FINAL REPORT ON
AIR POLLUTION EFFECTS ON WOODY PLANTS
Environmental Protection Agency
Grant No. R800865
by
Donald D. Davis
Principal Investigator
Center for Air Environment Studies
The Pennsylvania State University
University Park, PA
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FINAL REPORT ON
AIR POLLUTION EFFECTS ON WOODY PLANTS
Project Period: May 1, 1972 - March 31, 1976
Environmental Protection Agency
Grant No. R800865
by
Donald D. Davis
Principal Investigator
Centre for Air Environment Studies
The Pennsylvania State University
226 Fenske Laboratory
University Park, Pennsylvania 16802
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I. Stated Objectives:
The overall or long range objectives of the proposed research
were:
A. The development of air quality criteria with respect to the
effects of photochemical air pollutants on woody vegetation.
B. An understanding of theinfluence of various environmental
factors on the response of plants to photochemical air pollutants.
C. The development of a list of woody ornamental plants that are
resistant to photochemical air pollutants under a variety of enviro-
nmental conditions which could be used as a basis for recommendations
to landowners, homeowners, nurserymen, foresters, municipal and
governmental agencies in areas of potential air pollution problems.
D. Ultimately, the development of models that could be used to
predict the occurrence of vegetation injury and damage in a given area.
II. Research Conducted to Meet Objectives:
A. Research conducted under guidance of F. A. Wood and N. L. Lacasse
(5-1-72 to 4-30-73]
The following research was conducted under the supervision of the
first two principal investigators on the grant. Portions of this
research were initiated in previous years under the previous EPA grant,
but were completed during the present project period. Therefore, this
information was included by Dr. Lacasse in the first project report.
Since Dr. Davis was not involved in this phase of the research, the
information is reported exactly as written by Dr. Lacasse in the first
project report.
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Continuation page
1. Screening of ornamentals for resistance to ozone. (1972)
The following species of ornamentals were exposed to various
concentrations of ozone for varying time periods to determine
the dosage required to produce visible injury. The plants
were from either seedling or cutting origin and were exposed
under controlled conditions in a growth chamber. Prior to
fumigation these plants were potted in a peat-perlite mixture
and maintained either in the greenhouse or in a nursery bed
outdoors. A standard procedure was adopted for all fumigations
and consisted of: 2 hours of light treatment prior to exposure,
exposures at an air temperature of 75° F, relative humidity at
75%, and light intensity at approximately 2,200 ft. c. Time
of exposure was varied to determine response threshold, as
was the concentration of the ozone.
Table 1
Response of ornamentals to ozone exposure 1
—' —i
— ; rr
2 4 6 8
Ailanthus
10
• X
X
25
X
Azalea (campfire)
10
X
X
25
X X
Azalea (Mollis)
10
X
X
25
X
Azalea (snow)
10
X
25
X X X X
Cotoneaster (rock)
10
X
X
25
XXX
Cotoneaster (snow)
25
X
Cotoneaster (spreading)
10
X
X
25
X X X X
Euonymus (dwarf winged)
25
X
X
50
X
X
Firethorn (Lalands)
25
X
50
X X X X
Forsythia (Lynwood gold)
25
X
Gingko
25
X
X
Ivy (English)
25
X
X
50
X
X
Juniper (Andora)
25
X
X
50
X X X X
X
Lilac
25
X
x
Pachysandra
25
X
X
50
X
X
Periwinkle
25
X
X
50
x x x 8
Pieris (Japanese)
25
X
X
50
X
Sensitive
x
x
x
X
X
X
X
X
X
X
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Continuation page
Table 1 (continued)
Species
Time hrs.
O3 conc.,pphm 2 4 6 8
Response
Resistant Sensitive
Rhododendrum (Nova Zembla)
10
X
X
25
X
X
Rhododendron (Roseum elegans)
10
X
X
Rhodo. Cataw. album
25
X
X
50
X
X
Rhododendrum (Carolina)
25
X
X
50
X X X X
X
Viburnum (Koreanspice)
25
X
X
50
X X X X
X
Yew (compact spreading)
25
X
X
50
X
X
Yew (Hicks upright)
25
X
X
50
X X X X
X
*A total of 1,824 plants were exposed in these fumigations.
Relative amount of sensitivity within populations. In addition
to the data in Table 7, a log is kept on the number of sensitive
individuals within a given species or variety. Such information
is useful in determining the amount of variability present. Such
information would be useful, for example, for geneticists in
selecting resistant stock of a particular variety or cultivar
for propagation. This information is presented in Table 2.
Table 2
Percent of fumigated population sensitive to ozone at 25 pphm for 8 hrs.
Species
# of plants exposed % of plants sensitive
Ailanthus
14
100
Azalea (campfire)
52
98
Azalea (Mollis hybirds)
33
33
Azalea (Snow)
82
85
Cotoneaster (Rock)
79
94
Cotoneaster (Spreading)
85
62
Firethorn (Laland's)
63
14
Forsythia (Lynwood Gold)
47
62
Rhododendron (Catawbeiense album)
34
9
Rhododendron (Nova Zembla)
38
66
Rhododendron (Roseum elegans)
34
53
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¦3
"2. Response of petunia and chrysanthemum to PAN.
Petunia. Twenty-eight F-l hybrids of petunia were exposed to 15
pphm ±1.5pphm of PAN for 1 hour at a temperature of 24 °C,
relative humidity of 70%, and a light intensity of 3400 ft. c.
The plants were potted in a 1:1:1 soil, peat, perlite mix and
fertilized. These plants were maintained in the greenhouse. All
plants received a minimum of 3 hours pre- and post-exposure to
light following the PAN fumigation. The plants were exposed in
which the PAN concentration was established prior to placing the
plants within it.
PAN was generated by irradiating ethyl nitrate and oxygen with black
lights. Monotoring of PAN within the chamber was accomplished by injecting
a grab sample into a modified gas chromatograph.
A symptom evaluation scheme was developed based on symptom type and
all leaves on the main stem of the plants were evaluated. The leaves were
consecutively numbered beginning with the top-most leaf greater than 1 cm
in length. The results are presented in Table 3 below.
Table 3
The relative sensitivity of F-l varieties of petunia (Petunia hybrids)
exposed to PAN for 1 hr. at 24°C. and 70% RH.
Variety name
Colora
Severity
index
Leaf # with
most injury
Snowdrift
W.Y
219.9
6
White cascade -1
W
209.9
6
White Sails
w
167.4
6
Popeye
R,W
122.5
6
Pink Cameo
P
120.6
6
White Joy
W
115.2
7
Pink Paradise
P,W
113.4
5
White Cascade -2
w
101.8
6
Red cap
s
100.2
7
Pink Bountiful
p
89.8
6
Snow Magic
w
68.3
7
Blue Magic
B
63.8
6
Festival
R,W,Y
47.4
5
Apollo
W
46.2
7
Black Magic
B1
39.3
6
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S GPO* 1970 -
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Table 3 (continued)
Variety name
Color a
Severity
Leaf # with
index
most injury
Red Joy Improved
R
38.6
7
Rose Joy
P
37.8
5
King of Diamonds
0,S
36.9
6
White Delight
W
21.9
7
Pink Cascade
P
25.5
6
Zig Zag
R,W
25.5
5
Glacier
W
25.0
7
Red § White Delight
R,W
7.9
6
Mariner
P
7.4
6
Coral Cascade
C
7.1
6
Happyness
P
1.4
7
Candy Apple
R
0.4
6
Coral magic
C
0.0
a.
B=blue
Bl=black
O=orange
P=pink
R=red
S=scarlet
W=white
Y=yellow
Additional work was done with PAN on petunias to determine the dosage
response. The objective of this study was to provide basic information on
the response of plants to PAN by exposing petunias to PAN at different
concentrations for different time intervals.
In these studies plants were grown from seed in the same type of soil
mix listed above. PAN was generated in the usual manner. In these experi-
ments, the plants were placed in the chamber a minimum of 24 hours prior
to exposure. They were maintained at 24° C, 75% RH, and 3400 ft. c. for
a 15 hour photo-period.
At the beginning of the exposure sequence 24 plants were placed in
the exposure chamber and 24 m the control chamber. After 30 minutes of
exposure, six plants were removed from the exposure chamber and placed in
the control chamber. These six plants were replaced by six unexposed plants.
The plants that were removed from the exposure chamber represented a 30-minute
treatment, while the six plants placed m the treatment chamber were exposed
for the next 120 minutes. After 60 minutes had elapsed, six additional plants
were exchanged in the same fashion. This process was repeated after each
30 minutes of elapsed time up to 120 minutes. This procedure resulted in
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Continuation page
two exposure replications at each time interval of 30, 60, 90 and 120 minutes.
Plants were exposed to PAN at 5, 10, 20, 30 and 40 pphm. Plants exposed to
5 and 40 pphm were only exposed at 120 and 30 minutes, respectively.
The plant response was evaluated by the same scheme used in the previous
experiments. The results are presented in Table 4 below.
Table 4
Average severity index resulting from exposure of petunias to PAN
at different concentrations and times.
Time of exposure
minutes
PAN cone., pphm
5
10
20
30
40
30
-
0
74
214
279
60
-
97
231
272
-
90
-
153
283
300
_
120
116
222
305
304
-
The results of these experiments suggest that the dose response is
linear after the threshold is exceeded and before the amount of susceptible
tissue remaining becomes a limiting factor. These results also suggest that
threshold values for doses obtained over long periods of time are somewhere
between 5 pphm-hr at which no damage was observed and 10 pphm-hr where sub
stantial injury was noted.
Chrysanthemum. Eight cultivars of chrysanthemums were exposed
to 20 and 60 pphm of PAN for 4 hours. Of the 256 cuttings ex-
posed, symptoms developed on one plant exposed to 60 pphm for
4 hours.
Chrysanthemum (chrysanthemum morifolium ramat.1 is one of the most widely
used herbaceous ornamental plants in the U.S. today and it represents a major
source of cut flowers. During the mid-1950's chrysanthemum was reported to be
generally resistant to ambient smog in California. However, several varieties
have been reported to develop PAN-like symptoms in the eastern U. S. The symp-
toms reported consisted of an abaxial leaf surface glazing and bronzing.
These experiments reported below were done to determine the relative
sensitivity of eight different cultivars to PAN.
The cultivars used were: Gaiety, Delight, Forty-niner, Golden Gate,
Princess Ann, Streamer, Torch, and Tuneful. The cuttings were obtained by
Yoder Brothers, Inc. of Barberton, Ohio. The cuttings were maintained in a
regularly fertilized peat-perlite mixture and grown in the greenhouse prior to
and immediately following exposure to PAN. Four cuttings of each cultivar
were used in the exposure, and the eight varieties were subjected to eignt
different exposures. During the initial exposures, PAN dosages of 20 pphm for
4 hours were used, dosages of 60 pphm for 4 hours were used in subsequent ex-
posures. Following exposures, cuttings were transferred to the greenhouse
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where they were examined daily for symptoms and final symptom evaluation was
made five days following exposure.
The following results were obtained. A total of 256 cuttings were ex-
posed to dosages ranging from 20 pphm for 4 hours to 60 pphm for 4 hours.
Symptoms were only observed on one plant which had been exposed to 60 pphm.
The symptoms consisted of a bifacial bronzing of the leaves and they occurred
on the leaves at the third node down from the terminal bud.
The failure of PAN-like symptoms to develop on eight varieties exposed
to these concentrations suggests that the symptoms observed in the field in
the eastern U.S. are probably due to something other than PAN.
3. Influence of atmospheric temperature and moisture regimes on
the response of sensitive plants to ozone on PAN.
Only a portion of the work proposed has been completed, other work is in
progress and additional information will be available by the end of the present
budget period. The work that has been done so far consisted of determining the
influence of temperature, relative humidity, and absolute humidity on the re-
sponse of petunias to PAN.
White cascade petunias (Petunia hybrida Vilm.) were grown in the soil mix
described above and fertilized and watered as needed. The plants were grown in
the greenhouse. At a minimum of four days prior to exposure, 48 plants were
placed in a growth chamber under one of the following conditions:
Temperature,°C. Relative humidity,% Absolute humidity
21.0
75
(14.0 gm 3)
26.5
7S
(19.0 gm"3)
32.5
75
(26.0 gm"3)
26.5
55
(14.0 gm 3)
32.0
41
(14.0 gm"3)
26.5
65
(16.5 gm"3)
Absolute humidity is the mass of_water vapor per unit volume of dry air,
expressed as grams per cubic meter (gm~3). An absolute humidity of 14 gm"3
was chosen as the constant absolute humidity since at the three temperatures
tested it was possible to establish, with available growth chambers, the
appropriate relative humidities necessary to maintain this level.
On the day of the exposure the conditions within the fumigation cham-
ber were adjusted to match the pre-treatment environmental conditions under
which the test plants were being maintained. Each experiment consisted of
four consecutive replications of ten plants. The plants were exposed to 151
1.5 pphm of PAN for 1 hour. The temperature was checked at the beginning of
each exposure with a standard mercury thermometer and was monitored during
exposures with copper constantan thermocouples and recorded. The relative
humidity was periodically checked with an aspirated psychrometer and was
continuously monitored with hygrodynamic sensors.
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^VSCPO 1070 - 379-595
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Continuation
An evaluation scheme for symptoms similar to that reported in the
experiments described above was used.
The results of these experiments are presented below in Table 5.
Table 5
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Average severity indices of petunias exposed to 15 pphm of
PAN for one hour under varying temperature and atmospheric moisture conditions
Temperature, C
21.0
26.5
32.0
Relative humidity
75% 55% 41°
184*
261
281
242*
253*
~Severity index at the temperature and relative humidity combinations necessary
to maintain an absolute humidity of 14 gm~3.
As can be seen from Table 5 there was progressive increase in the mean
severity index as temperature increased from 21 to 32°C. The lowest sensi-
tivity occurred at 21 C.
The pollutant flow rate necessary to maintain the same concentration
was much higher at 32 C and 41% RH than during exposures at 32°C and 75% RH.
This difference did not occur during any other combination of exposures, all
of which had flow rates similar to the exposure at 32°C and 75% RH. This
suggests a rapid breakdown of PAN occurring under the conditions of high
temperature and low relative humidity.
The results of this study indicate that temperature is an important
factor in the response of petunia to PAN. This relationship was consistent
regardless of how moisture was expressed, and resulted in an increase in
injury as temperature increased. These results differ with data reported
from our laboratory which showed a decrease in sensitivity of Virginia pine
seedlings to ozone with an increase in temperature.
The results also show that if, in addition to increasing the temperature,
there is a concomitant increase in the amount of atmospheric moisture, there
may be an even greater increase in the amount of plant injury indicating that
injury is also directly related to absolute humidity.
4. Response of hybrid poplar to sequential exposures of ozone
and PAN.
The work reported below was completed during the present budget period
but was done over a period of two years. The objectives of this study was to
(1) determine if prior exposure to ozone increases the sensitivity of plants
to PAN, and (2) to determine if prior exposure to PAN increases the sensitivity
of plants to ozone.
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Continuation page
For this study, cuttings of hybrid poplar (Populus maximowiezii X
trichocarpa) were chosen because of their relative sensitivity to ozone and
genetic homogeneity. Cuttings were collected in December, 1970, and stored
until May through September. Beginning with the first week in May and con-
tinuing until the first week in September cuttings were rooted in a peat-
perlite mix. One week after bud break the pots were fertilized and held for
an additional five weeks in the greenhouse. Plants were exposed to ozone or
PAN in growth chambers as described above. Symptoms were evalu-
ated 5 to 10 days after exposure by examining individual leaves. Each leaf
(15-20 per plant) was numbered by position on the stem beginning at the base
of the plant.
The following results were obtained. A synergistic effect was observed
in eight of the 11 sequences with plants that had been placed at pre- and
post-exposure conditions in the growth chamber. The visible injury on the
plants exposed to both pollutants was greater than the sum of the injury values
on the plants exposed to each gas separately. The most pronounced synergistic
effect occurred in two sequential exposures when plants were exposed to 30
to 40 pphm of PAN for 4 hours in late afternoon and to 25 pphm of ozone for 3
hours the following morning.
Sequential exposures with 15 to 25 pphm of ozone for 2 to 4 hours and 20
to 25 pphm of PAN for 3.5 to 4 hours on the same day resulted in a synergistic
effect in five of the six cases.
Subjecting the plants to darkness immediately following the PAN segment
of the sequential exposures reduced the effect of the previous or subsequent
exposure to ozone.
These results indicate that synergistic effects due to ozone and PAN
interactions are possible. Such information should be considered in establishing
air quality standards.
The response of hybrid poplar to simultaneous exposure to ozone and PAN
was also investigated. In these studies the plants were exposed in groups of
25. On the day of exposure the potted cuttings were removed from the green-
house after receiving 3 to 4 hours of light and placed directly into the
exposure chamber and control chamber. Three treatments were used: ozone alone,
PAN alone, and ozone and PAN simultaneously. Concentrations of 25 pphm of 0^
and 15 pphm of PAN were utilized in both the individual and simultaneous
exposures. All exposures were of 4 hour duration. Symptoms were evaluated 3
days after exposure.
The following results were obtained. The levels of injury produced by
ozone in three replications were relatively uniform and were not significantly
different. In one replication, however, the level of injury produced by the
combined pollutants was significantly less than the sum of the injury levels
produced by the ozone and PAN individually. This effect represents an antag-
onistic interaction. An additive effect was observed in one of the replications.
In a third replication a synergistic effect was observed, i.e. the injury
produced by the combined pollutants increased.
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~u^CPO- 1970 - 379-696
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B. Research Conducted Under the Supervision of D. D. Davis:
1. Screening of plants for resistance to ozone
a. 1973 growing season
During the 1973 growing season, more than 1,000 plants
representing 15 species and/ or cultivars of woody ornamentals
were exposed to 0.25 ppm ozone for 8 hours at bi-weekly intervals.
A different set of plants was used in each bi-weekly exposure;
thus each plant was exposed to a single, acute dose of ozone.
The results of this study have been published as shown in Appendix I
(Hort Science 9: 537-539).
b. 1974 growing season
1) Relative susceptibility
The 1974 screening study was initiated to determine the
the relative ozone susceptibility of 900 plants of various azalea
cultivars. (The 1973 study revealed that certain azalea cultivars
were very susceptible to ozone). Materials and methods were as
previously described (Appendix I). The results of the 1974 study
revealed that one azalea cultivar was extremely susceptible to
ozone, while others were more resistant (Table 6). Ozone symptoms
on sensitive varieties were primarily a reddish stipple on the
upper leaf surface. Bifacial necrosis and premature defoliation
was also observed on "Louise Gable".
Table 6. The incidence and relative susceptibility of azalea cultivars
exposed to 0.25 ppm ozone for 8 hours during 1973.
Cultivar
No. plants
exposed
%
Susceptible
Severity
index
Louise Gable
90
97
173
Delaware Valley White
90
68
64
Rose Greeley
90
43
23
Stewartonian
90
12
10
Fedora
90
31
9
Orange Beauty
90
14
8
Hinocrimson
90
14
4
Hershey Pink
90
11
1
Rosebud
90
2
o b-
Springfield Crimson
90
3
0 b-
"Based on [(severity factor) X (% foliage injured) X (°6 population
susecptible)]/ 100
k'Rounded off
Page 10
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2) Relationship between stomatal resistance and relative
ozone susceptibility of azalea cultivars
Five of the azalea cultivars representing a range in
susceptibility were selected for further studies to determine if
degree of stomatal opening was related to susceptibility. A
diffusion porometer was utilized to determine the degree of
stomatal resistance (which is negatively correlated with degree
of stomatal opening). Weekly measurements were taken on selected
leaves on five plants of each cultivar. Replicated data was
gathered at mid-day (11 a.m. to 1 p.m.) on greenhouse grown plants
in February - March and June - July, 1975.
This data is currently being analyzed; however, Tables
7 and 8 summarize the findings. More complete data cannot be
presented until all the statistical analyses have been conducted.
In general, the degree of stomatal opening was not related to the
relative susceptibility of a cultivar. In fact, the most sensitive
cultivar "Louise Gable" had the highest resistance (most closed
stomata) of all the cultivars. Findings from this study will be
forwarded to HortScience for publication consideration.
Table 7. The resistance of azalea stomata, as measured by a diffusion
porometer (The higher the resistance value, the more closed
the stomata).
Cultivar
Louise
Del.Valley
Orange
Date
Gable
White
Fedora
Beauty
Rosebud
(cm/sec.)
Feb.
25
11.9a
13.8
8.4
9.6
11.1
Mar.
3
10.7
9.6
8.1
9.5
8.5
Mar.
12
5.9
4.7
5.1
6.3
4.9
Mar.
21
28.0
21.5
13.9
15.9
14.8
Mar.
28
18.2
14.6
12.4
12.2
11.5
June
25
6.0
3.8
4.9
3.7
5.1
July
8
6.2
5.1
6.3
2.7
5.2
July
22
5.0
3.5
4.7
3.9
4.1
July
23
6.5
3.6
7.2
3.9
5.3
£
'Average of readings on five plants
3) Relationship between number of stomata and relative
ozone susceptibility of azalea cultivars
Silicon impressions have been made of both leaf surfaces
of selected azalea varieties. The number of stomata/cm^ will be
compared to the relative ozone susceptibility. This research is
presently being conducted.
Page 11
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c. 1975 Growing Season
During the past growing season, 415 plants representing 14 woody
shrubs or vines were exposed to 0.25 ppm ozone for 8 hours. Common
understory species and/or species important in wildlife habitat were
considered. The results of this study are in manuscript form
and are included in this report as Appendix II.
2. Environmental studies
a. Influence of needle age on ozone susceptibility of ponderosa
pine
This study was initiated to determine if the age of current
ponderosa pine needles influenced ozone susceptibility in a
manner similar to the eastern species which we had previously
studied. To summarize, the influence of current needle age was
essentially similar for Ponderosa pine and eastern species, as
exemplified by Virginia pine. This finding is discussed in detail
in Appendix III (Plant Dis. Rptr. 58: 660-663).
b. Influence of PAN on ponderosa pine
One hundred sixty-five young ponderosa pine seedlings in the
cotyledon and primary needle stage were exposed to 0.08, 0.20,
or 0.40 ppm PAN for 8 hours. All plants failed to develop symptoms,
indicating a high degree of resistance to PAN.
This study also served as a preliminary study to the ozone/PAN
interaction research. The study is in Appendix IV (Plant Dis. Rptr.
59: 183-184).
c. Influence of PAN on 10 bean varieties
This study was originally initiated to study the influence of
low-level ("sub-threshold") concentrations of photochemical oxidants
on drought stress of trees. Initial studies were conducted with
hybrid poplar but two problems arose: 1) hybrid poplar was found to
be extremely resistant to PAN and 2) the measurements of water
potential were extremely variable with hybrid poplar.
Therefore, bean was selected as a model plant to perfect the
technique, to be used in future studies with woody plants. It was
desired to use a susceptible bean variety as well as a resistant
bean variety to study PAN predisposition to drought stress. Thus,
10 bean varieties were selected and their susceptibility to PAN
determined (Appendix V). From these results, two varieties were
selected for the water stress studies.
Page 12
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d. Influence of PAN on plant water relations
This study revealed that very low concentrations of PAN, well
below the threshold dosage needed to cause visible injury, signifi-
cantly upset the water relations of the PAN-sensitive variety. In
contrast, the water relations of the PAN-resistant variety was not
affected by the same dosage of PAN. This work is currently being
pursued with other plants and other air pollutants. A manuscript
describing the research has been prepared for publication in
Phytopathology (Appendix VI).
e. Simultaneous exposure of plants to ozone and PAN
1) Ponderosa pine
Ponderosa pine is very resistant to PAN and is susceptible
to ozone. Simultaneous exposure of seedlings to both ozone and
PAN usually decreased the amount of visual injury (Appendix VII).
2) Hybrid poplar
Hybrid poplar is also very resistant to PAN and sensitive
to ozone. However, with this plant, simultaneous exposure generally
resulted in more visual injury than with ozone alone (Appendix VIII).
3) Pinto bean
a) Visual symptoms
Pinto bean was selected for further interaction
studies because: it is sensitive to both ozone and PAN;
the symptoms caused by each pollutant are distinctly
different; and the symptoms generally appear on opposite
leaf surfaces. Simultaneous exposure to both ozone and
PAN completely suppressed the PAN symptom on the lower
leaf surface, while the ozone symptom on the upper leaf
surface was either unaffected or slightly enhanced
(Appendix IX).
b) Microscopic symptoms
A technique was developed to separately count
palisade or spongy mesophyll cells from macerated plant
leaves which were exposed to photochemical oxidants. This
method was devised to better estimate the amount of actual
damage occurring at the cellular level, and to enable one
to compare visual injury with cellular injury. Although
the technique was developed using photochemical oxidants
and bean leaves, it is applicable for correlating visual
injury with cellular injury on woody or other plants
injured by any air pollutant. The technique and its
application are described in detail in Appendix X.
Page 13
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APPENDIX I
"Relative Ozone Susceptibility of
Selected Woody Ornamentals"
(HortScience 9: 537-539)
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Relative Ozone Susceptibility of
Selected Woody Ornamentals1
D. D. Davis and J. B. Coppolino2
The Pennsylvania State University, University Park
Abstract More than 1000 plants
representing IS species and/or cultivars of
woody ornamentals were exposed to 0.25
ppm ozone for 8 hours at bi-weekly intervals
throughout (he 1973 growing season. A
different set of plants were utilized in each
bi-weekly exposure. Plants injured at this rate,
m descending order of susceptibility, were
Rhododendron obtusum Planch. 'Hinodegtri'
(Hinodegiri Hiryu azalea), Rhododendron
poukhanenns Leveille (Korean azalea),
Ailanthus altissima Swingle (tree-of-heaven),
Ulmus parvtfolta J acq. (Chinese elm),
Ph iladelphus coronarius L. (sweet
mock-orange), Viburnum setigerum Hance
(tea viburnum), and Viburnum dtlatatum
Thunb. (Unden viburnum). Plants resistant at
this rate were Hex crenata Thunb. 'Hetzu'
(Hetz Japanese holly), Hex optica Ait
(staminate and pistillate American hotly),
Katmia latifolta L. (mountain-laurel kalmia),
Ltngustrum amurense Can. (amur privet),
Nyssa sylvatica Marsh, (black gum), Taxus x
media Rehd. 'Densiformis' (dense Anglojap
yew), Taxus x media Rehd. 'Hatfieldii'
(Hatfield Anglojap yew), and Tilia amertcaru
L. (American linden). The most common
ozone symptom on the broadleaved plants
was a tan or dark red to black stipple on the
upper leaf surface. Premature defoliation
occurred on susceptible plants. Plants were
more susceptible to ozone in mid- to late
summer than in early spring.
Several authors have already
reviewed the Literature concerning the
influence of ozone on vegetation (7, 8,
10, 14, 16) Ornamentals are especially
vulnerable to damage from air
pollutants such as ozone, because injury
as slight as discolored leaves may render
an ornamental undesirable or
unmarketable. This report provides a list
of ozone-resistant ornamentals
recommended for planting in areas of
the Northeast having high ozone levels.
Susceptible species or cultivars should
not be planted in these areas This list
may also help explain why ozone
sensitive plants fail in certain areas for
previously unknown reasons, and
symptom descriptions may aid in
diagnosing ozone injury in the field.
Fifteen species or cultivars of
1-ye'ar-pld rooted cuttings and 2-year
old seedlings commonly grown in the
' Received for publication, May 22, 1974
Authorized for publication as The
Pennsylvania Agricultural Experiment Station
Journal Series Papei No 4685 Contribution
No 777, Department of Plant Pathology and
No 342-73, Center for Air Environment
Studies Funds provided by the
Environmental Protection Agency, grant no
RS00865
^Assistant Professor and Research Assistant,
respectively. Department of Plant Pathology
and Center for Air Environment Studies
3l ppm ozone is equivalent to 1960 Mg/m^ at
2S°C and 760 mm Hg
Northeast were used in this study (Table
1). Plants were potted in a peat perlite
mix (2 1 by vol) in 10 or 15 cm pots
(depending on plant size) in early May,
1973 and maintained in outdoor beds
In early Jure, one 12 g Agnform
(Agnform International Chemical,
Newark, California) slow release
fertilizer tablet (analysis 14N-4P-6K)
was placed in each 15 cm pot and three
1.5 g tablets were placed in each 10 cm
pot. All plants were watered as needed
throughout the summer. One
tablespoon of 50% Malathion
(emulsifiable concentrate) in 4 liters of
water was sprayed on infested plants in
late summer to control Japanese beetles
and aphids. Plants subjected to
malathion were not immediately
utilized in ozone exposures for two
weeks, during which time rainfall or
water from the sprinkling system
washed the insecticide off the plants.
Ten plants of each species or cultivar
growing in 10 cm pots and 5 of each
growing in 15 cm pots were brought
inside at bi-weekly intervals beginning 4
weeks after budbreak. A different set of
plants were utilized at each bi-weekly
interval. The plants were preconditioned
in the exposure chamber (19) for 1 day
at 23°C, 75% relative humidity (r.h.)
and a 12-hr photopenod from 6 AM to
6 PM of 25 klux. On the following day,
plants were exposed to 0.25 ppm3
ozone at
conditions
Ozone concn
continually dunng
REM chemilumenescent ozone monitor
(REM, Inc , Santa Monica, California)
connected to a strip chart recorder. The
ozone monitor was calibrated using the
1% neutral buffered potassium iodide
method (11) Temperature and relative
humidity during exposures were
monitored continually using wet- and
dry-bulb copper-constantan
thermocouples connected to a 24-point
recorder.
After exposure plants were
transferred to another controlled
environment chamber maintained at the
same environmental conditions as the
exposure chamber The plants were held
in this chamber for 5 days, at which
time symptoms were evaluated on the
current foliage
The symptom was described and
each plant was assigned a seventy factor
from 0 (no symptoms) to 5 (very severe
symptoms) based on the overall
appearance of the plant and potential
marketability. In addition, the
percentage of injured foliage was
estimated and the location of symptoms
the above environmental
were measured
exposures with a
wim lopcvt tcoi ^aiiiuii auu aunavo
was noted. A seventy index was
developed by multiplying (seventy
factor) x {percentage of foliage injured)
x (percentage of plant population
affected) The seventy index was
utilized as the cntenon for estimating
the overall ozone susceptibility of a
species or cultivar
Relative susceptibility of species and
cultivart Seven of the 15 species or
cultivars were injured whereas 8 were
resistant (Table 1) The no of plants
exposed to ozone, percentage of
susceptible plants (incidence), and
seventy index are also given in Table 1
Hinodegiri, Korean azalea, and
tree-of-heaven were the most sensitive
species of those tested
The incidence of ozone-injured
plants within a susceptible species or
cultivar of species varied from 5-100%
Species with the highest incidence of
susceptible individuals usually showed
the most severe symptoms. An
exception was the Chinese elm
population, which had a high percentage
(80%) of susceptible individuals, but the
symptoms were generally a very
uniform, mild, stipple
Symptoms Both azalea cultivars
exhibited a uniform, dense, dark-brown
to reddish-brown stipple Stipples
coalesced on severely affected leaves,
resulting in a uniform brown necrosis,
followed by premature defoliation
The first symptom observed on
tree-of-heaven was a uniform, light tan
stipple. Most leaflets were affected on
injured leaves. Exposures later in the
growing season induced the most severe
symptoms, which appeared as large,
scatterea, black stipples as well as
chlorosis. Chlorotic leaves ranged in
color from light green to yellow Also,
leaflet tips became necrotic, necrotic
patches appeared between the veins, and
occasionally an entire leaflet became
brown and desiccated. All types of
symptoms were present on severely
affected plants Leaflets with chlorotic
or necrotic symptoms defoliated
prematurely. However, the rachis of the
entire leaf did not abscise.
A very uniform, mild, tan stipple
covering the entire leaf surface was the
most common symptom observed on
Chinese elm. As the growing season
progressed, ozonation produced a dark
brown stipple and necrosis on the older
foliage, accompanied by premature
defoliation on severely affected leaves.
Chinese elm seedlings also form leaves at
the base of branches or on the main
stem itself. Complete chlorosis occuned
on these leaves after exposure, followed
by premature defoliation Interveinal,
necrotic patches of tissue also appeared
near the leaf midrib
A uniform, sparse, tan stipple was
observed on sweet mock-orange Tissue
adjacent to the veins became chlorotic,
especially along the midrib Premature
defoliation occasionally occurred after
rt J
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Table 1 The incidence and relative susceptibility of plants exposed to 0 25 ppm ozone for 8 hr
during the summer of 1973 The primary source for scientific names was Bailey (I)
No plants
%
Severity
Common name7
exposedy
susceptible
index*
azalea, Hinodegiri
95
71 5
94 8
azalea, Korean
100
81 O
70 0
tree-of-heaven
35
74 3
65 3
Chinese elm
35
80.0
24.5
mock-orange, sweet
35
62 8
17 2
viburnum, tea
90
17 8
5 4
viburnum, linden
90
6 7
2 2
American holly (d)
45
0
0
American holly (9)
45
0
0
American linden
35
0
0
amur privet
40
0
0
black gum
35
0
0
dense Anglojap yew
90
0
0
Hatfield Anglojap yew
80
0
0
Hetz Japanese holly
70
0
0
mountain-laurel kalmia
90
0
0
Cultivars or species of azalea, viburnum, holly, privet, and yew were 1-year old rooted cuttings
Remaining plants were 2-year old seedlings
ypiants were exposed in groups of S or 10, depending on size, at bi-weekly intervals throughout
the growing season A different group of plants was used in each exposure, each plants was ex-
posed once during the growing season
xBased on |(severity factor) x (% foliage injured) x (% population susceptible)]/100.
ozonation.
The most common symptom on the
2 viburnums was a scattered, black
stipple. However, these species were
only slightly susceptible and symptoms
were very mild
Relationship between plant age and
sensitivity Plants were sensitive to
ozone for varying time periods during
the growing season, and the age at
which sensitivity of the current foliage
was initiated varied with species or
cultivars (Table 2) All susceptible
species were injured at some time during
the 8th to 14th week. Sensitivity of
azaleas continued until the end of the
growing season.
Fig 1 illustrates the influence of
time of growing season or age of foliage
on the relative sensitivity of the two
azalea cultivars. Data from azalea
exposures was utilized because of the
completeness of the data. The plants
were fairly resistant early in the growing
season, and became less so as time
progressed. A curve was not drawn for
the conifers, since these plants were
resistant in the present study
Discussion Tan or dark brown to
black stipples on the upper leaf surface
were the most common symptoms
observed on susceptible plants. This
symptom produced by ozone was first
described for grape (IS) and has since
been described for numerous other
species (8, 9, 10, 18). Stipple appears to
be one of th£ most common symptoms
on woody ornamentals injured by
ozone, and is a useful tool for
diagnosing ozone injury in the field.
General chlorosis, necrotic patches of
tissue, and premature defoliation were
also occasionally observed. These
symptoms have also been reported for
other woody plant species injured by
ozone (18), but would be expected to
occur in the field only on extremely
sensitive plants during periods of high
ozone concentration.
Coniferous species used in this
experiment were resistant to ozone.
However, symptoms on susceptible
conifers have been previously described
as a chlorotic mottle of needles and/or a
needle tip necrosis (2, 3, 6, 13).
Seven of the 15 species used in this
study were susceptible to 0.25 ppm
ozone for 8 hr while 9 were resistant
(Table 1), indicating definite
interspecific and possible intraspecific
differences in susceptibility. In previous
comparable studies, the incidence of
injured plants within various susceptible
species or cultivars of species was 13 out
of 24 (17), 9 out of 18 (3), and 12 out
of 21 (18). Thus, it appears that the
Table 2 -Relationship between age of current foliage and the occurrence of symptoms on plants
sensitive to ozone
dose of 0.25 ppm ozone for 8 Hr,
applied under the environmental
conditions described herein, represents a
useful dose at which about half of the
exposed populations were susceptible to
ozone and half were resistant.
The incidence of ozone-injured
plants within each susceptible species of
cultivar population ranged from 6 7 to
70.0%. In previous studies with
coniferous trees, the incidence ranged
from 6 to 69% (3) while with deciduous
trees it was 41 to 100% (18) These
wide ranges represent a tremendous
amount of variability in ozone
sensitivity. This variability in no. of
plants injured may represent inherent
genetic differences in sensitivity to
ozone, and/or differential response to
environmental factors such as
temperature or humidity, which in turn
affects sensitivity (4, 7). In addition,
variable response was observed among
various leaves on the same plant.
Immature leaves were fairly resistant to
ozone, while recently mature leaves
were more susceptible. This is consistent
with observations on non-woody species
(12, 14). However, additional variability
was noted among leaves of the
approximately same age, apparently due
to subtle microclimatic or physiological
differences among the various leaves
The broadleaved ornamentals,
represented by the azaleas in Fig. 1,
were fairly resistant to ozone early in
the growing season, becoming more
sensitive as the growing season
progressed The high degree of
sensitivity extended into early Oct We
have also observed this trend with other
hardwood trees and deciduous
ornamentals studied 'in our laboratory.
The sensitivity curve is sometimes
bi-modal, with mid-summer and late
summer to early fall peaks, but the
general trend shows increasing
sensitivity with increasing age.
We have also determined the
ozone-susceptibility of numerous other
woody plants (3, 17, 18). Plants listed
as resistant in previous reports and in
this report were not injured by an 8-hr
exposure to 0 25 ppm ozone This dose
represents a high ozone stress to place
Age of current foliage (weeks)
Species
12 3 4 5
6 7 8
9 10 11 12 13 14 15 16 17
18
19 20 21
22 23
Hinodegiri Korean
azalea
R*
Korean azalea
linden viburnum
R -
R - R
R
R
R
tea viburnum
R R
R
R
R
sweet mock-orange
tree-of-heaven
R
Chinese elm
R
ZR indicates plants were resistant to ozone at that age The horizontal bars represent the period
of time the tissue was susceptible Blank spaces indicate that exposures were not conducted dur-
ing that period of time
ig J Relationship between ozone sensitivity
and age from budbreak of azalea foliage
The smooth curve was drawn using a
non-statistical, computer plotting program
based on the method of least squares
i Of£.\ H rrrn nrD 1 07/1
-------
upon the plants. Consequently, those
plants which remained resistant should
remain relatively free of ozone uyury
when ' planted in areas such as the
Megalopolis along the East coast, or
other areas suffering from
photochemical smog. However, plants
listed as resistant to ozone may not be
resistant to other pollutants such as
sulfur dioxide, or to mixtures of ozone
with other pollutants. Plants listed as
susceptible, especially those with high
severity indices, should not be planted
in urban areas where photochemical air
pollution occurs
Literature Cited
1 Bailey, L H 1949 Manual of cultivated
plants MacMillan, N Y
2 Berry. C R , and L A Ripperton 1963
Ozone, a possible cause of white pine
emergence tipburn. Phytopathology
S3 S52-557.
3 Davis, D D., and F A Wood 1972 The
relative susceptibility of eighteen
coniferous species to ozone
Phytopathology 67 14-19
4 and 1973 The
influence of environmental factors on the
sensitivity of Virginia pine to ozone.
Phytopathology 63 371-376.
S. , and 1973 The
influence of plant age on the sensitivity
of Virginia pine to ozone.
Phytopathology 63 381-388
6 and J B Coppohno 1974
Relationship between age and ozone
sensitivity of current needles of
ponderosa pine Plant Dis Rptr
58 660-663
7 Heck, W W 1968 Factors influencing
expression of oxidant damage to plants.
Annu Rev Phytopath 6 165-1 SB
8 Heggestad, H E 1968 Diseases of crops
and ornamenlal plants incited by air
pollutants Phytopathology
58 1089-1097
9 Hibben, C R 1969 Ozone toxicity to
sugar maple Phytopathology
59 1423-1428
10 Hill, A. C , M. R Pack, M Treshow, R. J
Downs, and L G Transtrun 1961 Plant
injury induced bi ozone. Phytopathology
51.356-363
11 Intersociety Committee, American
Public Health Assoc. 1972. Tentative
method for the manual analysts of
oxidizing substances in the atmosphere
Washington, DC p 351-355
12 Menser, H A , H E Heggestad, and O E.
Street 1963 Response of plants to air
pollutants II Effects of ozone
concentrations and leaf maturity on
injury to Nicotiana tabacum
Phytopathology S3.1304-1308
13. Miller, P. R., J R Parmeter, O C Taylor,
and E A Cardiff 1963. Ozone injury to
the foliage of Pinus ponderosa
Phytopathology 53 1072-1076
14. Rich, S 1964. Ozone damage to plants
Annu Rev Phytopath 2 253-266
15 Richards, B L , J T Middleton, and W
B Hewitt 1958 Air pollution with
i elation to agronomic crops V Oxidant
slipple of grapes Agron J 50 559-561
16 Treshow, M 1970. Ozone damage to
plants Environ. Pollution I 115-162
17 Wood, F A , and ) B Coppohno. 1971
The influence of ozone on selected
woody ornamentals Phytopathology
61 133
18 and 1972 The
influence of ozone on deciduous forest
tree species Effects of air pollutants on
forest trees VII International symposium
of forest fume damage experts, Vienna,
September 1970 p 233-253.
19 , D B Drummond, R G
Wilhour, and D D Davis 1973 An
exposure chamber for studying the
effects of air pollutants on trees Pa Agr
Expt Sta Prog. Rpt No 33S
HOGT^urvc I VO' w n rri'M nr R 1974
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APPENDIX II
"Ozone Susceptibility of Woody
Shrubs and Vines"
(Manuscript form)
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
31) ft - b t a .lit !~' 11) S
Osone Suaceptlblllty of Shrubs and Vines
Donald D. Davia, Assistant Profesoor of Plant Pathology, and John
fl. Goppollno, Research Aaalatant; Departaent of Plant Pathology and
Canter for Air Environment Studlea.
Ozona la an air pollutant produced In tha atmosphere aa a raault
of reactiona between compounds originating directly or Indirectly
from various industrial and transportation-related aourcaa. Currently
the combustion of foaall fuels, such aa gasoline, repreaenta the
major aourca of ingredients for this so-called photochemical air
pollutant. Osone develops principally In the atmosphere over cltlaa;
however, Its effects are not confined to urban areas: For example,
ozona develops In the atmosphere over Los Angeles. The pollution
cloud drlfta from Los Angelea to the San Bernadlno Mountains 60-70
miles away where It Is responsible for tha decline of ponderosa pine.
Recent monitoring data In the Northeast Indicate that osone concen-
trations In rural areas nay actually exceed that In urban areas.
These facta Indicate that photochenlcal air pollution of the ozone
type Is not solely a city problea.
Results o4 early fumigation studies by this Station suggest
that many of our woody plant apeclea are sensitive to 0.25 parta per
million (ppa) of osone for A to 8 hours, tfa previously used this
dosage of osone to differentiate sensitive and resistant apecles of
coniferous trees, deciduous hardwood trees, and woody ornamentals.
We Initiated this present study to determine the ozone susceptibility
of woody shrubs which are native to, or planted In, our northeaatem
TO TYPIST—Begin typing flush wuh the left hand marginal line, and end typ
mg so the average length of Ime corresponds with the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Divli & Coppollno
Selene* In Ag. - 2
forests. Species ware selected primarily on their Importance for
the wildlife sector of the forest ecosystem.
Methods. - TVo-year old seedlings of the various species were
potted In a peat:perlite mix In early May. 1975 and wuHntHned In
outside beds. Beginning 4 weeks after budbreak, ten plants of each
species were brought inside at bi-weekly Intervals (until the supply
of plants was exhausted) and exposed to 0.25 ppn osone for 8 hours
In controlled environment chambers. Five days after exposure, the
ozone symptom was described and each plspt was assigned a severity
factor from 0 (no symptoms) to 5 (very severe symptoms) based on the
overall appearance of the plant. In addition, the percentage of
Injured foliage was estimated and the location of symptoms was re-
corded. A "severity Index" developed by moltlplylng (severity
factor) x (percentage of foliage Injured) x (percentage of plant
population affeeted). The severity index waB utilised as the crlterJ
for estimating the overall osone susceptibility of each species.
Results. - Symptoms. - The most conaon symptom appearing on
ozone injured leaves was a "stipple" on the upper leaf surface.
This symptom was comprised of numerous, small, discrete reddish-
brown spots. Each spot was about the size of the head of a pin.
When viewed from a distance, the entire lesf appeared to have e
reddish cast. Leaves on very sensitive plants became completely
red, or at tlaea yellow, and absclssed prematurely from the plant.
The entire symptom complex often resembled premature fall coloration
and defoliation.
TO TYPIST—Begin typing flush with the- left hand marginal line, and end typ-
ing so the average length of line corresponds with the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Davis & Coppolino
Science In Ag. - 3
g»l»tlve susceptibility. - F^urfceeu of the 15 npecles exposed to
ozone developed synptoos, whereas one did not. The number of plants
exposed to osone, percentage of Injured plants (Incidence), and
severity index are given in Table 1. Sassafras was the mat sus-
ceptible species of those OBonatad. The Incidence of injured plants
vithln a species varied froa 3 to 93%, and the oost severe symptoms
usually occurred on those species with the highest Incidence.
TO TYPIST—Begin typing flush with the left hand marginal line, and end typ-
ing so the average length of line corresponds with the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Davis & Coppollno
Science fn Ag - 4
Table 1. Tha incidence and relative susceptibility of plants exposed
to 0.25 ppra oscme for 8 hours during the 1975 growing
season.
No. plants
Cosqdoq nana Exposed
Z
Susceptible
Severity
Index
Sassafras
15
93.3
186.1
Sumac, ataghorn
35
68.6
73.2
Virginia creeper
25
64.0
69.6
Indian currant (coral berry)
25
68.0
63.8
Elderberry (American elder)
30
51.4
62.6
Dwarf nlnebark
35
57.1
46.7
Multiflora rose
25
36.0
30.4
Sumac, smooth
35
45.7
18.9
Dogwood, rad-osier
30
33.3
18.6
Dogwood, silky
35
37.1
14.0
Autumn olive
35
17.1
12.3
Uhlte snowberry
35
11.4
0.8
Bittersweet
25
8.0
0.3
Honeysuckle, Morrow
30
3.3
0.0
TO TYPIST—Begin t\oing flush with the left hand margiml line, and end t>p
ing so the average length of line corresponds unh the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
2-4
25
Davis & Coppollno
Science In Aff - S
Relationship between foliage age and sensitivity - Plants were
sensitive to ozone for various time periods during the growing season
(Table 2). The most susceptible period of growth was front 4 to 8
weeks of age. By the 12th week, 10 out of 13 species were resistant.
Only the two suaae species were still sensitive at 16 weeks of age.
TO TYPIST—Begin t\ping flush with (he left hand marpinal ! ne, and end t\p
ing so (he a>eragc length of line corresponds with the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Davit & Coppollno
SttftWW fa Aft. - 6
Table 2. Relationship between age of current foliage and the occur-
rence of synptoas on plants sensitive to ozone. The bare
represent the age at which the plants were susceptible; the
'H' Indicates that that particular species was resistant at
that age; species were not exposed where blank spaces appear.
Species
Age
A
of current foliage (weeks)
6 8 10 12 14
16
Sassafras
Sumac, staghorn
R
Virginia-creeper
R
Indian currant (Coral berry)
R
Elderberry (American elder)
R
R
Dwarf nlnebark
R
R
Multiflora Rose
R
R
R
Sumac, seooth
R
R
Dogwood, red osier
R
R
R
Dogwood, silky
R
R
R
Autuem olive
R
R
R
R
R
White snowberry
R
R
R
R
R
R
Bittersweet
R R
R
R
R
R
Honeysuckle, Morrow
R
R
R
R
R
R
TO TYPIST—Begin tjping flush with ihe left hand marginal lint, and end
ing so the average length of itne corresponds with the right hand marginal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
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22
23
24
25
Davis 6 Coppollno
Solmca la A|. - J
Discussion. - The taost common symptom on woody plants exposed
to osone Is s dark stipple on the upper leaf surface. This symptom
appears to be the most conmon and useful diagnostic symptom Induced
by ozone on woody plants. Osone also causes premature defoliation on
sensitive species. Premature defoliation could adversely affect a
voody plant In numerous ways, Including loss of vigor, growth, fruit,
etc. These factors are all Important In shrubs being managed for
game food and cover.
The Incidence of sensitive plants within each susceptible
species population ranged from 3 to 932. He previously reported
that the Incidence of sensitive conifers ranged from 6 to 692,
deciduous trees from 41 to 1001, and woody ornamentals from 7 to 70%.
These findings illustrate a tremendous amount of variation within
an ozone sensitive species. This variability may represents a useful
gene pool from which to select tolerant Individuals for further
propagation.
The plants used in this study were susceptible to ozone at a
fairly early age, and generally became progressively more resistant as
the season progressed. This is the general trend that we have
noticed for Virginia pine, white ash, hybrid poplar, Jack pine, and
Scotch pine. In contrast, certain oxalea cultlvars generally become
sore susceptible as the season progresses, and retain their suscep-
tibility into early Fall. Thus, the age at which the current foliage
is nost sensitive to osone varies between plant species.
TO TYPIST—Begin typing flush with the left hand marginal line, and end typ-
ing so the average length of line corresponds with the right hand marginal line
-------
APPENDIX III
"Relationship Between Age and Ozone
Sensitivity of Current Needles of
Ponderosa Pine"
(Plant Disease Reporter 58: 660-663)
-------
660
Vol. 58, No. 7--PLANT DISEASE REPORTER--July 1974
RELATIONSHIP BETWEEN AGE AND OZONE SENSITIVITY
OF CURRENT NEEDLES OF PONDEROSA PtNE
Donald D. Davis and John B Coppolino
Assistant Professor and Research Assistant, Department of Plant Pathology and Center for Air Environ-
ment Studies, The Pennsylvania State University, University Park 16802
Contribution No 773 from the Department of Plant Pathology Paper No 4632 in the Journal Series of
the Pennsylvania Agricultural Experiment Station and Contribution No 33C-73. Center for Air Environment
Studies The Pennsylvania State University, University Park
This research was supported by Environmental Protection Agency Grant No R800865 Space was provided
by the School of Forest Resources in the Forest Resources Laboratory
ABSTRACT
One hundred and ninety 2-year-old ponderosa pine seedlings were exposed to
ozone in groups of ten at weekly intervals throughout the summer. The current
needles exhibited maximum sensitivity to ozone during the third to ninth week of
age. Chlorotic mottle was the most common symptom, tip necrosis, and banding
were also observed to a lesser degree.
Plant Dis. Reptr. 58 660-663.
Additional key words air pollution, Pinus.
Previously, we have reported on the relative ozone susceptibility of the current foliage of
various conifer species (3) and of factors affecting this sensitivity (4,5). The bge of current
foliage was determined to be an important factor affecting sensitivity. When Virginia pine
(Pinus virginiana) seedlings were exposed to acute doses of ozone, the most severe injury oc-
curred on the current-year, young elongating needles mlate spring to early summer (5) Similar
observations have been made on eastern white pine (Pinus strobus) in the field (1, 6).
In contrast, Miller (7) in California recently reported that ponderosa pine (Pinus ponderosa)
was most severely injured by continued ozone exposures begun in mid-August, and that 1-year-
old needles were injured three times as severely as were current year needles.
To compare the seasonal influence of sensitivity on the current needles of Virginia and
eastern white pines with ponderosa pine, we exposed ponderosa pine seedlings to single, acute
doses of ozone at weekly intervals throughout the summer.
MATERIALS AND METHODS
Two- to three-year-old pondersoa pine (P ponderosa var ponderosa) seedlings were potted
in a 2 1 (v/v) peat perlite mix in early May 1973, and maintained in outside beds where they
were watered regularly. One 12 g Agriform (Agriform International Chemical, Newark, Cali-
fornia) slow release fertilizer pellet (analysis 14N ¦ 4P :6K)was placed in each pot in early June.
No pesticides were applied.
Each week, one day prior to ozone exposure, ten seedlings were brought inside and placed
in a controlled environment exposure chamber (10). The chamber was maintained at 24° C. 75%
relative humidity (RH) and a 12-hr photoperiod from 6 am. to 6 p.m. of 25 K lux. These
ennronmental regimes are hereafter referred to as "standard conditions. " A different group of
seedlings was exposed each week
The following day seedlings were exposed in the same chamber to 0.25 ppm ozone for 8 hr
beginning at 9 a m. under standard conditions. Exposure techniques were as previously de-
scribed (2).
Following exposure to ozone, the plants were placed in a post-exposure controlled environ-
ment chamber, maintained at standard conditions for 5 days, at which time symptoms were
evaluated.
Each seedling was assigned a severity factor of 1 to 5, depending on the overall severity
of the ozone injury. In addition, the percentage of current needles affected was estimated, the
symptom for each seedling described, and the average needle length measured. A severity
index was calculated by multiplying the severity factor times the percentage of needles affected
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Vol. 58, No. 7 — PLANT DISEASE REPORTER--July 1974
661
for each seedling. An average severity index was calculated for the 10 plants used in each
weekly exposure.
RESULTS
Symptoms appeared on seedlings within 24 hr after exposure to ozone. The most common
symptom was a chlorotic mottle, but banding and tip necrosis were also observed. Chlorotic
mottle occurred on the tips of immature needles, and was widespread on mature needles.
Banding occasionally occurred near the tips of young needles, and was located progressively
toward the needle base as the needle length increased. Some degree of tip necrosis was observed
after most exposures.
Sixty-seven percent of the ponderosa pine seedlings exposed during the fourth to fourteenth
week of age exhibited symptoms. This percentage is essentially equal to that reported for
Virginia pine (3). Because Virginia pine was the most ozone sensitive of 18 conifers, we
conclude that the current needles of ponderosa and Virginia pines are among the most sensitive
of many conifers, at least when grown in Eastern United States.
Figure 1 illustrates the influence of age on the susceptibility of the current needles of
ponderosa pine to ozone. The sensitivity peaked in early summer at about the fifth to seventh
week (mid-June), then declined. By the fifteenth week only slight symptoms were present.
Symptoms were absent from the sixteenth to nineteenth week.
The relationship between average needle length and sensitivity is also illustrated by Figure
1. Maximum sensitivity appeared at the time when the needles were 60-90 mm long and still
elongating. The sensitivity decreased as the needles matured and approached their terminal
length of 100-120 mm.
160
NEEDLE LENGTH -
SEVERITY INDEX -
140
2 X 120
— UJ
f §100
60
llJ
40
20
AGE (WEEKS)
FIGURE 1. The influence of age and needle length on the ozone sensitivity of current foli-
age of ponderosa pine seedlings Ten different seedlings were exposed each week to 0. 25 ppm
ozone for 8 hours. Smooth curves were drawn through the data by using the method of least
squares.
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662
Vol. 58, No. 7 —PLANT DISEASE REPORTER--July 1974
DISCUSSION
The ozone symptoms observed on the current year's needles ofpondjrosa pine were similar
to those described for various other conifer species <3), and for older needles of ponderosa
pine (8, 9).
The relationship between needle length or age and ozone sensitivity was similar to that
observed on Virginia pine and other eastern species (1. 3, 5, 6). Immature needles were generally
injured most severely at the tips, injury on mature needles was more general. The sensitivity
of current Virginia pine needles (5) reached a peak slightly earlier than did current needles of
ponderosa pine (Fig. 1). The slight difference in the trend of the response curve of the two
species may be explained in part by the cool growing conditions during the spring and early summer
of 1973 which delayed typical development, elongation, and subsequent ozone sensitivity of pon-
derosa pine needles. Growth rate differences between the two species might also be involved.
We feel that the overall trend of the curve relates to age of the current foliage, and that
variability along the curve probably reflects environmental influences. Conditions such as
temperature, RH, light, and ozone concentration are uniform in our exposure chamber from
week to week. The major variable is the outdoor growing conditions in which the seedlings are
maintained prior to exposure. It is apparent that during certain pe-iods of time specific weather
conditions affect plant sensitivity to ozone.
Miller (7) conducted experiments in California with ponderosa pine grown for 2 years in
charcoal-filtered air and then exposed to 0.45 ppm ozone, 12 hr per day, for time periods of
up to 40 days. He found that the 1-year-old needles were three times as sensitive as the
current needles, and were most sensitive to exposures begun in mid-August. Although we did
not quantify symptoms on previous years' needles, we did observe these needles to be fairly
resistant to acute doses of ozone under our experimental conditions. Resistance of previous
years' needles has alsobeen noted in our previous research concerning 18 coniferous species (3)
and unpublished data concerning numerous ornamental evergreens, as well as with field obser-
vations concerning ozone injury to eastern white pine (1,6). Also, we have found that current
needles of various conifer species subjected to acute doses of ozone were most sensitive in late
spring to early summer (3, 5). Linzon (6) and Berry and Ripperton (1) also have observed that
current foliage of eastern white pine is most sensitive to ozone during time of needle emergence
in late spring or early summer.
In summary, this report supports Miller's findings (7,8,9) that ponderosa pine is very
sensitive to ozone, however, we found that the period of maximum sensitivity to acute exposures
is in late spring to early summer, while Miller reported maximum sensitivity to continual,
chronic exposures is in late summer. His findings are similar to findings concerning ozone
injury to ponderosa pine in the forests of California. Our findings are similar to field obser-
vations concerning ozone injury to eastern white pine m the East. Other differences in such
reports may be due to inherent differences in experimental design, or to differing environmental
conditions during exposure of plants to ozone. Miller subjected the same group of plants to daily
exposures of ozone, and we exposed a different set of plants each week. Although Miller used
fairly high doses of ozone, his study consisted of a continual, chronic exposure, whereas ours
was a single, acute exposure each week. Also, the ambient environmental conditions in Cali-
fornia differ markedly from those in the East. Miller exposed plants at ambient conditions of
relatively high temperatures and low humidities, both of which tend to reduce injury. In
contrast, we exposed seedlings at moderate temperatures and fairlv high humidities, which
tend to increase the amount of injury (4).
Literature Cited
1. BERRY, C. R , and L. A. RTPPERTON. 1963. Ozone, a possible cause of white
pine emergence tipburn. Phytopathology 53 552-557.
2. D4VIS, D. D. , andS. H. SMITH 1974 Reduction of ozone-sensitivit\ of pinto bean by
bean common mosaic virus. Phytopathologv 64- 383-385.
3. DA\ IS, D. D. , and F. A. WOOD. 1972. The relative susceptibility of eighteen
coniferous species to ozone. Phytopathologv 62 14-19
4. DA\ [S, D D , and F. A. WOOD. 1973. The influence of environmental factors on
the sensitivity of Virginia pine to ozone. Phytopathology 63 371-376.
5. DA\ IS, D. D., and F. A. WOOD 1973. The influence of plant age on the sensi-
tivity of Virginia pine to ozone. Phytopatholog"> 63 381-387.
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Vol. 58. No. 7--PLANT DISEASE REPORTER--July 1974
663
6. LINZON, S. N. 1967. Ozone damage and semimature-tissue needle blight of eastern
white pine. Can. J. Bot. 45-2047-2061.
7. MILLER. P. R. 1973. Susceptibility to ozone of selected western conifers. 2nd Int.
Cong, Plant Pathol. Abstr. No 0579.
8. MILLER, P. R., J. R. PARMETER, B. H. FLICK, and C. W. MARTINEZ. 1969.
Ozone dosage response of ponderosa pine seedlings. J. AirPollut. Control Assoc.
19: 435-438.
9. MILLER, P. R., J. R. PARMETER, Jr., O. C. TAYLOR, and E. A. CARDIFF. 1963.
Ozone injury to the foliage of Pmus ponderosa. Phytopathology 53 1072-1076.
10. WOOD, F. A.. D. B. DRUMMOND, R. G. WILHOUR, and D. D. DAVIS. 1973.
An exposure chamber for studying the effects of air pollutants on plants. Pa.
State Univ. College Agric. Prog. Rep. 335. 7 pp.
-------
APPENDIX IV
"Resistance of Young Ponderosa Pine
Seedlings to Acute Doses of PAN"
(Plant Disease Reporter 59: 183-184)
-------
RESISTANCE OF YOUNG PONDEROSA PINE SEEDLINGS
TO ACUTE DOSES OF PAN
D. D. Davis
Assistant Professor, Department of Plant Pathology and Center for Air Environment Studies, The Penn-
sylvania State University, University Park, Pennsylvania 16802
Contribution No. 813 from the Department of Plant Pathology. Paper No 4662 in the Journal Series of
the Pennsylvania Agricultural Experiment Station and Contribution No. 378-74, Center for Air Environmental
Studies, The Pennsylvania State University, University Park, Pennsylvania 16802. Funds provided by the
Environmental Protection Agency, Grant No. H800865, administered through the Center for Air Environ-
ment Studies.
The author gratefully acknowledges receipt of seed from Mr Leroy C Johnson, U.S. Forest Service
Institute of Forest Genetics, Placerville, California. Gratitude is expressed to C. L Martin for technical
aid.
ABSTRACT
One hundred sixty-five young ponderosa pine seedlings in the cotyledon and pri-
mary needle stage were exposed to 0.08, 0.20, or 0.40 ppm peroxyacetyl nitrate
(PAN) for 8 hr. All plants failed to develop symptoms, indicating a high degree
of resistance to PAN.
Plant Dis. Reptr. 59 183-184.
Additional key words: air pollution, Pinus ponderosa.
It is well documented that ponderosa pine trees (Pinus ponderosa) in the San Bernardino
Mountains of California have been injured by photo-chemical smog (4, 5, 6, 7). Ozone is apparently
responsible for a high percentage of the observed injury (6,7); however, the influence of
PAN, another major phytotoxic component of smog, on ponderosa pine has not been reported.
Therefore, we exposed young seedlings of ponderosa pine to acute levels of PAN. The results
of this study are reported here.
MATERIALS AND METHODS
Seeds of ponderosa pine (P. ponderosa var. ponderosa) collected on the San Jacinto Ranger
District, San Bernardino National Forest at an elevation of 5,300 feet were used in this study.
Seeds were soaked in water for 24 hr, blotted dry, and maintained at 4° C for a minimum of
2 weeks before the initial planting.
Beginning on April 29, 1974 ten seeds were planted for 11 consecutive weeks in each of
five 10-cm pots containing a 1 -1 (by volume) peat:perlite mix. After emergence, seedlings
were thinned to five per pot, leaving those plants representing the most uniform stand. Five g
of slow release fertilizer (analysis 14N * 14P • 14K) were applied to each pot.
Seedlings were maintained on a greenhouse bench until the first week of August 1974. The
oldest seedlings at that stage were approximately 10 cm tall, maintaining approximately 100
primary needles which had a maximum length of 4.0 cm. The youngest seedlings, 1 week
after seedcoat shedding, were approximately 5 cm tall with a small tuft of 10-12 primary
needles 0. 2-1. 0 cm long. Cotyledons were present on all seedlings.
-------
RESISTANCE OF YOUNG PONDBROSA PINE SEEDLINGS
TO ACUTE DOSES OF PAN
D. D. Davis
Assistant Professor, Department of Plant Pathology and Center for Air Environment Studies, The Penn-
sylvania State University, University Park, Pennsylvania 1 6802.
Contribution No. 813 from the Department of Plant Pathology. Paper No. 4662 in the Journal Series of
the Pennsylvania Agricultural Experiment Station and Contribution No. 378-74, CenterforAir Environmental
Studies, The Pennsylvania State University, University Park, Pennsylvania 16802. Funds provided by the
Environmental Protection Agency, Grant No. R800865, administered through the Center for Air Environ-
ment Studies.
The author gratefully acknowledges receipt of seed from Mr. LeroyC. Johnson, U.S. Forest Service
Institute of Forest Genetics, Placerville, California. Gratitude is expressed to C. L. Martin for technical
aid.
ABSTRACT
One hundred sixty-five young ponderosa pine seedlings in the cotyledon and pri-
mary needle stage were exposed to 0.08, 0.20, or 0.40 ppm peroxyacetyl nitrate
(PAN) for 8 hr. All plants failed to develop symptoms, indicating a high degree
of resistance to PAN.
Plant Dis. Reptr. 59- 183-184.
Additional key words: air pollution, Pinus ponderosa.
It is well documented that ponderosa pine trees (Pinus ponderosa) in the San Bernardino
Mountains of California have been injured by photo-chemical smog (4, 5, 6, 7). Ozone is apparently
responsible for a high percentage of the observed injury (6,7), however, the influence of
PAN, another major phytotoxic component of smog, or ponderosa pine has not been reported.
Therefore, we exposed young seedlings of ponderosa pine to acute levels of PAN. The results
of this study are reported here.
MATERIALS AND METHODS
Seeds of ponderosa pine (P. ponderosa var. ponderosa) collected on the San Jacinto Ranger
District, San Bernardino National Forest at an elevation of 5,300 feet were used in this study.
Seeds were soaked in water for 24 hr, blotted dry, and maintained at 4° C for a minimum of
2 weeks before the initial planting.
Beginning on April 29, 1974 ten seeds were planted for 11 consecutive weeks in each of
five 10-cm pots containing a 1 1 (by volume) peat perlite mix. After emergence, seedlings
were thinned to five per pot, leaving those plants representing the most uniform stand. Five g
of slow release fertilizer (analysis 14N : 14P • 14K) were applied to each pot.
Seedlings were maintained on a greenhouse bench until the first week of August 1974. The
oldest seedlings at that stage were approximately 10 cm tall, maintaining approximately 100
primary needles which had a maximum length of 4.0 cm. The youngest seedlings, 1 week
after seedcoat shedding, were approximately 5 cm tall with a small tuft of 10-12 primary
needles 0.2-1.0 cm long. Cotyledons were present on all seedlings.
-------
APPENDIX V
"The Susceptibility and Symptoms of Ten
Bean Varieties Exposed to Peroxyacetyl
Nitrate (PAN)"
(Submitted to Plant Disease Reporter)
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
.'0
21
22
23
24
25
THE SUSCEPTIBILITY AND SYMPTOMS OF TEN BEAN VARIETIES
EXPOSED TO PEROXYACETYL NITRATE (PAh)
T. E. Starkey, D. D. Davis and W. Merrill
Graduate Assistant, Assistant Professor, and Professor;
Department of Plant Pathology and Center for Air Environment Studies,
The Pennsylvania State University, University Park, Pennsylvania 16802
Contribution No. from the Department of Plant Pathology.
Paper No. in the Journal Series of the Pennsylvania Agricultural
Experiment Station and Contribution No. 414-75 Center for Air
Environment Studies. The Pennsylvania State University, University
Park, Pennsylvania 16802.
This publication has been financed in part with Federal funds
from the Environmental Protection Agency under grant numbers T900011
and R800865. The contents do not necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recom-
mendation for use. This study was also partially funded under grant
FS-NE-26 from the United States Department of Agriculture, Forest
Service, Northeastern Forest Experimental Station.
ABSTRACT
Ten bean varieties in the primary or trifoliate leaf stage were
3 3
exposed to 593 ug/m (0.12 ppm) and 742 ug/m (0.14 ppm), respectively.
of peroxyacetyl nitrate (PAN) for 2 hr to determine the relative PAN
susceptibility of each leaf stage. The percentage of abaxial leaf
surface bronzing or glazing, adaxial leaf surface stipple or fleck,
TO TYPIST—Bft;in t>ptng flush with ihe Ml hand marginal line, and end t>p-
ing so the average length of hne corresponds wirh the right hand margtnal line
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1
2
3
4
5
6
7
8
9
10
11
12
13
1-4
IS
16
17
18
19
20
21
22
23
2-1
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 2
bifacial tissue collapse and total injury were statistically analyzed
to determine which symptom-type was the best indicator of PA suscep-
tibility. Bifacial tissue collapse consistently gave the best correla
tion between the primary and trifoliate leaf stage. The ten varieties
were ranked in the following descending order of susceptibility:
Provider, Astro, Harvester, Sanilac, Bush Blue Lake 274, Tendercrop,
Pinto 111, Stringless Black Valentine, Tempo and Eagle. Provider may
be useful as a bioindicator and experimental plant in future PAN
studies because of its apparent tolerance to ozone but susceptibility
to PAN.
Plant Dis. Reptr. 60:
In a previous study (2) ten bean (Phaseolus vulgaris L.)
varieties were ranked as to their relative susceptibility to ozone.
The objectives of this current study were to compare the relative sus-
ceptibility of these same varieties to PAN and to compare the response
between the primary and trifoliate leaves. Results from this latter
comparison could provide a basis for predicting the response of a
variety in the trifoliate leaf stage based on knowledge of its response
in the earlier primary leaf stage. This study also may furnish in-
formation to growers concerning resistance of these varieties to PAN
and provide researchers with a basis for choosing experimental plants
for future PAN research.
MATERIALS AND METHODS
The ten bean varieties selected for this study were: Astro,
TO TYPIST—Beqin tjpmg flush with tlu left hand mirijnil Imp and end t>p
ing io ihe average Icn^tb of 1 nc corrc *uh iIil ri^ht hand marginal Itnc
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
95
Starkey, Davis & Merrill
Plant Dis. Reptr. - 3
Bush Blue Lake 274, Eagle, Harvester, Pinto 111, Provider, Tempo,
Tendercrop, Sanilac and Stringless Black Valentine. The basis for
the variety selection, as well as the source of seed, was previously
described (2).
The first part of this study dealt with the response of the
primary leaves to PAN, while the second dealt with the response of
the trifoliate leaves. Different plants were used for each experiment
The materials and methods used for each experiment were the same unless
otherwise noted.
3
Four seeds of each variety per 16 ounce pot (589 cm ) were
planted approximately 3 cm deep in aerated steam treated 1:1:1 peat:
perlite: clay-loam soil mix on two consecutive days. Cotyledons
emerged 5-6 days after planting. One or two seedlings per pot were
removed two days after the seed coats were shed, leaving two plants
representing the most uniform stand. All plants were maintained in
the greenhouse prior to and following PAN exposures. The temperature
within the greenhouse varied from a night low of 22° C to a daily
high of 34° C. Humidity varied naturally and was not monitored.
Natural light was supplemented with General Electric Power Groove
Cool White Lamps during a 12-hour photoperiod beginning at 6:00 A.M.
In the primary leaf study, ten plants of each variety were
exposed to PAN 8 days after cotyledon emergence. At this age the
first trifoliate leaf was beginning to expand. The primary leaves
o 3
were exposed to 593 ug/m + 74 ug/m (0.12 ppni + 0.015 ppm) PAN for
2 hour. In the trifoliate leaf study, ten plants of each variety
TO TYPIST—Begin t\pmg flush with ihe left hand imrpinjl line, and end tvp
ing so ihe average lcn^ib of line corresj^nds w.ih ihe r pht hand marginal line
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1
2
3
4
5
6
7
&
9
10
11
12
1J
14
IS
16
17
18
19
20
21
22
23
24
Starkey, Davis & Merrill
Plant Dis. Reptr. - 4
3 3
were exposed to 742 ug/m + 74 ug/m (0.15 ppm + 0.015 ppm) PAN for
2 hour. Plants were exposed 20 days after cotyledon emergence. At
this age the first trifoliate leaf of each variety was completely
expanded, the second 80-100% expanded and one or two others in the
early stages of expansion. Since the primary leaves are more suscep-
tible than the trifoliate leaves, two concentrations of PAN were
used in these studies to differentiate varietal susceptibility.
The plants were exposed to PAN in a specially modified con-
trolled environment chamber (13). During the exposures, temperature
was maintained at 24^ C, relative humidity at 70%, and light intensity
at 25 Klux. The pots were arranged randomly within the chamber. All
plants received a minimum 3 hour pre- and post-exposure light treat-
ment considered necessary for symptom development (4, 10, 11).
PAN was generated, collected and stored as described by
Stephens (8). During exposure the PAN concentration was monitored at
approximately 10 minute intervals by taking samples from the chamber
and injecting them into an electron capture gas chromatograph (6),
calibrated as described by Stephens (7).
Following exposure, the plants remained in the greenhouse for
7 2 hours at which time the symptoms were evaluated. The symptoms
were rated in terms of total percentage of leaf or leaflets injured,
as well as the percentage of leaf or leaflets injured within each of
the following classes of symptoms: bifacial necrosis, abaxial leaf
surface bronzing and glazing, and adaxial stipple or fleck. Data were
subjected to analysis of variance with a completely random experimental
TO TYPIST—Beun taping eSe left hand marptnnl line, and rn
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 5
design and a factorial treatment design using varieties and replica-
tions as factors (1,5). The mean percentages of injury within each
symptom class were compared using a Duncan's Modified (Bayesian)
Least Significant Different Test value (k=100) approximating P=0.05
(12).
RESULTS
Symptoms. — The amount of injury and symptom types varied
between the primary and trifoliate leaves. The primary leaves were
more susceptible and exhibited more symptom types than did the
trifoliate leaves. No symptoms were observed on the trifoliate leaves
in the primary leaf study. Similarly, in the trifoliate leaf study
no symptoms were observed on the primary leaves. In this latter study,
symptoms were confined to the two oldest trifoliate leaves of all
varieties with the exception of Sanilac. In this variety abaxial
glazing did occur on the third oldest trifoliate leaf.
Bifacial necrosis and abaxial bronzing and glazing were the
predominant symptoms on both leaf types on all varieties. On leaves
where both bifacial necrosis and abaxial bronzing and glazing were
present, the latter symptom was generally restricted to the immature
portions of the leaf. Pinto 111 showed the greatest variation in
symptom types, particularly in the primary leaf stage. In addition
to bifacial necrosis and abaxial bronzing and glazing, Pinto 111 ex-
hibited adaxial ozone-like stipple and fleck, and adaxial glazing.
All varieties showed some abaxial bronzing and glazing on either
the primary or trifoliate leaves. Only the intensity of this symptom
TO TYPIST—Begin tvpmg flush »iih the left hanrj marRinal Jinr, and end
ing so the average lengib of line corusponds wuh the right hand marginal line
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1
2
3
4
S
6
7
8
9
10
11
12
13
14
15
16
17
13
19
20
21
22
23
24
tz.
Starkey, Davis & Merrill
Plant Dis. Reptr. - 6
varied among varieties. The primary leaves showed two different types
of bifacial necrosis, a dull green and an ivory white necrosis. The
green necrosis was usually interveinal, and when severe, all the tissue
between the veins became necrotic. The ivory coloration appeared
either as isolated necrotic flecks 2-10 mm in diameter or as inter-
veinal necrosis. The necrotic flecks often coalesced resulting in
widespread interveinal necrosis. Although the intensity of the
bifacial necrosis varied among varieties, there was no apparent corre-
lation between varietal susceptibility in the primary leaf stage and
the presence of either one of these types of necrosis.
The trifoliate leaves also showed two basic types of bifacial
necrosis. Provider, Harvester and Astro consistently showed a dark
tan bifacial necrosis. The other seven varieties occasionally showed
this dark tan bifacial necrosis in addition to a dull green necrosis
similar to that described for the primary leaves.
The time required for tissue collapse to develop differed
among varieties. The dark tan collapse of the trifoliate leaves
was generally evident within 18 hours following exposure to PAN.
The dull green and ivory white tissue collapse required up to three
days for full development. The dull green collapse required the
longest period of time to develop.
Relative susceptibility. — Table 1 shows the relative ranking
of the ten varieties in the primary leaf stage based on abaxial
bronzing and glazing. The ten varieties have been arbitrarily
separated into two groups. The five above the line were designated as
TO TYPIST—Begin tvpmg flu
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J
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2J
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 7
relatively susceptible and the five below the line as relatively toler-
ant. The ranking of these same varieties in the trifoliate stage is
shown in Table 2. The number preceding the variety indicates the
relative ranking in the primary stage- Table 3 lists the relative
ranking of the varieties in the primary leaf stage based on bifacial
necrosis. The ranking of the ten varieties is again arbitrarily sep-
arated into two groups. Table 4 shows the relative susceptibility of
the varieties in the trifoliate leaf stage based on bifacial necrosis.
The number preceding each variety indicates its position in the
primary stage.
DISCUSSION
Abaxial bronzing and glazing have been reported as PAN symptoms
for many plant species. Another PAN symptom less commonly reported
but frequently observed in the laboratory is bifacial necrosis. This
symptom has been reported as the predominate PAN symptom on petunias
and tomatoes (3,9).
Peroxyacetyl nitrate can cause many types of symptoms on one
leaf. If one symptom is predominate, the amount of other symptoms
may be reduced. For example, Sanilac had 64% bifacial necrosis in the
primary leaf stage (Table 3). Since such a high percentage of tissue
was necrotic, very little abaxial bronzing and glazing could occur.
Thus, only 19% of the abaxial surface of Sanilac showed bronzing and
glazing in the primary leaf stage (Table 1).
Abaxial bronzing and glazing was not a consistent indicator of
PAN susceptibility when comparing the primary and trifoliate leaves.
TO TYPIST—Begin flush *ith the left harv) marpmal line, anrt end »>p.
ing so the avrrsgc length of l:nc corre^iom'a w.iih 1 he right hand marginal line
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 8
Using this criterion, only two varieties (Pinto 111 and Tendercrop)
were relatively susceptible and two other varieties (Sanilac and
Harvester) were relatively tolerant in both Table 1 and 2. The other
six varieties differ in their relative susceptibility between the
primary and trifoliate leaves using this criterion.
Bifacial necrosis (Tables 3 and 4) gave a more consistent
indication of PAN susceptibility in both the primary and trifoliate
leaf stages than did abaxial bronzing and glazing. Sanilac, Provider,
Harvester, Bush Blue Lake 274 and Astro were all relatively susceptible
Tendercrop, Eagle, Pinto 111, Stringless Black Valentine and Tempo
were all relatively tolerant in both the primary and trifoliate leaf
stage.
The primary leaves of bean have been used extensively in air
pollution research. Generalizations concerning the susceptibility
of the trifoliate leaves have frequently been based solely on the
response of the primary leaves- If the effects on the trifoliate
leaves are to be extrapolated from the response of the primary leaves,
the susceptibility of the variety should be similar in both growth
stages. These results indicate that bifacial necrosis is a better
indicator of PAN susceptibility than is bronzing or glazing.
Table 5 shows a comparison of the relative susceptibility
of the ten bean varieties in the trifoliate leaf stage in response to
ozone or PAN. The numbers next to the varieties exposed to PAN
represent their position in the ozone ranking. Provider, which is
relatively tolerant to ozone, is very susceptible to PAN. This
TO TYPIST—Bei;in upinp flush wnh the left hand marpmal line and end l>p
ing so the a>rrasc Itngth of line correspond* v.irh ihe right hand marginal line
-------
1
2
3
4
S
6
7
S
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 9
inverse relationship may prove useful in using Provider as an exper-
imental plant for PAN research. By using Provider, instead of a less
susceptible variety such as Pinto 111, the researcher could use con-
centrations of PAN similar to ambient levels. Provider may also be
useful as a bioindicator for ambient levels of PAN once knowledge
of its suceptibility to both ozone and PAN together are determined.
Stringless Black Valentine is relatively tolerant to both PAN and
ozone. However, the relative susceptibility of this variety to simul-
taneous exposures of ozone and PAN should also be determined. Sanilac
and Astro are relatively susceptible to both ozone and PAN, and may
be injured by ambient concentrations of either pollutant.
TO TYPIST—Beqm ^ping flup-
ing so the average length of line corre
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2•>
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 10
Table 1. — The relative susceptibility of ten bean varieties in the
primary leaf stage based on abaxial bronzing and glazing.
~~ Percentage of"
Variety Tissue Injured
1.
Tempo
53
b
a
2.
Pinto 111
52
a
3.
Provider
39
b
4.
Stringless Black Valentine
38
be
5.
Tendercrop
32
c
6.
Eagle
23
d
7.
Astro
22
d
8.
Harvester
22
d
9.
Sanilac
19
de
10. Bush Blue Lake 274 15 e
aEach number represents an average of 40 plants (4 replications,
10 plants per replication).
^Numbers followed by the same letter are not significantly different
according to a Duncan's Modified (Bayesian) Least Significant
Test value (k=100) approximating P=0.05.
TO TYPIST—Begin r\pinp flu
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 11
Table 2. The relative susceptibility of ten bean varieties in the
trifoliate leaf stage based on abaxial bronzing and glazing.
Percentage of"
Variety Tissue Injured
2. Pinto 111 23
b
a
5. Tendercrop 20
b
7. Astro 18
b
10. Bush Blue Lake 274 18
b
6. Eagle 17
be
4. Stringless Black Valentine 14
cd
9. Sanilac 12
de
1. Tempo 11
de
8. Harvester 10
e
3. Provider 5
f
aEach number represents an average of 40 plants (4 replications,
10 plants per replication).
^Numbers followed by the same letter are not significantly different
according to a Duncan's Modified (Bayesian) Least
Significant
Test Value (k=100) approximating P=0.05.
TO TYPIST—Benin t>ping flup-
ing so the average length of line corres-omh unh the r fiht hand marginal line
-------
1
2
3
4
5
6
7
8
9
10
II
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 12
Table 3. The relative susceptibility of ten bean varieties in the
primary leaf stage based on bifacial necrosis.
¦ ¦ i— ii i i —
Percentage of
Variety Tissue Injured
b
1. Sanilac 64 a
2. Provider 47 b
3. Harvester 44 be
4. Bush Blue Lake 274 43 be
5. Astro 40 cd
6. Tendercrop 35 d
7- Eagle 34 d
8. Pinto 111 26 e
9. Stringless Black Valentine 19 e
10. Tempo 9 f
Each number represents an average of 40 plants (4 replications
10 plants per replication)
^Numbers followed by the same letter are not significantly different
according to a Duncan's Modified (Bayesian) Least Significant
Test value (k=100) approximating F=0.05.
TO TYPIST—Bcqin t>pmp flu*h wnh ihi left hand rmrpimt lint, and end M
ing so the average lengih of line corntjoiiih *ith the xifrht hand marginal Jme
-------
1
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 13
Table 4. The relative susceptibility of ten bean varieties in the
trifoliate leaf stage based on bifacial necrosis.
¦ - - ¦ - ~' 3.
Percentage of
Variety Tissue Injured
2.
Provider
35 ab
5.
Astro
30 ab
3.
Harvester
29 b
1.
Sanilac
27 b
4.
Bush Blue Lake 274
*
vO 1
CM 1
6. Tendercrop
8. Pinto 111
9. Stringless Black Valentine
10. Tempo
7. Eagle
aEach number represents an average of AO plants (4 replications,
10 plants per replication)
^Numbers followed by the same letter are not significantly different
according to a Duncan's Modified (Bayesian) Least Significant
Test Value (k=100) approximating P=0.05.
20 b
16 cd
15 cd
14 cd
13 d
TO TYPIST Beqin flush with the left hand nnrpiml 'inc. and end t>p
ing so the average lenyiti of line corrc^i'onds wjtb the right hand marginal line
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 14
Table 5. A comparison of the relative susceptibility of ten bean
varieties in the trifoliate leaf stage exposed to ozone (2)
or PAN. Varieties are placed in order of susceptibility.
Numbers preceding varietal names indicate order of suscepti-
bility to ozone.
Ozone
1. Sanilac
2. Pinto 111
3. Tempo
4. Astro
5. Bush Blue Lake 274
6. Harvester
7. Eagle
8. Stringless Black
Valentine
9. Provider
10. Tendercrop
PAN3
9. Provider
4. Astro
6. Harvester
1. Sanilac
5. Bush Blue Lake 274
10. Tendercrop
2. Pinto 111
8. Stringless Black
Valentine
3. Tempo
7. Eagle
aBased on bifacial necrosis.
TO TYPIST—Begin t)pmg flush with the left hand marginal line, and end t)p
*ng so the a\erage length of line corre$i«onds <*iih the right hand marginil line
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 15
Literature Cited
1. DAUBERT, N. C. 1972. ANOVES/ANOVUM. A statistical package
program for analysis of variance. Unpublished Computation
Center memo. The Penna. State Univ., University Park.
2. DAVIS, D. D., and L. KRESS. 1974. The relative susceptibility
of ten bean varieties to ozone. Plant Dis. Reptr. 58:14-16.
3. DRUMMOND, D. B. 1972. The effect of peroxyacetyl nitrate on
petunia (Petunia hybrida Vilm.). Center for Air Environment
Studies No. 260-72. The Penna. State Univ., University Park
70 pp.
4. DUGGER, W. M., Jr., 0. C. TAYLOR, W. H. KLEIN, and W. SHROPHIRE,
JR. 1963. Action spectrum of peroxyacetyl nitrate damage
to bean plants. Nature 198:75-76.
5. NETER, J., and W. W. WASSERMAN. 1974. Applied linear statistical
models, regression, analysis of variance and experimental
designs. Richard D. Irwin Pub. 842 pp.
6. SMITH, R. G., R. J. BRYAN, M. FELDSTEIN, B. LEVADIE, F. A. MILLER,
and E. R. STEPHENS. 1972. Tentative method of analysis for
peroxyacetyl nitrate (PAN) in the atmosphere (gas chromato-
graphic method), in: Methods of Air Sampling and Analysis,
Amer. Public. Health Assoc. Washington, D. C. pp. 215-219.
7. STEPHENS, E. R. 1964. Absorptivities for infrared determination
of peroxyacetyl nitrates. Anal. Chem. 36:928-929.
8. STEPHENS, E. R. 1965. The production of pure peroxyacetyl
nitrates. J. Air Pollut. Control Assoc. 15:87-89.
TO TYPIST—Brtjin t\ping flu«h uith the lefi >and mirpinnl Itne, and end t>p
mg so the average length of Itne corrr^j-or Ja mth ihe right hand marginal line
-------
1
2
3
4
S
6
7
8
9
10
tl
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Starkey, Davis & Merrill
Plant Dis. Reptr. - 16
9. STOLZY, L. H., 0. C. TAYLOR, J. LETEY, and T. E. SZUSZKIEWICZ.
1961. Influence of soil-oxygen diffusion rates on suscepti-
bility of tomato plants to air-borne oxidants. Soil Sci. 91:
151-155.
10. TAYLOR, 0. C. 1969. Importance of peroxyacetyl nitrate (PAN)
as a phytotoxic air pollutant. J. Air Pollut. Control Assoc.
19:347-351.
11. TAYLOR, 0. C., W. M. DUGGER, JR., E. A. CARDIFF, and E. F. DARLEY
1961. Interaction of light and atmospheric photochemical
products ('smog') within plants. Nature 192:814-816.
12. WALLER, R. A., and D. B. DUNCAN. 1969. A Bayes rule for the
symetric multiple comparisons problem. J. Amer. Stat. Assoc.
64:1484-1503.
13. WOOD, F. A., D. B. DRUMM0ND, R. G. WILHOUR, and D. D. DAVIS.
1974. An exposure chamber for studying the effects of air
pollution on plants. The Penna. State University, Pa. Agr.
Expt. Sta. Prog. Rpt. No. 335, 7 pp.
TO TYPIST—Beun tjping flush with thi Irfi hami marginal line, and end t>p
ing so the average length of hire com »iih the right hand marginal line
-------
APPENDIX VI
"Influence of Sub-threshold Concentrations
of Peroxyacetyl Nitrate (PAN) on Post-exposure
Water Stress in Bean"
(Manuscript form)
-------
INFLUENCE OF SUB-THRESHOLD CONCENTRATIONS OF PEROXYACETYL
NITRATE (PAN) ON POST-EXPOSURE WATER STRESS IN BEAN
T. E. STarkey, D. D. Davis, E. J. Pell and W. Merrill
Graduate Assistant, Assistant Professors, and Professor;
Department of Plant Pathology and Center for Air Environment Studies,
The Pennsylvania State University, University Park, Pennsylvania 16802.
Contribution No. from the Department of Plant Pathology.
Paper No. in the Journal Series of The Pennsylvania Agricultural
Experiment Station and Contribution No. Center for Air Environment
Studies. The Pennsylvania State University, University Park, Pennsylvania
16802.
This publication has been financed in part with Federal funds
from the Environmental Protection Agency under grant numbers T900011
and R800865. This study was also partially funded under grant FS-NE-
26 from the USDA Forest Service, Northeastern Forest Experiment Station.
The contents do not necessarily reflect the views and policies of
E.P.A. or U.S.D.A. nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
Accepted for publication:
-ABSTRACT
Provider, a relatively susceptible bean variety and Stringless
Black Valentine, a relatively tolerant variety were exposed to a sub-
3
threshold concentration of 395 ug/m (0.08 ppm) peroxyacetyl nitrate (PAN)
for 0.5 hours. Both varieties were subjected to drought stress follow-
ing exposure. Peroxyacetyl nitrate predisposed the susceptible variety
Provider to post-exposure water stress, but did not predispose the
tolerant Stringless Black Valentine to post-exposure water stress.
-------
T. E. Starkey et al. - 2
Phytopathology
Provider showed a significant increase in transpiration following PAN
exposure as measured by a more negative water potential, decreased
soil moisture and visible willing. Although stomatal function may
be involved, it is hypothesized that PAN effects either the abaxial
cuticular membrane or the abaxial epidermal cells thus increasing the
transpiration rate.
Phytopathology 65:
Additional key words: air pollution, Phaseolus, cuticle, water potential
Foliar injury has often been used as the criterion for assessing
economic damage to vegetation caused by air pollution. However, it is
probable that sub-threshold concentrations of air pollutants (i.e. below
that needed to cause visible injury) also impart significant reductions
in crop value. In addition, low levels of air pollution may predispose
plants to other physiological stresses such as drought. In these cases,
the initial causal agent usually goes unnoticed, and the economic loss
is assigned to the obvious secondary causal agent.
We have made casual observations that plants exposed to photochemical
oxidants are predisposed to drought stress. However, there are few
published reports dealing with the effects of sub-threshold concentrations
of oxidizing air pollutants on water uptake and transpiration by plants.
Ting et_ al_. (29) reported that cotton leaves exposed in vitro to sub-
threshold concentrations of ozone incurred approximately a 50% increase
in sucrose uptake compared to the controls; these changes are thought to
reflect altered cell permeability. Koritz and Went (11) reported that
tomato plants exposed to a sub-threshold concentration of ozone plus
-------
T. E. Starkey et al. - 3
Phytopathology
vapors of 1-n-hexene had a decreased water uptake and transpiration rate.
They hypothesized that the decrease in water uptake rnd transpiration
rate was due to a decreased permeability of cell membranes. "There have
been no reports dealing directly with the influencfe of PAN on water stress.
Therefore, the objective of this study was to determine if two varieties
of bean (Phaseolus vulgaris L.) exposed were more susceptible to water
stress following an exposure to a low dose of PAN which did not cause
visible symptoms.
MATERIALS AND METHODS. — Bean varieties — Provider was selected
because of its high susceptibility to PAN in both the primary and trifoliate
leaf stage, based on bifacial necrosis as an indicator of susceptibility
(23). Stringless Black Valentine was selected because of its relative
tolerance to PAN in both leaf stages based on bifacial necrosis (23). Two
varieties differing in PAN susceptibility were used to determine if the
response to water stress following exposure to PAN was different between
susceptible and a tolerant variety.
Cultivation. — One hundred and sixty seeds of each variety were
planted in vermiculity treated with 5.3 g/liter solution of 20-20-20
fertilizer. The vermiculite was moistened with water as required.
Cotyledons emerged 5 days after planting; two days later 48 plants
uniform in size, of each variety were transplanted into 16 ounce (589 cc)
pots (one plant per pot) and approximately 360 cc of aerated-steam
treated 1:1:1 (v/v) peat:perlite:clay-loam soil mix were added. The soil
in each pot was packed down uniformly by Ughtly tapping each pot
five times on the table top and 200 ml of water were slowly added to each
pot. Two days following transplanting an additional 200 ml of water were
added to each pot. All plants were grown in a controlled environment
-------
T. E. Starkey et al. -4
Phytopathology
chamber maintained at 24 C, 70% relative humidity (RH) and a 12-hour
photoperiod with a light intensity of 25 Klux, hereafter referred to as
"standard conditions".
Exposure to PAN. — Three days after transplaAting, 24 plants of
3 3
each variety were exposed to 395 ug/m + 74 ug/m (0.08 ppm + 0.015
ppm) PAN for 0.5 hour at standard conditions in a modified controlled
environment chamber (80). The pots were arranged randomly within the
chamber. All plants received the 3-hour pre- and post-exposure light
treatment considered necessary for symptom development (4, 27, 28).
PAN was generated, collected and stored as described by Stephens
(25). During exposure the PAN concentration was monitored at approximately
10 minute intervals by taking air samples from the chamber and injecting
them into an electron capture gas chromatograph (22) calibrated as
described by Stephens (24).
Post-exposure conditions. — Immediately following PAN exposure, the
plants were returned to the controlled environment chamber and maintained
at standard conditions and groups of 12 plants of each variety were treated
as follows: exposed to PAN, water withheld following exposure (+PAN
-WATER) and exposed to PAN, water supplied following exposure (+PAN
-WATER). Control plants not exposed to PAN also had water withheld
following exposure (-PAN -WATER) or water supplied following exposure
(-PAN +WATER). Plant in the +WATER treatments received 200 ml of water
every other day rendered water potential measurements less variable than
did maintaining the pots at or above container capacity (31). Water was
withheld from the -WATER treatments for the duration of the experiment.
-------
T. E. Starkey et al. - 5
Phytopathology
Water potential and soil moisture. — Water potential and soil
moisture measurements were determined on the day of exposure, and the
second and fourth day following exposure. Measurements were taken at the
same time of day. Four plants of each variety in each treatment were
used to determine water potential and soil moisture on each day.
Bean stems were cut 1 cm below the cotyledons and the water potential
of the upper portion was determined by the pressure bomb technique (1).
Pressure was applied to the chamber with nitrogen until the xylero sap appeared
at the cut stem surface projecting from the chamber. Measurements were
recorded as pounds pressure per square inch and then converted to negative
bars. The accuracy of this pressure bomb technique was approximately +
0.17 bars.
Following the water potential measurements the lower stem and roots
were removed from the soil and the percentage soil moisture determined
on a volume basis. The volume percentage of water at container capacity
was 47.71%. The volume percentage of water at the permanent wilting point
was estimated to be 3%. Measurements of soil moisture were within these
limits of available soil moisture.
Data analysis. — The study was replicated four times. Data were
subjected to analysis of variance using a four way completely crossed
design with variety, PAN, water, and time of analysis as factors (17, 21).
Mean separations for water potential and soil moisture measurements were
conducted using a Duncan's Modified (Bayesian) Least Significant
Different (DLSD) Test Value (k=500) approximating P=0.01 (30).
-------
T. E. Starkey et al. "6
Phytopathology
RESULTS. — Neither Stringless Black Valentine nor Provider
sustained macroscopic symptoms following exposure to 395 ug/m3 PAN for
0.5 hour. Provider, the relatively susceptible variety, and Stringless
Black Valentine, the relatively tolerant variety, responded differently
to water stress following PAN exposure.
Provider. — (PAN-Susceptible). - Plants in the +PAN -WATER
treatment had a significantly greater negative water potential on the second
and fourth days following exposure than did plants in the -PAN -WATER
treatment (Table 1). There was no statistical difference at any time
between the plants in the +PAN +WATER treatment and those plants in the
-PAN +WATER treatment (Table 1). Plants exposed to PAN had a significantly
lower percentage of soil moisture on the second and fourth days than the
plants not exposed to PAN (Table 3). The effect of PAN on water stress
was not apparent on the first day since there were no significant
differences among any of the four treatments. Visible wilting was
stimulated by PAN exposure. Plants in the +PAN -WATER treatment began to wilt
on the second day following exposure whereas the plants in the -PAN -WATER
treatment did not begin to wilt until the third day following exposure.
Stringless Black Valentine (PAN-Resistant). — Plants in the +PAN
-WATER treatment had a significantly less negative water potential on
the fourth day following exposure than the treatment -PAN -WATER. There were
no statistically significant differences in these treatments on the first
two days (Table 2). There was no significant difference at any time period
between the treatments +PAN -WATER and -PAN +WATER, nor in the percentage
of soil moisture at any time between the plants exposed to PAN and those
not exposed to PAN (Table 3). PAN did not stimulate visible wilting in
-------
T. E. Starkey, et al. - 7
Phytopathology
this variety. On the second day following exposure, equal numbers of plants
in the treatments +PAN -WATER were wilted.
Both varieties treated with either +PAN +WATER or -PAN +WATER regime
exhibited a greater negative water potential on the day of exposure and less
negative water potential four days after exposure.
DISCUSSION. — Many researchers have studied the effects of drought
stress on the subsequent susceptibility of plants to various air pollutants
(10, 11, 13, 26). It has been consistently observed that water stress prior
to exposure decrease plant-injury. In addition, this study revealed that
an air pollutant may predispose plants to drought stress. Provider was
much more susceptible to PAN than was Stringless Black Valentine in both
the primary and trifoliate leaf stage (23). Provider was also more susceptible
to drought stress following exposure to PAN than was Stringless Black
Valentine. This indicates that PAN or other air pollutants may be more likely
to predispose a susceptible variety to drought stress.
PROVIDER. — Exposure of Provider to sub-threshold concentrations
of PAN resulted in a significantly greater negative water potential; a
significantly lower percentage of soil moisture; and an earlier visible
wilting of exposed plants compared to the controls. It has been concluded
from these observations that PAN stimulated an increase in the transpiration
rate of Provider. The plants exposed to PAN and denied water following
exposure were under a greater drought stress than those not exposed to PAN
under a similar watering regime. These results agree with Ting et al. (29)
who found an increase in uptake of sucrose at sub-Lhreshold pollutant con-
centrations. The results of Koritz and Went (11) differ from the results of
this study. They found a decreased rate of transpiration in plants exposed
-------
T. E. Starkey, et al. - 8
Phytopathology
to sub-threshold pollutant concentrations.
The increased water uptake of relatively susceptible bean varieties
exposed to sub-threshold concentration of PAN has several implications
to laboratory studies and field observations. The'volume of soil in
which laboratory plants are grown in generally much smaller than the
available volume of soil in the field. Subsequently, an increase in
water at a much faster rate than plants not exposed to PAN. The plants
exposed to PAN would require more frequent watering to prevent wilting.
If wilting should occur, the use of these plants in laboratory experiments
incolving daily sequential exposures to pollutants may be limited due
to possible irreversible cellular damage.
Increased water uptake in the field could be of economic importance.
If plants are removing water from the soil faster than it can be replenished,
wilting may occur. More frequent irrigation would be required to prevent
permanent plant damage and subsequent reduction in yield. Possible
ozone-induced drought stress has been implicated in the mid-summer death
of Poa annua on golf courses (16).
Stringless Black Valentine. — Stingless Black Valentine, the more
PAN-tolerant variety, did not appear to be more susceptible to drought
when exposed to sub-threshold concentrations of PAN. Although the plants
in the treatment +PAN -WATER had a significantly less negative water
potential on the 4 day after exposure than those in the treatment -PAN
-WATER, this difference was so small that it would not be biologically
significant. In order that the means be statistically different, the
difference between the means had to exceed a DLSD value of 0.39 bars.
The mean water potential for the treatment -PAN -WATER was -9.06 bars.
The mean water potential for the treatment +PAN -WATER was -8.63 bars.
-------
T. E. Starkey, et al. - 9
Phytopathology
The difference between these two treatment (0.43 bars) only exceeded the
DLSD value by 0.04 bars. Due to the inherent measurement error in the
pressure bomb technique (+ 0.17 bars), it must be concluded -that although
the difference between the two treatments was statistically different,
it would not be biologically significant. The soil moisture measurements
and visual observations of the wilted plants confirms this lack of biological
significance.
The change in water potential of the +PAN +WATER and -PAN -WATER
treatments, from a greater negative value on the day of exposure to a lower
negative value four days after exposure may be explained by the root-soil
contact relationship. Since PAN exposure occurred 3 days after transplanting,
the roots may not have become well established in the soil; this could
result in the greater negative water potential observed at first. By the
fourth day the roots would have been more completely established in the
soil and a less negative water potential measured as a result. This change
in negative water potential was not seen in the treatments in which water
was withheld, probably due to the masking effect of the drought stress.
Possible Mechanism of Action. — In attempting to explain the mechanism
of action of air pollutants on plants, researchers have drawn correlations be-
tween amatomical and biochemical differences of tolerant and susceptible
paints. In this discussion another possible mechanism of action to explain
the results will be hypothesized.
In order to account for the PAN initiated increase in water uptake,
it is reasonable to hypothesize an alteration in stomatal behavior and/or
epidermal transpiration.
Stomatal transpiration. — Stomates must be open prior to exposure to
allow diffusion of pollutants into the plant (19). However, many researchers
have shown that a significant increase in stomatal resistance (decreased
-------
T. E. Starkey et al, - 10
Phytopathology
opening) occurs during and after the plants are exposed to air pollutants
(3, 5, 6, 12, 19). If the increase in transpiration observed in plants
exposed to PAN is due to increased stomatal transpiration, either the stomates
must be open longer or wider than the control non-exposed plants.
One possible mechanism to explain such stomatal behavior would be
an inhibition of the starch hydrolysis translocation process. Hanson and
Stewart (9) have indicated that plants exposed to low concentrations of ozone
and PAN exhibit an inhibition in sta-ch hydrolysis resulting in an accumula-
tion of starch in the leaf. If high levels of starch do accumulate, the
stomates could possibly remain partially open during the night allowing for
an increased transpiration rate. Whether this actually occurs is questionable
since stomatal regulation is governed by other factors which could negate
the effect of the increased starch leaves. Although the authors stated that
such a phenomenon existed for low levels of PAN, they did not present data
to support their allegation.
In contrast, Dugger e£ al. (3) indicated that stomatal opening is
not the primary controlling factor in predisposing plants to PAN damage.
Injury to plants by PAN is also governed by a series of light dependent
reactions between PAN and the.plant, as well as physiological age of the
plant (5).
Cuticular transpiration. — The second major pathway through which
transpirational water may be lost is through the epidermis and cuticle.
Cuticular transpiration comprises the loss of water through the cuticle
of epidermal cells and peristomatal guard cells (14). An interaction between
PAN and the cuticular membrane might explain the increase in transpiration
observed in this study.
-------
T. E. Starkey - 11
Phytopathology
The cuticular membrane may be Important in determining the response
of plants to air pollutants, particularly PAN. Bystrom trt al^. (2)
have indicated that the amount of wax on the surface of garden beet may be
responsible for the susceptibility of this species to damage by irradiated
auto exhaust. PAN injury has previously been reported on garden beet
(15, 18). The immature leaves, which were most susceptable to irradiated
auto exhaust, had an incomplete waxy layer of the leaf surface (2).
Exposures to irradiated auto exhaust resulted indefinite changes in the
surface texture of the leaf within 24 hours to 1 week after exposure.
Excessive extrusions of wax were produced in response to the pollutant.
The mechanism of action of PAN may be in part related to reactions
with either the cuticular membrane or the epidermis itself. Immature leaves,
which are susceptible to PAN, have a thin and incomplete cuticle on the
abaxial leaf surface (14). The amount of wax per unit area is less in a young
leaf than in mature leaves (41) which are generally resistant. PAN may
act either directly on the epidermal cells resulting in collapse of the
tissue or on the cuticle, exposing the epidermal cells to the atmosphere
and making them vulnerable to desication and autoxidation. The collapse
of the abaxial epidermal cells could result in the common PAN symptom of
bronzing and glazing on that surface (7). As the leaf matures, the cuticle
thickens and the amount of wax per unit area increases (8, 14). It is possible
that the increased amounts of wax on older plants may act as a site of break-
down of PAN or that the thickened cuticle of older leaves may act as a barrier
preventing PAN injury to the epidermal cells.
Plants require approximately 3 hours of light prior to PAN exposure
to obtain maximum symptom development (4, 27, 28). Sargent and Blackman (20).
-------
T. E. Starkey, et al. - 12
Phytopathology
reported that light has a significant effect on the penetration of various
compounds (2,4-D, dalapon, picloram, chloride) through the leaf surfaces of
bean. In darkness, penetration through both surfaces occured at a constant
rate. Exposure to relatively high light intensities (16 Klux) significantly
enhanced the penetration of compounds through the abaxial leaf surface, but
not through the adaxial surface. Initially the enhancement was small, but
after 2-3 hours of light the compounds "surged" into the leaf at a high
rate (20). Peroxyacetyl nitrate may be taken through the cuticle in this
manner. The frequent occurrence of abaxial bronzing and glazing and the
pre-exposure light requirements supports this hypothesis.
Provider showed an increase in transpiration when exposed to sub-
threshold concentration of PAN. PAN may possibly have attacked the epidermal
cells or altered the composition of the cuticular membrane, thus increasing
the surface area for transpirational loss of water. Stringless Black
Valentine did not show an increase in transpiration. Morphologically this
variety was larger than Provider although chronologically they were the same
age. Stringless Black Valentine may have had a thicker, more complete cuticle
or more wax per unit area than Provider on the day of exposure, thus
providing a degree of resistance to PAN.
Structural differences in the cuticular membrane (including superficial
wax) occur among plant species and among varieties of the same species, vary
with position on the plant, age or environment and may be induced by chemicals
(14). It is possible that the variations in plant sensitivity due to cultural
practices, and time of year may also be related to differences in the cuticular
membrane.
-------
T. E. Starkey et al. -
Phytopathology
TABLE 1. The influence of PAN and water on the water potential
of Provider (PAN-susceptible).
Water potential (negative bars)
Treatment " Days after composure
PAN WATER 0 2 4^
+ - 6.52 a 6.89 a 8.42 a
6.48 a 5.96 b 7.09 b
+ + 6.73 a 5.31 c 4.44 c
- + 6.65 a 5.35 c 4.38 c
z Numbers followed by the same letter in each time column are
not significantly different according to a Duncan's
Modified (Bayesian) Least Significant Test Value (k=500)
approximating P=0.01. Values represent the average of
16 plants.
-------
T. E. Starkey et al. -
Phytopathology
TABLE 2. The influence of PAN and water on the water potential
of Stringless Black Valentine (PAN tolerant).
Water potential (negative bars)
Treatment
Days after
exposure
PAN WATER
0^
2
4
+
6.15
a
5.96
a
8.63
-
6.14
a
6.32
a
9.06
+ +
6.05
a
5.05
b
4.38
+
5.92
a
5.16
b
4.56
Numbers followed by the same letter in each time column are
not significantly different according to a Duncan's
Modified (Bayesian) Least Significant Test Value (k=500)
approximating P=0.01. Values represent the average of
16 plants.
-------
T. E. Starkey et al.
Phytopathology
TABLE 3. The influence of PAN on soil moisture of two bean
varieties, Stringless Black Valentine (SBV) and
Provider (PROV).
Soil
r*-
:—~~ , " gi i ¦ i ¦
moisture (% by volume) _
Days after exposure
Variety Treatment
0
2
4
PROV +PAN
43.30 a
24.40 a
19.87 a
-PAN
43.42 a
30.40 b
22.25 b
SBV +PAN
43.19 a
27.61 a
20.27 a
-PAN
42.91 a
26.56 a
21.22 a
Numbers followed by the same letter in each time column for
each variety are not significantly different according
to a Duncan's Modified (Bayesian) Least Significant Test
Value (k=500) approximating P=0.01.
-------
T. E. Starkey et al. - 16
Phytopathology
LITERATURE CITED
1. Boyer, J. S., and S. R. Ghorashy. 1971. Rapid field measurement
of leaf water potential in soybean. Agron. J. 63:344-345.
2. Bystrom, B. G., R. B. Glater, F. M. Scott, and E. S. C. Bowler. 1968.
Leaf surface of Beta vulgaris - electron microscope study. Bot.
Gaz. 129:133-138.
3. Dugger, W. M., Jr., 0. C. Taylor, E. Cardiff, and C. R. Thompson.
1962. Stomatal action in plants as related to damage from
photochemical oxidants. Plant Physiol. 37:487-491.
4. Dugger, W. M., Jr., 0. C. Taylor. W. H. Klein, and W. Shrophire, Jr.
1963. Action spectrum of peroxyacetyl nitrate damage to bean
plants. Nature 198:75-76.
5. Dugger, W. M., Jr., 0. C. Taylor, C. H. Thompson, and E. Cardiff. 1963.
The effect of light on predisposing plants to ozone and PAn damage.
J. Air Pollut. Control Assoc. 13:423-428.
6. Engle, R. L., and W. H. Gabelman. 1966. Inheritance and mechanism
for resistance to ozone damage in onion, Allium cepa L. Proc.
Amer. Soc. Hort. Sci. 89:423-430.
7. Glater, R. B., R. A. olberg, and F. M. Scott. 1962. A developmental
study of the leaves of Nicotiana glutinosa as related to their smog-
sensitivity. Amer. J. Bot. 49:954-970.
8. Hallam, N. D., and B. E. Juniper. 1971- The anatomy of the leaf
surface, in T. F. Preece and C. H. Dickinson eds. Ecology of
Leaf Surface Micro-organisms. Academic Press, p 3-37.
9. Hanson, G. P., and W. S. Stewart. 1970. Photochemical oxidants.
effect on starch hydrolysis in leaves. Science 168:1223-1224.
10. Heck, W. W. 1968. Factors influencing expression of oxidant damage
to plants . Ann. Rev. Phytopathol. 6:165-188.
-------
T. E. Starkey et al. - 17
Phytopathology
11. Koritz, H. G., and F. W. Went. 1953. The physiological action of
smot on plants. I. Initial growth and transpiration studies.
Plant Physiol. 28:5j3-62.
12. Lee, T. T. 1965. Sugar content and stomatal width as related to ozone
injury in tobacco leaves. Can. J. Bot. 43:677-685.
13. Macdowall, F. D. H. 1965. Predisposition of tobacco to ozone damage.
Can. J. Plant Sci. 45:1-12.
14. Martin, J. T., and B. E. Juniper. 1970. The cuticle of plants.
Edward Arnold Pub. 347 p.
15. Middleton, J. T., J. B. Kendrick, Jr., and H. W. Schwalm. 1950. Smog
in the southern coastal area. Calif. Agr. 4:7-10.
16. Moyer, J., H. Cole, Jr., and N. L. Lacasse. 1974. Reduction of ozone
injury on Poa annua by benomyl and thiophante. Plant Dis. Reptr.
58:41-44.
17. Neter, J., and V. W. Wassertnan. 1974. Applied linear statistical models,
regression, analysis of variance and experimental designs. Richard
D. Irwin Pub. 842-p.
18. Pell, E. J. 1972. Economic impact of air pollution on vegetation in
New Jersey and an interpretation of its annual variability.
Environ. Pollut. 8:23-33.
19. Rich, S., and N. C. Turner. 1972. Importance of moisture on stomatal
behavior of plants subjected to ozone. J. Air Pollut. Control
Assoc. 22:718-721.
20. Sargent, J. A., and G. E. Blackman. 1970. Studies on foliar
penetration VII. Factors controlling the penetration of chloride
ion into the leaves of Phaseolus vulgaris. J. Exp. Bot. 21:933-942.
21. Scott, D. T., M. W. Carter, G. R. Bryce, B. L. Joiner. 1974. RUMMAGE,
a general data analysis system. Unpublished Statistical Dept.
memo. The Penna. State Univ., University Park.
-------
T. E. Starkey et al. - 18
Phy topa tholo gy
22. Smith, R. G., R. J. Bryan, M. Feldstein, B. Levadie, F. A. Miller,
and E. R. Stephens. 1972. Tentative method of analysis for
peroxyacetyl nitrate^(PAN) in the atmosphere (gas chromatographic
method). In Methods of Air Sampling and Analysis. Amer. Public.
Health Assoc. Washington, D. C. p. 215-219.
23. Starkey, T. E., D. D. Davis, and W. Merrill. 1976. Susceptibility and
Symptoms of ten bean varieties exposed to peroxyacetyl nitrate (PAN)
Plant Dis. Reptr. (in press).
24. Stephens, E. R. 1964. Absortivities for infrared determination of
peroxyacetyl nitrates. Anal. Chem. 36:928-929.
25. Stephens, E. R. 1965. The production of pure peroxyacetyl nitrates.
J. Air Pollut. Control Assoc. 15:87-89.
26. Taylor, G. S., H. G. DeRoo, and P. E. Waggoner. 1960. Moisture and
fleck of tobacco. Tobacco Sci. 4:62-68.
27. Taylor, 0. C. 1969. Importance of peroxyacetyl nitrate (PAN) as a
phytotoxic air pollutant. J. Air Pollut. Control Assoc. 19:347-351.
28. Taylor, 0. C., W. M. Dugger, Jr., E. A. Cardiff, and E. F. Darley. 1961.
Interaction of light and atmospheric photochemical products
('smog') within plants. Nature 192:814-816.
29. Ting, I. P., J. Perchorowicz, and L. Evans. 1974. Effect of ozone on
plant cell membrane permeability. In Air Pollution Effects on Plant
Growth. Symp. Series No. 3. Amer. Chem. Soc. p. 8-21.
30. Waller, R. A., and D. B. Duncan. 1969. A Bayes rule for the symmetric
multiple comparisons problem. J. Amer. Stat. Assoc. 64:1484-1503.
-------
T. E. Starkey et al. - 19
Phytopathology
31* White, J. W., and J. W. Mastalerz. 1966. Soil moisture as related to
"container capacity". Proc. Amer. Soc. Hort. Sci. 89:758-765.
32. Wood, F. A., D. B. Drummond, R. G. Wilhour, and D. D. Davis. 1974.
An exposure chamber for studying the effects of air pollution on
plants. Pa. Agr. Expt. Sta. Prog. Rpt. No. 335. 7 p.
-------
APPENDIX VII
"Interaction of Acute Doses of Ozone
and PAN on Young Ponderosa Pine
Seedlings"
(Manuscript form)
-------
INTERACTION OF ACUTE DOSES OF OZONE AND PAN ON
YOUNG PONDEROSA PINE SEEDLINGS
D. D. Davis
Assistant Professor, Department of Plant Pathology and Center for Air
Environment Studies. The Pennsylvania State University, University Park,
Pennsylvania 16802.
Contribution No. from the Department of Plant Pathology. Paper
No. in the Journal Series of The Pennsylvania Agricultural Experiment
Station and Contribution No. , Center for Air Environmental Studies,
The Pennsylvania State University, University Park, Pennsylvania 16802.
Funds provided by the Environmental Protection Agency, Grant No. R800865,
administered through the Center for Air Environment Studies.
The author gratefully acknowledges receipt of seed from Mr. Leroy
C. Johnson, U. S. Forest Service Institute of Forest Genetics, Placervllle,
California. Gratitude Is expressed to J. B. Coppolino and H. J. Smith for
technical aid.
ABSTRACT
Younc ponderosa pine seedlings In the primary needle stage were exposed
to acute dcsages of ozone, PAN, or the pollutants combined at their respec-
tive concentrations. Ozone injured the foliage while PAN did not Induce
symptoms. The combined pollutants caused less Injury on six-week-old plants
than did ozone alone. The same trend was observed for 10-week-old plants,
but the reduction In Injury caused by the combined pollutants was non-
significant. These results Indicate that the simultaneous exposure of pon-
derosa pine seedlings to ozone and PAN results In an antagonistic reaction
on very young primary needles. However, the magnitude of the Interaction may
be affected by plant or needle age, as well as dosage.
Plant Dls. Reptr.
-------
0. D. Davis - 2
Plant DIs. Reptr.
Additional key words: air pollution, Plnus ponderosa.
Miller and his co-workers (6, 7, 8, 9) previously reported that ponderosa
pine (Plnus ponderosa) trees were declining In the San Bernardino Forest of
California due to the ozone component of photochemical smog. Ponderosa pine
seedlings were reported to be very resistant to peroxyacetyl nitrate (PAN),
the other major phytotoxic component of photochemical smog (2). However,
since these two pollutants often co-exist in the polluted air of the Los
Angeles Basin, it is important to determine the response of plant species to
simultaneous exposure of the two pollutants.
Kohut (5) reported that a PAN-resistant clone of hybrid poplar (Populus
trIchocarpa x P. maxlmowizci i) developed more foliar Injury when exposed to
ozone and PAN simultaneously than when exposed to ozone alone. However,
when he simultaneously exposed a PAN-susceptible variety of bean (Phaseolus
vulgaris 'Pinto III') to ozone and PAN, the PAN-type symptom was inhibited
and the ozone-type symptom was slightly enhanced. The study reported herein
was designed to determine the influence of a simultaneous exposure of ozone
and PAN on the primary foliage of young ponderosa pine seedlings.
MATERIALS AND METHODS
Ponderosa pine seeds from the 197^* crop were collected on the Arrowhead
Ranger District, San Bernardino National Forest at an elevation of 5,000
feet. Seeds were soaked in water for 2A hr, blotted dry, and maintained at
k° C for a minimum of two weeks before the initial planting. Ten seeds were
planted in each of five 10-cm pots containing a 1:1 (v/v) peatrperlite mix
and placed in the greenhouse. After emergence, seedlings were thinned to
five per pot, leaving those plants representing the most uniform stand. Five
g of slow release fertilizer (analysts 14N: 14P: 1 *»K) were applied to each pot.
-------
D. D. Davis - 3
Plant DIs. Reptr.
Seedlings were maintained on a greenhouse bench until exposed to ozone
and/or PAN at 24° C, 75% relative humidity, and 25 Klux light Intensity In
a previously described exposure chamber (13)*
PAN was generated as described by Stephens (10). Exposure and moni-
toring techniques for PAN were as described by Oar ley et al. (!) and Wood
and Drummond (12). Ozone was generated and monitored as described by Davis
and Coppolino (3). Data were analyzed using the student's t-test.
RESULTS
Experiment 1. - This initial experiment was designed to determine the
age of maximum ozone susceptibility for the primary needles of ponderosa
pine. This information would be used as a basis for further experiments.
Seeds were planted at weekly Intervals beginning In June, 197^ and seedlings
emerged approximately two weeks later. Plants were maintained In the green-
house until November, at which time 310 seedlings were exposed to 0.25 parts
per million (ppm) ozone on two consecutive days. At this time, seedling
age ranged from 1 to 16 weeks. Following exposure, plants were returned
to the greenhouse for one week at which time symptoms were evaluated. Symptom
severity was rated In a 0-5 scale, progressing from '0' which represented no
Injury, to 5 which indicated complete needle necrosis. Figure 1 Illustrates
the results of this initial experiment. Based on these results, experiments
2 and 3 were conducted using primary needles within 3-10 weeks of age, and a
slightly higher dose of ozone.
Experiment 2. - This study was designed to determine the Influence of
simultaneous ozone and PAN exposures on ponderosa pine seedlings. In this
first Interaction study, 2k0 6-week old seedlings were exposed to 0.^0 ppm
ozone, 0.20 ppm PAN, or a combination of the two at their respective concen-
trations, for 4 hrs. We had previously determined that this dosage of ozone
-------
D. 0. Davis -
Plant DIs. Reptr.
gave a measurable response on pine seedlings. Although ponderosa pines
are resistant to PAN, I chose 0.20 ppm as an experimental concentration,
the maximum ever recorded In ambient air.
The seedlings were exposed to ozone In the morning and to the combined
pollutants In the afternoon of the same day. The following morning, the
plants were exposed PAN alone. The basal 10 needles were evaluated five
days later and percent Injured tissue recorded. The results of this first
Interaction experiment are given In Table 1.
A similar experiment was then conducted using 10-week-old plants. This
study was designed to determine If seedlings which were becoming resistant
(Figure 1) responded similarly to the simultaneous exposure as did those
in their peak of ozone susceptibility. Results of this study are given in
table 2.
-------
D. D. Davis - 5
Plant DIs. Reptr.
Figure 1. Influence of age of plant from germination the ozone-
susceptibility of ponderosa pine juvenile needles. The smooth curve
was drawn using the method of least squares. Each dot represents
the average value of 20 seedltngs.
-------
0. D. Davis - 6
Plant DIs. Reptr.
Percent
Follar
• t a
1njury
ReplIcat ion
Ozone
PAN
Ozone/PAN
1
5.2b **
0.0
l.M»
2
17.20 **
0.0
0.39
Each number represents the average percent injury
on the foliage of 40 plants.
Significantly greater than the ozone/PAN Injury
at .01 level of confidence (t-test).
Table 1. Percent foliar injury on six-week-old ponderosa
pine seedlings exposed to 0.^0 ppm ozone, 0.20 ppm PAN,
or ozone and PAN combined at their respective concentrations.
-------
D. D. Davis - 7
Plant Dis. Reptr.
Rep 11cat Ion
Percent Foliage
Injured
Ozone
PAN
Ozone/PAN
1
10.358
0.00
5.85
2
0.95
0.00
0.1»8
3
2.50
0.00
3.10
l»
k.(>2
0.00
0.12
Each number represents the average percent injury
on kO plants. All comparisons were non-significant
at the 0.1 level of confidence.
Table 2. Percent foliar Injury on 10-week-old ponderosa
ptne seedlings exposed to 0.^0 ppm ozone, 0.20 ppm PAtl,
or ozone and PA!) combined at their respective concentrations.
-------
D. D. Davis - 8
Plant Dts. Reptr.
Experiment 3. - In the Initial Interaction experiments, plants were
exposed to ozone or PAN In the morning and ozone/PAN In the afternoon.
To ensure that the results were not biased by a tlme-of-day effect, and
to determine If the antagonistic Interaction also occurred at various
ozone dosages, the following experiment was designed.
A constant level of 0.05 ppm PAN and 0.40 ppm ozone was maintained
In the exposure chamber. On two consecutive days, 120 pine seedlings
were exposed to this combined dosage for 2, k, 6, or 8 hours beginning at
9 AM. To minimize the time-of-day effect, all plants were scheduled Into
the chamber at various tines of the day. For example, half of the plants
exposed for two hours were exposed from 9 AM to 11 A!1 and the other half
from 3 PM to 5 PM. This same exposure scheme was used for each dosage.
Plants were grouped for evaluation of symptoms. Figure 2 illustrates the
results of this study. The percent injury induced by ozone/PAil at A, 6,
and 8 hours was significantly less (antagonistic reaction) than that pro-
duced by ozone alone at the 0.01 level of confidence. Injury from the two-
hour exposures were statistically similar for all pollutants.
DISCUSSION
The Influence of needle age on susceptibility to acute doses of ozone
(Figure 1) was similar to that reported previously (3). These previous
results and those reported herein were based on single acute fumigations,
rather than continual, chronic exposures. Thus these studies both reveal
the time of maximum needle susceptibility, but not necessarily the time at
which the maximum amount of tissue Injury might be observed In the field.
That is, the injury would accumulate during chronic .exposures, such as those
reported by Miller et al. (6, 7, 8) and maximum tissue injury would likely
appear late in the growing season. Indeed, this exact trend was reported
by Miller (6).
-------
D. D. Davls^- 9
Plant DIs. Reptr.
This experiment and a previous report (2) indicates that ponderosa
pine seedlings are very resistant to acute levels of PAN. In similar
studies, Drummond (4) exposed 30 tree species to exceedingly high doses
of PAN and only Induced Injury once at a very unrealistic dose of PAN.
Thus, based on these experiments, one may conclude that many tree species
are very resistant to PAH. This Is difficult to explain, since Immature
tree leaves certainly pass through the chronological stage of development
of maximum PAH susceptibility. However, there must be a more general
physiological difference between the leaf of a tree and that of highly
susceptible plants such as leafy vegetables. A major phenomenon which
may be related to PAN susceptibility may be the chemical and physical
nature of the cuticle. Perhaps the cuticle of tree species lends Inherent
resistance to PAN in leaves of all ages.
Experiments 2 and 3 both indicated that a simultaneous exposure to
ozone and PAN together under sensitive conditions caused significantly less
damage than to ozone alone. (PAN alone caused no injury). We have pre-
viously determined that exposures using combined pollutants do not affect
our ozone concentration. Also, the two components probably do not react
In the atmosphere of the exposure chamber. This would indicate that PAN
or a breakdown product of PAN may be having a reacting physiological effect
on the stomates or with ozone or a by-product within the leaf tissue.
-------
D. D. Davis - 10
Plant DIs. Reptr.
Figure Percent foliar injury on primary needles of ponderosa pine
exposed to 0.^0 ppm ozone, or 0.^0 ppm ozone + 0.05 ppm PAN for 2, *», 6
or 8 hrs. Each dot represents the average value determined for 30 seedlings
in two replications. PAN alone Induced no Injury.
-------
0. D. Davfs - 11
Plant Dls. Reptr.
Kohut ( ) reported that the simultaneous exposure of a PAN-reslstant
hybrid poplar clone to ozone and PAN yielded a significant synergistic
reaction. In contrast, a simultaneous exposure of pinto bean suppressed the
PAN symptom on the abaxial leaf surface and had essentially no effect on
the ozone symptoms on the upper leaf surface ( ).
Apparently, the simultaneous exposure of various plant species to
ozone and PAN will yield varying results for different species of plants.
The results will also depend upon the dosage and ratio of the combined
pollutants, age of plant or leaf, leaf surface, species, and method of
evaluation.
If extrapolated to the field, these results indicate that young ponderosa
pines exposed simultaneously to acute doses of ozone and PAH would probably
suffer less injury than if exposed only to ozone. However, at times other
than the period of naxinun susceptibility, the antagonistic Interaction may
not be significant. Acute combined exposures should not result in synergistic
levels of injury.
-------
D. D. Davis - 12
Plant DIs. Reptr.
Literature Cited
1. DARLEY, E. F., K. A. KETTNER, and E. R. STEOHENS. I963. Analysis of
peroxyacetyl nitrates by gas chromatography with electron capture
detection. Anal. Chem. 35:589-591.
2. DAVIS, D. D. 1975. Resistance of young ponderosa pine seedlings to
acute doses of PAN. Plant DIs. Reptr. 59:183-184.
3. DAVIS, D. D., and J. B. COPPOLINQ. 197^. Relationship between age and
ozone sensitivity of current needles of ponderosa pine. Plant DIs.
Reptr. 58:660-663.
k. DRUMMOND, D. B. 1971. Influence of high concentrations of peroxyacetyl-
nitrate on woody plants. Phytopathology 61:128 (Abstr.).
5. KOHUT, R. 1975. Unpub. Ph.D. Thesis, The Pennsylvania State University,
pp.
6. MILLER, P. R. 1973. Oxidant-Induced community change in a i.iixed conifer
forest. In Air Pollution Damage to Vegetation, Adv. in Chem. Series
No. 122. pp. 101-117.
7. MILLER, P. R., II. :1. ilcCUTCHAN, and 3. C. RYAil. 1972. Influence of climate
and topography un oxidant air pollution concentrations that damage
conifer forests in southern California. Kite. Der Forstlichen Bundes -
Versuchsanst., Vienna, pp. 535-607.
c. MILLER, P. R., J. R. PARMETER, Jr., 0. C. TAYLO.-;, and E. A. CARDIFF. 1963.
Ozone Injury to the foliage of Pinus ponderosa. Phytopatholo^y 53"1072-1076.
9. RICHARDS, B. L., Sr., 0. C. TAYLOR, and G. F. EDMUNDS, Jr. i960. Ozone
needle mottle of pine in southern California. J. Air Pollut. Control.
Assoc. 13:73-77.
10. STEPHENS, E. R. 1965. The production of pure peroxyacyl nitrates. J. Air.
Pollut. Control Assoc. 15:87-89.
-------
D. D. Davis - 13
Plant DIs. Reptr.
11. TAYLOR, 0*. C. 1969. Importance of peroxyacetyl nitrate (PAN) as a
phytotoxlc air pollutant. J. Air Pollut. Control Assoc. 19:3^7-351 .
12. WOOD, F. A., and D. G. DRUMMOND. 197^*- Response of eight cultlvars of
chrysanthemum to peroxyacetyl nitrate. Phytopathology 6^:897-898.
13. WOOD. F. A., D. B. DRUMMOND, R. G. WILHOUR, and D. D. DAVIS. 1973.
An exposure chamber for studying the effects of air pollutants on
plants. Penn. State Univ., Agrlc. Exp. Stn. Prog. Rep. 335. 7 pp.
-------
APPENDIX VII
"The Interaction of Ozone and PAN
on Hybrid Poplar"
(Submitted to Plant Disease Reporter)
-------
The Interaction Of Ozone And PAD On Hybrid Poplar
R. J. Kohut, D. D. Davis, and W. Merrill
Former graduate student, Assistant Professor and Professor,
Department of Plant Pathology and Center for Air Environment Studies,
The Pennsylvania State University, University Park, Pennsylvania 16802.
Contribution No. from the Department of Plant Pathology.
Paper No. in the Journal Series of the Pennsylvania Agricultural
Experiment Station and Contribution No. , Center for Air Environment
Studies, The Pennsylvania State University, University Park, Pennsylvania
16802.
ABSTRACT
Hybrid poplars were exposed for 4 hr under controlled environmental
3 3
conditions to either 353.2 mg/m ozone, 890.1 mg/m PAN or the two pollutants
combined at these concentrations. No visible symptoms were produced by
exposure to PAN alone. The ozone and ozone/PAN exposures produced a dark
brown bifacial necrosis. The pollutant interaction was evaluated in each
experiment by a statistical comparison of the injury level produced by the
ozone/PAN treatment with the sum of the injury levels produced by the
individual pollutant treatments. In four of the six experiments, ozone
and PAN interacted synergistically. In two experiments their effects were
additive. This is the first demonstration that ozone and PAN can interact
synergistically in the production of injury to vegetation.
Plant Disease Reporter
The photochemical oxidants ozone and peroxyacetylnitrate (PAN are
produced by chemical reactions in the atmosphere which involve oxides of
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nitrogen, hydrocarbons, and ultraviolet light. The phytotoxic nature of
ozone and PAN individually has been clearly established. In addition,
the ability of ozone to interact with sulfur dioxide when producing injury
to vegetation has been examined (1, 2, 3, 5, 8, 9, 10). However, ozone
and PAN interactions have not been reported.
Since ozone and PAN are frequently found together in the atmosphere,
it is important to determine if they are capable of interacting in pro-
ducing injury to vegetation. To examine their ability to interact, a series
of controlled exposures was conducted utilizing treatments of ozone, PAN
and the ozone/PAN combination. A hybrid poplar clone was chosen as the
model plant since it was of known ozone sensitivity and could be propagated
asexually. Asexual propagation yields cuttings of similar genetic sensitivity,
avoiding the variation in sensitivity encounted in plants derived from sexual
combinations, i.e. as in seeds.
MATERIALS AND METHODS
Cuttings of hybrid poplar (Populus maximowiczii x trichocarpa) clone
No. 388 were grown in plastic pots filled with a 2:1 (v/v) peat:perlite mixture.
The cuttings were maintained in outdoor beds throughout the summer. After
budbreak, all lateral buds but one were removed to allow only one branch
to develop. Each plant received biweekly applications of one liter of a
solution of 112 grams of 20-190-18 fertilizer in 95 liters of water.
Plants were selected at random and exposed in groups of 12 to either
353.2 ug/m^ (0.18 ppm) ozone, 890.1 ug/m"* (0.18 ppm) PAN, or the two
pollutants combined at these concentrations. The exposures began when the
plants were three weeks old from budbreak and were repeated six times at
two week intervals throughout the summer. All exposures were 4 hours in
duration and conducted from approximately 10 a.m. to 2 p.m. Ozone was
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monitored continuously during the exposures with a chemiluminescent ozone
monitor using the buffered 1% KI technique (6). PAN was ge&Sfiated using
the technique described by Stephens (7) and monitored with a gas chromato-
graph with an electron capture detector. The ozone and Pan monitors were
pollutant specific and not subject to interference during the combined
pollutant exposures.
The possibility that ozone and PAN might interact chemically in
the exposure chamber atmosphere was investigated prior to conducting the
exposures. A stable ozone concentration was established in the exposure
chamber and PAN was then introduced. The REM monitor provided a con-
tinuous reading of ozone concentration and indicated that PAN had no
effect on the ozone concentration. The procedure was then reversed by
establishing a stable PAN concentration and introducing ozone. No
change in PAN concentration was detected. Therefore, it was possible to
discount an atmospheric reaction between the two pollutants as an
explanation for any changes in injury levels produced by the combined
pollutants.
All exposures were conducted in a specially modified growth chamber
(11) at environmental conditions of 23C, 75% relative humidity (RH), and
25 Klux light intensity. A similar growth chamber housed the control
plants at the above environmental conditions.
On the third day after exposure, symptoms were evaluated on the 15
most distal leaves on each plant. The procedure to determine the percentage
of the leaf surface affected involved the use of two reference charts.
The first chart (Fig. 1) represented the gross area of the leaf showing
symptoms and the second chart (Fig. 2) represented the intensity of symptom
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development within the affected area. Each leaf was compared to the two
charts and factors were determined for both gross area affected and symptom
intensity. Multiplication of these two factors resulted in an estimation
of the percentage of the leaf surface which was comprised of symptomatic
tissue. The injury estimates for the 15 leaves on each of the 12 places were
averaged to produce a treatment injury level.
The pollutant interaction was evaluated in each replication. The
procedure employed to evaluate the interaction is based on the following
hypothesis: if there is no pollutant interaction, synergistic or antagonistic,
the injury level produced by the combined pollutant exposure, minus the sum of
the injury levels of the individual pollutant exposures should not be signifi-
cantly different from zero.
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Fig. 1, Reference ch^rt used to estinate the gross area of
the leaf shcvdng synptons produced ty the pollutant
exposures.
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10%
20%
30%
40%
50%
80%
90%
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Fig, 2. Heference chart ussd. to estimate the intensity of
synpton development frora the pollutant expos'jres
on the affected area.
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If this difference is significantly different from zero, the form of the
interaction is indicated by the sign associated with the difference. The
interaction is synergistic if the sign is positive and antagonistic if the
sign is negative. An F-test was used to determine the significance of
the difference.
RESULTS
None of the plants exposed to PAN developed visible symptoms. The
plants exposed to either ozone or the ozone/PAN mixture developed areas of
dark brown to black bifacial necrosis. There was no difference in the
time of symptom initiation with either treatment or in the distribution of
symptoms on the individual leaves. The recently mature leaves on the plant
developed the most severe symptoms in both treatments. Visible symptoms were
not found below the 15 most distal leaves on the plant.
The injury level produced by the ozone/PAN exposure was signifi-
cantly greater than the sum of that produced by the ozone and PAN exposures
in four of the six experiments (Table 1); in two experiments the injury
levels were not significantly different.
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TABLE 1. Visual estimates of the percentage of adaxial injury produced
3
on hybrid poplar by exposure for 4 hours to either 353.2 ug/m
3
(0.18 ppm) ozone, 890.1 ug/m (0.18 ppm) PAN or the two pollutants
combined at these concentrations.
Exposure
Dates
% Injury3
Confidence
level of the
synergistic
interaction
Ozone
PAN
Ozone/PAN
June
20-22
0.8+ 1.1
0.0
3.7+
5.7
95%
July
5- 7
0.0
0.0
0.6+
1.4
N.S.b
July
19-21
0.4+ 0.6
0.0
2. 1+
3.2
90%
Aug.
3- 5
5.4+ 7.3
0.0
5.8+
6.3
N.S.
Aug.
16-18
28.8+ 21.6
0.0
52.6+
18.2
99%
Aug.
29-31
0.0
0.0
2.8+
3.3
99%
aThe mean and standard deviation of the percentage of foliar injury on the
15 most distal leaves on each of the 12 plants in the treatment.
k{Jo significant pollutant interaction at the 90% confidence level.
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DISCUSSION
The introduction and monitoring experiment provided evidence that
the two pollutants were not interacting chemically in the chamber
atmosphere. Thus, it appears that the pollutant interaction was a plant
response to both pollutants. The results of these exposures provide the
first evidence that the photochemical oxidants ozone and PAN can interact
when producing injury to vegetation.
The symptom type produced by the combined pollutants was the same
as that produced by ozone alone. This augmentation of the ozone-type
symptom has been observed by others (1, 2, 3, 5, 8, 9, 10) who have examined
the ability of ozone to interact with sulfur dioxide. The recently
matured foliage of a plant is recognized as being most sensitive to ozone
(4). The symptoms observed in both the ozone and ozone/PAN treatments were
most severe onthis recently matured tissue.
Although all of the cuttings utilized in the exposures were obtained
from only a few trees, there was a high degree of variation in their
responses in each ozone or ozone/PAN exposure. Since the plants were
treated as uniformly, it appears that there were physiological differences
in the clonally reproduced plant materials which affected their susceptibility
to the air pollutants.
These interactions were observed on a clone of hybrid poplar which
is highly resistant to PAN. Additional studies, not reported here, show
that exposures to 1978.0 ug/m3 (0.40 ppm) PAN for 4 hours do not produce
visible symptoms on this hybrid poplar clone. These results illustrate that
a plant does not have to be susceptible to both pollutants for them to
interact and produce injury. This should be a consideration when evaluating
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the importance of an air pollutant in the field. It must be remembered
that in addition to the pollutant of interest, a plant in the field may
also be subjected to other pollutants. While these pollutants may be
considered innocuous on their own, their potential to enter into inter-
actions must be considered.
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Literature Cited
1. DOCHINGER, L. S. and C. S. SELISKAR. 1970. Air pollution and the
chlorotic dwarf disease of eastern white pine. For. Sci.
16:46-55.
2. HODGES, G. H., H. A. MENSER, Jr. and W. B. OGDEN. 1971.
Susceptibility of Wisconsin Havana tobacco cultivars to air
pollutants. Agron. J. 63:107-111.
3. MACDOWALL, F. D. H. and A. F. W. COLE. 1971. Threshold and
synergistic damage to tobacco by ozone and sulfur dioxide.
Atmos. Environ. 5:553-559.
4. MENSER, H. A., H. E. HEGGESTAD and 0. E. STREET. 1963. Response
of plants to air pollutants. II. Effects of ozone concen-
trations and leaf maturity on injury to Nicotiana tabacum.
Phytopathology 53:1304-1308.
5. MENSER, H. A., G. H. HODGES and C. G. McKEE. 1973. Effects of air
pollution on Maryland (Type 32) tobacco. J. Environ. Quality
2:253-258.
6. SALTZMAN, B. E. 1965. Determination of oxidants (including ozone):
Neutral buffered-potasium iodide method, in Selected methods
for the measurement of air pollution, U. S. Dept. Health,
Education and Welfare. Public Health Service Pub. No. 99-AP-ll.
Cincinnati, Ohio, 60 p.
7. STEPHENS, E. R., F. R. BURLESON and E. A. CARDIFF. 1965. The
production of pure peroxyacyl nitrates. J. Air Pollut. Control
Assoc. 15:87-89.
8. TINGEY, D. T., W. W. HECK and R. A. REINERT. 1971. Effect of low
concentrations of ozone and sulfur dioxide on foilage, growth
and yield of radish. J. Amer. Soc. Hort. Sci. 96:369-371.
9. TINGEY, D. T., R. A. REINERT, J. A. DUNNING and W. W. HECK. 1973.
Foliar injury responses of eleven plant species to ozone/sulfur
dioxide mixtures. Atmos. Environ. 7:201-208.
10. TINGEY, D. T., R. A. REINERT, C. WICKLIFF and W. W. HECK. 1973.
Chronic ozone or sulfur dioxide exposures, or both, affect the
early vegetative growth of soybean. CAn. J. Plant Sci. 53:875-879.
11. WOOD, F. A., D. B. DRUMMOND, R. G. WILH0UR and D. D. DAVIS. 1974.
An exposure chamber for studying the effects of air pollution on
plants. Pa. Agr. Expt. Sta. Prog. Rept. No. 335. 7 p.
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APPENDIX IX
"The Interaction of Ozone and PAN
on Pinto Bean"
(Manuscript Form)
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Ilia Interaction of Osone and PAH on Pinto Bean.
R. J. Kohut and D. D. Davla
Former graduate student and Assistant Professor,
ABSTRACT
Pinto Bean plants, when 10 days old from germination, were ex-
posed for 4 hour to either 588.6 mg/m3 (0.30 ppn) ozone, 247.3 mg/m3
(0.05 ppo) PAN, or the tvo pollutant* combined at these concentrations.
The plants were grown and exposed under controlled environmental
conditions. Exposure to 03 produced adaxlal fleck while PAN produced
primarily abaxlal bronslng. Exposure to the combined pollutants In-
dicated that and PAN Interacted synergistically In the production
of adaxlal injury. The combined pollutants Interacted antagonistically
in the production of abaxlal injury with nearly a complete suppresslot
of symptoms. The overall foliar response Indicated an avlagonistlc
pollutant interaction when the percentage of the leaf surface injured
was utilised as the criteria for evaluation.
Phytopathology.
Osone and peroxyacetylnltrate (PAN) have been shown to Interact
synerglstlcally when producing visible injury on a clone of hybrid
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R. J. Kohttt*& D. D. Dei
Phytopathology - Page 2
poplar (5). The Interaction occurrad even though the hybrid poplar
wma reslatant to PAN. This study was initiated to tha Inter-
action of ozone and PAN on a plant species susceptible to both
pollutants.
Pinto bean (Phaaeolus vulgaris cv. Pinto) la known to be sensltiv
to both ozone (2,3) and PAN (3). The symptom response of pinto bean
to either pollutant is quite distinctive. Osone produces adaxial
fleck while PAN produces primarily abaxlal bronzing. The spatial
separation and visual distinctiveness of the synptoms would be useful
in evaluating tha pollutant Interaction.
MATERIALS AND MgTBQD3. — Pinto bean seeds were planted in
perilte on three consecutive days to provide plant material of the
same chronological age for three sequential exposures. One day after
emergence, the plants were transplanted into individual pots con-
taining steam treated 1:1:1 (v/v) peattperlite:soli. After trans-
planting. all plants were maintained in growth chambers at 23C, 75Z
RH with a 14 hour photoperiod at 25 Klux light intensity. Preliminary
exposures had shown that the first trifoliate leaf on plants 10 days
old from emergence was sensitive to both 0? and PAN. Therefore, the
plants were exposed at this age.
The pollutant production and monitoring procedures were as
previously described (5). The environmental conditions during ex-
posure were the same as those at which the plants had been grown.
All exposures were 4 hours In duration and commenced at approximately
10 after the plants had received at least 4 hours of light.
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ft. J. Kotut & D. 0. Dv
Phytopathology. VaM 3
The plants win exposed to either S58.6 ug/m (0.30 ppa) osoae,
247.3 ug/a3 (0.05 ppa) PAN* or the two combined at these concentration
TVenty plants were utilised In each exposure. The eerles of three ex-
posures was conducted on consecutive days and replicated five tines.
Twenty plants were selected on the basis of uniformity of height
and foliar development for exposure In that day's cohort. Control
plants were maintained in another chaaber at the seas environmental
conditions during the exposure. After the exposure, all plants were
returned to their original growth chambers during symptom development.
The first trifoliate leaf on each plant was evaluated for injury
three days after exposure. The adaxial and abaxial surfaces of each
leaflet were evaluated separately. The synpton types and the per-
centage of the leaflet surface affected by each type ware recorded.
The procedure used to determine the percentage of the leaf surface
comprised of symptomatic tissue was the two factor system previously
described (5). The adaxial and abaxial evaluations of the leaflets
were used to calculate a plant injury level and a treatment injury
level for each surface. A treatment injury level was also calculated
for the combined leaf surfaces. This was done by averaging the
adaxial and abaxial injury levels for the leaflets and then using
these values to calculate plant and treatment injury levels.
Using these procedures, the injury produced In each exposure
was evaluated in terms of the adaxial, abaxial, and combined surface
responses.
The pollutant interaction In each repllcetion was evaluated for
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R* J. Robot & D. D. Da-r
Phytopathology . pag> 4
the adaxlal, abaxlal, and combined loaf surface responses. The
atatiatlcal procedure employed to evaluate the inter at Hon was pre-
viously described (5). The significance of the Interaction was
determined with aa F-test attthe 95Z confidence level.
B&SULT8. — The symptom type produced by the Individual pollutant
syssasB
exposures van thoaa connouty aaaoolated with each pollutant. The
oaotta exposures produced a light tea fleck on the adaxlal surface
while PAH produced prlaarlly abaxlal bronslns vlth sotae light tan
bifacial aeeroala. The symptoms produced by the combined pollutant
expoaores sere alnoat exclusively reatrlcted to the adolal leaf sur-
face with a nearly couplets suppression of symptoms on the abaxlal
surface. The ******1 syqptoa produced by the combined pollutants
vaa a fleck similar in appearance to that produced by ozone but
slightly more yellow. In the one replication where abaxlal symptoms
were produced by the combined pollutants, the symptom was a bronzing
alallar to that produced by PAN.
In each replication, the interaction was evaluated for the
adaxlal, abaxlal, and combined leaf aurfacea (Table 1). In the
evaluations of the adaxlal response, three of five replications In-
dicated a synergistic interaction in the combined pollutant exposures.
The response vaa additive In the other two repllcationa.
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R» J. Kohut i D. D. Dv
Phytopathology. paff, s
Table 1. Visual evaluation* of the percentages of foliar injury and
the pollutant ^.interactions on pinto bean produced by 4-hour
3
exposures to either 558*6 ug/a (0.30 ppa) osone, 247.3
3
ug/a
(0.05 ppm) PAN or the two pollutants coabined at
these concentrations.
Leaf
Z Injury®
Sep.
Surface
Osone
PAN oO*onfe/PAN Interact"
1
Adaxial
Abaxial
Coabined
2
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R. J. Kohut & D. D. Da*
Phytopathology - Page 6
The abaxial leaf surface responses Indicated an antagonistic inter-
action In all five replications. When the combined leaf surface
response was evaluated, all of the replications Indicated an antagon-
istic Interaction.
DI8CPS8I0H. — Since the responses of pinto bean to osone and
PAH were easily differentiated visually and separated spatially, the
plant provided a unique opportunity to evaluate the pollutant inter-
action. The combined pollutant exposures resulted in an Increase in
the osone type symptoms on the adaxlal leaf surface. The predominance
and accentuation of the adaxlal osone response has been observed in
Interaction studies Involving osone and sulfur dioxide (1, 4, 6, 7, 8,
9, 10) and in an interaction study with osone and PAH on hybrid poplar
(5). While the interaction of osone and PAH on the leaf surfa:
response was not always synergistic in this study, the antagonistic
Interaction of the pollutants In the abaxlal response was consistent
and quite dramatic. In four of five replications the combined
pollutant exposures did not produce abaxlal Injury while in the re-
maining replication the incidence of bronzing averaged only 4X.
Bronzing produced by the exposures to PAN ranged from 23 to 66Z.
Although the concentrations of pollutants employed In this
study nay be slightly higher than those found in the urban photo-
chemical complex, their ratio of 1:6 (PAHtosone) approximates that at
which they are found in the atsosphere. These studies indicate that
although PAN may be present at phytotoxlc concentrations in the
atmosphere, it may Interact with 0^ without producing the characterise
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E. J. Kohue A D. D. Da*
Phytopathology - Page 7
abaxlal bronslng associated with PAH Injury to vegetation.
Evaluation of the pollutant Interaction* was based on the
estimated percentage of visible ayaptoos on the adaxlal, abaxlal,
and combined leaf surfaces. When an evaluation was performed for
either the adaxlal or abaxlal surface, the comparison was being made
within a similar symptom type: either fleck ontthe adaxlal or bronslng
on the abaxlal surface. Because the symptoms were similar, they
could be compared quantitatively. However, when the adaxlal and abaxl
Injury levels were used to calculate an Injury level for the combined
leaf surfaces, comparisons within similar symptom types were no
longer possible. Although these comparisons can be made strictly
on the basis of the percentage of the leaf Burfaca affected, the
impact of equal percentages of flecking and bronslng on the plant
at the cellular level may be different. The final evaluation of
the ability of 0^ and PAN to interact will require an Injury
evaluation system which Is Independent of symptom description.
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ing so the average length of line corresponds with the right hand marginal line
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Phytopathology ¦ Pago 8
LITERATURE CITED
1* DOCHINGER, L. S. and C. S. SEL1SKAH> 1970. Air pollution «««<
the ehlorotlc dwarf disease of eastern white pine. For.
Scl. 16146-55.
2. DUGGER, V. M. Jr., 0. C. TAYLOR, E. CARDIFF and C. &. THCMBSON.
1962. Stomatal action In plants as related to damage from
photochemical oxidants. Plant Physiol. 37:487-491.
3. DUGGER, W. M. Jr., 0. C. TAYLOR, C. &. THOMPSON and E. CARDIFF.
1963. The effect of light on predisposing plants to osone
and PAN damage. J. Air Pollut. Control Assoc. 13:423-428.
4. HODGES, G. H,, H. A. MEHSE&, Jr. and U. B. OGDEN. 1971.
Susceptibility of Wisconsin Havana tobacco cultlvars to
air pollutants. Agron. J. 63:107-111.
5. KOHUT, R. J. and D. D. DAVIS. 1976. The interaction of ozone
and PAH on hybrid poplar. Pland Dis. Reptr. (In Press).
6. MACDOWALL, F. D. H. and A. F. W. COLE. 1971. Threshold and
synergistic damage to tobacco by ozone and sulfur dioxide.
Atnos. Environ. 5:553-559.
7. MENSER, H. A., G. H. HODGES and C. G. McKEE. 1973. Effects of
air pollution on Maryland (Type 32) tobacco. J. Environ.
Quality 2:253-258.
8. TINGEY, D. T., W. W. HECK and R. A. REIHERT. 1971. Effect of
low concentrations of ozone and sulfur dioxide on foliage,
growth and yield of radish. J. Amer. Soc. Hort. Scl.
96:369-371.
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ing so the average length of line corresponds with the right hand marginal line
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&. J, Kohut & D. D. Dav:
Phytopathology - Page 9
9. TINGE!, D. T., R. A. RE INERT, J. A. DUNNING and W. W. HECK. 1973.
Foliar iivjury responses of eleven plant species to ozone/
sulfur dioxide mixtures. Atoos. Environ. 7:201-208.
10. TINGEY, D. T., R. A. REINERT, C. WICKLIFF and W. W. HECK. 1973.
Chronic osone or sulfur dioxide exposures, or both, affect
the early vegetative growth of soybean. Can. J. Plant Scl.
53:875-879.
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ing so the average length of line corresponds with the right hand marginal line
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APPENDIX X
"The Development and Application of a
Tissue Maceration Process to Evaluate
Air Pollution Injury to Plants"
(Ph.D. Dissertation Format)
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CHAPTER III
THE DSVEICPSNT A\'D APPLICATION OF A
TISSUE MACERATION PROCESS TO EVALUATE
AIR POLLUTION INJURY TO PLANTS
INTRODUCTION
Air pollution researchers have developed various foliar injury
evaluation systexs to serve their specific research objectives.
These systems are often highly subjective, and the data generated
through their use do rot lend ther^elves to quantitative analyses.
The evaluation of foliar injury is also conplicated by the problem
of comparing different synpton types. Visual estimates of foliar
injury may be adequate when riakins conparisons within a single
synpton type. When the foliar effects of several pollutants are
to be compared and their synpton: tyres differ, it is difficult to
make these comparisons on the basis of visual evaluations.
To overcome these problems, a quantitative method of injury
assessment is required which will be independent of symptom type.
It was decided to evaluate the pollutant impact on the basis of
the percentage of the leaf rescphyll cells killed since the foliar
symptoms observed are primarily a result of cell death in the
palisade and spor.jy r.esorhyll.
The evaluations of tr.e overall response of -sinto bean to
simultaneous exposure tc C^ and PAN in the previous chapter
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required comparing different symptom types. Evaluating plant injury
on the basis of the.percentage of mesophyll cells killed by each
treatment would allow quantitative comparison of all symptom types.
By using the visual and cellular processes to evaluate the pollutant
interactions, it would be possible to determine whether the cross-
symptom comparisons made were valid. It would also allow compari-
sons to be made between the visual estimates of the percentage of
foliar injury and the percentage of mesophyll cells killed.
MATERIALS AND METHODS
Verification of the maceration rrocess. Cells can be separated
from foliar tissue with a maceration solution comprised of
ethylenediar.inetetraacetic acid (SDTA) and pectinase (h, 19). To
insure that the tissue maceration process would have quantitative
applications, it was necessary to establish that the percentages
of injured r.esophyll cells determined through its use were equiva-
lent to the percentages in the injured tissue prior to maceration.
Thus, uniformly injured tissue was evaluated by maceration and by
fixing and sectioning.
Pinto beans were grown under controlled environmental cor.di-
3 3
tions and exposed to either 558.6 pg/n (30 prhn) C^, 2^7.3 ug/n
(5 ppha) FAN, or both pollutants cc-.bined at these concentrations
as described in Chapter II, Plants were exposed to each treatment
for either 2, or 6 hours to provide a range of symptom
intensities.
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Three days after exposure, several plants in each treatment
whose tip leaflet of the first trifoliate displ.-.ved a uniform
level of injury were selected for evaluation. I-'ach leaflet was cut
in half along the midrib; one half was macerated while samples of
tissue from the other half were fixed and sectioned for comparison.
Four levels of injury produced by both the 0^ amd PAN exposures and
five levels of injury from the 0^/PAM exposure vere evaluated.
Tissue from unexposed plants was also evaluated using both
procedures.
The differences between the maceration and sectioning evalua-
tions of the percentage of injury in the palisade and spongy
raesophyll were examined for significance with Student's t-tests at
the 95^ confidence level.
laceration. The maceration solution was a tricine buffer
(0,5 M sorbitol in 0.02 M tricine buffer, pH 6.0) to which the
macerating raterials (3^ (v/v) fungal pectinase, 0.05 M EDTA,
1 mM KgClg) were added. The foliar tissue sarrie was first cut with
a razor blade into pieces approximately 0.5 erf. Minor veins were
included in the sample, but the n>idrib was avoided. The tissue
was placed into 25 ml of naceration solution in a 50 ml test tube
and infiltrated in a vacuun chamber. The infiltrated tissue and
maceration solution were transferred to 125 *-1 flasks and agitated
for U hours at room temperature. After this p-.-riod, the cell sus-
pension was strained through cheesecloth and thf cheesecloth washed
with tricine buffer to recover as '- = ry cells ar ccssible. The
filtered cell suspension was centrifuced at 3000 ?F>< for 5 ninutes,
-------
the supematent was discarded, and the cells resuspended in buffer.
The cells were maintained at 4C until sampled and counted.
The percentage of the palisade and spongy mesophyll cells
killed by the pollutants was determined. A sample was drawn from
the cell suspension and mixed with a 0.01% (v/v) solution of
neutral red stain in "buffer at a 1»5 ratio of stain to cell
suspension. A drop of the stained cells was placed on a microscope
slide and covered with a 22 una coverslip,
A systematic pattern of scanning the coverslip under the light
microscope was used when counting. The total and injured cell
counts were maintained on manual counters. Each saraDle was scanned
twice, once to count the palisade and once to count the spongy
mesophyll. At least 200 cells of each type were counted in each
sample. The percentages of injured palisade and spongy mesophyll
cells were determined for five sanples taken from the cell suspension
produced from each leaflet.
Sectioning. Sanples of tissue were selected frcm each leaflet
being evaluated. They were fixed (32) ar.d embedded in Spurr's
low-viscosity epoxy resin (34) prior to sectioning.
To determine the percentages of palisade and spongy nesophyll
cells injured in the tissue, 2.5 y sections were cut on a Porter
Slum Ultraraicrotor* yodel XT-2. Every fifteenxh section was
selected as a sample and placed on a nicrcscota slide. Twenty such
sample sections were collected fron each piece of tissue. The
sections were heat rounted ard stair.ed with O.QM (v/v) trypan blue.
The percentage of the palisade and spor.ry resophyll cells injured
-------
in each section was determined by counts performed with the aid of a
light microscope. The average injury level in each piece of tissue
for both the palisade and spongy nesophyll was determined from the
20 sections examined. Five tissue sanples from each injury level
were examined.
The sections were also used to identify the specific foliar
tissues affected and to develop general descriptions of the cellular
patterns of Injury from the 0y FAN, and O^/PAN exposures.
Evaluations of the -pollutant interaction. Pinto bean plants
were grown, exposed, and maintained until evaluation as described
in Chapter II. Ten plants were used in each treatment, and the
series of treatments was replicated three tines.
The injury levels on the adaxial, abaxial, and combined
surfaces of the terminal leaflet of the first trifoliate leaf of
each exposed plant were evaluated visually using the two factor
system presented in Chapter I. The statistical procedure also
described in Chapter I was used to evaluate the pollutant inter-
action on these surfaces in each replication.
After the visual evaluation, each leaflet was icacerated and
the percentage of injured cells in the palisade and spongy ir.esorhyll
was determined. These injury levels were averaged to calculate a
response for the overall nesophyll tissue. The pollutant inter-
actions were evaluated in the palis2.de, sror.ry, and overall mesophyll
using the statistical procedure described in Chapter I.
The visual evaluations of the pollutant interactions could then
be compared with the cellular evaluations of the interactions for
each replication.
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39'-
Comparison of the vls'jal ar.d cellular estimates of injury.
Visual estimates of the -percentage of adaxial and abaxlal injury ard
cellular estimates of injury in the palisade and spongy nesophyll had
teen made for each leaflet used in the interaction study. To compare
the visual estimates of foliar injury with the cellular estimates
of injury, the values frcn the 30 leaflets in the three replications
of each treatment were considered as a group. Each pair of values
was placed intc an injury category on the "basis of the severity of
the visual estimate of injury and the leaflet surface affected. The
categories were forced by injury increxents for each leaflet surface
of 1.0% for 0^, 25% for PAN, and 203 for 0^/PAN. The increments were
selected to balance the nunber of observations in each category,
A Student's t-test was performed to determine whether the visual
and corresponding cellular injury estimates in each category were
significantly different. All tests were conducted at the 95#
confidence level.
RESULTS
Verification of the "-^ceratlor. rrccess. The unexposed tissue
which had been evaluated by both processes showed r.o plassolyzed
cells. The tissue maceration indicated that those cells which had
been killed by the pollutants were severely pias-olyzed with tneir
protoplasts appearing contracted ard condensed. The neutral red
stain was taken up ard concentrated in the space between the cell
wall and the protoplast ar.d aided in identifying the plasir.olyzed
cells.
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40
Examination of the sectioned tissue provided an opportunity to
describe the patterns of injury which developed in the leaflets.
Ozone injury was restricted primarily to the palisade nesophyll.
However, injury extended into the spongy nesophyll when the palisade
injury was severe (Fig. 4). Tissue which had been exposed to FAN
exhibited abaxial enidemal collapse (Fig. 5). Injury in the spongy
mesophyll appeared adjacent to the epiderrais and was always asso-
ciated with an area of epidermal collapse. Cellular injury which
resulted from exposure to the combined pollutants occurred primarily
in the palisade mesophyll in a manner similar to 0^ injury. However,
the spongy mesophyll cells adjacent to the palisade were frequently
injured even though the palisade injury was not severe (Fig. 6).
The comparison of the percentages of injured cells as determined
by the tissue iraceration ar.d sectioning processes showed no signifi-
cant differences In either the palisade or spongy nesophyll for
three of the four 0^ injury levels examined (Table 4). The percent-
ages of both the palisade and spongy mesophyll cells injured were
significantly different only at the highest injury level where the
maceration estinate was significantly less than the section estinates.
The comparisons of the injury levels from the 0~/?AN exposures
also indicated that the estinates fros the two processes were in
agreement (Table 5), In two instances the estinates were signifi-
cantly different. In one case the ulceration estimate of palisade
injury was significantly greater than that determined by sectioning,
and in the ether instance the raceration estimate was significantly
less than the sectioning estimate of injury in the spongy nesophyll.
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4. Section of pinto "bean foliar tissue showing injury
in the spongy nesophyll adjacent to severe injury in the
palisade mesophyll.
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Fig. 5. Section of pinto bean foliar tissue showing collapse
of abaxial epidemis ar.d plasmolysis of adjacent spongy
rcesophyll cells as a result of exposure to FAN.
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<*3
Fig, 6. Section of pi-to bean foliar tissue after exposure to
O^/PAN showi-_- plasraolysis of spongy mesophyll cells
adjacent to palisade nesophvll without severs
palisade injury.
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44
TABUS 4. Comparison of the percentage of mesophyll injury
produced on pinto bean by 0^ as evaluated by
sectioning (Sec) and macerating (Mac) injured tissue.
Injury
level
Palisade
Spongy
*
Diff.b
%
Diff.
Light
Sec
6*5
NS
1*1
NS
Mac
4*2
1*1
Moderately
Sec
16*5
NS
2*1
NS
light
Mac
23*5
2*1
Moderate
Sec
2115
NS
3*2
NS
Mac
35^7
3*12
Severe
Sec
94*4
SIG
97*2
SIG
Mac
77*6
48*3
aThe mean and standard deviation of the percentage of plas^solyzed
cells observed.
^Significant differences determined at the 95^ confidence level
for the section and maceration evaluations of injury within
either the palisade or sparer/ nesophyll. 13 indicates that the
evaluations are not significantly different. SIG indicates that
the estinates are significantly different.
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*5
TABIE 5. Comparison of the percentage of mesophyll injury
produced on pinto bean by 0^/PAN as evaluated by
sectioning (Sec) and macerating (Mac) injured tissue.
Injury
level
Ifelisade
Spongy
<
Diff.b
%
Diff.
Light
Sec
4* k
NS
1* 1
NS
Mac
5*1
1*1
Moderately
Sec
17- 7
NS
12* 5
NS
light
Mac
17*3
3* 2
Moderate
Sec
28* 8
NS
22*18
NS
Mac
43*6
31* 4
Moderately
Sec
25*15
SIG
40* 7
NS
severe
Mac
60* 6
31* 8
Severe
Sec
99- 1
NS
99* 1
SIG
Mac
95*3
861 3
The mean and standard deviation of the percentage of plasnolyzed
cells observed.
^Significant differences detemir.ed at the 95^ confidence level
for the section and r.aceration evaluatiors cf injury within eithsr
the palisade or spor.ey nesoTivll. indicates that the evaluations
are not significantly different, SIG indicates tr.at the evaluations
are significantly different.
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46
These differences occurred in different injury levels which were
examined.
Significant differences were most common in the comparisons
performed on the PAN injured tissue (Table 6). Five of the eight
comparisons indicated that the injury levels determined "by the two
processes were significantly different. The maceration estimates
were significantly higher than the section estimates in two
comparisons and significantly lower in three.
Evaluation of the -pollutant Interaction. The quantitative
values for the visual and cellular estimates of injury and the
results of the evaluations of the pollutant interactions in the
three replications are presented in Tables 7-9. A summary of
the interaction evaluations with the two processes is presented
in Table 10.
The evaluations of the pollutant interactions "based on the
visual foliar symptom estimates indicated a consistent antagonistic
interaction in the production of abaxial surface symptoms. Two
of three evaluations of the pollutant interactions based on
cellular evaluation of injury in the spongy mescphyll also indi-
cated an antagonistic interaction while the third evaluation
indicated an additive response.
In two of the three replications, there was a co-rplete suppres-
sion of visible abaxial symptoms by tne combined pollutants. Visible
abaxial symptoms w*?re not produced in any of the 0^ exposures.
Evaluation of these tissues by the maceration -rocess indicated that
up to 12f5 of the spongy mesophyll had been plasrrolysed (Table 8).
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4?
TABLE 6. Conparison of the percentage of mesophyll injury
produced on pinto bean by PAN as evaluated by
sectioning (Sec) and macerating (Mac) injured tissue.
Injury
level " b
light
I&lisade Spongy
Dlff. % Diff,
light Sec 1*1 SIG 14*11 NS
Mac 29*6 10+ 3
Moderately Sec 9-7 SIG 41*11 NS
Mac 30*5 34* 8
Moderate Sec 99*1 SIG 99* 1 SIG
Mac 25*3 40* 6
Severe Sec 99*1 NS 99* 1 SIG
Mac 93*5 87* 2
The nean ar.d standard deviation of the percentage of plasmolyzed
cells observed,
^Significant differences determined at the 95^ confidence level
for the section ar.d r.aceration evaluations of injury within either
the palisade or snoivrv -esorhyll. N3 indicates tr.at the evaluations
are not significantly different. SIG indicates that the evaluations
are significantly different.
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48
TABLE 7. Injury levels and pollutant interactions on pinto bean
in replication 1 as a result of a 4-hour exposure to
either 558.6 yg/s? (30 pphn) 0y 2^7.3 pg/n^ (5 ppha) PAN
or the two pollutants combined at these concentrations
as evaluated with the visual and cellular systems.
Visual Evaluation4
°3
PAN
O^/PAN
Interact
Adaxial
28*10
y* 2
43^L7
NS
Abaxial
0
42*29
0
ANT
Combined
14*" 5
22*16
20* 8
ANT
g
Cellular Evaluation
°3
PAN
0^/PAN
Interact
Palisade
31*11
1* 1
3^12
NS
Spongy
2- 2
17*1^
5* 5
ANT
Overall
16* 6
9* 8
19* 8
NS
£
The mean and standard deviation of the percentage of each surface
injured using the visual evaluation system,
^The pollutant interaction is evaluated for each surface. NS
indicates no significant rolluta-it interaction, A synergistic
interaction is indicated by SYN and an antagonistic interaction
by ANT.
C,Hie mean and standard deviation ef the percentage of the nesophyll
tissues plasmolyzed using the cellular evaluation system.
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TABLE 8, Injury levels and pollutant Interactions on pinto bean
In replication 2 as a result of a 4-hour exposure to
either 558.6 fig/(30 pphsi) 0^# 247,3 pg/m^ (5 pphm) PAN
or the two pollutants combined at these concentrations as
evaluated with the visual and cellular systems.
Visual
£
Evaluation
°3
PAN
Oj/PAN
Interact**
Adaxial
23*13
6* 4
48* 9
SYM
Abaxial
0
57*34
0
ANT
Combined
12* 6
31*19
24* 5
ANT
Cellular
Evaluation0
°3
PAN
o^/fah
Interact
f&lisade
29*13
39*27
^7*15
KS
Spongy
5* 5
55*31
12* 6
ANT
Overall
16* 9
47*28
29* 9
ANT
aThe mean and standard deviation of the percentage of each surface
injured using the visual evaluation system.
^The pollutant interaction is evaluated for each surface. NS indicates
no significant Tiollutant interaction. A synergistic interaction is
indicated by SYN' and an antagonistic interaction by ANT,
CThe aean and standard deviation of the percentage of the cesophyll
tissues plasrolyzed using tne cellular evaluation system.
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30
TABLE 9. Injury levels and pollutant interactions on pinto bean in
replication 3 as a result of a 4-hour exposure to either
558.6 yg/rP (30 pphm) 0^, 2^7.3 yg/vP (5 pphm) PAN or
the two pollutants combined at these concentrations as
evaluated with the visual and cellular systeos.
Visual Evaluation3,
°3
PAN
(Xj/PAN
Interact
Adaxial
35*10
5*11
56*22
NS
Abaxial
0
15*15
1* 3
ANT
Combined
18* 5
10*12
29*12
NS
Cellular Evaluation6
°3
FAN
O^/PAK
Interact
Palisade
53*13
10*25
61*14
NS
Spongy
9* 5
11*18
10* 9
NS
Overall
31* 9
10*21
35* 9
NS
aThe mean and standard deviation of the percentage of each surface
injured using the visual evaluation system.
^The pollutant interaction is evaluated for each surface. NS
indicates no significant pollutant interaction. A synergistic
interaction is indicated by SYN and an antagonistic interaction
by ANT.
cHie Bean and standard deviation of the percentage of the sesophyll
tissues plasmolyzed using the cellular evaluation systea.
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•51
TAB1E 10. Summary of the visual and cellular evaluations of
the interaction of 0^ ar.d PAN on pinto beam
Interaction Evaluations3,
Rep. Surface Visual Cellular Tissue
1
Adaxial
NS
NS
felisade
Abaxial
ANT
ANT
Spongy
Coabined
ANT
NS
Overall
2
Adaxial
SYN
NS
felisade
Abaxial
ANT
ANT
Spongy
Combined
ANT
ANT
Overall
3
Adaxial
NS
NS
Palisade
Abaxial
ANT
NS
Spongy
Combined
NS
NS
Overall
aThe pollutant interactions vere evaluated for each s-jrface and
tissue in each replication. N3 indicates no significant
pollutant Interaction. A synergistic interaction is indicated
by SYN and an antagonistic interaction by ANT.
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52
Evaluations of the interactions by the visual estimates of
adaxial injury indicated an additive response to the combined
pollutants in two of the three replications and a synergistic
interaction in the third. The cellular evaluations of the injury
in the palisade nesophyll indicated an additive response In all
replications.
The interaction on the combined leaf surfaces was antagonistic
in two replications and additive in the third when visually
evaluated. The cellular evaluations of the overall mesophyll
response indicated an additive effect in two replications and an
antagonistic interaction in the third.
Comparison of the visual and cellular estlrates of Injury.
The estimates of the percentage of foliar injury as determined
through the use of the visual and cellular evaluation systems
were generally not significantly different, The visual and
cellular estinates of injury were significantly different in only
one of the four levels of adaxial 0^ injury (Table 11). This
difference occurred in the 31-^0^ injury increment where the
cellular estimate was significantly greater than the visual estimate.
Three levels of abaxial injury from the FAN exposures were
examined with the visual and cellular evaluation systems (Table 12).
The injury estimates were not significantly different in any of
the levels evaluated. The cellular estimate of injury was signifi-
cantly greater than the visual estir^ate for the single level of
adaxial Injury produced by FAN.
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53
TABUS 11. Comparisons of the visual and cellular estimates of
injury on pinto bean produced by a 4-hour exposure to
558.6 ug/a^ (30 ppha) Oy
% Injury*
Surface
Level^
Visual
Cellular
Diff.C
Adaxial
( 0-20)
13-6
22*13
NS
(21-30)
25*3
29± 9
NS
(31-^0)
36*3
45*11
SIG
(41-50)
45*1
52*14
NS
Ataxia!
(0)
0
5* 5
d
aThe leean and standard deviation of the percentage cf surface
injury determined with the visual evaluation system and the
percentage of neso-.hyll injury determined with the cellular
evaluation system.
^Category increcents tased on the visual estijsates of injury,
CSignificant differences were determined for the visual and cellular
estimates of injury. NS indicates that the estimates are not
significantly different. SIG indicates that the estimates are
significantly different.
dThe difference cannot "be statistically evaluated since one value
is zero.
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TABLE 12. Comparisons of the visual and cellular estimates of
injury on pinto bean produced by a hour exposure
to 247.3 ps/m3 (5 PFtai) PAN.
£ Injury3,
Surface Level* Visual Cellular
Adaxial ( 0-13) ^ & 17-26
Abaxial
( 0-25)
& 9
6± 8
NS
(26-50)
35±11
22±25
NS
(5>75)
66± 8
45*23
NS
aThe mean and standard deviation of the percentage of surface
injury determined with the visual evaluation systea and the
percentage of nesop'nyll injury determined with the cellular
evaluation system.
^Category increments based on the visual estimates of injury.
cSignificant differences were detemined for the visual ar.d
cellular estiratss of injury, "S indicates that the estir.ates
are not significantly different. SIG indicates that the
estimates are significantly different.
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55
There were no significant differences between the visual and
cellular estimates of injury for the three levels of adaxial injury
produced by the conbined pollutants (Table 13). However, the
cellular estimate of injury was significantly greater than the
visual estimate for the single level of abaxial injury rroduced,
DISCUSSION
The percentages of the foliar mesophyll cells plasnolyzed "by
exposure to oxidant air pollutants as determined by macerating or
sectioning injured tissue were generally similar. The significant
differences in the incidences of plasmolysis in the PAN exposed
tissue did not indicate a fault in the maceration process, but
rather were functions of the sympto-n type being examined and the
process of selecting tissue sar-.ples for fixation.
Most differences were found in the evaluations of tissue
exposed to PAN, The PAN exposure produced adaxial necrosis and
abaxial bronzing while the ana O^/PAN' exposures produced adaxial
fleck. The necrosis and bronzing occurred in relatively large,
isolated, discrete lesions while the fleck was more randomly
distributed across the leaf surface. The random distribution
of the fleck symptom dictated that a sar.ple of tissue being fixed
for embedding should have contained plas-olyzed mesopiyll cells
in a proportion representative of tnat throughout the leaf. However,
when samples of PAN injured tissue were selected for fixation,
they came from either inside or outside the affected area.
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56
TABLE 13• Comparisons of the visual and cellular estimates of
injury on pinto bean produced by simultaneous exposure
to 558.6 yg/r? (30 pphm) Oj and 24?.3 (5 pphm) PAN.
% Injury1
Surface
Level*5
Visual
Cellular
Diff.c
Adaxial
(0-40)
30*10
35*15
NS
(41-60)
49* 6
NS
(61-80)
6?i 8
63*11
NS
Abaxial
(0- 8)
1* 2
8* 6
SIG
The oean and standard deviation of the percentage of surface
injury detemir.ed with the visual evaluation syster. and the
percentage of resophyll injury deternined with the cellular
evaluation system,
^Category increments based on the visual estimates of Injury.
c
Significant differences were determined for the visual and
cellular estimates of injury, >:S indicates that the estimates
are not significantly different. SIC indicates that the estimates
are significantly different.
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57
The percentages of plasmolyzed cells subsequently calculated from
these samples were not representative of the incidence of injury
throughout the leaf because a randomizing process was not employed
to insure that injured and u.iinjured tissue samples would be
selected in the same ratio at which they occurred in the leaflet.
The tissue maceration, by its nature, is a randomizing process.
If the maceration itself has no effect on the percentage of
plasmolyzed nesophyll cells, the determinations of cellular injury
from the cell suspension should represent the average percentages
of mesophyll injury for the leaf* Thus, the significant differences
observed between the estimates of mesophyll plasr.olysis produced
by PAN were a result of comparing the maceration estimates of injury
with the section estimates which were not representative of the
actual level of foliar injury due to the nature of the syr.ptom
and the nonrandon process of selecting the tissue samples. The
agreement between the maceration and section estimates of nesophyll
plasmolysis produced by 0^ and C^/FAN indicates f,hat the rs.ceration
process can be used to evaluate air pollution injury on a cellular
basis in the palisade and spor.gv nesophyll.
The maceration process did not allow the epideiral tissue
to be isolated and recovered. Subsequently, no comparisons of
epidemal injury were made.
The evaluations of the pollutant interactions by the visual
and cellular systens were in close agreement. The differences
observed in the evaluations were between deterrdnations of an
additive or an antagonistic response or between an additive or
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synergistic response (Table 10). These differences were partly a
result of the fact that in instances where there was no visible
abaxial injury, up to 12% of the spongy mesophyll cells were found
to be plasmolyzed. These differences may also have resulted from
the snail number of plants which were used in each treatment.
Examination of the interaction date (Tables 7-9) revealed
that in those exposures where abaxial injury was slight or not
visible but where plasmolysis was observed in the spongy parenchyma,
the percentage of plasmolyzed cells in the spongy parenchyma was
directly related to the percentage of plasmolyzed cells in the
palisade mesophyll. On the basis of the descriptions of the
mesophyll tissue response to 0y PAN, and 0^/FAN previously given
and tne relationship discussed above, it appears that the
plasnolyzed spongy mesophyll cells were adjacent to the palisade
mesophyll and therefore not expressed as visible injury on the
abaxial leaf surface.
The summary of the visual and cellular evaluations of the
pollutant interactions (Table 10) provides additional information
on which to detemine the general responses of pinto bean to the
combined pollutants. The evaluations by both systems of the
response on the abaxial foliar surface indicated an antagonistic
pollutant interaction. This is in agreement with the response
reported in Chapter II.
The cellular injury evaluations indicated that the adaxial
response was additive in all three replications. The visual injury
evaluations indicated an additive response in two replications
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59
and a synergistic interaction in one. When considered with the
results presented in Chapter II, the visual evaluations provide no
clear indications of adaxial foliar response to the combined
pollutants. T.-.e cellular evaluations provide a clear indication
that the respc-.se is additive, however.
The evaluations of the cor.bir.ed surface and overall mesophyll
responses and the results of the previous interaction study all
provide evidence that general 0^ and PAN interaction is antagonistic
when producing injury to pinto bean.
This study has demonstrated that a visual evaluation system,
when carefully employed, can be used to appraise the interaction
of Cj and PAN on pinto bean even when different symptom types
must be compared.
The visual estimates of the percentage of the foliar adaxial
surface exhibiting oxidant symptoms were in rood agreement with
the cellular estizates of the percentage of the palisade nesophyll
cells plasraolyred. Since the fleck observed on the adaxial surface
is a result of injury in the palisade mesoph;. 11 which is a monolayer
of cells in pinto bean, it is predictable that percentage of visible
adaxial surface injury is a direct result of an equivalent amount
of plasmolysis in the palisade mesophyll.
The reason for the close association between the visual and
cellular evaluations of abaxlal surface bronzing on tissue exposed
to PAN is different from that presented above sincej (l) the
visible symptom is primarily a result of e^idomal collapse and
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60
(2) the spongy mesophyll is a multilayer of cells. The association
can "be explained by an observation frc- the previous sectioning
work. As the incidence of abaxial brc-.zing increased, the depth
into the spongy mesophyll in which pls.s no lysis was observed also
increased. Since the bronzing synptcr. is a discrete lesion, that
portion of the spongy mesophyll beneath the center of the lesion
has a greater percentage of plasrnolyzed cells than those portions
near the margin. Thus, there is a direct relationship developed
between the visual estimate of the percentage of the abaxial
surface which is bronzed and the percentage of spongy mesophyll
cells which are plasmolyzed.
The present study has demonstrated that if a system to evaluate
the percentage of visible foliar injury is well designed and
uniformly applied, the injury levels determined for 0^ and PAN
injury on the adaxial and abaxial leaf surfaces will be closely
associated with the percentages of foliar palisade and spongy
mesophyll which have been plasmolyzed.
In summary, oxidant injury on pir.to bean can be evaluated on
a cellular basis by the use of a tissue maceration process. Ihe
injury percentages obtained by the visual and cellular evaluation
systems are essentially the same. The interactions'o'f " 0^ and PAN
on pinto bean were evaluated with both systens. The visual
evaluations of the interactions agreed with those obtained with the
cellular system even when cross-symptom comparisons were involved
in the visual assessments.
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