition series design. In the Nelder experiments, plant density and spatial
arrangement are varied systematically often in an arc or circle. The advantage
of this design is that an array of densities can be studied without changing the
plant arrangement pattern and can be done in a smaller area than other designs.
However, the design does not allow for the partitioning of density and proportion
effects, nor can the effects of intraspecific or interspecific competition be
separated.
The addition series experimental design changes both density and proportion.
The density of two species increases along perpendicular gradients producing a
range of monoculture density, total densities, and proportions.
8
Neighborhood Experiments
When individual responses to the proximity of other plants is of interest, the
neighborhood design can be used. With this design, the growth of one target
species is evaluated as a function of the number, biomass, cover, or distance of
its neighbors.
AN 03 / PLANT COMPETITION MODEL
When one considers the number of experiments required to evaluate the
environmental and biological factors which influence the effects of both 03 and
competition, the need for selecting an experimental design and species which act
as a model for 03 / competition effects studies is obvious. The results from such
a model should provide general conclusions about 03 effects on plant
competition which would be generally applicable to a wide variety of ecological
settings.
Experimental Design Selection
The primary consideration in selecting an experimental design for the analysis
of competition is the study objectives. In a study designed to understand and
predict 03-caused changes in a plant community, partitioning responses into
9
intra- and interspecific competition components is important, because both forces
are responsible for shaping community structure. Therefore, a substitutive
experimental design would be most appropriate.
Combining 03 and Competition Experiments
When applying a competition experimental design to 03 studies, one aspect
which must be considered is that of space allocation. The systematic designs
(Nelder and addition series) require large amounts of space to incorporate the
range of densities and proportions which they generate. Thus, these designs are
not adaptable to a controlled environment chamber system where space is
limited. However, either of the substitutive designs can be easily incorporated
within a chambered system.
Recovery of plant roots also needs consideration when adapting competition
experiments to 03 studies. When species are grown in mixtures it is often
impossible to recover and separate the roots of individual plants. In these
situations, the effects on root growth cannot quantified. This is important
because the effects of 03 are often first shown by reduced root growth (Miller,
1988) which in turn would have an effect on competition.
Species Selection
A general hypothesis is 03 would cause the greatest change in intraspecific
competition in an 03 sensitive species. The reductions in growth and changes in
10
carbon allocation from 03 would influence intraspecific competition by
consuming resources for defense instead of growth. Thus, smaller plants would
have less intraspecific competition.
Another general hypothesis is would cause the greatest changes in
interspecific competition when an 03 tolerant species grows in competition with
an 03 sensitive species. In this case, the 03 tolerant species may show increased
growth in 03 due to reduced competition from the 03 sensitive species.
To test these hypotheses, I have designed studies with two co-occurring
grass species that have either C3 or C4 photosynthetic pathways. C3 plants
generally have higher stomatal conductance rates than C4 plants and thus,
differing 03 absorption capacities (Winner and Mooney, 1980), and therefore,
should have different responses to 03 assuming their internal sensitivities do not
differ significantly.
RESEARCH OBJECTIVES
Western wheatgrass (Agropyron smithit), a C3 species, and Sideoats grama
(Bouteloua curtipendula), a C4 species, co-occur over a broad geographic range
across most of the U.S. (Hitchcock, 1971). They are of similar size and
morphology, and they grow together in the same habitat in midwestern U.S.
(Christie and Detling, 1982). These two species were planted in a modified
replacement series design (Jolliffe et. al., 1984), also known as a synthetic nointeraction design (Rousch et. al., 1989).
11
The general experimental objectives were to:
1) determine the effects of 03 on intra- and inter- specific competition under
conditions of limiting light and nutrients; and
2) relate 03 uptake to the changes in competition.
12
REFERENCES
Armentano, T.V., and J.P. Bennett, 1992. Air Pollution Effects on the Diversity
and Structure of Communities. IN: Air Pollution Effects on Biodiversity.
Edited by J.R. Barker and D.T. Tingey. Van Nostrand Reinhold, NY. 322pp.
Berrang, P., D.F. Kamosky, R.A. Mick ler, and J.P. Bennett, 1986. Natural
selection for ozone tolerance in Populus tremuloides. Can. J. For. Res. 16:12141216.
Christie, E.K., and J.K. Detling, 1982. Analysis of interference between C3 and
C4 grasses in relation to temperature and soil nitrogen supply. Ecology 63:12771284.
Cowling, E.B., and W.W. Heck, 1988. Air pollution effects on vegetation,
including forest ecosystems. Proceedings of the Second US-USSR Symposium.
Edited by Reginald D. Noble, Juri L. Martin, and Keith F. Jensen. Broomall, PA.
Northeastern Forest Experiment Station, 1989. p. 65-70.
Grace, J.B., and D. Tilman, 1990. IN: Perspectives on Plant Competition.
Edited by J.B. Grace and D. Tilman. Academic Press, Inc., San Diego. 484 pp.
Harper, J.L., 1977. Population biology of plants. Academic Press, NY. 892 pp.
Hitchcock, A.S., 1971. A manual of the grasses of the United States. Dover
Publications, Inc. 999pp.
Hogsett, W.E., D. Olszyk, D.P. Ormrod, G.E.Taylor, Jr., D.T. Tingey, 1987. IN:
Air pollution exposure systems and experimental protocols: Volume 1: A review
and evaluation of performance. EPA 600/3-87/037a.
Hogsett, W.E., D.T. Tingey, and E. Henry Lee, 1988. Ozone exposure indices:
Concepts for development and evaluation of their use. IN: Assessment of crop
loss from air pollutants. W.W.Heck, O.C.Taylor, and D.T.Tingey editors.
Elsevier Applied Science. New York.
Johnson, A.H., 1989. Decline of red spruce in the northern appalachians:
Determining if air pollution is an important factor. IN: Biologic markers of airpollution stress and damage in forests. National Academy Press. pp 91-104.
13
Jolliffe, P.A., A.N. Minjas, and V.C. Runeckles, 1984. A reinterpretation of
yield relationships in replacement series experiments. J. Appl. Ecol. 21:227-243.
Miller, P.R., O.C. Taylor, R.G. Wilhour, 1982. Oxidant air pollution effects on
a western coniferous forest ecosystem. USEPA-600/D-82-276. l0pp.
Miller, J.E., 1988. Effects on photosynthesis, carbon allocation, and plant growth
associated with air pollution stress. IN: Assessment of crop loss from air
pollutants. W.W.Heck, O.C.Taylor, and D.T.Tingey editors. Elsevier Applied
Science. New York.
Odum, E.P., 1985. Trends expected in stressed ecosystems. Bioscience 35:419422.
Radosevich, S.R., 1987. Methods to study interaction among crops and weeds.
Weed Technology. 1:190-198.
Rousch, M.L., S.R., Radosevich, R.G. Wagner, R.D. Maxwell, T.D. Petersen,
1989. A comparison of methods for measuring effects of density and proportion
in plant competition experiments. Weed Science 37:269-275.
Smith, W.H., 1975. Air pollution - Effects on the structure and function of the
temperate forest ecosystem. Environ. Pollut. 6:111-129.
Smith, W.H., 1992. Air pollution effects on ecosystem processes. IN: Air
Pollution Effects on Biodiversity. Edited by J.R. Barker and D.T. Tingey. Van
Nostrand Reinhold, NY. 322pp.
Winner, W.E. and H.A. Mooney, 1980. Ecology of SO2 resistance: III.
Metabolic changes of C3 and C4 Atriplex species due to SO2 fumigations.
Oecologia 44:296-302.
Wit, C.T. de, 1960. On competition. Versiagen van landbouwkundige
Onderzoekingen. 66(8):1-82.
Woodwell, G.M., 1970. Effects of pollution an the structure and physiology of
ecosystems. Science 168:429-433.
14
CHAPTER 2: ANALYSIS OF PRODUCTIVITY.
INTRODUCTION
Ozone Effects
Tropospheric ozone (03), a regional pollutant which occurs across the US
(Wolff and Lioy, 1980), can change the growth rate and form of plants. For
example, the National Crop Loss Assessment Network (NCLAN) found that
ambient concentrations reduced yields of soybean c.v. 'Corsoy' (10%), peanut
(14 - 17%), turnip (7%), head lettuce (53-56%), and red kidney bean (2%)
(Heck, et. al., 1982). These data have led to the development of several
economic models which indicate that a 25% reduction of ambient 03 levels
across the U.S. would lead to an economic benefit of almost $2 billion per year
(Adams et. al., 1988).
03 has also been shown to cause ecological changes in forests. The mixed
conifer forest in the San Bemadino National Forest of southern California has
been exposed to air pollution for several decades. Ponderosa pine mortality in
this area has been extensive, and changes in species composition are correlated
with this species mortality. For example, white fir and incense cedar will tend
to become the dominant species, replacing ponderosa pine (Miller, 1984).
North American temperate grasslands are also exposed to 03. The Pawnee
National Grassland in northern Colorado has hourly 03 concentrations as high as
88ppb (Zeller and Hazlet, 1989). While this concentration is below the National
Ambient Air Quality Standard of 120ppb, it is a significant concentration when
one considers the nearest towns are 30 km away and the largest metropolitan
15
area is more than 110km away. Acute 03 response studies on grasses have been
conducted on numerous turfgrass cultivars. Sensitivity to 03 varied greatly
among C3 species and cultivars while C4 species were generally less sensitive to
03 than the C3 species (Youngner and Nudge, 1980). Similar variability in the
responses of grasses to 03 was found by Elkiey and Ormrod (1980).
03 absorbed into foliage is known to affect virtually all physiological and
biochemical processes of plants. The mechanism of phytotoxicity is based on
the capacity for 03 to oxidize organic compounds and increase free radical
production in cells (Floyd et. al., 1989). Two mechanisms of 03-caused changes
in productivity are known to include reduction in photosynthetic rates and
changes in whole plant resource partitioning. Physiological effects of 03 have
been studied extensively since the 1950's and have been reviewed by Guderian
et. al. (1985), and Reich (1987).
One important conclusion from past research is the effects of 03 on plant
physiology and growth can be modified by other environmental factors including
nutrient level and soil moisture. For example, water stress protects plants from
03 by reducing stomatal aperture and hence, 03 absorption (Tingey and Hogsett,
1985).
Competition Effects
In both agricultural and natural ecosystems, plants grow in competition with
other plants for many resources including light, water, and nutrients. A plant's
presence changes the environment of its neighbors and alters their growth rate
16
and form (Harper, 1977). Competition can be viewed as a suite of stresses
affecting plants. Since a plant rarely has only one stress to cope with at a time,
much work is currently focused on plant responses to multiple stresses (eg.,
Mooney et. al., 1991), including 03 (Pell et. al., 1990). Thus, it is important to
know the effects of air pollutants on plants grown both in monocultures where
intraspecific competition occurs, and in mixtures where interspecific competition
occurs.
Several studies have looked at the combined effects of competition and 03.
When ryegrass and clover were grown in competition, ryegrass total dry weight
yield, leaf area and tiller numbers were less depressed by 03 in mixtures than in
monocultures (Bennett and Runeckles, 1977). In this case, interspecific
competition reduced the effects of 03.
In a two year-old clover-fescue pasture, clover was the dominant species
where 03 was kept below ambient concentrations using open-top chambers.
Conversely, fescue was dominant in chambers with 03 maintained above ambient
concentrations (Rebbeck, et. al. 1988). In another study, the effects of 03 on
fescue and clover grown together were different than the effects of 03 on the
two monocultures. Clover was more sensitive when grown in a mixture than a
monoculture, while the fescue monoculture was more sensitive than fescue
grown in a mixture (Montes et. al. 1982). While these studies are important
they do not attempt to explain the role of 03 in modifying inter- and intraspecific competition.
17
Competition in North American Temperate Grasslands
The analysis of competition between grass species and changes in
competition due to 03 has both ecological and economic importance. As an
example of ecological importance, the Northern Great Plains Grasslands alone
covers more than 25 million hectares in Montana, North Dakota, South Dakota,
and Wyoming and extends well into Canada. Competition is particularly
important in grasslands where water and other resources needed for growth can
be limiting. This grassland is not only ecologically important, but also supports
an industry based on grazing.
Over much of the North American temperate grasslands, C3 and C4 species
grow together (Christie and Detling, 1982). C3 and C4 species differ in many
ways. C3 plants have their optimum growth rates at lower temperatures than C4
plants (15-25 °C compared to 30-47°C) (Salisbury and Ross, 1985). C4 plants
generally have lower stomatal conductance than C3 plants which reduces their 03
absorption capacity (Winner and Mooney, 1980), and should, therefore, have
different responses to 03 assuming their internal sensitivities do not differ
significantly.
Research Objectives
Using two grasses with differing 03 absorption capacities growing in both
monocultures and mixtures the specific objectives of this study were to:
18
1) determine whether monocultures and mixtures were equally sensitive to
03; and
2) determine whether intra- and inter- specific competition were equally
sensitive to 03.
19
MATERIALS AND METHODS
Experimental Design
Two experiments were conducted using Western wheatgrass (Agropyron
smithii), a C3 species, and sideoats grama (Bouteloua curtipendula), a C4
species. These species were chosen for several reasons:
1) they co-occur over a broad geographic range (Hitchcock, 1971).
2) they are of similar size and morphology, and
3) they have different photosynthetic pathways and stomata' conductance
rates (Winner and Mooney, 1980) and should therefore have differing
responses to 03.
Monocultures and mixtures of each species were planted in a modified
Replacement Series Design (Jolliffe et. al., 1984), also known as a synthetic nointeraction design (Rousch, et. al., 1989).
Plant Culture
A. smithii seeds were collected by Stock Seed Farm (Murdock, Nebraska)
from field sites in northern Nebraska and southern South Dakota. Seeds of B.
curtipendula were collected at the Stock Seed Farm in eastern Nebraska, (Dave
Stock, pers. comm.).
20
A. smithii seeds were sown onto brown blotter paper which had been soaked
in 0.2% KNO3" solution. Seeds were germinated in the dark, in growth
chambers maintained at 25/15 °C day/night temperatures until the shoots were
approximately 5 cm tall (approximately 10 days). These procedures were used
to break dormancy based on techniques used by the Oregon State Seed
Laboratory (Corvallis, OR.).
Five days after A. smithii seeds were sown, B. curtipendula seeds were sown
into four 50cm x 38cm x 7.5cm wooden flats filled with vermiculite. Flats were
placed into growth chambers maintained at 30/27 °C day/night temperatures, and
16 hour photoperiod at 500 /moles m-2
Seeds were misted with tap water
daily until the shoots were approximately 4 cm tall (approximately 5 days).
Seedlings of uniform size from both species were then individually
transplanted into pots (10cm x 20cm) filled with a promix:perlite mixture (1:1 by
volume). Monocultures at densities of 2, 4, 6, and 8 plants per pot, and
mixtures of 2 C3's with 6 C4's, 4 C3's with 4 C4's, and 6 C3 's with 2 C4's were
replicated twice in each of 6 chambers (Figure 2). Plants were held in a
greenhouse maintained at a constant 25°C temperature, ambient light levels (Oct.
- Nov. for Experiment #1, and Jan. Feb. for Experiment #2) supplemented with
1000 Watt high pressure sodium lamps, and watered with tap water. Five weeks
after transplanting, pots were placed into exposure chambers.
21
Exposure System
A plant exposure system, located in Weniger Hall on the Oregon State
University campus, was constructed to conduct research on the effects of air
pollutants on plants. The system is capable of delivering and monitoring 03 to
12 Continuously Stirred Tank Reactors (CSTR's). Additionally, the system can
control and monitor day/night temperature and humidity, and photoperiod.
Continously Stirred Tank Reactor
(CSTR)
6
Chambers
3 Control + 3 Ozone
Figure 2. Planting Density (C3:C4 Plants/Pot). Note: Pots were randomly
arranged within each chamber.
Plants were exposed to 03 for 28 days in Experiment 1 and 42 days in
Experiment 2. Environmental conditions for both experiments were set to 27/20
°C day/night temperatures, 50/50 % day/night relative humidity, and 16 hour
22
photoperiod at 400 moles
ni2 S-1
photosynthetically active radiation (PAR)
(Table 0). This temperature regime was selected so that neither grass species
was grown at its optimum temperature.
Throughout the exposure, data on hourly
03
averages, and daily maximum,
minimum, and average air temperature were collected in each chamber.
Additionally, data on daily maximum, minimum, and average relative humidity,
and total solar radiation were collected in two chambers. Plants were watered to
pot capacity with tap water daily and allowed to drain.
Table 1. Experimental conditions and parameters measured for analysis of
productivity.
EXPERIMENT #2
EXPERIMENT #1
OZONE EXPOSURE DURATION
28 DAYS
42 DAYS
n=6
n=3
n=3
n=6
n=6
DRY WEIGHTS
Shoot Dry Weight (pots ch
Root Dry Weight
(pots ch
Dark Regrowth
(pots ch
-1.
03-1)
-1 03-1)
-1
03-1)
ENVIRONMENTAL PARAMETERS
Ozone SUM (ppm-hr)
Day Temperature °C
Night Temperature °C
Day Humidity %
Night Humidity %
Solar Radiation Sum (E m-2)
1 Mean ± SE
44
27.0
21.9
46.2
49.7
451
±
±
±
±
71
0.11
0.1
0.2
0.2
26.7
18.2
47.0
47.1
542
±
±
±
±
0.1
0.1
0.3
0.2
23
Plants in three chambers received charcoal-filtered air, while plants in three
other chambers were exposed to an episodic 03 profile which is repeated every
28 days (Figure 3).
0.3
0.25
0.2
P
0.05
A\
.
9
11
13
13
17
19
21
23 23
2.7
29
DAY
Figure 3. 28-day repeating episodic 03 profile.
The 03 profile, developed at the Environmental Protection Agency's (EPA) Corvallis Environmental Research Laboratory, was modified from a "base"
profile originally developed utilizing the EPA's 1978-1983 Storage and Retrieval
of Aerometric Data (SAROAD) databases from 6 mid-Western states (Illinois,
Indiana, Ohio, Michigan, Wisconsin, and Minnesota). This "base" profile was
designed to have similar characteristics to ambient 03 in this region. These
characteristics include:
1) the daily minimum 03 concentration occurring between 0300 and 0500
hours,
2) the maximum concentration occurring between 1400 and 1700 hours,
24
3) a 7 hour seasonal (May
Sept.) mean of 0.044 ppm,
4) equal frequency of peak days (defined as days during which the hourly
concentration exceeded 0.070 ppm).
The episodic profile used in this experiment was modified from this "base"
profile by increasing all hourly concentrations by sigmoid weights which
increased the total exposure by 50%.
At the end of both experiments, all shoots were harvested leaving
approximately 5 cm of stubble. (This allowed the roots to maintain their
structural integrity during washing, and then be easily identified and separated
by species). Initial concerns about the ability to accurately separate the roots in
the mixtures, and an interest in root carbohydrate storage led to using a
technique which indirectly estimates root carbohydrate storage by regrowing
plants in the dark. Grown under dark conditions, plant growth is dependent
upon root carbohydrate storage only and not photosynthate (William E. Hogsett,
pers. comm.). In experiment #1, roots from half of the pots in each chamber
were washed, while the remaining pots were allowed to regrow in the dark,
without 03, for 30 days after which shoot biomass was harvested again and roots
were washed. In the second experiment, this technique was not used due to the
highly variable regrowth (data not shown), instead, all roots were washed. Both
shoots and roots were oven dried at 70 °C for at least 48 hours until constant
weight was obtained.
25
Data Analysis
The effects of intraspecific and interspecific competition were determined by
the methods proposed by Jolliffe et. al. (1984). The relationship between yield
and density in the absence of any competition (intra- or inter- specific) is termed
projected yield (Ye). The projected yield is calculated as:
Yp- (
1.)x(N)
where:
Ye
is the Projected Yield (grams/pot),
Ym. is the maximum monoculture yield (grams/pot),
K.
is the density at 50% maximum yield (plants/pot), and
N
is density (plants/pot).
At any given planting density, the difference between the projected yield
(Ye) of a species and the actual monoculture yield (YM) is a measure of
intraspecific competition. For comparison of two experiments, it is useful to
compare the effects of competition on a relative basis. Thus, the relative effect
of intraspecific competition on the yield of one species in a monoculture is
expressed by:
Riv-
YP -YM
Yp
26
where:
RM is the relative monoculture response (intraspecific competition),
Yp is the projected yield, and
Y. is the monoculture yield.
Similarly, at any given planting density, the difference between the actual
monoculture yield (YM) of a species and the actual mixture yield (Yx) is a
measure of interspecific competition. Thus, the relative effects of interspecific
competition are defined by:
Y -Y
Rx m x
where:
Rx is the relative mixture response (interspecific competition),
YM is the monoculture yield, and
Yx is the mixture yield.
Treatment means (for each species, 03 treatment, and planting mixture) were
fitted to rd degree polynomial equations for shoot (shoot + stubble), root, and
total (shoot + stubble + root) dry weights. Modified replacement series
diagrams, relative monoculture response, and relative mixture response graphs
were constructed using these fitted curves following the methods of JoLliffe et.
al. (1984). Shoot/root ratios were computed for each pot and then regressed (for
each species, 03 treatment, and planting mixture) against planting density.
27
RESULTS
Protocols were adjusted between the two experiments to improve various
techniques. For example, in the first experiment, seedlings of B. curtipendula
exhibited transplant shock several days after transplanting. The dead seedlings
were immediately replaced with vigorous ones, however this process may have
disturbed the roots of adjacent plants and reduced the yield of B. curtipendula in
both monocultures and mixtures, especially at the densities 6 and 8 where the
greatest numbers of seedlings needed to be replaced. Therefore, for the second
experiment, additional pots of these densities were planted and when a seedling
died, the entire pot was replaced thereby eliminating any disturbance of adjacent
roots. Exposure duration for the second experiment was increased to 42 days
since the plants were smaller at the start of the experiment.
Analysis of both experiments showed consistent monoculture response to 03,
however, results differed between experiments for plants raised in mixtures
(Table 1 -Table 3 ). This is probably a result of the reduced yields obtained by
the transplanting technique used in Experiment 1. Therefore, only graphs from
Experiment 2 are shown.
Total Plant Yield
In the control chambers, total plant monoculture and mixture yields were
greater for A. smithii than B. curtipendula at all densities. For example, in the
monocultures, maximum final total plant yield was 6.9 grams/pot for A. smithii
and 5.1 grams/pot for B. curtipendula (Figure 4A).
g 52 52 52 52 52 52 52 52 52 52 52 52 52 52 ED ED CI
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4) 4) 4..) W W oa W 4.) CA CA W ca) 4) 42 4) 43 41 (.0 W (.1) W 4.) W oa 43
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NO
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8R gN 80 8R
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tai
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3
Table 3. Shoot dry weight means and predicted means for Experiments 1 and 2.
SPECIES
0
C3
0
0
C3
0
0
0
0
0
0
0
C3
0
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
TRIM'
MIXTURE
DENSITY
N
MEAN
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
MONO
MONO
MONO
MONO
2
4
6
6
MIX
MIX
MIX
2
4
6
2
4
6
0.81
1.79
2.11
2.52
0.85
1.09
1.98
0.83
1.16
1.81
EXPERIMENT #1
SE.'
PREDICTED
g/pot
Standard Error
MONO
MONO
MONO
MONO
MIX
MIX
MIX
MONO
MONO
MONO
MONO
8
8
2
4
6
2
4
6
8
MIX
MIX
MIX
2
4
6
MONO
MONO
MONO
MONO
2
MIX
MIX
MIX
2
4
6
4
6
8
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
2.34
0.50
1.04
1.38
0.97
2.22
1.16
1.16
0.62
1.44
1.89
1.79
2.11
1.41
2.03
0.37
1.23
1.28
0.15
0.27
0.26
0.13
0.07
0.19
0.12
0.17
0.16
0.15
0.21
0.09
0.10
0.24
0.27
0.57
0.18
0.22
0.19
g/pot
0.93
1.65
2.18
2.51
0.65
1.29
1.91
0.67
1.27
1.82
2.32
0.57
0.96
1.18
1.16
1.71
1.66
1.00
0.41
0.71
1.35
0.33
0.46
0.54
0.58
0.42
0.08
0.18
0.23
1.92
1.27
1.97
2.11
1.70
0.58
1.02
1.35
EXPERIMENT #2
S.E.
PREDICTED
N
MEAN
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
1.06
2.68
3.31
2.96
0.93
1.08
1.99
1.43
1.78
2.15
3.01
0.49
0.21
1.51
0.27
0.93
0.52
0.17
2.53
3.06
3.10
0.68
1.32
1.41
0.11
1.55
1.16
2.50
2.88
3.54
1.02
1.68
2.00
1.95
2.51
3.38
3.75
0.95
1.77
2.71
0.16
0.15
g/pot
6
6
6
6
6
6
6
6
g/Pot
0.11
0.39
0.26
0.19
0.20
0.42
0.04
0.41
0.58
0.89
0.14
0.39
0.33
0.17
0.32
0.58
0.80
0.29
0.45
0.41
1.91
1.04
1.85
2.45
2.82
0.68
1.22
1.62
1.28
2.29
3.03
3.50
1.02
1.68
2.00
1.59
2.73
3.43
3.68
0.91
1.81
2.70
Table 4. Root dry weight means and predicted means for Experiments 1 and 2.
EXPERIMENT #2
EXPERIMENT #1
SPECIES
IRTMT
MIX
DENSITY
N
MEAN
S.W.
g/pot
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
OZONE
Standard Error
MONO
MONO
MONO
MONO
2
4
MIX
MIX
MIX
MONO
MONO
MONO
MONO
MIX
MIX
MIX
MONO
MONO
MONO
MONO
MIX
MIX
MIX
6
3
3
3
8
3
2
4
6
2
4
6
3
3
3
3
3
3
3
3
3
3
3
3
8
2
4
6
2
4
6
8
2
4
6
MONO
MONO
MONO
MONO
2
MIX
MIX
MIX
2
4
4
6
8
6
3
3
3
3
3
3
3
3
3
3
3
3
0.63
1.35
1.86
2.15
0.38
1.15
1.57
0.65
0.85
0.95
1.30
0.20
0.48
0.88
0.27
1.09
0.72
0.59
0.19
0.70
1.12
0.73
0.83
0.91
1.32
0.15
0.59
0.41
0.19
0.32
0.14
0.33
0.12
0.39
0.30
0.19
0.21
0.15
0.28
0.07
0.08
0.19
0.13
0.31
0.16
0.14
0.12
0.10
0.29
0.25
0.43
0.50
0.32
0.03
0.22
0.08
PREDIL 1 ED
N
g/pot
0.72
6
1.32
1.81
2.18
6
6
6
0.50
6
6
6
6
6
6
1.03
1.61
0.49
0.86
1.10
1.22
0.17
0.51
1.04
0.53
0.81
0.82
0.58
0.30
0.59
0.89
0.49
0.86
1.10
1.22
0.30
0.45
0.46
MEAN
SEi.
g/pot
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
1.24
3.32
3.74
3.67
0.65
1.26
2.25
1.08
1.95
1.86
2.99
0.33
1.42
1.52
0.53
1.31
1.43
1.64
0.39
0.84
1.20
0.89
1.17
1.52
1.68
0.37
0.87
1.61
0.23
0.42
1.02
0.69
0.11
0.21
0.34
0.11
0.20
0.08
0.31
0.06
0.08
0.27
0.06
0.17
0.28
0.47
0.08
0.22
0.18
0.08
0.13
0.22
0.33
0.13
0.18
0.31
PREDICTED
g/pot
1.77
2.98
3.65
3.77
0.58
1.32
2.23
0.94
1.71
2.32
2.76
0.61
1.14
1.61
0.67
1.16
1.49
1.63
0.42
0.82
1.21
0.74
1.25
1.56
1.64
0.35
0.88
1.60
31
A.
8.0
7.0
C3 Monoculture
6.0
C-
4 Monoculture
C3 Mixture
C-
4 Mixture
C3 Projected
C4 Projected
0.0
0:8
2:6
4:4
6:2
8.0
C3:C4 PLANTS/POT
7.0
C3 Monomiture
6.0 -
C4 Monomittre
oe
N
-.35.0
C3 Mixture
i4.0
C4 Mixture
Mixture
C3 Projected
C4 Projected
2.0
1.0 -
0.0
0:8
2.6
4:4
6:2
80
C3:C4 PLANTS/POT
Figure 4. Modified Replacement Series Diagrams for total dry weight
(grams/pot) in control (A) and 03 (B). A. smithii= C3, B. curtipendula =
C4.
32
A. smithii monoculture yields were more depressed by 03 than the mixture
yields. 03 depressed monoculture yields across all densities an average of 31%
while mixture yields were depressed an average of 10% (Figure 4B). Growth
reductions of this magnitude are known for other species. In B. curtipendula,
03 caused an increase in maximum final monoculture yield from 5.1 grams/pot
in control chambers to 5.3 grams/pot with 03 (Figure 4B).
The monoculture response (ie., intraspecific competition) increased with
increased planting density for both species. With 03, the intensity of the
monoculture response decreased in A. smithii and increased slightly in B.
curtipendula (Figure 4).
A. smithii was affected more by interspecific competition than B.
curtipendula - that is, it had a greater mixture response. Additionally, the
mixture response in A. smithii was reduced by 03 (Figure 6). Yields of B.
curtipendula appeared to increase in response to 03 in the 2:6 mixtures thereby
reducing the mixture response at this density (Figure 4B). However, it is more
likely that B. curtipendula was responding to the reduced yields of A. smithii
rather than to 03. When grown with B. curtipendula, A. smithii actual yields
under 03 were approximately 50% of the controls, however, the predicted means
showed the yields to be equal. (Table 3 shows predicted shoot dry weights for
A. smithii to be 0.68 grams/pot in both the control and 03 treatments while
actual means were 0.93 grams/pot in control and 0.49 grams/pot with 03).
In
this mixture, A. smithii yields were reduced by nearly half under 03 which may
have allowed B. curtipendula to capture more resources and increase its growth.
Thus, it appears that for sensitive species, 03 has a greater impact than
competition on plant growth, however, for insensitive species, interspecific
33
competition has a greater impact on plant growth than 03 except when the
proportion of 03 sensitive species is low.
9
1.0
0.9
C3 CONTROL
0.8
C- 4
CONTROL
0.7
C- 3 0201E
1 0.6
C4 MOPE
0.5
0.4
0.3
0.2
0.1
0.0
2:6
4:
8:0
Rots / Pot
C3:C4
Figure 5. Intraspecific Competition (Relative monoculture response) for total
dry weight in control and 03. A. smithii = C3, B. curtipendula = C4.
1.0
0.9
C3 CONTROL
-4-
0.8
C4 CONTROL
0.7
C3 OZONE
O-
ZONE
10.6
0.4
0.3
0.2
0.1
0.0
61
4
2:6
C&C4
80
Plants / Pot
Figure 6. Interspecific competition (Relative mixture response) for total dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
34
Shoot Yield
The effect of 03 or competition on plant growth is not uniform for all plant
parts. For example, 03 typically inhibits root growth to a greater extent than
shoot growth (Miller, 1988). In addition, root competition is generally more
intense than shoot competition (Wilson, 1988). Thus, the separate analysis of
root and shoot dry weights may contribute towards understanding where biomass
reductions are occurring and which stress is biologically most important.
Monoculture maximum shoot yield was greater for B. curtipendula than A.
smithii in the control chambers. Conversely, at lower densities, A. smithii yields
were slightly greater than B. curtipendula. Thus, A. smithii reached a maximum
fmal yield at a lower density than B. curtipendula. Yields of B. curtipendula
were greater than A. smithii in mixtures (Figure 7A).
Monocultures of A. smithii were affected by 03 more than the mixtures. The
monocultures averaged a 22% ± 0.10 growth reduction across all densities while
mixtures averaged only a 8% ± 0.08 reduction (Figure 7B). An increase in
growth occurred in B. curtipendula monocultures (15% ± 0.04) and mixtures
(8% ± 0.1) with 03. 03 reduced the monoculture response for A. smithii which
corresponds with their decrease in growth. The monoculture response increased
slightly for B. curtipendula with 03 as did its growth (Figure 8). Interspecific
competition in A. smithii was reduced by 03. B. curtipendula showed an
increase in growth with 03 and a subsequent increase in interspecific except at
the 2:6 density where A. smithii yields were reduced thereby reducing
interspecific competition (Figure 9).
35
A.
4.0
-B-
3.5
C3 Monoculture
3.0-
C4 Monoculture
*
C3 Mixture
CA Mixture
12.0-
C3 Projected
C4 Projected
0.0:8
6:2
8.0
C3:C4 PLANTS/POT
84.0
-s-
3.5-
C3 Monoculture
3.0-
C4 Monoculture
am
-KC3 Mixture
s2.5-
C4 Mixture
1'2.0
C3 Projected
C4 Projected
0.0
0:8
2A
4:4
6:2
80
C&C4 PLANTS/POT
Figure 7. Modified Replacement Series Diagram for shoot dry weight
(grams/pot) in control (A) and 03 (B). A. smithii = C3, B. curtipendula = C4.
36
1.0
- 8-
0.9
C.3 CONTROL
0.8
C4 CONTROL
CONTROL
0.7
C3 OZONE
ak--
0.6
C4 MOPE
0.5
0.4
0.3
0.2
0.1
0.0
6:2
4
C3Z4
Rants / Po1
Figure 8. Intraspecific competition (relative monoculture response) for shoot
dry weight in control and 03. A. smithii = C3. B curtipendula = C4.
C3 CONTROL
-MC4 CONTROL
C3 OZONE
C4 OZOPE
Figure 9. Interspecific competition (relative mixture response) for shoot dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
37
Root Yield
The greatest difference in yield between these two species occurred in the
roots. A. smithii root yield was more than twice that of B. curtipendula at all
densities in monocultures (Figure 10A). Similar to total and shoot yields,
monocultures of A. smithii were more affected by 03 than the mixtures
(Figure 10B). Additionally, root growth was reduced by 03 more than shoot
growth (Figure 10A and B).
03 reduced the monoculture response for A. smithii and increased it slightly
for B. curtipendula (Figure 11). Root yield of B. curtipendula did not respond
to 03, but like the total yields, responded to the reduced A. smithii growth in the
2:6 mixtures (Figure 12). A. smithii was still more affected by interspecific
competition than B. curtipendula.
In summary: A. smithii is more sensitive to 03 than B. curtipendula; and for
A. smithii, 03 reduced total, shoot, and root growth more in the monocultures
than in mixtures; and root growth was more sensitive to 03 than total or shoot
growth (Table 4).
Table 5. Mean percent change ± 1 S.E. in growth across all densities due to 03.
A. smithii
B. curtipendula
Monoculture
Mixture
Monoculture
Mixture
Shoot
-15% t 0.05
-11% ± 0.04
+15% ± 0.04
+11% ±0.13
Root
-38% ± 0.04
-12% ± 0.09
+6% ±0.02
+8% ±0.14
Total
-31% ± 0.05
-10% ± 0.07
+12% ±0.03
+10% ±0.13
38
e
A. 4.0
C3 Monoculture
el
3.5
x
C4 Monoculture
3.0
C3 Mixture
32.5
Mixitre
C4 Mixture
C3 Projected
C4 Projected
1.0
0.5
0.0
0:8
2:6
4:4
6:2
80
C3:C4 PLANTS/POT
B.
0
4.0
o
C3 Monoculture
3.5 -
ii
C4 Monoculture
3.0
-
C3 Mixture
.315
C4 Mixture
C3 Projected
C4 Projected
1.0
0.5 0.0
0:8
2:6
4:4
6:2
8.0
C3C4 PLANTS/POT
Figure 10. Modified Replacement Series Diagrams for root dry weight
(grams/pot) in control (A) and 03 (B). A. smithii = C3, B. curtipendula = C4.
39
1.0
0.9
C3 CONTROL
0.8
C4 CONTROL
0.7
C3 MOPE
O- ZONE
10.6
0.5
0.4
0.3
0.2
0.1
0.0
26
4
C3C4
Plants / Poi
Figure 11. Intraspecific competition (relative monoculture response) for root
dry weight in control and 03. A. smithii = C3, B. curtipendula = C4.
1.0
- El-
0.9
C3 CONTROL
0.8
C4 CONTROL
0.7
C3 OZONE
1 0.6
C4 OZONE
0.5
0.4
0.3
0.2
0.1
0.0
26
62
4
C3C4
80
Plants / Pot
Figure 12. Interspecific competition (relative mixture response) for root dry
weight in control and 03. A. smithii = C3, B. curtipendula = C4.
40
Shoot /Root Ratios
03 increased shoot/root ratios in both A. smithii monocultures and mixtures
(Figure 13) by reducing root growth more than shoot growth. In monocultures
and mixtures of B. curtipendula, shoot/root ratios increased as 03 increased shoot
growth more than root growth (Figure 14).
Co- ntrol Monoct Itire
---
Control MIxtwo
- --
Ozone Monocult ure
--Ozone Mixture
Figure 13. 03 caused changes in A. smithii shoot/root ratios in
monocultures and mixtures.
4.5
- --
Control Monom lture
4.0
0
Control Mixture
g
Ozone Monocult Lre
---
.0
Ozone mixtire
5
2.0
1.5
1.0
1
3
5
7
9
Density
Figure 14. 03 caused changes in B. curtipendula shoot/root ratios in
monocultures and mixtures.
41
DISCUSSION
The modified replacement series design has allowed the effects of 03 on both
intra- and inter- specific competition to be determined. While fitting a curve to
the data provides a useful means of interpreting the replacement series diagrams,
it can also obscure some of the results as seen in the 2:6 mixtures. Additionally,
without statistical methods of determining differences between two lines,
interpretation of results can be biased towards the researcher's own expectations.
The two species allocated their carbon differently with A. smithii having
less absolute shoot yield and more absolute root yield than B. curtipendula.
However, in the controls, B. curtipendula affected A. smithii shoot, root and total
yield more than A. smithii affected B. curtipendula. With 03, interspecific
competition changes in ways which are difficult to generalize, but is an
indication that the "system" itself has changed.
03 reduced both intra- and inter-specific competition in A. smithii. The
changes in intraspecific competition in B. curtipendula were small in comparison
with changes in intraspecific competition in A. smithii. Reductions in
interspecific competition in B. curtipendula were most likely a response to the
03-caused reduced growth of A. smithii while increases in interspecific
competition was due to its greater increase in growth than biomass reduction in
A. smithii with 03.
These results do not agree with the general conclusions of Armentano and
Bennett (1992) that intraspecific competition is more sensitive to pollution than
interspecific competition. Rather it appears that the sensitivity of intraspecific
42
competition to 03 is dependent upon the species sensitivity to 03. In addition,
changes in interspecific competition appears to be dependent upon the proportion
of 03 sensitive-to-insensitive species. Thus, air pollution caused changes in
ecosystem structure and function would also be dependent upon each species 03
sensitivity and the ratio of sensitive to insensitive species.
The yields of A. smithii, the C3 species, were reduced by 03 with the
monocultures being more sensitive than the mixtures. This difference in mixture
sensitivity has also been found using monocultures and mixtures of ryegrass and
clover when exposed to 03 (Bennett and Runeckles, 1977), and acid rain
(Menchaca and Hornung, 1989). The role that stomatal conductance and ozone
uptake plays in causing a difference in sensitivity between monocultures and
mixtures is the basis of the next chapter.
Extension of Laboratory Results to the Field
Laboratory experiments provide the best way to analyze components of
stress and competition by controlling environmental conditions, and allowing for
experimental controls and replication. However, often with laboratory
experiments, extrapolation to field conditions is risky. In this study,
experimental temperature did not mimic normal growing temperatures or
duration. These species have growing periods which extend from spring to
summer with western wheatgrass actively growing in the spring while sideoats
grama waits until summer. Additional problems include potting media effects,
reduced root zone volume, and increased root zone temperatures.
43
Results from laboratory and greenhouse studies can provide useful
information that may or may not be verified with field studies. For example,
Evans and Ashmore (1992) used laboratory studies to examine the effects of 03
on several grass and forb species found in a semi-natural grassland. The grasses
were insensitive to 03 relative to the forbs. The researchers then went to the
field where they placed open-top chambers over the grassland to filter out
ambient 03. Predictions of changes in community structure based on the
laboratory results would have been incorrect. In the field, grasses responded to
filtered air with increased growth while the forbs showed reduced growth despite
their greater sensitivity to 03. The decrease in forb growth was due to the
decreased penetration of light through the grasses. Thus, when attempts are
made to predict air pollution effects on competition, inferences should be based
upon studies which utilize a mixture of plants and not on those designed to
determine individual plant responses.
General Conclusions
This model for 03/competition studies which utilizes an 03 sensitive and
insensitive species has provided some conclusions which may be generally
applicable to many ecosystems. However, without additional studies using a
variety of species, the results cannot be generally applied. Additional
information concerning whether the increase in growth in 03 insensitive plants
under 03 exposure is dependent upon the proportion of 03 sensitive to insensitive
can be further evaluated by expanding the number of mixtures in the
experimental design. This would allow for more than three points to be plotted
on the mixture curves, and thereby verify this hypothesis. Grasslands across the
temperate U.S are exposed to 03, and these experiments indicate that changes in
44
community structure are possible. Further investigations of 03 caused changes
in competition utilizing the lower 03 levels found in grasslands are needed to
assess this risk.
45
REFERENCES
Adams, R.M, J.D. Glyer, B.A. Mc Carl, 1988. The NCLAN economic
assessment: Approach, finding and implications. IN: Assessment of crop loss
from air pollutants. Edited by W.W. Heck, O.C. Taylor, and D.T.Tingey. 552pp.
Armentano, T.V., and J.P. Bennett, 1992. Air Pollution Effects on the Diversity
and Structure of Communities. IN: Air Pollution Effects on Biodiversity.
Edited by J.R. Barker and D.T. Tingey. Van Nostrand Reinhold, NY. 322pp.
Bennett, J.P., and V.C. Runeckles, 1977. Effects of low levels of ozone on plant
competition. J. Appl. Ecol. 14:877-880.
Christie, E.K., and J.K. Detling, 1982. Analysis of interference between C3 and
C4 grasses in relation to temperature and soil nitrogen supply. Ecology 63:12771284.
Elkiey, T., and D.P.Ormrod, 1980. Response of turfgrass cultivars to ozone,
sulfur dioxide, and nitrogen dioxide, or their mixture. J. Amer. Soc. Hort. Sci.
105:664-668.
Evans, P.A., and M.R. Ashmore, 1992. The effects of ambient air on a seminatural grassland community. Agriculture, Ecosystems and Environment 38:9197.
Floyd, R.A., M.S. West,W.E.Hogsett, and D.T.Tingey, 1989. Increased 8hydroxyguanine content of chloroplast DNA from ozone-treated plants. Plant
Physiol. 91:644-647.
Guderian, R., D.T. Tingey, and R. Rabe, 1985. Effects of photochemical
oxidants on plants. IN: Air pollution by photochemical oxidants: Formation,
transport, control, and effects on plants. Edited by Robert Guderian. SpringerVerlag. Ecological Series 52. New York.
Harper, J.L., 1977. Population biology of plants. Academic Press, NY. 892 pp.
Heck, W.W., O.C. Taylor, R. Adams, G. Bingham, J. Miller, E. Preston, L.
Weinstein, 1982. Assessment of crop loss from ozone. J. Air Pollut. Control
Assoc. 32:353-361.
46
Hitchcock, A.S., 1971. A manual of the grasses of the United States. Dover
Publications, Inc. 999pp.
Jolliffe, P.A., A.N. Minjas, and V.C. Runeckles, 1984. A reinterpretation of
yield relationships in replacement series experiments. J. Appl. Ecol. 21:227-243.
Menchaca, L., and M. Hornung, 1989. Response of white clover (Trifolium
repens L.) and ryegrass (Lolium perenne L.) to acid rain is situations of species
interferences. New Phytol. 111:483-489.
Miller, P.R., O.C. Taylor, R.G. Wilhour, 1982. Oxidant air pollution effects on
a western coniferous forest ecosystem. USEPA-600/D-82-276. lOpp.
Miller, 1984. Ozone effects in the San Bemadino National Forest. IN: Air
pollution and the productivity of forests. Eds. A.A. Millen and L. Dochinger.
Izaak Walton League of America. Arlington, VA. pp 161-197.
Montes, R.A., U. Blum, and A.S. Heagle, 1982. The effects of ozone and
nitrogen fertilizer on tall fescue, ladino clover, and a fescue-clover mixture.
I. Growth, re-growth, and forage production. Can. J. Bot. 60: 2745-2752.
Mooney, H.A., W.E. Winner, and E.J. Pell, 1991. Responses of plants to
multiple stresses. Academic Press, Inc. 422pp.
Pell, E.J., W.E. Winner, C. Vinten-Johansen, and H.A. Mooney, 1990. Response
of radish to multiple stresses. I. Physiological and growth responses to changes
in ozone and nitrogen. New Phytol. 115:439-446.
Rebbeck, J., U. Blum, and A.S. Heagle, 1988. Effects of ozone on the regrowth
and energy reserves of a ladino clover-tall fescue pasture. J. of Appl. Ecology.
25:659-681.
Reich, P.B., 1987. Quantifying plant response to ozone: A unifying theory.
Tree Physiology 3:63-91.
Rousch, M.L., S.R., Radosevich, R.G. Wagner, R.D. Maxwell, T.D. Petersen,
1989. A comparison of methods for measuring effects of density and proportion
in plant competition experiments. Weed Science 37:269-275.
Salisbury, F.B., and C.W. Ross, 1985. Plant Physiology. Wadsworth Publishing,
Inc., Belmont CA., 540 pp.
47
Tingey, D.T., and W.E. Hogsett, 1985. Water stress reduces ozone injury via a
stomata' mechanism. Plant Physiol. 77: 944-947.
Wilson, J.B., 1988. Shoot competition and root competition. J. of Appl. Ecol.
25:279-296.
Winner, W.E. and H.A. Mooney, 1980. Ecology of SO2 resistance: III.
Metabolic changes of C3 and C4 Atriplex species due to SO2 fumigations.
Oecologia 44:296-302.
Wolff, G.T. and P.J. Lioy, 1980. Development of an ozone river associated with
synoptic scale episodes in the eastern United States. Environ. Sci. Technol.
14:1257-1260.
Youngner, V.B., and F.J. Nudge, 1980. Air pollution oxidant effects on coolseason and warm-season turfgrasses. Agronomy Journal 72:169-170.
Zeller , K.F., and D.L.Hazlet, 1989. Ozone deposition and leaf area index at the
Pawnee National Grassland for the 1988 growing season. IN: Effects of air
pollution on western forests. Transactions of the Air and Waste Management
Association. Edited by R. K. Olson, and A. S. Lefohn. 577pp.
48
CHAPTER 3: ANALYSIS OF OZONE UPTAKE.
INTRODUCTION
The relationship between ambient 03 concentrations and effects on plants is
directly related to 03 absorption. Absorption is defined as the product of the
flux rate of 03 through leaf stomata and the duration of exposure.
Absorption
and adsorption (03 adhered to leaf surfaces) are sometimes combined to define
total 03 uptake, however, for the purposes of this paper, I will use the term
uptake synonymously with absorption. Uptake differs from the more generic
term, 03 exposure, which is simply the concentration of the pollutant and the
amount of time the organism was subjected to the pollutant (Hogsett, et. al.,
1988). Uptake should be a more accurate predictor of plant responses to 03 than
exposure. However, determining uptake is more difficult than determining
exposure since it requires knowledge of ambient 03 concentrations, stomatal
conductance, and an approach to calculating 03 flux into the stomata which is
based upon assumptions which are not testable. Additional problems arise
because total plant leaf area and stomatal conductance are constantly changing
and cannot be monitored continuously.
Research Objectives
Since competition causes changes in plant growth and form, it is
possible that it would also cause a change in 03 uptake. Possible mechanisms
49
for changes in 03 uptake due to competition could include changes in plant leaf
area and stomatal conductance. In the previous Chapter, 03 was shown to cause
changes in competition between two grasses. This study was designed to
determine if the amount of 03 uptake is related to the changes in competition.
The specific objectives of this study were to:
1) quantify 03 uptake in two grasses with differing 03 absorption rates
growing in both monocultures and mixtures;
2) determine how intra- and inter- specific competition modified 03 uptake;
3) determine if 03 uptake is related to changes in competition.
50
MATERIALS AND METHODS
Experimental Design
Two experiments were conducted using western wheatgrass (Agropyron
smithii), a C3 species, and sideoats grama (Bouteloua curtipendula), a C4 species.
These species were chosen for several reasons:
1) they co-occur over a broad geographic range (Hitchcock, 1971).
2) they are of similar size and morphology,
3) they have different photosynthetic pathways and stomatal conductance
rates (Winner and Mooney, 1980) and should therefore have differing
responses to 03.
Monocultures and mixtures of each species were planted in a modified
Replacement Series Design (Jolliffe et. al., 1984), also known as a synthetic nointeraction design (Rousch, et. al., 1989).
Plant Culture
A. smithii seeds were collected by Stock Seed Farm (Murdock, Nebraska)
from field sites in northern Nebraska and southern South Dakota. B.
curtipendula seeds were collected at the Stock Seed Farm in eastern Nebraska,
(Dave Stock, pers. comm.).
51
A. smithii seeds were sown onto brown blotter paper which had been soaked
in 0.2% KNO; solution. Seeds germinated in the dark, in growth chambers
maintained at 25/15 °C day/night temperatures until shoots were 5 cm tall
(approximately 10 days). These procedures were used to break dormancy based
on techniques used by the Oregon State Seed Laboratory (Corvallis, OR.).
Five days after A. smithii seeds were sown, B. curtipendula seeds were sown
into four 50cm x 38cm x 7.5cm wooden flats filled with vermiculite. Flats were
placed into growth chambers maintained at 30/27 °C day/night temperatures,
and 16 hour photoperiod at 500 pmoles ni 2 s-1. Seeds were misted with tap
water daily until shoots were 4 cm tall (approximately 5 days).
Seedlings of uniform size from both species were then individually
transplanted into pots (10cm x 20cm) filled with a promix:perlite mixture (1:1 by
volume). Monocultures at densities of 2, 4, 6, and 8 plants per pot, and
mixtures of 2 C3's with 6 C4's, 4 C3's with 4 C4's, and 6 C3 's with 2 C4's were
replicated twice in each of six chambers (Figure 15). Plants were held in a
greenhouse maintained at approximately 25°C, ambient light levels (Oct. - Nov.
for Experiment #1, and Jan. - Feb. for Experiment #2) supplemented with 1000
Watt high pressure sodium lamps, and watered with tap water. Five weeks after
transplanting, pots were placed into exposure chambers.
Exposure System
A plant exposure system, located in Weniger Hall on the OSU campus, was
constructed to conduct research on the effects of air pollutants on plants. The
52
system is capable of delivering and monitoring 03 to 12 Continuously Stirred
Tank Reactors (CSTR's) chambers. Additionally, the system can control and
monitor day/night temperature and humidity, and photoperiod.
Plants were exposed to 03 for 28 days in Experiment 1 and 42 days in
Experiment 2. Environmental conditions for both experiments were set to 27/20
°C day/night temperatures, 50/50 % day/night relative humidity, and 16 hour
photoperiod at 400 pmoles iff2
photosynthetically active radiation (PAR)
(Table 6). This temperature regime was selected so that neither grass species
was grown at its optimum temperature.
Cont inouely Stirred Tar& Reactor
(CSTR)
C3 Monocuttures
41k.
0 ® 0 01
g 0 0®
g 0 IC>.
C4 Mcoclihres
C34 kixturas
e go@
g @oo
6
Chambers
oeo
3 Control + 3 Ozone
Figure 15. Planting Density (C3:C4 Plants/Pot). Pots were randomly arranged
within each chamber.
53
Table 6. Experimental conditions and parameters measured for analysis of 03
uptake.
Experiment #1 Experiment #2
OZONE EXPOSURE DURATION
WEEKLY POT LEAF AREA
All pots in 1 03 & 1 Control Chamber
Density = 4 pots in a 2nd 03 & Control Chamber
GAS EXCHANGE
Baseline Diurnal (on 1 leaf)
Week 1 Diurnal
Week 2 Diurnal
Leaf Age (young to old)
Leaf Position (base to tip)
Whole Plant Week 1
Whole Plant Week 2
Whole Plant Week 3
Whole Plant Week 4
Whole Plant Week 5
Whole Plant Week 6
28 DAYS
42 DAYS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
DRY WEIGHTS
Shoot Dry Weight (pots ch 0;1)
Root Dry Weight
Dark Regrowth (Shoot & Root)
ENVIRONMENTAL PARAMETERS
Ozone SUM (ppm-hr)
Day Temperature °C
Night Temperature °C
Day Humidity %)
Night Humidity %
Solar Radiation Sum (E m-2)
'Mean ± SE
n=6
n=6
n=6
n=3
n=3
44
27.0 t 0.11
21.9 ± 0.1
46.2 ± 0.2
49.7 ± 0.2
451
71
26.7 ± 0.1
18.2 ± 0.1
47.0 ± 03
47.1 ± 0.2
542
Throughout the exposure, data on hourly 03 averages, and daily maximum,
minimum, and average air temperature were collected in each chamber.
54
Additionally, data on daily maximum, minimum, and average relative humidity,
and total solar radiation were collected in two chambers. Plants were watered to
pot capacity with tap water daily and allowed to drain.
Plants in three chambers received charcoal-filtered air, while plants in three
other chambers were exposed to a 28-day repeating episodic 03 profile
(Figure 16).
0.3
0.25
0.2-
t
0.05
3
5
7
9
11
13
15
17
19
21
23 25 27
29
DAY
Figure 16. 28-day repeating episodic 03 profile.
The 03 profile, developed at the Environmental Protection Agency's (EPA)
Corvallis Environmental Research Lab, was modified from a "base" profile
originally developed utilizing the EPA's 1978-1983 Storage and Retrieval of
Aerometric Data (SAROAD) databases from 6 mid-Western states (Illinois,
Indiana, Ohio, Michigan, Wisconsin, and Minnesota).
55
This "base" profile was designed to have similar characteristics to ambient 03 in
this region.
These characteristics include:
1) the daily minimum 03 concentration occurring between 0300 and 0500 hours,
2) the maximum concentration occurring between 1400 and 1700 hours,
3) a 7 hour seasonal (May - Sept.) mean of 0.044 ppm,
4) equal frequency of peak days (defined as days during which the hourly
concentration exceeded 0.070 ppm).
The episodic profile used in this experiment was modified from this "base"
profile by increasing all hourly concentrations by sigmoid weights which
increased the total exposure by 50%.
Leaf Area Measurements
An additional set of 4:0, 4:4, and 0:4 pots (3 pots species"' mixture' 03
treatment-1 week') were planted as described above for use in a weekly
destructive harvest. Once per week, all leaves from these pots were placed
individually onto a LI-COR 3000A Leaf Area Meter which measures and records
leaf length and leaf area. Weekly leaf area regression curves were then derived
by regressing leaf length on leaf area. Then, leaf lengths were recorded for all
plants in all pots in one control chamber and one 03 chamber. Replicate
samples were taken on all plants in the 4:0, 4:4, and 0:4 pots in second control
chamber and a second 03 chamber. Total pot leaf area was then determined
from the established regression curves. Due to an unbalanced leaf length
sampling design repeated measures analysis was used to determine leaf area
56
differences between 03 treatments, monocultures and mixtures (mixture effect),
and their interaction (ozone x mixture) on the 4:0, 4:4, and 0:4 pots only.
Stomatal Conductance Measurements
A LI-COR 6200 portable photosynthesis system was used to monitor
conductance (gam) rates. To minimize disturbance of the plants while continuing
03 exposure, measurements were made inside the chambers by placing a
plexiglass door, with a 25cm x 15cm opening, in place of the chamber door.
The opening then allowed for handling of the plants and operation of the
equipment while the plants were being exposed to 03.
For both experiments, one plant in the density 4 monoculture and 4:4
mixture pots was measured. During the first experiment, diurnal patterns were
measured on days 0 (baseline), 7, and 14 by monitoring the most fully expanded
leaf every other hour during the daily photoperiod using a 'A liter cuvette. For
the second experiment, whole plant conductance was measured by placing all the
leaves within a 4 liter cuvette. These measurements were conducted at mid-day
once per week throughout the exposure period. Conductance values were
assumed to be representative for all plants within the pot, for plants in the other
planting densities, and for the entire interval between sampling periods. For
example, I assumed that the C3 monoculture pots at density 2, 4, 6, and 8 had
one conductance value for an entire week, while C3 mixture pots had a different
value that week. Repeated measures analysis was used to determine conductance
differences between 03 treatments within a species.
57
Ozone Uptake Calculation
03 uptake (pot total) was calculated for the second experiment only using
predicted leaf area, whole plant conductance, and actual 03 concentrations in
each chamber. 03 concentrations in leaf intercellular air spaces were assumed to
be zero, as was boundary layer conductance (Laisk, et. al., 1989). Uptake was
calculated as:
z.
003
1 . 68
where:
Q03 = 03 uptake
za = ambient 03 concentration
gip, = gas phase conductance to water vapor
1.68 = ratio of diffusion rates for water and 03.
58
RESULTS
Leaf Area
In A. smithii, there was a significant 03 effect (p=0.040), mixture effect
(p<.001), and 03 x mixture interaction (p4.006) on leaf area in the density 4
pots. While not statistically analyzed, differing results occurred at the other
densities. For example, an 03 effect was observed at all densities, but there was
no mixture effect or interaction at the lowest density (Figure 17A-D). In the
density 4 pots of B. curtipendula, only the mixture effect was significant
(p4.015) with the total pot leaf area of mixtures being less than monocultures.
Similar results occurred in the other densities (Figure 18A-D). Thus, the
reduction in leaf area, for both species, could potentially reduce photosynthetic
capacity as well as surface area for 03 uptake.
Conductance
Stomatal conductance was measured because of its role in regulating 03
uptake. For example, 03-caused decreases in leaf area would not necessarily
lead to reductions in 03 uptake if conductance increased. Assessing stomatal
conductance can be complex because conductance rates change with time of day,
leaf age, environmental factors and even 03 uptake.
59
After 14 days of 03 exposure in the first experiment, single leaf stomatal
conductance was significantly depressed (p4.002) in A. smithii. There was no
significant mixture effect (p>0.5) or 03 x mixture interaction (p>0.5).
Conductance averaged 0.447 Moles In-2 sec' in control chambers and 0.163
Moles Ma see in 03 chambers. In B. curtipendula, there was no significant 03
effect (p>0.5), mixture effect (p>0.5), or 03 x mixture interaction (p=0.19). B.
curtipendula conductance averaged 0.056 Moles ni2 sec"' (Figure 19).
After following the decline in conductance on a single leaf, it was obvious
that it was not representative for the whole plant since other leaves had various
stages of visible injury and therefore the conductance would not be a
representative value to use in uptake calculations. Additional conductance
measurements showed that leaf conductance varied depending upon its position
on the plant. However, there was no difference in conductance with plant
density (data not shown). Therefore, in Experiment 2, whole plant conductance
was measured instead of a single leaf to give a more accurate approximation of
conductance and hence 03 uptake. Except for a mid-day depression in A.
smithii, conductance was relatively constant and therefore, a diurnal trend in
stomatal conductance was not used in calculating 03 uptake (Figure 19).
A. smithii whole plant conductance was significantly greater (p=0.03) for 03
exposed plants than for control plants by week 3 and remained that way
(Figure 20). In A. smithii, there was also a significant mixture effect (p=.003)
with monocultures have greater whole plant conductance rates. There was no
significant 03 x mixture interaction (p>0.5). Thus, A. smithii monocultures had
higher conductance rates than mixtures, and 03 increased conductance rates in
both (Figure 21).
For B. curtipendula, no significant 03 effect (p=0.44),
mixture effect (p>0.5), or 03 x mixture interaction (p4.27) was found , and
60
therefore, all data were pooled and a mean conductance value calculated to be
0.030 pmoles m 2 sec"' (Figure 19).
Ozone Uptake
If 03 uptake was a function of leaf area only, 03 uptake by B. curtipendula
would have been greater than A. smithii since total pot leaf area in B.
curtipendula is greater. However, due to the large differences in whole plant
conductance, 03 uptake was actually greater in A. smithii than B. curtipendula
(Figure 22). Monocultures of A. smithii generally had greater conductance rates
and leaf areas than mixtures, resulting in higher 03 uptake. Monoculture uptake
was also higher in B. curtipendula, however, this was a function of increased
leaf area only since monoculture and mixtures were assumed to have the same
conductance rates.
61
so
t°
v
I
440
ri
1
k2.
10
Control Mona
1/0nOCIJI UN'S 0
MI xttro
111
AA
0
0
2
1
4
3
5
7
6
Week
Figure 17A. 03 caused leaf area changes over time in A. smithii monocultures
and mixtures. Density=2, Error Bars = 1 S.E., n=4.
130
120
%ADO
ga
1 8°
1 70
BO
& 30
40
30
30
Control Mona
Monccu I tiro 0 .
All num
10
1
A
2
5
6
7
Week
Figure 17B. 03 caused leaf area changes over time in A. smithii monocultures
and mixtures. Density=4, Error Bars = 1 S.E., n=4.
62
140
130
L0110
100
90
31"
270
& so
50
40
30
Contra I Ozone
Monoculture
Witty*
AA
20
0
1
2
Week
Figure 17C. 03 caused leaf area changes over time in A. smithii monocultures
and mixtures. Density=6, Error Bars = 1 S.E., n=4.
180
170
6160
X130
ti
i2a
70 80
Contra I Ozone
Monoculture
Mixture
50
AA
40
0
1
7
2
Week
Figure 17D. 03 caused leaf area changes over time in A. smithii monocultures.
Density=8, Error Bars = 1 S.E., n=4.
63
1
2
4
3
5
6
7
Week
Figure 18A. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =2, n=4, Error Bars= 1 S.E.
1E10
170
150
t5°
tA40
$130
.120
1110
woo
t 90
BO
70
50
50
40
30
20
0
1
2
4
3
5
6
7
Week
Figure 18B. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =4, n=4, Error Bars= 1 S.E.
64
300
290
WM°
el3a
1.190
314:
120
k100
BO
60
40
Control Ozone
Monaco !tiro°
81xture
AA
20
0
0
2
1
3
6
5
7
Week
Figure 18C. 03 caused leaf area changes over time in B. curtipendula
monocultures and mixtures. Density =6, n=4, Error Bars= 1 S.E.
200
Contra I Ozone
Monoculture
Mixture
1130
0
E
U
ra
1140
O
2120
k
100
80
800
1
2
3
4
5
6
7
Week
Figure 18D. 03 caused leaf area changes over time in B. curtipendula
monocultures. Density =8, n=4, Error Bars= 1 S.E.
65
0
Contra 1 Ozone
U
C3
164
A
A
i'3
0.1
00
6
8
-10
12
14
16
18
20
22
How
Figure 19. Single leaf diurnal conductance after 14 days of 03 exposure. Error
bars represent 1 standard error. n=8. A. smithii = C3, B. curtipendula = C4.
0 40
1
8.24
115
8
0 00
0
1
2
3
4
WEEK
Figure 20. Whole plant conductance during 6 weeks of 03 exposure. Error bars
represent 1 standard error. n=8. A. smithii = C3, B. curtipendula = C4.
66
Contra I Ozone
1x
1
2
3
4
5
6
A
7
WEEK
Figure 21. 03 caused changes in A. smithii conductance in monocultures and
mixtures. Error bars = 1 standard error. n= 4.
300
250
110
I
i00
I50
00
50
0
2
4
6
8
Density
2
4
6
Figure 22. 03 uptake in A. smithii (C3) and B. curtipendula (C4) grown in
monocultures and mixtures.
67
DISCUSSION
By estimating whole pot leaf areas, whole plant stomatal conductance rates,
and knowing hourly 03 concentrations, total 03 uptake per pot has been
calculated. While 03 reduced leaf area in A. smithii, it increased the stomatal
conductance rates. The increased conductance rate is a result of older leaves
senescing, leaving the younger leaves which have an inherently higher
conductance rate. Interspecific competition also reduced leaf area and
conductance in A. smithii. However, the increase in conductance due to 03 was
greater than the reductions in leaf area and conductance due to competition and
thus, the net effect was to maintain a high level of 03 uptake.
In B. curtipendula, interspecific competition reduced leaf area but had no
effect on conductance. Also, 03 had no effect on leaf area or conductance.
Thus, for B. curtipendula, changes in 03 uptake were a function of leaf area
while in A. smithii, changes in uptake were a function of conductance, leaf area
and competition.
As seen in the previous chapter, A. smithii monocultures showed a
greater reduction in growth due to 03 than mixtures. Due to the higher
conductance rates of the monocultures, 03 uptake is greater than in mixtures
even though leaf area is reduced by 03, and interspecific competition. Thus,
changes in plant competition are directly related to 03 uptake.
In this experiment, plants were watered daily with tap water and while not
quantified, it did not appear that mixtures were drier than monocultures. If they
were, then stomatal closure would account for the reduced conductance rates and
68
reduced 03 sensitivity. Otherwise, investigations into the mechanism of reduced
stomatal conductance of plants grown in mixtures are required.
General Conclusions
The relationship between 03 uptake and reductions in dry weight showed a
positive correlation. Thus, knowing 03 uptake can provide some estimation of
potential growth reduction. However, a plant's internal sensitivity and response
to 03 (eg., its ability to repair damage) can vary by species, and therefore makes
the uptake-growth response relationship more complex.
The relationship between 03 uptake and the subsequent changes in
competition is difficult to define because both are represented on a per pot basis.
Comparing dry weight and uptake on a per plant basis does not provide any
better understanding of the relationship. Additionally, as seen in the previous
Chapter, the influence of 03 may be dependent upon the proportion of sensitive
to insensitive individuals. Thus, quantifying 03 uptake provides little insight to
the potential changes in either intraspecific or interspecific competition.
69
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