EFFECTS OF OZONE 0N
Mnium cuspidatum (L. ) “Leyss

Thesis for the Degree of M. S,

MECHiGAN STME UNIVERSITY '

SO-YUNG JANE CHAI
1974

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ABSTRACT

EFFECTS OF OZONE 0N Mnium cuspidatum (L.) Leyss

 

BY

So-Yung Jane Chai

Controlled exposures to ozone at concentrations of 0.75 ppm
or more for 3 hr caused permanent injury to leaves of the moss species,

Mnium cuspidatum (Grout, 1965). Affected areas became distinct with

 

a transparent appearance within 12 hr after exposure. Chloroplasts
in ozone affected cells became shrunken and resembled distinct green
dots in the injured cells. Gradually, the green chlorOplast dots
became smaller and less distinct. Eventually, affected areas became
almost colorless. There was usually a sharp demarcation line between
healthy and injured cells.

Four phases of cytological changes progressively developed
in ozone injured cells of moss leaves. 1) ChlorOplasts became dis-
tinctly shrunken soon after eXposure. They were round or elliptical
in shape, and were more sharply defined than were healthy ones.
2) Damaged chloroplasts continued to shrink to 1/3 - 1/2 the size of
the normal ones. Nuclei disintegrated into a mass of membraneous
fragments. 3) Plasmolysis was accompanied by shrinkage and breaking
of plasmalemma. As affected chlorOplasts further shrunk and/or Split,
they gradually broke down. A) Plasmalemma and cytOplasmic contents,
including chloroplasts, collapsed completely into a diffuse, jelly-
like mass and injured cells ceased to function and did not recover.

Since Mnium cuspidatum does not possess stomata, its ozone

 

sensitivity may be less affected by various environmental conditions.

So-Yung Jane Chai

It responded to ozone promptly and consistently. Hence, Mnium cuspidatum

 

is believed to be potentially a good bioassay material for gross rapid

detection of ozone toxicity.

EFFECTS OF OZONE 0N Mnium cuspidatum (L.) Leyss

 

By

So-Yung Jane Chai

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

197k

ACKNOWLEDGEMENTS

The author wishes to express her sincere gratitude to
Dr. W. J. Hooker for his guidance, suggestions, and encouragement in
this study.

Thanks are also due to the other members of her committee,
Drs. E. J. Klos, A. L. Jones, and A. W. Saettler for their real
interest and assistance.

She would also like to thank Drs. H. A. lmshaug and w. 8. Drew
for their helpful information in the identification of moss plants.

Grateful recognition is extended to Mr. T. C. Yang, Mr. T. K. Wu

and Mr. Paul Wei for their kind assistance and encouragement.

TABLE OF CONTENTS

Page

LIST OF FIGURES . . . . . . . . . . ,., . . . . . . . . . . iv

Chapter

I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1

II. LITERATURE REVIEW A
A. Effects of Ozone on Higher Plants A
8. Effects of Ozone on Fungi 9
III. MATERIALS AND METHODS . . . . . . . . . . . . . . . . 1h
IV. RESULTS . . . . . . . . . . . . . . . . . . . . . . . 19

V. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 30

VI. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 32

BIBLIOGRAPH . . . . . . . . . . . . . . . . . . . . . . . . 3h

Figure

LIST OF FIGURES

Apparatus for fumigating Mnium cuspidatum to ozone. .

 

Estimation of ozone concentration produced by the

ozonator from data supplied by the manufacturer .

Leaf of Mnium cuspidatum, 3 weeks after exposure to
Damaged areas were almost
completely bleached while border remained apparently

 

1 ppm ozone for 3 hr.

unaffected. (Bar equals 100 u.) . . . . . . . . . . .

Normal cells in non-ozonated leaf of M. cuspidatum,
Shows the nucleus with nucleolus.
streaming was evident. (Bar equals 10 u.) . . . . . .

Ozone injury -- phase 1.

ProtOplasmic

 

Leaf cells of M. cu5pidatum

 

Immediately after exposure to 1 ppm ozone for 3 hr.

ChlorOplasts have shrunk noticeably. (Bar equals 10 u.

Ozone injury -- phase 2.

‘fl. cuspidatum leaf to 1 ppm ozone for 3 hr.

 

of the normal size. (Bar equals 30.u.). . .

Ozone injury -- phase 3.

12 hr after exposure of
Chloro-
plasts aggregated in groups and were about 1/3 - 1/2

 

after exposure to 1 ppm ozone for 3 hr.

equals 10 u.) . . . . . . . . .

Ozone injury -- phase 4.

Leaf of M. cuspidatum,

 

2-3 weeks after exposure to 1 ppm ozone for 3 hr.

a) Breaks devel0ped in the limiting membrane of chloro-
b) Cytoplasmic contents
have broken down and collapsed, and cells have degener-

plasts. (Bar equals 10 u.)

ated. (Bar equals 30 u.). . .

Leaf of M, cu5pidatum, 1 day

a) Plasmolysis
with disintegration of plasmalemma. Cyt0plasmic contents
clumped in the center of cells. (Bar equals 30 u.)

b) ChlorOplasts may be splitting into two halves. (Bar

Page

. 27

I. INTRODUCTION

Since 1957. the year ozone was first recognized as a major
phytotoxicant to crap plants, it has proved to be the most persistent,
if not the most difficult, atmospheric phytotoxicant to control
(Richards et al., 1965). Ozone damage has been reported on almost
every type of ornamental and agronomic crOp (Ledbetter, 1959; Hill
et al., 1961). The problem has become increasingly serious in the
U.S., during recent years.

Even though intensive research has been carried out to under-
stand and solve the problems associated with ozone damage, many
questions still remain unanswered. Among these is the role of stomata
in controlling sensitivity of higher plants to ozone. Generally, it
has been accepted that ozone injury to higher plants is regulated
to a considerable degree by the action of stomata. Some investigators
have shown that ozone sensitivity was positively related to Opening
of stomata (Rich, 196A; Lee, 1965; Macdowell, 1965; Engle and Gableman,
1966; Hill and Littlefield, 1969; Fletcher et al., 1972; Adedipe, et
al., 1973). This was accomplished by regulating stomata Openings
with either environmental factors including light, temperature, water
stress, or CO2 (Heath, 1959; Zelitch, 1961; Ketellaper, 1963), or
with chemicals such as phenylmercuric acetate (PMA) (Zelitch et al.,
1962) or abscisic acid (Fletcher et al., 1972). However, other
investigators failed to observe such a relationship with stomata
(Dugger et al., 1962; Dugger et al., 1963; Menser et al., 1963a;

Heck et al., 1965; Ting et al., 1968). These discrepancies may have

been due to the several differences in techniques (porometer, silicone

rubber impressions, cellulose acetate impressions, transpiration,
etc.) adapted by different investigators for studying stomata
(Adedipe et al., 1973). It seemed plausible then that studying
responses to ozone would be greatly simplified using plants which
lack stomata. Since with such plants, the influence of stomatal
activity could not be involved in the final response.

Moss leaves lack stomata and are but one cell in thickness,
except at the midrib and occasionally around the leaf margin (Grout,
1965). Hence, photosynthetically active cells may be readily observed
directly under a microsc0pe. Moss plants have only rhizoids instead
of true roots. Although most moss Species possess central strands
of narrow elongated cells to carry water lengthwise through the stems,
they do not have the true vascular bundles of the higher plants in
their stems. Furthermore, moss plants are fast-growing and easy to
maintain in a small amount of space.

These distinct characteristics make the response of moss to
ozone an interesting subject to study. Such a study may add knowledge
to our understanding of phytotoxicant action since little is known
concerning the response of lower green plants to ozone, environment
relations influencing toxic action, and levels of toxicant required
to injure moss.

In preliminary studies, several Species of mosses were collected
and tested for their sensitivities to ozone. Of the group, fiflium
cuspidatum was chosen for the present study.

External injury symptoms and progressive cellular changes will

be described in the present study. Where possible, responses of moss

to ozone will be compared to those of the higher plants. The possi-
bility of using moss as a bioassay system for rapid gross detection

of ozone toxicity will also be discussed.

II. LITERATURE REVIEW

Numerous reports have discussed ozone plant damage in relation
to sources (Rich, 1964; Treshow, 1970), species susceptibility (Rich,
1964; Hill et al., 1970), symptoms (Hill et al., 1961; Rich, 1964;
Treshow, 1970), growth suppressions (Ormrod, 1971; Adedipe et al.,
1972), histological changes (Ledbetter, 1959; Hill et al., 1961;
Rich, 1964), metabolic effects (Rich, 1964; Treshow, 1970), environ-
mental predisposition (Heck, 1968), and synergisms with other gases
(Menser and Heggestad, 1966; Heck, 1968) etc. (Above are listed the
most important discussions selected from a much larger group of
reports.) However, since the present study concerns mainly ozone
damage observed on moss leaves, only closely related literature is

reviewed.
A. Effects of Ozone on Higher Plants

General symptomatology

The degree of injury to susceptible plants is directly related
to the concentration of ozone to which they are exposed and to the
duration of exposure (Rich, 1964). Although symptoms of ozone injury
vary with plant species, there are certain characteristics which
appear to be typical of the ozone toxicity syndrome. However, not
all of these symptoms are produced consistently on all susceptible
plant species.

The earliest macrosc0pic indication of ozone injury on broad-

leaved, herbaceous species is the appearance of shiny, oily spots

due to intercellular water congestion (Taylor et al., 1960; Hill et
al., 1970). These water soaked spots are usually visible on the upper
leaf surface, and may or may not become permanent lesions. If the
damage is not too severe, injured cells may recover.

When tissue is irreversibly damaged, affected areas become
water-soaked and dull, gray-green in appearance (Hill et al., 1961).
Gradually the water-soaked areas become chlorotic to bleached. Iso-
lated groups of palisade cells between the smallest veinlets are
affected first, forming the characteristic punctate or flecked
pattern. Flecks are usually white or light tan in color, but on
some plants, they are brown to black (Richards et al., 1958). It is
generally agreed among investigators that leaves with adaxial palisade
parenchyma show the flecking symptoms chiefly on the upper surface
of the leaf (Heggestad and Middleton, 1959; Hill et al., 1961; Rich,
1964; Hill et al., 1970). With more severe injury, chlorotic or
discolored areas may: 1) become necrotic and collapse; 2) extend com-
pletely through the leaf; and/or 3) form bifacial necrotic lesions
(Hill et al., 1961; Rich, 1964). Sometimes the entire leaf was killed.

Another symptom of ozone injury is yellowing or premature
senescence of older leaves, accompanied by abscission (Ledbetter, 1959;
MenSer and Street, 1962). The outgrowth of enations on the under
surface of broccoli leaves subjected to ozone is an unusual response
not seen on other plants (Hill et al., 1961).

The pattern of injury on plants with undifferentiated meso-

phyll (i.e. grasses) varies only slightly. Damage usually extends

completely through the leaf (Ledbetter, 1959; Hill et al., 1961).
Symptoms develop randomly on both upper and lower leaf surfaces. The
mild symptom on such parellel-veined monocotyledonous plants as corn,
wheat, etc, is a fine chlorotic stippling between the largest veins.
When injury is more severe, the bleached flecks coalesce into elongated,
chlorotic, interveinal streaks often extending through the leaf blade.
Lesions are characteristically pale tan to bleached in color.

Sensitive conifer species may develop pinkish spots in reSponse
to ozone. The Spots coalesce into bands of dead tissue, and cause a
chlorotic or necrotic banding. Tips of the needles beyond this zone
generally die, producing a needle tip necrosis (Berry and Ripperton,
1963; Costonis and Sinclair, 1969).

Young plants are usually more sensitive to ozone and mature
plants are relatively resistant (Hill et al., 1961; Rich, 1964).
In general, on a single plant, the very youngest leaves are the most
resistant (Ledbetter, 1959; Menser et al., 1963b). As the leaves
begin to eXpand, they become sensitive at their tips. With continuing
expansion, more and more of the leaf area becomes susceptible. The
most sensitive stage is when the leaves are about three-quarters
fully expanded (Ting and Dugger, 1968). Expanded mature leaves again
become resistant, with many of the older leaves sensitive only at

their bases (Ledbetter, 1959; Menser et al., 1963b).

Histopathologjcal effects

 

Usually, palisade tissue comprising the upper leaf meSOphyll
is most sensitive to ozone and the first injured; next are the interior

spongy meSOphyll cells and finally, the lower meSOphyll cells (Hill

et al., 1961; Rich, 1964). When 2-3 palisade layers were present as
in spinach, the outermost was usually, but not invariably, the first
to show injury. 0n leaves without palisade cells, the outer spongy
meSOphyll cells were most readily affected. Epidermal cells over—
laying ozone-injured tissues may or may not show damage, depending
on the type and severity of injury. Vascular tissues are most
tolerant of ozone, becoming damaged only when the associated tissues
are killed (Ledbetter et al., 1959; Hill et al., 1961; Rich, 1964).

Cellular responses to ozone vary with different types of
symptoms. The only apparent changes with chlorotic symptoms were
disrupted chlorOplasts and reduced chlorOphyll content of affected
cells (Hill et al., 1961).

Dark punctate stipples were caused by thickening and pigmen-
tation of the walls in groups of palisade cells before their collapse
(Ledbetter et al., 1959; Richards et al., 1958). The overlaying
epidermal and surrounding mesophyll cells appeared to be unaffected.
There was a sharp demarcation, with no gradation, between affected
and adjacent unaffected cells.

Light-colored upper-surface flecks were produced by collapse
of islands of palisade cells (Ledbetter et al., 1959; Hill et al.,
1961; Rich, 1964). Epidermal tissues and Spongy mesophyll adjacent
to injured cells generally remained normal unless injury was severe.
Breakdown of chlorOplasts was accompanied by plasmolysis of the proto-
plast. However, plasmodesmatal connections were retained. Cell wall
collapse followed plasmolysis and produced large intercellular spaces.

This probably gave the lesions the bleached appearance.

Bifacial necrotic lesions were formed because of simultane-
ous collapse of cells through the affected areas -- including the
epidermal layers. The adaxial and abaxial surfaces were drawn close
together by the desiccating meSOphyll, resulting in thin, papery
areas (Hill et al., 1961).

In ozone-injured tobacco palisade cells, Povilaitis (1962) .
found that nuclei were greatly shrunken and distorted. The nuclear
membrane became clearly discernible in some injured cells. Some
chlorOplasts were disrupted apparently because of excessive swelling.

He believed that nuclei and cytOplasm were damaged before the chloro-
plasts showed abnormalities.

Thomson et al. (1966) reported that effects of ozone on fine
structure of palisade cells in bean leaves were of two phases. The
first phase involved changes in the chloroplast stroma, which was
almost entirely granulated. In some cells, ordered arrays of granules
and fibrils also developed in the stroma. In the second phase, nuclei
of the damaged palisade cells became shrunken to spherical or irregu-
larly ellipsoid shapes. The plasmalemma was pulled away from the
wall and the cellular contents aggregated in the center of the cells.
Most of the membrane system, including membranes of plasmalemma, tono-
plast, and limiting membranes of chloroplasts and mitochondria, gradually
disintegrated into small fragments. A general disruption of cell
organelles followed and remnants of the degenerated organelles scattered
throughout the cytoplasmic mass. Thomson (1966) also indicated that
these changes were probably related to the oxidation properties of

ozone .

Effects of ozone on duckweed (Lemna spp.)

Feder and Sullivan (1969) reported that ozone caused depression
of frond multiplication and floral production in duckweed Lemna per-
pusilla. Feder (personal communication) indicated that stomata of

Lemna perpusilla were non-functional. Plants grown in an environ-

 

ment charged daily with a low concentration of ozone (10 pphm) for

over 2 weeks were slower to begin multiplying. They had a signifi-
cantly lower rate of frond doubling and required longer to produce
fewer flowers than control plants. Treated plants produced smaller,
slightly yellow fronds but had no symptoms of acute injury. Control
plants produced four times as many fronds and six times as many flowers
as plants continuously exposed to ozone.

Later, Craker (1971) studied effects of ozone on Lemna minor,

 

another duckweed species, and found significant loss of chlorophyll.
He suggested that chlorOphyll loss could be used as an indicator of
injury to Lemna plants following ozone fumigation.

In further studies, Craker (1972) investigated the influence

of ozone on RNA and protein content of Lemna minor plants. He found

 

immediate decrease in the amount of RNA and protein in the ozone
treated plants, as compared with those not treated. Differences

remained detectable throughout a 24-hr sampling period.

8. Effects of Ozone on Fungi

For a long time ozone has been known to have fungicidal activi-
ty and has been used in meat and fruit storage to retard fungal

development (Richards, 1949). Fungicidal effects of high ozone

10

concentrations have also been noted both by Magie (1963) and Ridley
et al. (1966). However, not until very recently were the effects of
ozone determined on additional types of fungi.

Some fungi are as sensitive to ozone as are higher plants.
Rich and Thomlinson (1968) first reported effects of ozone on conidi-

Ophores and conidia of Alternaria solani. They found that 10 pphm

 

ozone for 4 hr or 100-pphm for 2 hr stOpped the elongation of conidi-
Ophores, caused apical cells to swell, and cell walls often collapsed
at their tips. If removed from ozone and exposed to light to prevent
sporulation, injured conidi0phores usually resumed elongation. How-
ever, the collapsed end wall was sloughed and the new cells often
grew at an angle to the original axis of the conidiOphores. Sometimes
breaks appeared in the pigmented layers of walls of the swollen cells.
Given the necessary dark period for sporulation, ozone-damaged conidi-
Ophores sporulated normally. If sporulating cultures were exposed to
100 pphm ozone for 30 min, conidia began to germinate while still
attached to the conidiophores.

Hibben (1969) exposed detached spores of 14 fungi on agar to
1-100 pphm ozone for 1 to 6 hr and found considerable variation in

germination. Larger pigmented spores of Chaetomium sp., Stemphylium

 

sarcinae-forme, S. loti and Alternaria sp. were insensitive to 100 pphm.

 

 

Spores of Trichoderma viride, Aspergillus terreus, A. niger, Penicilli-

 

 

gm egyptiacum, Botrytis allii and Rhi20pus stolonifer were reduced in

 

 

germination by 50 and 100 pphm ozone over longer exposures. Small
hyaline spores of Fusarium oxysporum, Collectotrichum lagenarium,

Verticillium albo-atrum and V. dahliae were the most sensitive, as

 

11

fl M’Yfi- ‘1‘.“

germination was prevented or reduced by most exposures at 50 and 100
pphm and were occasionally reduced by doses as low as 25 pphm for

4 and 6 hr. Lower doses sometimes stimulated Spore germination.

They also reported abnormal growth characteristics of fungus colonies
maintained in an ozone atmOSphere. However, on air dried spores or
spores in a liquid medium, ozone had little inhibitory effect.

Somov et al. (1972) also reported that Verticillium dahliae,

 

Fusarium sp., Penicillium purpurogenum and Alternaria tenuis died in

 

media exposed to certain doses of ozone while Escherichia coli and

 

Candida tropicalis were stable to ozone.

 

Kormelink (1967) found that the refractive globules in ozone-
fumigated mycelia were larger and more numerous than in the controls.
The same was also detected by Treshow et al. (1969). Furthermore,
they also found that radial growth and spore production of Collecto-

trichum lindemuthianum were suppressed when fumigated daily for 4 days

 

with ozone, 10 pphm or more for 4 hr. Macrosc0pic differences includ-
ed loss of the characteristic violet color of the cultures; formation
of growth rings (or annulations) which resulted from the alternation
of aerial and ”subterranean” growth, coinciding with fumigation
periods. The other two species tested, Helminthosporium sativum and
Alternaria oleraceae, were more tolerant to ozone. Neither radial
growth nor mass growth was inhibited by 60 pphm ozone. While sporu-
lation of the former was slightly suppressed, that of the latter was
greatly stimulated. In all cases, ozonated cultures had a lower

lipid content than controls. However, no distinctive differences were

noted in fatty acid composition.

12

Heagle (1970) exposed ten crown rust differential varieties

of oats (Avena Spp.), infected with Puccinia coronata, to 10 pphm

 

ozone for 6 hr in the light for 10 days. On all varieties tested,
rust pustles were significantly smaller on leaves exposed to ozone
than on nonexposed leaves. This suggested that ozone treatment sig-
nificantly reduced the growth of uredial sori. However, urediospores
produced on the ozone-exposed plants germinated as well as those
produced on nonexposed leaves. The former also produced as many
appressoria as did the latter. EXposure of dry leaves at 20 pphm
for 3 hr for 1-5 days did not affect urediospore germination,
appressoria formation, or penetration.

Heagle and Strickland (1972) also studied effects of ozone on

all stages of the asexual growth cycle of Erysiphe graminis f. sp.

 

hordei. Barley plants infected with powdery mildew were exposed to
low levels of ozone. Germination of exposed conidia was not signifi-
cantly affected. However, the number of normal infections was sig-
nificantly reduced when maturing or germinating Spores were exposed
to ozone. 0f conidia that failed to infect, the germ tube protOplasm
appeared to be released at tips of the germ tubes which indicated
germ tube rupture. After two exposures, colonies on exposed plants
were significantly smaller than on those not exposed. However, after
4, 6 and 8 exposures, colony lengths on ozone exposed leaves were
usually significantly larger than those on the controls. Ozone did
not significantly affect the number of conidiOphores after 4 exposures.
Once infection had been established, vegetative growth of Erysiphe

graminis hordei was tolerant to ozone or was even slightly stimulated

 

13

by ozone treatments. Heagle and Strickland (1972) suggested that
either a lack of penetration of hyphae by ozone or a physiological
resistance of hyphae protOplasm to ozone may account for this toler-
ance. They felt that ozone may have reacted with the hyphal cell
walls; decomposed; and hence failed to penetrate the hyphae. Other-
wise, had ozone penetrated hyphae of powdery mildew, injury should

be anticipated.

Ill. MATERIALS AND METHODS

The apparatus for exposing plants to ozone was prepared as
illustrated (Figure 1). Ambient laboratory air was drawn through a
variable speed Masterflex Tubing Pump (Figure 1a) with a suitable
head (Catalogue No. 7018) to give pr0per air flow. A Lab-Crest
Century Flowmeter (Figure 1b) was connected to the air pump for measur—
ing and adjusting the air flow rate. Air was then hydrated, (possibly
”saturated” (Treshow et al., 1969)), by bubbling it through distilled
water (Figure 1c). Various concentrations of ozone were produced by
a Pen-Ray SOG-2 ozonator (Figure 1d) by varying the rate of airflow
and the length of the tubular bulb exposed for irradiation of the
air passing over the tube. The latter was accomplished by an adjust-
able aluminum sleeve which fitted over the bulb. The ozonated air
was then passed into a glass chamber, 10 cm in diameter and 43 cm
long, which served as a fumigation chamber (Figure 1e). Another
glass jar was fitted inside the tubing to support the plants for
fumigation. The inlet of the fumigation chamber was maintained
about 20 cm above the plants so that exposure of plants would be as

uniform as possible.

Estimation of ozone concentration exposed to_plants

The relationships between ozone concentration (in ppm) and
length of bulb exposed at a flow rate of 1 L/min are given in Figure 2.
Theoretical concentrations of ozone produced from the ozonator were
estimated. The actual ozone concentrations within the fumigation
chamber were not determined due to the lack of necessary quantitative
analyzing equipment.

14

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17

3.01
2.54
2.04
71.54 \ L/ml“

1.01

Ozone in ppm

.Si

 

ol 2 z , , , - . - - - -
0 1 2 3 4 5 6 7 8 9 10 11

Length of bulb exposed (in inches)

 

Figure 2.-- Estimation of ozone concentration produced by the ozonator
from data supplied by the manufacturer.

Portions of Mnium cuspidatum gametOphytes, bearing rhizoids

 

and/or leaves, were planted in glass dishes (diam. 95 x 72 mm) con-
taining vermiculite and covered with a glass plate. They were grown
in a growth chamber with ambient laboratory air which was apparently
ozone-free. Growth conditions were 18°C, light intensity of 1500 ft-
candle at plant surface, and relative humidity of 95-100%. Distilled
water was added to the dishes when needed. Some dishes were supple-
mented with a diluted nutrient solution (Plant Marvel Laboratories,
Chicago 28, Illinois; 12, 31 and 14 percent of N, P and K respectively.)
Prior to fumigation, uniform branches of moss leafy gametOphytes
(hereafter referred to as ”moss plants”), with 6-7 leaves, were select-
ed. They were kept in petri dishes filled with water, and examined
under a dissecting microsc0pe for any pre-existed injury. Healthy
plants were then transferred carefully with forceps to glass vials
(diam. 1.7 x 5.8 cm), standing vertically in water moistened vermicu-

lite and immediately placed into the fumigating chamber.

18

Control plants were exposed either in the light or in the dark
to non-ozonated ambient laboratory air in the fumigation chamber for
2 to 4 hours at an air flow of 1 L/min. Before and after exposure,
plants in the fumigating chamber were sprayed with water vapor from
an atomizer to prevent dehydration.

After control plants were prepared, similar moss plants were
exposed to an atmosphere containing ozone, following the same pro-
cedure. The bulb of ozonator was usually eXposed at lengths of 4-8
inches so that 0.7 - 1.3 ppm of ozone was introduced at the same flow
rate as for control plants, i.e. 1 L/min,

After exposure, plants were returned to the water-filled petri
dishes and maintained in the laboratory. Injury was observed under
an American Optical Co. Spencer Cycl0ptic Stereoscopic microsc0pe (25x)
and a Wild-Heerbrugg microsc0pe at 100x, 400x and 1000x. Sequential

comparisons were made between treated and untreated plants up to 30 days.

IV. RESULTS

Moss identification
The moss plant used for fumigation in this study was identified

according to the key and descriptions in MOSSES With Hand-Lens and

 

Microsc0pe by Grout (1965), as Mnium cuspidatum (L.) Leyss. Because
no capsules were produced from the leafy gametOphytes when grown under
artificial conditions, identification of the moss species was based
chiefly on the characteristics of the sterile shoots. The following
are general descriptions of this moss plant:

1. Leaves broadly oblong to obovate, acute, decurrent. Strongly
bordered and serrate in the upper half with one-celled single
teeth.

2. Costa stout, vanishing in or just below the cuSpidate apex, some-
times confluent with the border and appearing excurrent.

3. Leaf cells hexagonal, smaller near the apex, larger near the middle
and the base of the leaf, somewhat collenchymatous, not papillose.

4. Leaves strongly shriveled when dry.

5. Sterile shoots prostrate or suberect.

6. Synoicous, capsules single. (This was not confirmed due to the

previously mentioned reason.)

Comparisons between dark and light fumigation

In preliminary studies, two sets of experiments were initially
conducted, one with light (500 ft-candle at plant surface), the other
without. Results were compared to evaluate qualitative differences

in injury between light and dark fumigation. However, desiccation

l9

20

injury was sometimes found on moss plants exposed to ozone in the
light. In order to eliminate possible complication of light and its
effect upon humidity, temperature, etc., subsequent experiments were

carried out in the dark.

Symptoms of external injury

Degree of injury was directly related to concentration of ozone
and duration of the exposure to which M. cuspidatum gametophytes were
exposed. Due to the lack of apprOpriate analytical equipment for
determining the actual ozone concentrations applied to moss plants,
the ”threshold” concentration necessary for causing evident and perma-
nent ozone injury on moss leaves could not be critically determined.
An exposure of approximately 0.75 ppm ozone (theoretical value esti—
mated according to Figure 2) for 3 hr was required.

The fIrst indication of ozone injury on moss was evident immedi-
ately after exposures as chlorotic, water-soaked areas on both the
upper and the lower leaf surfaces. If moss leaves were permanently
Injured, affected cells were conspicuous with a tranSparent appearance.
Chloroplasts became distinctly shrunken and resembled well defined
green dots. Irregularly shaped affected areas gradually became more
clearly defined with very clear-cut boundaries. Within the damaged
areas, almost every cell was injured. As the chlor0plasts were dis-
rupted and cells degenerated, both the upper and lower surfaces of
affected areas were slightly depressed below the normal surface of
the leaf. The dot-like injured chloroplasts progressively became
smaller and less distinct. Eventually, damaged areas became complete-

ly bleached (Figure 3). However, there was no apparent rupture of

21

 

Figure 3.-- Leaf of Mnium cuspidatum, 3 weeks after exposure to
1 ppm ozone for 3 hr. Damaged areas were almost
completely bleached while border remained apparently
unaffected. (Bar equals 100 u).

 

 

Figure 4.-- Normal cells in non-ozonated leaf of M. cuspidatum,
shows the nucleus with nucleolus. ProtOplasmic
streaming was evident. (Bar equals 10 u.

22

cell walls nor dissolution of middle lamella. Cells around lesions
did not deveIOp the reddish brown pigment as was usually observed in
desiccation-injured moss leaves.

Usually, desiccation injury could be distinguished from the
typical ozone injury by the second or the third day after treatment.
It was characterized by the rapid break down of chlor0plasts and
complete collapse of cells including the cell walls. Injured areas
gradually became papery and finally holes appeared. The cells sur-
rounding the holes were usually reddish-brown.

When moss leaves were exposed to sub-lethal concentration,
cells occasionally recovered from injury 12 to 24 hrs after fumigation
and the leaf returned to normal appearance.

Since only one layer of cells is present in most of the leaf
area of Mnium cuspidatum, injury was visible from both sides. The
third to fifth leaves from the shoot apex were most prone to injury.
In a single leaf, cells adjacent to and interior to the elongate,
marginal cells (borders) were most sensitive and were evidentally the
first injured. Next in sensitivity were the cells close to the tip
and at the base of the leaves. And finally, the more mature cells
between the midribs and the edges of the leaves. To some extent this
order of sensitivity varied due possibly to uneven exposure to ozone,
and the age of the tissues. Borders of the leaves usually remained
normal unless injury was severe. Midribs and stems were most tolerant

of ozone, and were damaged only when injury was very severe.

23

Cytological changes

ChlorOplasts in the younger cells of healthy moss leaves were
somewhat square shaped with the margins rounded. Some were constrict-
ed in the middle and were dumb-bell shaped. CytOpIasm appeared as
a turgid, fine granular matrix. Under high magnifications (1000x),
numerous granules and small Spheruoles were present, apparently
within the chlorOplasts (Figure 4). The plasmalemma, apparently un-
affected, was usually not detectable. In most cells, nuclei and nu-
cleoli (Figure 4), vacuoles, cytoplasmic strands and protOplasmic
streaming were readily apparent when focused prOperly.

As the leaf matured, chloroplasts enlarged and filled the Space
within the cell. Limited by one another, they became irregularly
shaped. In very old leaves, the sides of chlor0plasts almost touched
each other. Dumb-bell shaped chlorOplasts (Figure 4) constricted in
the middle were commonly found in most cells. The uninjured control
plants normally remained healthy and exhibited no apparent changes
for a period of time until they degenerated naturally.

Sequential microscopic observations of ozone-injured moss
plants suggested that at least four stages of cellular changes were
involved. Because both the dosage (exposure time and concentration)
of ozone and the age of tissues affected the response patterns of
moss, injured cells did not necessarily go through all four stages.
For instance, if cells were only slightly damaged, chlorOplasts might
temporarily shrink into round or elliptical shapes, and spread less
evenly in the cytOplasm. But with time, they gradually expanded

again and regained their normal appearance. In contrast, cells

24

exposed to extremely high concentrations of ozone might be irreversi-
bly Injured. In these cells, chlorOplasts quickly disintegrated into
fragments, nuclei disappeared, plasmolysis occurred, plasmalemma
broke down, and cells began to degenerate.

The following are general descriptions of the progressive pattern
,of ozone injury. They were determined by comparative studies of the

cellular structures of slightly, moderately, and severely damaged

moss leaves.

Phases of ozone injury:

Phase 1.--Microsc0pic examination immediately after exposure to 1 ppm
ozone revealed that chloroplasts seemed to be the first organelle to
respond to ozone, becoming markedly shrunken, round or elliptical
(Figure 5). Some of the dumb-bell shaped chlorOplasts formed deep
furrows transversely in the middle. Affected chlorOplasts became
more sharply defined and less granulated than in healthy cells, and
were less evenly distributed in the cytOplasm. Injured cells were
more transluscent than the normal cells possibly because shrinking

of chlorOplasts exposed greater space between plastids.

Under higher magnifications (1000x), nuclei as well as cyto-
plasmic strands appeared to be normal. ProtOpIasmic streaming was
very active and distinct. Plasmalemma and tonoplasts also appeared
to be normal. The Spheruoles within chlorOplasts seemed to be smaller
and less distinct, possibly in a process of disappearing.

Phase 2, -- With time (12 hr after fumigation with 1 ppm ozone for
3 hr), damaged chloroplasts continued to shrink to about 1/3 - 1/2

the size of the normal ones (Figure 6). They aggregated in groups,

 

 

Figure S.-- Ozone injury -- phase 1. Leaf cells of M. cuspidatum
immediately after exposure to 1 ppm ozone for 3 hr.
Chloroplasts have shrunk noticeably. (Bar equals 10 u.)

 

Figure 6.-- Ozone injury -- phase 2. 12 hr after exposure of
M. cuspidatum leaf to 1 ppm ozone for 3 hr. Chloro-
plasts aggregated in groups and were about 1/3 - 1/2
of the normal size. (Bar equals 30 u.)

26

either in the center or near the edge of cells. Short connections
were sometimes detectable between adjacent chlorOplasts before they
separated completely. This suggests that the dumb-bell Shaped chloro-
plasts might have split along the constriction, and formed the abnomal
appearance. Another noticeable change within the chlorOplasts was
that Spheruoles were no longer visible. Instead, short dark lines
were prominent.

Meanwhile, nuclei underwent a series of changes, and eventual—
ly some of them disintegrated into a mass of membraneous fragments.

The plasmalemma and ton0plast showed no sign of breaking down
at this stage. CytOplasmic strands remained clearly visible, although
strands seemed to be shortened by the grouping together of shrunken
chloroplasts. Protoplasmic streaming seemed to be less active.

Phase 3. -- Plasmolysis was evident in the injured cells as early as
one day after exposure to 1 ppm ozone for 3 hr (Figure 7a). With
breaks devel0ping, the plasmalemma pulled away from the cell wall.
Highly granulated cytoplasmic contents clumped in the center of the
cells. TonOplasts broke down and vacuoles collapsed.

As a result of further shrinkage, affected chlor0plasts became
even smaller, about 1/4 the size of the normal ones. Some chlorOplasts
superficially resembled dividing animal cells in late-teIOphase
(Figure 7b), and might have been splitting into two halves. Occasion-
ally, tiny empty vesicles were attached to the chlorOplasts. Since
the numbers of total existing chlorOplasts in the affected cells
did not significantly exceed those in the healthy cells, what actually

happened to those chloroplasts is still in speculation. One possible

._ A“...

a.

27

 

(b)

Figure 7.-— Ozone injury -- phase 3. Leaf of M. cu5pidatum, 1 day
after exposure to 1 ppm ozone for 3Thr. a Plasmolysis
with disintegration of plasmalemma. Cytoplasmic contents
clumped in the center of cells. (Bar equals 30 u.)

b) Chloroplasts may be splitting into two halves (Bar
equals 10 u.)

ll—‘W—W .

28

explanation is that, after splitting, only some of the divided chloro-
plasts retained their integrity and turgidity. The others may have
disintegrated. ProtOplasmic streaming, though still detectable, was
very slow.

Phase 4. -- As injury progressed, more and more breaks appeared in the
limiting membrane of chloroplasts (Figure 8a). Losing their integrity,
the chlorOplasts began to break down. Eventually, plastids were
partially or completely bleached, and only a few plastids retained
color. The plasmalemma and cytoplasmic contents collapsed completely
into a diffuse, jelly like mass. The remnants of various degenerated
organelles and membranous fragments could hardly be distinguished
(Figure 8b). However, no apparent changes were observed in thickness
of cell walls nor in size of intercellular spaces between ozone injured

cells.

29

 

(b)

Figure 8.-- Ozone injury -- phase 4. Leaf of M. cuspidatum,
2-3 weeks after exposure to 1 ppm ozone for 3 hr.
a) Breaks developed in the limiting membrane of chloro-
plasts. (Bar equals 10 u.) b) CytOplasmic contents
have broken down and collapsed, and cells have degener-
ated. (Bar equals 30 u.)

V. DISCUSSION

Mnium cuspidatum is potentially a useful bioassay plant for

 

detection of ozone toxicity. Due to the fact that flfllflfl plants do
not have stomata, sensitivity to ozone may be less affected by
various environmental conditions, such as light, water stress,
temperature, etc. In my trials, similar responses were obtained
under different light conditions, provided that moisture was main-
tained throughout the experiment. The relatively low sensitivity

to variations in light intensity suggests that moss may be useful

in a bioassay system. In these trials, presence or absence of light
seemed to have minor influence on symptom deveIOpment and sensitivity

of plants. In addition, Mnium cuspjdatum reSponded to ozone promptly,

 

and injury was usually manifested within 24 hr after fumigation.
Severity of injury could be assessed directly by microsc0pic examina-
tion of both size of affected areas and damage to cell organelles.

The main disadvantage of such a bioassay system is the diffi-
culty in maintaining high moisture throughout the experiment so as
not to desiccate moss plants during exposure.

Practicability of moss as a bioassay plant could not be
established with certainty because the actual threshold concentration
for ozone injury could not be determined critically due to lack of
suitable analytical equipment. However, the threshold exposure for
injury of moss was approximately 75 pphm for 3 hrs (as estimated
according to Figure 2). This intensity of exposure to ozone is
considerably higher than that required to injure sensitive Bel W-3

tobacco (5 pphm for 4 hrs) or to injure eastern white pine (7 pphm

3O

31

for 4 hrs) (Hill et al., 1970). The possibility exists that the
actual ozone concentration to which moss plants were exposed may
have been somewhat less than that estimated (Figure 2) due to the
relatively slow rate of introduction of ozone into the system

(1 L/min) and sorption of ozone by the plants or by spontaneous
reversion to 02.

The presence of a leaf like structure a Single cell in thick-
ness permits sequential microsc0pic observation of cellular responses.
Furthermore, lack of stomata permits responses apparently less com-
plicated by environmental variation than is possible with the higher
plants. This advantage may more than offset the disadvantage of
relative insensitivity to ozone.

Even though the various cells of the moss leaf were apparently
equally exposed to ozone during eXposure, differences in tolerance
to ozone toxicity were apparent. Cells at the leaf border were
markedly more tolerant to ozone than were those in the central por-
tion of the lamina. Cells of the midribs and stems were also more
tolerant than those of the central leaf lamina. Tolerance in the
midrib and stem might well be associated with multiple layers of
cells because the midribs and stems are more than one cell in thick-
ness. Further work is necessary to explain these cellular differences.

Craker (1971) suggested that chlorOphyll loss could be used
as an indicator of ozone injury to Lemna plants. Perhaps this might

also be true for moss plants, and possibly ozone toxicity could be

measured quantitatively and correlated to ozone concentration.

VI. SUMMARY

A study was made of the effects of ozone on leaves of the moss
plant, Mgigm cuspidatum. If exposed to 0.75 ppm ozone (or higher)
for 3 hr, moss leaves suffered permanent injury. Affected areas were
extensive and evident, with a transparent appearance. ChlorOplasts
following substantial shrinkage were visible as numerous small green
dots from both sides of the injured leaves. As injury progressed,
those green dots gradually became smaller and less distinct. Eventu-
ally most of them became colorless, giving the affected area a bleached
appearance.

Cytological changes induced by ozone were in four successive
phases. 1) Immediately after ozone eXposure at 1 ppm for 3 hr, chloro-
plasts became markedly shrunken. Injured cells were more transluscent
than the normal cells. 2) Within 12 hr after ozone exposure, damaged
chlorOplasts shrunk to about 1/3 - 1/2 the size of normal chlorOplasts
and they were often aggregated in groups. Meanwhile, nuclei gradually
disintegrated, resembled a mass of membraneous fragments. 3) Plas-
molysis began in the injured cells one day after exposure. Plasma-
lemma pulled away from the cell wall and disintegrated. CytOplasmic
contents clumped in the center of the cells. ChlorOplasts continued
to shrink. Splitting of chlorOplasts into two parts was also suSpect-
ed. 4) ChlorOplasts disintegrated into fragments and the debris and
remnants of other disintegrated cell organelles were surrounded by a
diffuse, jelly-like mass. Eventually, the degenerated cells were
almost completely colorless.

Usually, the third to fifth leaves from the shoot apex were

32

33

most prone to injury. In a single leaf, cells adjacent to and interi-
or to the borders were most sensitive. Mature cells near the midribs
were more tolerant, borders and midribs of the leaves, and stems were

most tolerant of ozone.

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