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III

MECHANICAL INJURY 0F EPIDERMAL CELLS
IN LOCAL LESION VIRUS INFEGTION

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
IVAN PEDRO BUTZONITCH
1971

15"!!!» §

This is to certify that the

thesis entitled
Mechanical Injury of Epidermal Ce l ls

in Local Lesion Virus Infection

presented by

s
9

Ivan Pedro Butzonitch

has been accepted towards fulfillment
of the requirements for
Doctoral degree in Botany and Plant
Pathology

/ Major professor
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Date September 10, 1971

 

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ABSTRACT

MECHANICAL INJURY OF EPIDERMAL CELLS

IN LOCAL LESION VIRUS INFECTION

BY

Ivan Pedro Butzonitch

Following mechanical inoculation of upper leaf

surfaces of New Zealand spinach (Tetragonia expanse Murr.)

 

with tobacco necrosis virus, silicone rubber negative
replicas were made of epidermal cell surfaces at time
intervals for recording evidence of injury. Positive
replicas were then prepared with transparent nail polish.
Approximately 1.2% of the epidermal cells appeared injured
or had collapsed 45 minutes after inoculation. Of such
cells, approximately 20% recovered within 21.hr after
inoculation. Surfaces were again smooth and cells ap-
peared turgid. The remaining 80% of injured cells col-
lapsed further and appeared dead. Lesions did not become
apparent until 24-28 hr after inoculation. They were
first evident as small sunken areas where the epidermal

cells were usually turgid. Then a small group of

Ivan Pedro Butzonitch

epidermal cells within the sunken area become flaccid and
later collapsed. Adjacent cells quickly developed similar
symptoms and within 4 - 5 hr the lesion had become
macroscoPically evident. In approximately 27% of the
lesions observed, a cell which showed initial flaccidity
at the 45 minute observation and later recovered was pre-
sent in the lesion center. In approximately 30% of the
lesions observed, a cell that had collapsed irreversibly
at the 45 minute observation was present in the lesion
center. The other lesions were not preceded by cell
flaccidity and seemed to have developed from cells which
appeared uninjured during the period of observation.
Epidermal cells similarly treated without virus evidenced
either injury followed by recovery or collapse. Local

lesions did not form.

MECHANICAL INJURY OF EPIDERMAL CELLS

IN LOCAL LESION VIRUS INFECTION

BY

Ivan Pedro Butzonitch

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1971

To my son.

ii

ACKNOWLEDGMENTS

Deep appreciation is expressed to Dr. W. J.
Hooker for his guidance and assistance during the course
of this investigation and in the preparation of the
manuscript.

Thanks are also due to Dr. R. W. Chase, Dr. D. J.
deZeeuw, Dr. M. L. Lacy and Dr. H. H. Murakishi for their
critical evaluation of the manuscript.

To Dr. W. Tai I am appreciative for introducing
photographic techniques to me and for her generous en-
couragement and advice.

To Dr. W. G. Fields I am appreciative for his
generous encouragement in the unrestricted use of in-
struments at his laboratory.

I cherish the many friends I made here which made
my stay a most enjoyable experience.

To my wife, Alicia, for her understanding, un-
selfishness and patience and my son, Andres, for his
refreshing presence, I am profoundly grateful.

Acknowledgment is also made to the Agency for
International Development and the Instituto Nacional de
Tercnologia Agropecuaria (Argentina) for supporting this

study.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES O I O O O O O I O O O O O I O I O I V

LIST OF FIGURES O O O O O O C O D O O O O O O O 0 Vi

INTRODUCTION . . . . . . . . . . . . . . . . . . . 1
MATERIALS AND METHODS . . . . . . . . . . . . . . 8
EXPERIMENTAL RESULTS . . . . . . . . . . . . . . 13
DISCUSSION . . . . . . . . . . . . . . . . . . . . 43
SUMMARY . . . . . . . . . . . . . . . . . . . . . 49

LITEMTURE CITED O O O O O O O O O O O O O O O O O 52

iv

LIST OF TABLES
Table Page

1. Epidermal cell response after conventional
mechanical inoculation of T. expans
leaves with distilled water . . . . . . . . 24

2. Epidermal cells, located in sunken lesion
area, involved in early stages of TNV
local lesion development on T. expansa . . 27

3. Sequence of events preceeding local lesion
formation in epidermal cells following
mechanical inoculation of T. expansa
leaves with TNV . . . . . . . . . . . . . . 29

4. Frequency of non lesion-forming sequential
events in single epidermal cells in a
31 mm2 area of a T. expansa leaf after
mechanical inoculation w1t TNV . . . . . . 41

LIST OF FIGURES
Figure Page

1. Upper surface of a T; ex ansa leaf 3 days
after inoculation Wlth TNV infected
crude sap . . . . . . . . . . . . . . . . . l4

2. Upper leaf epidermis of T. expansa surface
replicas and cross sections of fresh
leaves . . . . . . . . . . . . . . . . . . 18

3. Replica evidence of the sequence of events
following mechanical injury by simulated
"inoculation" of upper leaf epidermis
surface of T. expansa . . . . . . . . . . . 20

4. Early symptoms and development of a TNV
esion on area of upper epidermis of
T. expansa leaf as evidenced by replicas
made sequentially . . . . . . . . . . . . . 25

5. Injury-recovery-lesion sequence. Replicas
of a lesion area on upper epidermis of
a T. expansa leaf . . . . . . . . . . . . . 31

6. Injury-injury-lesion sequence. Replicas of
a lesion area on upper epidermis of a
T. expansa leaf . . . . . . . . . . . . . . 34

7. 'Healthy'-injury-lesion sequence. Replicas

of a lesion area on upper epidermis of a
T. expansa leaf . . . . . . . . . . . . . . 37

vi

INTRODUCTION

There is general agreement among plant virus
workers that wounds are a prerequisite for introduction
of virus into plants. It is also generally agreed that
abrasives aid inoculation by injuring epidermal cells.

Cell damage ranging from very slight and super-
ficial to very severe and lethal have been either suggested
or demonstrated as suitable for successful infection. The
kind of injury most favorable for infection as well as the
extent of damage a cell can sustain without collapsing
rapidly and/or irreversibly is not known (Schneider 1965;
Bawden 1964).

The concept that inoculation injury must be non-
lethal for successful infection seems well established
(Bawden 1964; Fulton 1964; Matthews 1970). That cuticular
damage might be enough to allow virus infection has been
suggested by Matthews (1970). His suggestion is based in
the relationship between number of ectodesmata and degree
of susceptibility to infection (Brants 1963, 1964 and
1965; Thomas and Fulton 1968).

The concept that nonlethal injury is a prerequi-
site for virus infection is based largely on the assump-

tion that the virus multiplies in the initially infected

cell (Bawden 1964; Fulton 1964). Lethal injury has not
been defined in terms of the duration of time elapsing
from injury of the cell to the time of its death. Thus
death of a cell after injury could become less important
in limiting infection if we assume that the injured cell
allows the virus to move into uninjured cells whether
multiplication of the virus has occurred or not (Siegel
and Zaitlin 1964; Benda 1956; Ehara and Misawa 1968).
In addition, the possibility exists that infectible
wounds may be of different kinds and sizes (Mundry 1963).
Basal septa of broken trichomes have been postu-
lated as a point of entry for TMV (Kontaxis and Schlegel
1962; Herridge and Schlegel 1962). Although broken
trichomes have been shown to be of little importance for
virus infection by rubbing methods (Boyle and McKinney
1938), the possibility that severely damaged or even
dead cells could act as a point of entry for the virus
without being a primary infection site should be con-
sidered. It has been shown that infective southern bean
mosaic virus can be translocated through xylem cells of
Pinto bean before infecting the leaf parenchyma (Schneider
and Worley 1959). Tobacco necrosis virus (TNV) can in-
fect bean leaves after being translocated through the
xylem of scalded stems (Robert and Price 1967). In
these cases we have, in the same plant, both extremes

with respect to the types of possible damage that allow

virus infection—-dead xylem cells and apparently uninjured
leaf parenchyma cells.

Virus penetration of leaf cells through stomata
has been postulated (Duggar and Johnson 1933). Failure
in getting infection by spraying TMV on uninjured host
plants leaves (Sheffield 1936, Kalmus and Kassanis 1945)
and by vacuum infiltration of tomato tissue with TMV
(Caldwell 1932) provided evidence against infection of
uninjured cells.

The work of Duggar and Johnson has been criticized
mainly on the basis that epidermal cells were not intact
due to the methods used to inoculate the plants. Plants
were touched with the nozzle used for spraying the inocu-
lum and this way of manipulating the leaves could have
caused injuries to epidermal cells (Sheffield 1936).
Celite was also used and the abrasive injury could have
facilitated infection (Roberts and Price 1967). Never-
theless, these criticisms are not valid because in some
of the experiments (Duggar and Johnson 1933) the inoculum
was dropped on the leaves without the use of a sprayer
and without celite. The lack of correlation between
number of stomata (Boyle and McKinney 1938) in the upper
and lower sides of pepper leaves and the corresponding
number of infections when inoculated with TMV is a good

evidence against stomatal infection.

The possibility that undamaged cells in plant
tissue cultures may sometimes be infected with TMV has
been suggested (Kassanis et_al. 1958). In this type of
experiment, as well as in the inoculation of whole plants,
there is no proof that the cells were absolutely un-
wounded. In later trials (Kassanis 1967), the number of
permanent infections in tissue cultures was shown to de-
pend on the type and number of injuries.

Apparently the size and the type of wound a cell
can sustain without dying relatively soon after being in-
jured has not been investigated in detail. Hair cells
of tobacco and tomato can stand the wound produced by a
micropipette. This wounding is followed by protrusion
of a small amount of protoplasm (Hildebrand 1943).
Palisade and phloem single cells of Solanum nodiflorum,
tomato, tobacco, and Hyoscyamus niger have been inoculated
with aucuba mosaic virus. About 9% of the plants became
infected when one cell per plant was injected using a
micropipette 1-5 u in diameter (Sheffield 1936). It is
not clear in this case how damage to other cells was
avoided and no criteria for establishing survival of the
cells after the puncture was given.

About 60% of the inoculated leaf hair cells of

Nicotiana glutinosa survived puncturing with a glass

 

microneedle through a drOp of TMV inoculum placed on the

cell surface (Benda 1956). Tomato hair cells survived

puncturing with a microneedle 3-5 u in diameter (Hirai
and Hirai 1964).

Particles of carborundum, 600 mesh, have been ob-
served penetrating cauliflower epidermal cells during
mechanical inoculation with cauliflower mosaic virus
(Rawlins and Tompkins 1936). However, there is no evi-
dence that such injured cells survived those wounds nor
that infection originated in them.

The nature of the damage of cowpea epidermal cells
after rubbing with 600 mesh carborundum has been studied
with the electron microscope (Gerola gt_al. 1969). There
was no evidence in their study that the resulting damage
actually belonged to a successfully inoculated cell.

Wound healing of slightly injured cells seems to
be a relatively rapid process (Harrison 1961). It has
been studied more from the point of view of the duration
of the interval during which viruses can penetrate the
cell rather than as the first step in the somewhat longer
process of recovery (Sheffield 1936; Furumoto and Wildman
1963; Kalmus and Kassanis 1945; Jedlinski 1956, 1964).
The existence of this interval in which the presence of
"infective sites" is manifested suggests either than the
properties of the cell membrane change with time or that
some other barrier appears between the cell and the virus
particles. This barrier could be newly deposited cell

wall material. Also, the onset of cell wall regeneration

in protoplasts coincides with a diminishing pinocytic
uptake of virus (Cocking and Pojnar, 1969).

Wound healing and subsequent cell recovery as
evidenced by recovery of turgor and later by the survival
of cells for indefinite periods of time is probably as-
sociated not only with cell membrane integrity but also
with the regeneration of the cell wall. Experimental
cell wall regeneration has been obtained in suitably con-
trolled media and in the absence of contaminating enzymes
(Pojnar gt_al., 1967). In protoplasts of tomato fruit,
regeneration of the cell wall has been observed 8 hours
after cells were placed in a suitable medium (Cocking
1970). Cell wall regeneration usually requires the pre-
sence of the nucleus (Binding 1966).

Probably these conditions are fulfilled at least
in a portion of the epidermal cells when cell walls are
disrupted by mechanical inoculation using carborundum and
leaves are washed after inoculation.

Apparently the overall process of cell recovery
after mechanical injury has been relatively neglected by
plant virus workers and no references could be found.
This may be due in part to the lack of suitable nondestruc-
tive techniques for sequential studies of single cells.

This thesis is a study of the sequence of events,

(observable with a light microscope, that occur on the

external surfaces of epidermal New Zealand spinach leaf

cells when mechanically inoculated with TNV.

MATERIALS AND METHODS

A common strain of TNV was maintained and increased

on young cowpea plants: Vigna sinensis (Torner) Savi,

 

cultivar Black, strain SS (deZeeuw and Crum 1963, Price
1940). These plants were kept at 20-22 or at 23-27°C
under continuous light (500-600 foot candles) from
fluorescent tubes. From plants grown at 20-22°C, 10 day
old local lesions about-3 mm in diameter were cut from
the inoculated primary leaves and ground with mortar and
pestle. When COWpea has been grown at 23-27°C, 6 day old
lesions were used. A drop of distilled water was added
to every three local lesions and the resulting prepara-

tion was used to inoculate New Zealand spinach (Tetrogonia

 

expansa Murr.) grown under continuous cool white floures-
cent light (220-230 foot candles) at 23°C.

In other cases, for increased uniformity, frozen
inoculum was prepared by grinding local lesions from TNV
infected cowPea leaves in an ice-cooled mortar at a ratio
of 15 lesions per drop of water. The ground material was
centrifuged in a Lourdes table centrifuge at 7000 G for
20 minutes. The resultant clarified sap was kept frozen

in 5 mm diameter vials sealed with paraffin wax paper and

thawed rapidly by holding the vial in the hand just before
inoculation.

To mark the inoculated areas of the leaf, 4-5
small drops of nail polish were placed on the upper sur-
face of each 2. expanse leaf. The small lesions produced
by the nail polish served later as reference marks.

After 24 hr the leaves were dusted with silicon car-
bide, 400 mesh. Small drops of inoculum were then placed
on the upper surface and rubbed gently with a spatula
made from the somewhat flattened end of a glass rod, 2 mm
in diameter. Controls to evaluate mechanical injury were
similarly prepared with distilled water or healthy cowpea
sap. Experiments were usually made with leaves attached
to the plant. In some cases, detached leaves were placed
on top of glass slides, that had been wrapped with filter
paper. These were placed inside petri dishes 9 cm in
diameter containing 5 ml of distilled water. Water was
changed twice daily. After inoculation either detached
or undetached leaves were kept at 23-27°C under continuous
cool white fluorescent light (800-900 foot candles).

The sequence of events occurring on the epidermal
surface was recorded by making multiple negative replicas
after the method developed by Joan Sampson (1961) and
described by others (Bernstein and Jones 1967, Zelitch
1961). Replicas were usually made before and at suitable

intervals after inoculation. The technique involved

10

coating the leaves with a mixture of liquid silicone
rubber and a catalyst to obtain a negative replica. In
these experiments a mixture of 10 9 General Electric
RTV-ll liquid silicone rubber and 3 drops of Tenneco
Nuocure 28 (tin octoate) was used. The mixture remained
fluid no more than 3 minutes and hardened in about 15
minutes at 24°C. Then, after detaching, cleaning and
drying the negative replica, diluted nail enamel was
applied to the negative and a positive replica of the
leaf was obtained. Positive replicas were prepared using
nail enamel (Revlon Clear No. 61) diluted to 50% with
acetone. Some minor modifications of the method were
made. The nail enamel film usually became very brittle
and difficult to peel off, unless a drop of castor oil
was added to each 8 m1 of the diluted enamel. This de-
layed hardening of the film and made it somewhat sticky
and easier to handle. An atomizer powered by compressed
air was used instead of a paint brush for applying the
nail polish to the negative replica. The negatives were
Sprayed 3-4 times with enamel. This procedure gave fewer
air bubbles in the replicas than that obtained by brush
application. The enamel was allowed to dry for about one
hour, after which the replica was split along the leaf
vein, detached from the negative, applied to glass slides
with light pressure and allowed to dry for another hour.

Then a 22 x 50 mm cover slip was applied and a small

11

weight was left on the cover glass overnight. After the
replicas were completely dry the cover slips were fastened
to the glass slide with strips of masking tape.

The position of the reference marks as well as
that of the leaf veins was marked with water proof ink
on the cover slips to facilitate identification of local
lesion areas through the whole series of replicas. To
localize the lesion areas, the glass slide of the replica
where the lesions were first conspicuous was completely
covered with water proof ink of different color than the
reference marks. Then the ink was scratched away from
the glass surface over the lesion area with a dissection
needle. This replica was then used as a pattern to mark
the position of the lesion areas in the replicas made at
earlier observations. This was done by placing the pattern
replica on a slide sorter and the replica to be marked
was placed exactly on top of the pattern replica where
it was marked.

Observation and photographs of the enamel replicas
were made with an American Optical microscope with a 10 x
wide field ocular and a 10 X objective. Several trials
with different light sources were made. The best illumi-
nation for this material was provided by 4, cool white
40 watt, fluorescent tubes in a ceiling fixture covered
with frosted clear plastic, 7 feet away from the mirror

of the microscope and situated at approximately 30° from

12

the vertical axis of the microscope. The kind of shadows
produced in this way gave the replicas the desired tridi-
mensional appearance. An American Optical 35 mm Photo-
micrographic camera with Kodak Plux-X Pan, ASA 125, black
and white film at one second exposure was used for

photographs of the replicas.

EXPERIMENTAL RESULTS

Epidermal features of cotyledons and leaves of
several TNV hosts (Hollings 1959; Price 1940; Smith 1957)
were investigated in a search for a type of leaf appro-
priate for leaf epidermis study by surface replicas.
Hosts with trichomes on cotyledons or leaves were not
considered useful because leaf hairs would be destroyed
during preparation of replicas and because the wounds so
produced might also serve as infection sites.

2. expansa was chosen because it is susceptible
to TNV and responds to infection by development of con-
spicuous local lesions (Figure 1). Leaves lack trichomes
and the leaf epidermis consists of two types of compara-
tively large and regularly shaped epidermal cells. The
smaller of these two types of epidermal cells has a sur-
face area of approximately 50 x 75 u. The larger epidermal
cells are dome shaped with a surface approximately 100-
150 u across. Approximately 10% of the leaf epidermal
cells are of the large type. Injury as evidenced by lack
of turgor is easily observed in these large cells. The
stomatal guard cells because of their small size were not

useful in this investigation.

13

l4

 

Figure 1.

Upper surface of a T. expansa leaf 3 days
after inoculation with TNV infected crude
sap showing numerous local lesions. The
two larger spots (arrows) are reference
marks made with nail polish.

15

Other plants such as bean, beet, COWpea, cucumber,

Datura stramonium L., pepper and Vinca rosea L. do not

 

have trichomes in either cotyledons or leaves, but have
relatively small irregularly shaped cells. Chenopodium
album L. and zinnia have relatively large epidermal cells
but conspicuous lesions were not formed on zinnia and
leaves of 9. album were too tender and did not tolerate
manipulation.

Marking of leaves for reference purposes presented
particular problems when the marked leaf was to be repli-
cated sequentially. Ink could not be used because it
was removed during replication. Perforations in the leaf
were not satisfactory because liquid silicone rubber ex-
tended through to the opposite surface of the leaf and
formed "buttons." These prevented separation of the
replica from the leaf. After some experimentation with
several substances, fully developed leaves of young 2.
expansa were marked with a small drop of nail enamel.

The localized injury provoked by the enamel gave a point
of reference to find a definite area on the leaf surface
(Figure 1). In other experiements, 4-6 reference marks
per leaf were made by scratching the epidermal cells very
superficially with the end of a broken razor blade.

Experiments to establish improved conditions for
the development of TNV local lesions on T. expansa were

made. No obvious increase in the number of lesions nor

l6

advantages of any kind were found when T. expansa plants
were given a 24 hr dark period before inoculation. On
plants exposed at 15°C and continuous light after inocula-
tion, lesions developed slowly and were visible with the
naked eye approximately 40 hr after inoculation. At
24°C lesions were usually conspicuous about 24 hr
after inoculation.

TNV lesions were comparatively more abundant in
the younger fully developed leaves in the top of the T.
expansa plant than in the older mature leaves. Epidermal
cells were somewhat smaller in size when plants were
grown under intense illumination. Fresh inoculum from
TNV local lesions on cowpea was consistently much more
infective than clarified frozen inoculum from the same
host. Fresh inoculum produced approximately 10-50
lesions per 2. expansa leaf. Frozen inoculum usually
produced 1-10 lesions. Best results as measured by
number of lesions, size of epidermal cells, and resist-
ance to manipulation were obtained using fresh inoculum
and the younger fully developed undetached leaves from
75 day old plants.

Mechanical injury of epidermal cells in the ab-
sence of virus was studied in replicas of detached T.
expansa leaves that had been dusted with silicon carbide
and rubbed 4 times with a glass spatula dipped in dis—

tilled water. After rubbing, leaves were kept under

1?

continuous fluorescent light of 800-900 foot candles at
23-25°C.

Replicas of controls lacking virus were made be-
fore and at 1, l6 and 48 hr after rubbing. In no case
were lesions observed which resembled those obtained
after TNV infection.

All cells were turgid before rubbing (Figure 2-A,
D and 3-A). Effects of mechanical injuries were classi-
fied either as being flaccid or collapsed. Both types of
injury effects were observable with a 100 X total magnifi-
cation. Flaccidity was considered to be lack of turgor
which produced variable degrees of flattening and wrin-
kling of the cell surface (Figure 2-B, E and Figure 3-B).
The term collapse is used to describe an extreme type of
injury which was irreversible. Collapse was character-
ized by flattening of the cell surface and desiccation
which often exposed the end of the turgid palisade cells
immediately under the injured epidermal cells (Figure
2-C, F. and Figure 3-D, cells r and 3).

Most of the leaf epidermal cells were apparently
not affected by the rubbing treatment. Some of the cells
were permanently damaged, collapsing shortly after rub-
bing and these did not later recover turgor (Figure 3-B,
C, D).

The term recovery is used to designate the regain-

ing of turgor following mechanical injury by those cells

Figure 2.

18

Upper leaf epidermis of T. expanse surface
replicas and cross sections of fresh leaves.
(A) Surface replica of large and small cells
uninjured and turgid before mechanical inocu-
lation. (B) Surface replica flaccidity of
the cell (arrow) following mechanical inocu-
lation. (C) Surface replica showing
irreversibly collapsed cell (arrow). Note
ends of underlying palisade cells. (D)
Cross section of fresh leaf showing an intact
large epidermal cell. (B) Cross section of
unfixed leaf. Flaccidity of the large epi-
dermal cell was caused by manipulation and is
comparable to (B). (F) Cross section of un-
fixed leaf. The collapsed epidermal cell is
comparable to the one in (C).

19

 

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22

which showed effects of injury as soon as 0.75-l hr
after rubbing. Recovery was usually accomplished in less
than 24 hr.

Bubbles (Figure 3-C, cell v), darker areas (Figure
3-D, cell w) or small fractures (Figure 6-C) occasionally
produced artifacts on the replicas. Artifacts produced
by accidental attachment of small particles of foreign
material could be differentiated from injuries due to
mechanical inoculation by observing the replica with the
microscope focused at different depths.

A relatively small number of cells initially
showed severe damage one hour after rubbing. Some of them
later gradually recovered (Figure 3-C, D, cells t and v).

The work previously described was done with de-
tached leaves. The work to be described was done with
leaves still attached to the plant. Conditions and
methods were the same except that the plants bearing the
rubbed leaves were kept in the relatively dry laboratory
room environment (BO-40% R.H.). An area of 382 mm2 cor-
responding to approximately 90% of a leaf 27 mm long was
surveyed. Replicas made at 0.75 hr and 47 hr after
rubbing were compared cell by cell using two microscopes.
Of the cells which evidenced injury at 0.75 hour, 20% had
recovered 47 hr after rubbing. Large and small cells

recovered approximately in the same pr0portion. Some

cells did not show injury immediately after inoculation

23

but did show injury 47 .hr later (Table 1). Results were
similar with either detached leaves as previously de-
scribed or with leaves still attached to the plant.

Lesion develOpment following mechanical inocula-
tion with TNV was observed in replicas of T. expanse
leaves made after 15, 18, 22.5, 24, 28 and 40 hr
(Figure 4). Seven circular areas 13 to 18 mm in diameter
were surveyed and in these areas 9 lesions were studied
in detail (Table 2). The first suggestion of lesion
development was usually evidenced at the 24 hr observa-
tion, indicating that the onset of the lesion took place
in the interval between the 22.5 and the 24 hr observa-
tions. The only exception was lesion no. 7 (Table 2 and
Figure 4) which was present unusually early at 22.5 hr
after inoculation. First evidence of lesions usually
consisted of a sunken area comprising a group of 6-25
apparently uninjured cells (Figure 4-B). Exceptionally
at this early stage a sunken area contained one or a few
cells which seemed to lack turgor (Table 2, lesion no. 3
and Figure 7-C). These sunken areas always became evident
before any other macroscopic symptom was visible. Sunken
areas were spots of variable size in which epidermal cells,
whether injured or not, appeared to be at a lower plane
than epidermal cells from unaffected areas. This was due
possibly to collapse or lack of turgor of underlying

cells (Figure 4-B). Enlargement of the sunken area and

24

Table l. Epidermal cell response after conventional
mechanical "inoculation" of T. expanse leaves
with distilled water.

_ - -"
4— 4— _ :-

 

 

 

Response of the cell in area 382 mm2
Cell
Type Initial Recoveryb Delayed
injurya injuryc
No. No. % No.
Small 119 19 16 27
Large 107 27 23 15
Total 226 46 20 42

 

aEpidermal cells flaccid or collapsed in replica
made 0.75 'hr after mechanical "inoculation."

bCells that showed initial injury but evidenced
no injury in replica made 47 hr after "inoculation."

CCells apparently intact in the 0.75 hr replica
but evidenced injury in the 47 hr replica.

25

 

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mum maaoo can pomumaco mm: coflmma on» coaumasoocfl

Hmuwm A: ov Amy .ABOHHMV mmum :meSm may CH
mnemomaaoo uumum maaoo cowumasoocfl umumm H: mm
ADV .A3ounmv mufipfloomam 3ocm no» no: Op mono may

mpflmcfl maamo can uco©H>o mammoao muoE ma mono coxcsm
on» "coflumasoocfl umumm H: «N ADV .muflpfloomau
303m uoc Op mono many mpflmcfl maamo .Azouumv ucmpfl>m
mcfleoomn ma mono commoummp HHmEm m "coflumHsoocH
“mums us m.mm Amy .ABOHHMV pampfl>m mum coauomm

ucH mo mEoumewm Ho husncfl HMOHcmcooE m0 muomwmw on
">29 £DH3 coflumHsoocH HMOflcmnomE noumm n: ma can
n: ma Amy .waamHucmsoom some mMOAHmmH >3 poocop
Ifl>o mm mmoa mmcm xm .H mo mHEHocflmm Home: mo mono
so coamma >29 M NO ucwEQOHo>oU can mEoumfiwm haumm

.v mmsmflm

26

 

27

Table 2. Epidermal cells located in sunken lesion area
involved in early stages of TNV local lesion
development on T. expanse.

— — ‘_L
L _ j

 

 

 

Time after inoculation (hr)

 

 

Lesion
No. unin- injured unin- injured unin- injured
jured jured jured
No. No. No. No. No. No.
l -- -- -- -- 16 3
2 -- -- 7 0 8 9
3 -- -- 6 9 22 16
4 -- -- 13 0 34 8
5 -- -- 25 0 21 13
6 -- -- 14 0 23 7
7 15 0 15 0 45 18
8 -- -- 6 0 22 l

 

28

collapse of cells was typical at the 28 hr observation
(Figure 4-D). By this time the lesion was observable
macroscopically for the first time as a very small either
yellow or translucent dot. Further sinking of epidermal
surface and collapsing around the original infection site
resulted in a rapidly expanding lesion which by the 40

hr observatidn (Figure 4-E) was about 1 mm in diameter.
When the number of lesions per leaf was low, lesions were
broadly circular and they expended equally in all direc-
tions reaching 10-12 mm in diameter.

To determine the relationship between infection
as evidenced by lesion formation and the degree of cell
injury following inoculation, more extensive observations
were made soon after inoculation as well as before and
during local lesion development. Fully developed leaves
attached to the plant with the blade 45-50 mm long were
used. Replicas were made approximately 1 hour before
inoculation and at 0.75, 21, 24, 28, 33, and 44 hr
after inoculation. Whenever lesions developed earlier
and were well defined at 33 hr, the 44 hr replica was
omitted. This schedule worked relatively well most of
the time but occasionally a variable number of lesions
developed later than 44 hr and were not recorded by
the replicas. Silicon rubber was applied to approximately
80% of the leaf area usually around the central portion

of the lamina. In most cases 7-30 lesions developed on

29

eacih-leef. By the time the last replica was made, 59
lesions were well defined on the replicated areas of 10
leaves and these were studied in detail (Table 3).
Table 3. Sequence of events preceeding local lesion

formation in epidermal cells following mechani-
cal inoculation of T. expanse leaves with TNV.

 

 

 

 

Sequence of events Instances
0.75 hr ' 21 hr 24-44 hr Number
injury recovery lesion 16
injury injury lesion 18
'healthy' 'healthy' lesion 18
'healthy' injury lesion 7

 

After the replicas were obtained, the leaves were
saved as a reference to locate the lesions and for infec-
tivity assays of infected and healthy portions on cowpea.
TNV was recovered from lesions on either fresh or dried
leaves stored in the laboratory for 3 months at room
temperature (23-27°C). Portions of the same leaves taken
from green leaf areas between local lesions did not in-

fect COWpea.

30

In 16 instances, lesions developed around cells
that followed an injury-recovery-lesion sequence (Figure
5, Table 3). In this sequence a single epidermal cell
showed injury 0.75 .hr after inoculation as evidenced
by lack of turgor. At 21 hr the cell had recovered
partially or totally. Later between 21 and 28 hr, the
same cell was either turgid or collapsed in the center of
a sunken area. Usually after approximately 28 hr or
later the cell that followed the injury-recovery sequence
collapsed approximately in the center of the developing
lesion and was surrounded by other collapsed cells. A
single cell that appeared intact before inoculation (Figure
5-A, arrow) showed the effects of mechanical injury after
0.75 hr (Figure 5-B, arrow), as evidenced by pronounced
loss of turgor. The same cell (Figure 5-C, arrow), re-
covered 21 hours efter inoculation. At 24 hr, the same
single cell (Figure S-D, arrow) lost its turgor again and
began to collapse irreversibly in the center of a small
depressed area. Surrounding cells were slightly flaccid
and wrinkles on the surfaces suggest they were being sub-
jected to stretching forces. At 28 hr, the cell
(Figure S-E, arrow) that followed the injury-recovery
sequence collapsed approximately in the center of the ex-
panded local lesion and was surrounded by other collapsed
cells. At 33 hr, the local lesion was approximately

1 mm in diameter. The cell that followed the

Figure 5.

31

Injury-recovery-lesion sequence. Replicas of a
lesion area on upper epidermis of a T. ex ansa
leaf. (A) Before mechanical inoculation With
TNV all cells appear normal (arrow). (B) 0.75
hr after inoculation a flaccid cell is evident
(arrow). (C) 21 hr: the cell has almost re-
covered (arrow). (D) 24 hr: the cell col-
lapsed (arrow) and also surrounding cells have
begun to collapse. A small sunken area is evi-
dent. Flaccidity of cell (q) caused probably
because manipulation damage. (E) 28 hr:

the sunken area has enlarged around collapsed
cells. The cell that showed the recovery-injury
sequence is approximately in the center of the
sunken area (arrow). (F) 33 hr: the local
lesion approximates 1 mm in diameter. The cell
that showed the injury recovery sequence earlier
is now collapsed in the center of the lesion
(arrow).

32

 

33

injury-recovery sequence (Figure 5-F, arrow) and surround-
ing cells were flat and dry. The ends of palisade cells
were not apparent in the sunken area of any of these
replicas suggesting that underlying cells had also lost
their turgor. In this particular example another cell
was flaccid 24 .hr after inoculation (Figure S-D, cell
q) probably because of manipulation damage. This is the
type of injury referred to in Table 1 as "delayed injury."
Another example of this type of injury occurs in Figure
6-C, cell (Y).

In 18 instances, lesions developed around cells
that followed an injury-injury-lesion sequence (Figure 6,
Table 3). In this sequence a single epidermal cell col-
lapsed irreversibly during the first 0.75 hr after
inoculation. Later a typical local lesion developed
around this cell. Before mechanical inoculation cells
(Figure 6-A, cells x and y) were apparently uninjured.
At 0.75 hr after inoculation (Figure 6-B) one of the
cells (x) showed injury effects as evidenced by moderate
lack of turgor. The other cell (y) remained apparently
intact. At 21 hr (Figure 6-C), the previously damaged
cell (x) and the apparently undamaged cell (y) collapsed
irreversibly. The ends of turgid palisade cells were
evident under both cells (x) and (y). At 24 hr (Figure
6-D), the ends of palisade cells no longer were evident

under cell (x) that was injured 0.75 hr after inoculation

Figure 6.

34

Injury-injury-lesion sequence. Replicas of a
lesion area on upper epidermis of a T. ex ansa
leaf. (A) Before mechanical inoculation Wlth
TNV cells appear apparently uhinjured (x,y).

(B) 0.75 hr after inoculation a flaccid cell
is evident (x). (C) 21 hr: the previously
flaccid cell has collapsed and the ends of
turgid palisade cells are evident under the col-
lapsed cell (x). Another large cell which was
not obviously damaged at 0.75 hr has also col-
lapsed (y), probably because of manipulation
damage. (D) 24 hr: ends of underlying cells
cannot be distinguished under the cell that had
been injured at 0.75 hr, suggesting lack of
turgor of palisade cells (x). Ends of palisade
cells are evident under the more recently col-
lapsed large cell (y). (E) 28 hr: a sunken
area is evident around the cell that had been
injured at 0.75 hr (x). Additional cells are
now present in the sunken area. These have be-
come flaccid around the original cell (x). A
lesion did not develop around the other large
cells (c), and the shapes of underlying palisade
cells are still evident. (F) 33 hr: the le-
sion has enlarged, and contains additional
collapsed (s), and flaccid cells (t). The

cell (x) that had been injured at 0.75 hr is in
the center of the lesion.

35

'1'- ‘ “is“; r
6%
“56%).

f 35 7.
' ‘33:?a \‘b

E

 

36

suggesting loss of turgor of underlying cells. Ends of
palisade cells were evident under the more recently col-
lapsed large palisade cell (y). At .28 hr (Figure 6-E)
a sunken area was evident around cell (x) that had been
injured permanently at 0.75 ‘hr. Additional cells were
also involved in the sunken area. The cells became
flaccid around the earliest collapsed cell (x) and their
wrinkles (2) suggested that they were subjected to stretch-
ing forces originated by difference in level between
epidermal cells in the sunken and normal area. Cell (y)
was collapsed but no lesion as evidenced by a sunken area
developed around it. At 33 hr (Figure 6-F) the lesion
had enlarged and contained additional collapsed (s) and
flaccid cells (t). Cell (x) that had been injured per-
manently at 0.75 hr is in the center of the lesion.

In 7 instances lesions developed around a cell
that followed a 'healthy'-injury-lesion sequence (Figure
7, Table 3). In this sequence, no obvious injury effects
were recorded 0.75 hr after inoculation. At 21 hr,

a single epidermal cell appeared flaccid. This was
located in the center of the lesion which later developed.
Before mechanical inoculation cells were apparently in-
tact (Figure 7-A, cell x). The same cell was apparently
not affected 0.75 hr after inoculation (Figure 7-B,

cell x). At 21 hr (Figure 7-C), the cell showed very

slight flaccidity (x) and was situated in the center Of a

Figure 7.

37

'Healthy'-injury-lesion sequence replicas of a
large area on upper epidermis of a T. ex ansa
leaf. (A) Before mechanical inoculation Wlth
TNV cells appear uninjured. (B) 0.75 hr after
inoculation: no obvious injury is present.
Dark small bodies (arrows) are probably arti-
facts. (C) 21 hr: first evidence of an in-
cipient lesion is a slightly flaccid large cell
within a sunken area (x). (D) 24 hr: the
lesion area is clearly sunken and many cells
have collapsed. (E) 28 hr: the lesion is
defined. (F) 33 hr: the lesion has enlarged.

 

38

 

39

sunken area that had just become evident. At 24 hr
(Figure 7-D), the sunken area was conspicuous and cell

(x) was flaccid. Other cells (y), situated in the sunken
area also showed flaccidity. At 24 hr (Figure 7-E)

the cell that followed the healthy-injury sequence (x)

was situated in the center of small local lesion composed
of collapsed cells. At 33 hr (Figure 7-F), the lesion
had enlarged and cell (x) was located approximately in

the center of the lesion. In 2 instances no sinking of
the area adjacent to the injured cell was evident in the
21 hr replica. Dark small bodies (Figure 7-B, arrows)
probably artifacts, appeared on the cell surface as if
they were particles on or protruding from the surface of
large epidermal cells. They were not consistently associa-
ted with mechanical injury. In closer and more exhaustive
examination most of them proved to be loosely attached to
the surface of the cell and they usually did not appear

in succeeding silicone rubber casts.

The 'healthy'-'healthy'-1esion sequence was ob-
served in 18 lesions (Table 3). In this sequence no ef-
fects of mechanical injury were recorded at 0.75 hr.

The cells appeared to be normal at 21 hr and finally
lesions developed after 24 hr in the same way as in
the 'healthy'-injury-lesion sequence (Figure 7). In this
type of sequence usually no single cell could be identi-

fied as a possible primary infection site due to the

40

tendency of cells in the sunken area to become flaccid or
collapse simultaneously in a relatively short interval.

When all lesions which developed around cells
that followed the injury-recovery-lesion, injury-injury-
lesion and 'healthy'-injury-lesion sequences are con-
sidered as a single group, the ratio of number of lesions
following injury of large cells to the number of lesions
following injury of small cells was approximately 1:3.
If large and small cells were equally susceptible, the
above ratio should be closer to the 1:10 ratio of large
to small cells. Possibly the erumpent surface of the
large cells makes them more vulnerable to infection.

The frequencies of non lesion—forming sequential
events of single epidermal cells were also investigated
(Table 4). Identical areas, about 31 mm2, in both the
0.75 and the 21 hr replicas of one of the inoculated
leaves were compared photographically using composite
pictures measuring 39 x 54 cm. The surveyed area was
selected to include the spot where a lesion developed in
later replicas. Photographic observations were checked
with direct observation of the replicas under the micro-
scope if needed. Slightly more than 1% of the epidermal
cells were injured by the inoculation procedure and of
these injured cells 18% later recovered (Table 4). The
total number of mechanically injured large cells was es-

sentially similar to the number of injured small cells.

Table 4.

41

Frequency of non lesion-forming sequential
events in single epidermal cells in a 31 mm
area of a T. expanse leaf after mechanical
inoculation with TNV.

Sequence of events

 

Frequency

 

Type of cell

 

large small TOTAL

 

0.75 hr 21 hr 44 hr No. No. No.
injury recovery no lesion 5 4 ga
injury injury no lesion 15 25 40b
'healthy' 'healthy' no lesion 358 3,692 4,050
'healthy' injury no lesion 1 3 4
TOTAL 379 3,724 4,103°

 

% of mechanically injured cells (a + b) with reference to
the total (c)

1.2.

% of recovered cells (a) with reference to the injured
cells (a + b)

= 18.4.

42

Also, the number of large and small recovered cells was
roughly similar. Since the large cells were only 10% as
numerous as the small cells, frequency of injury of large
cells was approximately 6 times greater than expected.
This is in essential agreement with an earlier observation
(Table 1). Because the ratio of large to small cells is
approximately 1:10 (Table 4), results in Table 1 also sug-
gest that large cells are comparatively more vulnerable

to mechanical injury than smaller cells. This may be due
to their larger exposed area and raised position in the

epidermis of the leaves.

DISCUSSION

Interpretation of the sequence of events that
ended in the development of a local lesion was based on
two assumptions. First, that the primary site of infec-
tion later became the center of the lesion. Should this
be true, the infected epidermal cell should be situated
in the center of the lesion when the lesion later developed.
Second, that the primary infected cell was the first cell
or one among a small group of cells that collapsed early
in the sunken area produced by virus infection. Although
it was not feasible to establish the identity of the pri-
mary infected cell with absolute precision, sequential
observation of the replicas strongly suggested that these
2 assumptions were correct and that identification of the
primary infected cells was possible when infection was
accompanied by visible cell injury.

It was apparent that the large cells were
somewhat more susceptible to mechanical injury than the
smaller ones. This may have been due to the larger size
and raised position of the larger cells on the cell sur-
face. This result agrees with the suggestion of Matthews
(1970) that it is possible some types of epidermal cells

show different susceptibility to wounding than do others.

43

44

When numbers of lesions originating from wounded
cells were calculated, injured small cells were in the
center of approximately 3 times as many lesions as were
injured large cells. The ratio of small cells to large
cells on the leaf surface was 10 to 1. Thus, injury and
subsequent lesion development of large cells was relatively
higher than would have been anticipated if cells of both
sizes had been equally susceptible. This result and the
fact that establishment of infections depends largely on
cell injury suggest that epidermal cells of T. expanse
probably also differ in degree of susceptibility to in-
fection. Possibly the larger cells are more vulnerable
to infection because of their surface contour or possibly
because of their larger exposed surface.

Epidermal cells were in most cases apparently
unaffected and turgid in the sunken areas that appeared
as first evidence of the lesions. This result strongly
suggests differences in tolerance to virus infection.
Palisade cells were apparently less tolerant and collapsed
earlier than did the epidermal cells thus producing a
sunken area. Epidermal cells were apparently more toler-
ant to virus infection and collapsed sometime later.
Collapse of epidermal cells may have been more nearly the
result of death of underlying palisade cells than that of

direct necrotic effect of virus.

45

Cells which showed "delayed injury” probably re-
flected leaf damage during manipulation. Occasionally the
silicon rubber became sticky and some cells could have
been damaged during the separation of the replica. Bruis-
ing of the lower surface of the leaf produced some injured
cells on the upper epidermal layer of cells. Cells show-
ing "delayed injury" appeared collapsed between 21 and
24 hr. This is the time also when lesions show their
first symptoms. Thus an alternative hypothesis would be
that those cells were local lesions restricted to single
cells.

Existence of an injury-injury—lesion sequence of
events was strongly suggested by the sequential observa-
tions. In this case, injury of the cell was evident in
the 0.75 hr observation. This cell did not recover and
collapsed irreversibly sometime between 0.75 and 21 hr
after inoculation. If the lesion which later appeared
had originated in the cell showing early injury, the
virus apparently had had enough time to multiply in this
injured cell and spread to neighboring cells before the
death of the earlier injured cell.

It is evident that infective virus passed to ad-
jacent cells soon after inoculation. It is not certain
that the virus had multiplied in the initially infected
epidermal cell. Should there have been no multiplication

in the mechanically injured epidermal cell, it would have

./

46

acted as a 'distributor' similar to that suggested by
Benda (1956). Whatever the way virus succeeded in pro-
ducing a local lesion, it is apparent that the injury-
injury-lesion sequence contradicts the current concept
supported by Bawden (1964), Fulton (1964), Matthews (1970),
and many others that the injury to the cell must be "non
lethal" for virus infection. In general the results are
in better accordance with the suggestion of Mundry (1963)
that the possibility exists of infectible wounds being of
different kinds and sizes. Probably more important than
whether the injury is ultimately fatal or not is how long
the cell remained alive after the injury.

In the case of an injury-injury-lesion sequence,
unless the injured cell collapsed completely during the
first 21 hr,‘ we cannot be certain that the injury was
either fatal or that recovery would have later followed.
In the event of later recovery this process would have
been interrupted by the onset of the lesion. Thus, a cell
that probably was going to recover would be indistinguish-
able from a fatally injured cell. This is due to the
relatively long time necessary for cell recovery as evi-
denced by regaining of turgor. This recovery process may
still be going on after approximately 16-21 hr after
injury.

In the case of a 'healthy'-'healthy'-lesion se-

quence, no injury effect was observable preceeding the

47

.lesion formation. Infection may have followed submicro-
scopdc injury or very rapid recovery after slight injury.
In some instances the injury effect evidenced in the 0.75
hr replica was clear but indeed quite small. The most
probably interpretation is that a lesion forms after an
inconspicuous non lethal damage. Nevertheless, these
results suggest that epidermal cells can sustain consider-
able injury and yet recover. The relatively long recovery
process apparently occurred simultaneously with the infec-
tion process. These cells are probably simultaneously
subjected to at least two different types of stress after
mechanical inoculation.

The methods used in this research offer the pos-
sibility of investigating additional factors affecting
the early stages of the infection process by their action
on the host. Among those factors that are known to affect
the infection process are different types of abrasives,
certain buffers, inhibitors, several ions, washing of the
leaves, age of the plants, conditioning of the plant be-
fore inoculation by changing light intensity, temperature,
etc., time of day in which the inoculations are made and
supply of water to the host. Certain factors that in-
crease or decrease the number of infections appearing on

mechanically inoculated leaves in certain host virus

48

combinations can be studied directly in relation to the
increase or decrease in the number of permanently injured

and injured—recovered cells on the host epidermis.

SUMMARY

The sequence of events preceding lesion formation
was observed with a light microscope on the upper epider-

mal surface of Tetragonia expanse leaf cells mechanically

 

inoculated with tobacco necrosis virus (TNV).

Silicone rubber negative replicas of the upper
surface of T. expanse leaves were made before and at in-
tervals following mechanical inoculation with TNV. Posi-
tive replicas were then prepared with transparent nail
polish.

Mechanical injury of epidermal cells in the ab-
sence of virus was followed by injury effects evidenced
by flaccidity or collapse of epidermal cells. Approxi-
mately 20% of the injured cells recovered their turgor
sometime between 16 and 48 hr after inoculation. Col-
lapse was permanent in the rest of the injured cells.

The onset of virus caused lesions was studied on
replicas made between 15 and 40 hr after inoculation.
First evidence of lesions usually appeared between 22.5
and 24 hr after inoculation. It consisted of a sunken
area in which epidermal cells were usually intact. Be-
tween 24 and 28 hr after inoculation epidermal cells

collapsed and the lesion enlarged.

49

50

The relationship between infection and degree of
injury was studied making replicas before and at intervals
after inoculation. The S9 lesions studied were classified
in 4 types according to the sequence of events evidenced
in the replicas.

In 16 instances, a cell that presumably was the
primary point of infection followed an injury-recovery-
lesion sequence. In this sequence, a cell usually ap-
peared flaccid 0.75 hr after inoculation, recovered its
turgor by 21 hr, and later a sunken area appeared
around the cell. Finally, it collapsed surrounded by
other collapsed cells.

In 18 instances, lesions developed around a cell
that followed an injury-injury-lesion sequence. In this
sequence a single epidermal cell collapsed irreversibly
during the first 0.75 hr after inoculation. Later a
typical local lesion developed around this cell.

In 7 instances lesions developed around a cell
that followed a 'healthy'-injury-lesion sequence. In
this sequence, no obvious injury effects were recorded
0.75 hr after inoculation. At 21 hr a single epi-
dermal cell appeared flaccid. This cell was located in
the center of the lesion which later developed.

The 'healthy'-'healthy'-lesion sequence was ob-
served in 18 lesions. In this sequence no effects of

mechanical injury were recorded at 0.75 hr. Cells

51

appeared to be normal at 21 hr and finally lesions
developed after 24 hr.

The frequencies of non lesion-forming sequences
of events occurring in single epidermal cells were also
investigated in a 31 mm2 area containing 4,103 cells.
Slightly more than 1% of the epidermal cells were injured
by the inoculation procedure and of the se injured cells
18% later recovered. Larger epidermal cells were appar—
ently more susceptible to injury than the smaller cells
but, when injured, both types of cells recovered approxi-
mately in the same proportion.

These results suggest that epidermal cells are
capable of sustaining a great deal of injury and yet re-
cover and that infectible wounds can be of different kinds

and sizes.

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.. 1958. The introduction of tobacco mosaic virus
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Bernstein, E. C.; Jones, C. B. 1967. Skin replication

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Binding, H. 1966. Regeneration and Verschmelzung nackter
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Cooking, B. C.; Pojnar, E. 1969. An electron microsco-
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